CHAPTER 26 ENERGY METABOLISM AND MAINTENANCE OF ERYTHROCYTES
CHAPTER 26 ENERGY METABOLISM AND MAINTENANCE OF ERYTHROCYTES
The Utilization of Substrates other than Glucose as Energy Sources
Glutathione Metabolism of the Erythrocyte
Other Red Cell Enzymes
Red cells possess an active metabolic machinery that provides energy to pump ions against electrochemical gradients and to maintain hemoglobin in the reduced form. The main source of metabolic energy comes from glucose. Glucose is metabolized through the glycolytic pathway and through the hexose monophosphate shunt. Glycolysis catabolizes glucose to pyruvate and lactate, which represent the end- products of glucose metabolism in the erythrocyte, because it lacks the mitochondria required for further oxidation of pyruvate. ADP is phosphorylated to ATP and NAD+ is reduced to NADH in glycolysis. Bisphosphoglycerate, an important regulator of the oxygen affinity of hemoglobin, is generated during glycolysis. The hexosemonophosphate shunt oxidizes glucose-6-phosphate, reducing NADP+ to NADPH. In addition to glucose, the red cell has the capacity to utilize some other sugars and nucleosides as a source of energy. The red cell lacks the capacity for de novo purine synthesis but has a salvage pathway which permits synthesis of purine nucleotides from purine bases. The red cell contains high concentrations of glutathione, which is maintained almost entirely in the reduced state by NADPH through the catalytic activity of glutathione reductase. Glutathione is synthesized from glycine, cysteine, and glutamic acid in a two-step process that requires ATP as a source of energy. Catalase and glutathione peroxidase serve to protect the red cell from oxidative damage. The maturation of reticulocytes into erythrocytes is associated with a rapid decrease in the activity of several enzymes. However, the decrease in enzymatic activities of enzymes occurs much more slowly or not at all with ageing.
Acronyms and abbreviations that appear in this chapter include: ADA, adenosine deaminase; ADP, adenosine diphosphate; APRT, adenine phosphoribosyltransferase; ATP, adenosine triphosphate; 2,3-BPG, 2,3-bisphosphoglycerate; cAMP, cyclic adenosine 3′,5′-monophosphate; cGMP, cyclic guanosine 3′,5′-monophosphate; G-6-PD, glucose-6-phosphate dehydrogenase; GSH, reduced glutathione; GSSG, oxidized glutathione; HGPRT, hypoxanthine-guanine phosphoribosyltransferase; LDH, lactate dehydrogenase; NAD, nicotinamide adenine dinucleotide; PEP, phosphoenolpyruvate; R-1-P, ribose 1-phosphate; SCID, severe combined immunodeficiency; UDPG, uridine diphosphoglucose.
Although the binding, transport, and delivery of oxygen do not require the expenditure of metabolic energy by the red blood cell, a source of energy is required if the red cell is to perform its function efficiently and to survive in the circulation for its full life span of approximately 120 days. This energy is needed to maintain (1) the iron of hemoglobin in the divalent form, (2) the high potassium and low calcium and sodium levels within the cell against a gradient imposed by the high plasma calcium and sodium and low plasma potassium levels, (3) the sulfhydryl groups of red cell enzymes, hemoglobin, and membranes in the active, reduced form, and (4) the biconcave shape of the cell. If the red cell is deprived of a source of energy, it becomes sodium- and calcium-logged and potassium-depleted, and the red cell shape changes from a biconcave disc to a sphere. Such a cell is quickly removed from the circulation by the filtering action of the spleen and by a perceptive monocyte-macrophage system. Even if it survived, such an energy-deprived cell would gradually turn brown as hemoglobin is oxidized to methemoglobin by the very high concentrations of oxygen within the erythrocyte. The cell would then be unable to perform its function of transporting oxygen and carbon dioxide.
The process of extracting energy from a substrate, such as glucose, and of utilizing this energy is carried out by a large number of enzymes. Since the red cell loses its nucleus before it enters the circulation and most of its RNA within 1 or 2 days of its release into the circulation, it does not have the capacity to synthesize new enzyme molecules to replace those that may become degraded during its life span. The enzymes present in the red cells were formed largely by the nucleated marrow cell and to a lesser extent by the reticulocyte.
Glucose is the normal energy source of the red cell.1 It is metabolized by the erythrocyte along two major routes, the glycolytic pathway and the hexose monophosphate shunt. The steps in these pathways are essentially the same as those found in other tissues and in other organisms, including even relatively simple ones such as Escherichia coli and yeast. Unlike most other cells, however, the red cell lacks a citric acid cycle. Only the reticulocytes maintain some capacity for the breakdown of pyruvate to CO2 with the attendant highly efficient production of ATP. The mature red cell must content itself with extracting energy from glucose almost solely by anaerobic glycolysis. Before glucose can be metabolized by the red cell, it must pass through the membrane. The membrane contains a carrier2 that can combine with glucose and other sugars at the cell surface and release them at the interior surface of the membrane. The red cell membrane contains insulin receptors,3,4 but the transport of glucose into red cells is independent of insulin.5
PATHWAYS OF GLUCOSE METABOLISM
THE DIRECT GLYCOLYTIC PATHWAY
In the Embden-Meyerhof direct glycolytic pathway (Fig. 26-1), glucose is catabolized anaerobically to pyruvate or lactate. Although 2 moles of high-energy phosphate in the form of ATP are utilized in preparing glucose for its further metabolism, up to 4 moles of ADP may be phosphorylated to ATP during the metabolism of each mole of glucose, giving a net yield of 2 moles of ATP per mole of glucose metabolized. The rate of glucose utilization is limited largely by the hexokinase and phosphofructokinase reactions. Both of the enzymes catalyzing these reactions have a relatively high pH optimum; they have very little activity at pH levels lower than 7. For this reason, red cell glycolysis is very pH-sensitive, being stimulated by a rise in pH. However, at higher-than-physiologic pH levels, the stimulation of hexokinase and phosphofructokinase activity merely results in the accumulation of fructose diphosphate and triose phosphates, because the availability of NAD+ for the glyceraldehyde phosphate dehydrogenase reaction becomes a limiting factor.
FIGURE 26-1 Glucose metabolism of the erythrocyte. The details of the hexose monophosphate pathway are shown in Fig. 26-2.
FIGURE 26-2 The hexose monophosphate pathway of the erythrocyte: (1) glucose-6-phosphate dehydrogenase, (2) glutathione reductase, (3) phosphogluconate dehydrogenase, (4) ribulosephosphate epimerase, (5) ribosephosphate isomerase, (6) transketolase, and (7) transaldolase.
Branching of the metabolic stream after the formation of 1,3- bisphosphoglycerate provides the red cell with flexibility in regard to the amount of ATP formed in the metabolism of each mole of glucose. 1,3-Bisphosphoglycerate may be metabolized to 2,3-bisphosphoglycerate (2,3-BPG), also known as 2,3-diphosphoglycerate (2,3-DPG), thus “wasting” the high-energy phosphate bond in position 1 of the glycerate. Removing the phosphate group at position 2 by bisphosphoglycerate phosphatase results in the formation of 3-phosphoglycerate. Alternatively, 3-phosphoglycerate may be formed directly from 1,3-bisphosphoglycerate through the phosphoglycerate kinase step, resulting in phosphorylation of a mole of ADP to ATP. While metabolism of glucose through the 2,3-BPG step occurs without any net gain of high-energy phosphate bonds in the form of ATP, metabolism through the phosphoglycerate kinase step results in the formation of two such bonds per mole of glucose metabolized. This portion of the direct glycolytic pathway has been called the “energy clutch.”6 Regulation of metabolism at this branch point determines not only the rate of ADP phosphorylation to ATP but also the concentration of 2,3-BPG, an important regulator of the oxygen affinity of hemoglobin (see Chap. 28). The concentration of 2,3-BPG depends on the balance between its rate of formation from 1,3-BPG by bisphosphoglycerate mutase and its degradation by bisphosphoglycerate phosphatase. Hydrogen ions inhibit the bisphosphoglycerate mutase reaction and stimulate the phosphatase reaction. Thus red cell 2,3-BPG levels are exquisitely sensitive to pH: A rise in pH causes a rise in 2,3-BPG levels, while acidosis results in 2,3-BPG depletion. It may be that the ratio of oxyhemoglobin to deoxyhemoglobin also influences 2,3-BPG synthesis by virtue of the fact that only deoxyhemoglobin binds this compound, thus affecting the concentration of free 2,3-BPG that is available for feedback inhibition of the enzymes that lead to its formation. However, the available evidence suggests that the pH is the primary controlling factor.7
Metabolism of glucose by way of the Embden-Meyerhof pathway may also yield reducing energy in the form of NADH. The reduction of NAD+ to NADH occurs in the glyceraldehyde phosphate dehydrogenase step. If NADH is reoxidized in reducing methemoglobin to hemoglobin, the end product of glucose metabolism is pyruvate. If NADH is not reoxidized by methemoglobin, however, pyruvate is reduced in the lactate dehydrogenase step, forming lactate as the final end product of glucose metabolism.1 The lactate or pyruvate formed is transported from the red cell8 and is metabolized elsewhere in the body. Thus the erythrocyte has a flexible Embden-Meyerhof pathway that can adjust the amount of ADP phosphorylated per mole of glucose according to the requirement of the cell.
The regulation of red cell glycolytic metabolism is very complex. Products of some reactions may stimulate others. For example, the pyruvate kinase reaction is exquisitely sensitive to fructose diphosphate, the product of phosphofructokinase. Conversely, other metabolic products may serve as strong enzyme inhibitors. Attempts have been made to construct computer models that simulate this network of reactions,9,10,11 and 12 but the usefulness of such models has been limited by the fact that all of the interactions are not well understood.
THE HEXOSE MONOPHOSPHATE SHUNT
Not all the glucose metabolized by the red cell passes through the direct glycolytic pathway. A direct oxidative pathway of metabolism, the hexose monophosphate shunt, also functions. In this pathway glucose 6-phosphate is oxidized at position 1, yielding carbon dioxide. In the process of glucose oxidation, NADP+ is reduced to NADPH. The pentose phosphate formed when glucose is decarboxylated undergoes a series of molecular rearrangements, eventuating in the formation of a triose, glyceraldehyde 3-phosphate, and a hexose, fructose 6-phosphate (Fig. 26-2). These are normal intermediates in anaerobic glycolysis and thus can rejoin that metabolic stream. Because the glucose phosphate isomerase reaction is freely reversible, allowing fructose 6-phosphate to be converted to glucose 6-phosphate, recycling through the hexose monophosphate pathway is also possible. Unlike the anaerobic glycolytic pathway, the hexose monophosphate pathway does not generate any high-energy phosphate bonds. Its primary function appears to be the reduction of NADP+, and, indeed, the amount of glucose passing through this pathway appears to be regulated by the amount of NADP+ that has been made available by the oxidation of NADPH. NADPH appears to function primarily as a substrate for the reduction of glutathione-containing disulfides in the erythrocyte through mediation of the enzyme glutathione reductase, which catalyzes the conversion of oxidized glutathione (GSSG) to reduced glutathione (GSH) and the reduction of mixed disulfides of hemoglobin and GSH.13 NADP+ also strongly binds to catalase and may effect its activity.14,15
As in the case of anaerobic glycolysis, efforts have been made to construct a computer model of the hexose monophosphate pathway of red cells.16,17 and 18
ENZYMES OF GLUCOSE METABOLISM
Hexokinase catalyzes the phosphorylation of glucose in position 6 by ATP (Fig. 26-1). It thus serves as the first step in the utilization of glucose, whether by the anaerobic or the hexose monophosphate pathway. Mannose or fructose may also serve as a substrate for this enzyme.19 Red cell hexokinase does not phosphorylate galactose.20 The average normal activity of the hexokinase reaction (Table 26-1) is about 5 times the rate of glucose utilization by intact cells. Reticulocytes have much higher levels of hexokinase activity than do mature red cells.26,27 and 28
TABLE 26-1 THE ACTIVITIES OF SOME RED CELL ENZYMES
Hexokinase has an absolute requirement for magnesium. It is strongly inhibited by its product, glucose 6-phosphate, and is apparently released from this inhibition by the inorganic phosphate ion29 and by high concentrations of glucose.30 Inorganic phosphate enhances the rate of glucose utilization by red cells. It has been suggested that this effect is not exerted through hexokinase, but rather through stimulation of the phosphofructokinase reaction, resulting in a lowered glucose 6-phosphate concentration within the cell and thus releasing hexokinase from inhibition.31 GSSG32 and other disulfides and 2,3-BPG33,34 inhibit hexokinase.
The human enzyme resolves into two major bands by electrophoresis.35,36 Designated types IA and IF, both bands actually correspond to type I liver enzyme. Separated chromatographically, the two major fractions of red cell hexokinase have been designated HK and HKR, the latter fraction being unique to erythrocytes and particularly to reticulocytes.37 The hexokinase I gene has been cloned and its structure determined.38 Evidence of an alternative red-blood-cell-specific exon 1 located upstream of the 5′ flanking region of the gene has been obtained38 and a hexokinase cDNA that appears to be unique to erythrocytes has been isolated.39
Rabbit reticulocytes appear to contain hexokinase fractions that are membrane bound and free.40,41 It has been proposed that the ubiquitin-ATP proteolytic system selectively degrades the membrane-bound form of the enzyme, but the physiologic significance of such a process is not clear.42 A small amount of type III hexokinase is also present in erythrocytes.
Hexokinase deficiency is a rare cause of hereditary nonspherocytic hemolytic anemia43,44 (see Chap. 44).
GLUCOSE PHOSPHATE ISOMERASE
Glucosephosphate isomerase catalyzes the interconversion of glucose 6-phosphate and fructose 6-phosphate.1 Electrophoresis resolves the normal enzyme into three bands, all of which are products of the same gene45; mutations that affect electrophoretic mobility are known.45 The human GPI gene has been cloned and its structure and coding sequence determined.45,47 Glucose phosphate isomerase deficiency is one of the causes of hereditary nonspherocytic hemolytic anemia (see Chap. 45).
Red cells contain two distinct types of phosphofructokinase. The classical (or type I) form of the enzyme catalyzes the phosphorylation of the 1-carbon of fructose 6-phosphate by ATP. The type II enzyme, fructose 6-phosphate-2-kinase, phosphorylates the second carbon of fructose 6-phosphate.48 The product of this reaction, fructose 2,6-diphosphate, is a potent allosteric activator of type I phosphofructo kinase. The type I enzyme requires magnesium for activity and is stimulated by ADP, inorganic phosphate, ammonia, and fructose 2,6-diphosphate.49 The existence of the latter effector has been demonstrated in red cells.48
Red cell type I phosphofructokinase exists as a series of tetramers comprised of muscle (M) and liver (L) subunits. A platelet (P) subunit has also been identified.50 The M and L subunits of phosphofructo kinase I and phosphofructokinase II have been cloned and sequenced.51,52 and 53 Deficiency of type I phosphofructokinase, which may be associated with mild hemolytic anemia and with type VII glycogen storage disease, is discussed in Chap. 44.
Aldolase reversibly cleaves fructose 1,6-diphosphate into two trioses. The “upper” half of the fructose 1,6-diphosphate molecule becomes dihydroxyacetone phosphate (DHAP), and the “lower” half glyceraldehyde-3-phosphate. Red cells contain aldolase A, as is found in muscle, and no aldolase B (liver aldolase). On isoelectric focusing of hemolysates, however, five isoenzymes can be resolved, as is the case with other tissues.54 The isoenzymes presumably represent mixed tetramers of native a polypeptide chains and chains that have undergone posttranscriptional deamidation, a’ chains. Young red cells contain more of the nondeamidated isoenzymes. Aldose A has been cloned and sequenced.55,56 Aldolase deficiency is a rare cause of hereditary nonspherocytic hemolytic anemia (see Chap. 44).
Triosephosphate isomerase (TPI) is the enzyme of the anaerobic glycolytic pathway that has the highest activity. Its metabolic role is to catalyze interconversion of the two trioses formed by the action of aldolase—dihydroxyacetone phosphate and glyceraldehyde-3-phosphate.1 Although equilibrium is in favor of dihydroxyacetone phosphate, glyceraldehyde-3-phosphate undergoes continued oxidation through the action of glyceraldehyde phosphate dehydrogenase and is thus removed from the equilibrium. The gene encoding TPI has been cloned and sequenced.57 A polymorphism in the promotor region58 of uncertain significance has been identified.59 A deficiency of TPI has been found in patients with hereditary nonspherocytic hemolytic anemia associated with a severe neuromuscular disorder (see Chap. 44).
GLYCERALDEHYDE PHOSPHATE DEHYDROGENASE
Glyceraldehyde phosphate dehydrogenase performs the dual functions of oxidizing and phosphorylating glyceraldehyde-3-phosphate, producing 1,3-BPG. In the process, NAD+ is reduced to NADH. This enzyme is closely associated with the red cell membrane.60 A two- to threefold stimulation of activity by hemoglobin could have a regulatory role.61
Phosphoglycerate kinase effects the transfer to ADP of the high-energy phosphate from the 1-carbon of l,3-BPG to form ATP. The reaction is readily reversible. Electrophoretically detectable mutations of the enzyme have been described,62,63 and their transmission in families confirms that the structural gene for phosphoglycerate kinase is sex-linked. The amino acid sequence of phosphoglycerate kinase has been determined,64 the cDNA for phosphoglycerate kinase has been cloned and sequenced,65 and linkage relationships on the X-chromosome determined.66 Deficiency of phosphoglycerate kinase is a cause of nonspherocytic hemolytic anemia, often associated with neuromuscular abnormalities (see Chap 54).
The same protein molecule is responsible for both bisphosphoglycerate mutase and bisphosphoglycerate phosphatase activities in the erythrocyte.67,68 This enzyme is particularly important because it regulates the concentration of 2,3-BPG of erythrocytes. In its role as a bisphospho glyceromutase, the enzyme competes with phosphoglycerate kinase for 1,3-bisphosphoglycerate as a substrate. It changes 1,3-bisphosphoglycerate to 2,3-bisphosphoglycerate, thereby dissipating the energy of the high-energy acylphosphate bond.69 It is inhibited by its product, 2,3-bisphosphoglycerate, and by inorganic phosphate, and it is activated by 2-phosphoglycerate and by increased pH levels. It requires 3-phosphoglycerate for activity. Bisphospoglycerate phosphatase catalyzes the removal of the phosphate group from carbon 2 of 2,3-BPG.69 It is inhibited by its product, 3-phosphoglycerate, and by sulfhydryl reagents. It is most active at a slightly acid pH and is strongly stimulated by bisulfite and phosphoglycolate.
A deficiency of bisphosphoglyceromutase-bisphosphoglycerate phosphatase results in a marked decrease in red cell 2,3-BPG levels. The consequent left shift of the oxygen dissociation curve leads to polycythemia (Chap. 61). The cDNA of the enzyme has been cloned and sequenced.70,71
Phosphoglycolate, the most potent activator of phosphatase activity, is present in erythrocytes at very low concentrations,72,73 but the source of this substance in red cells is a mystery.74,75 and 76 Phosphoglycolate phosphatase, the enzyme that hydrolyzes phosphoglycolate has also been identified in erythrocytes.77,78
An equilibrium is established between 3-phosphoglycerate and 2-phosphoglycerate by phosphoglyceromutase.79,80 2,3-Bisphosphoglycerate acts as an essential cofactor for the transformation.
Enolase establishes an equilibrium between 2-phosphoglycerate and phosphoenolpyruvate (PEP).81 Electrophoresis of red cell enolase gives three bands, supporting the suggestion that it is composed of two different subunits that associate randomly into dimers.82
The transfer of phosphate from PEP to ADP, forming ATP and pyruvate, is catalyzed by pyruvate kinase.83 This is one of the energy- yielding steps of glycolysis. There are two major types of pyruvate kinase. The R type of enzyme found in erythrocytes closely resembles the L or liver enzyme; both are products of the same gene. The minor differences between the liver and red cell enzyme are due to differences in RNA processing84 (see Chap. 8). Leukocytes contain type M or muscle enzyme. It is quite different in its kinetic properties and is the product of a different gene. Red cell pyruvate kinase is an allosteric enzyme, manifesting sigmoid kinetics with respect to PEP in the absence of fructose diphosphate. Hyperbolic kinetics are observed in the presence of even minute amounts of fructose diphosphate,85,86 so that at low concentrations of PEP the enzyme activity is greatly increased by fructose diphosphate. Genes for both the L and M type enzymes have been cloned.87,88 Pyruvate kinase deficiency is the most common cause of nonspherocytic hemolytic anemia (Chap. 44).
Lactate dehydrogenase (LDH) catalyzes the reversible reduction of pyruvate to lactate by NADH. The enzyme is composed of H (heart) and M (muscle) subunits. In red cells the predominant subunit is LDH-H.89 However, hereditary absence of the H subunit seems to be a benign condition, usually without clinical manifestations,89,90,91 and 92 although one case with hemolysis has been reported.93 Absence of the M subunit has been reported as well89 and was unaccompanied by hematologic manifestations. Judging from the origin of the reports, LDH deficiency appears to be most common in Japan, where population surveys show a gene frequency of approximately 0.05 for each deficiency,94,95 and several frame-shift mutations have been identified.95
G-6-PD is the most extensively studied erythrocyte enzyme.94,96 It catalyzes the oxidation of glucose 6-phosphate to 6-phosphogluconolactone, which is rapidly hydrolyzed to 6-phosphogluconic acid. NADP+ is reduced to NADPH in the reaction. The enzyme has been crystallized,97 and its gene98 and cDNA99,100 and 101 cloned and sequenced. The structure of the Leuconostoc mesenteroides enzyme has been deduced from its crystal structure.102
Much information is available regarding substrate specificity, Michaelis constants, and pH optimum curves. The Mr of the highly purified enzyme has been reported to be 240,000 daltons,97 but in its natural state the Mr is probably approximately 105,000 daltons.103,104 In the absence of NADP+ G-6-PD dissociates into inactive subunits. The computed subunit Mr is 59,256. The enzyme is strongly inhibited by physiologic amounts of NADPH105 and, to a lesser extent, by physiologic concentrations of ATP.106,107 It is much more active in reticulocytes than mature red cells.27,28 Many electrophoretic mutations are known, as are others involving the activity, stability, and kinetic properties of the enzyme (see Chap. 44).
Although 6-phosphogluconolactone, the direct product of the oxidation of glucose-6-phosphate by glucose-6-phosphate dehydrogenase hydrolyzes spontaneously at a relatively rapid rate at a physiologic pH, enzymatic hydrolysis is much more rapid and is required for normal metabolic flow through the stimulated hexose monophosphate pathway.108,109 Partial deficiency of the enzyme has been observed110 and is probably benign.111
Phosphogluconate dehydrogenase catalyzes the oxidation of phosphogluconate to ribulose 5-phosphate and CO2 and the reduction of NADP+ to NADPH. Variability of electrophoretic mobility of the enzyme is common in humans and in several animal species.112 Deficiency of the enzyme has been observed only rarely and appears to be essentially innocuous.113
Ribosephosphate isomerase catalyzes the interconversion of ribulose 5-phosphate and ribose 5-phosphate.33,114
Ribulosephosphate epimerase converts ribulose 5-phosphate to xylulose 5-phosphate.33 The exact activity of this enzyme in human hemolysates has not been reported but seems to be less than that of ribosephosphate isomerase.
Transketolase effects the transfer of two carbon atoms from xylulose 5-phosphate to ribose 5-phosphate, resulting in the formation of the 7-carbon sugar sedoheptulose 7-phosphate and the 3-carbon sugar glyceraldehyde 3-phosphate.33,115 It can also catalyze the reaction between xylulose 5-phosphate and erythrose 4-phosphate, producing fructose 6-phosphate and glyceraldehyde 3-phosphate. Thiamine pyrophosphate is a coenzyme for transketolase, and the activity of erythrocyte transketolase has been used as an index of the adequacy of thiamine nutrition.116,117
The conversion of seduhepulose 7-phosphate and glyceraldehyde 3- phosphate into erythrose 4-phosphate and fructose 6-phosphate is catalyzed by transaldolase.115 This is another one in the series of molecular rearrangements that eventuate in the conversion of the 5-carbon sugar formed in the phosphogluconate dehydrogenase step to metabolic intermediates of the Embden-Meyerhof pathway.
Red cells contain L hexonate dehydrogenase, an enzyme that has the capacity to reduce aldoses such as glucose, galactose, or glyceraldehyde to their corresponding polyol (i.e., glucose to sorbitol, galactose to dulcitol, and glyceraldehyde to glycerol). NADPH serves as a hydrogen donor for this reaction.118 Aldose reductase,119 another enzyme that can catalyze this reaction, may also be present in red cells.
THE UTILIZATION OF SUBSTRATES OTHER THAN GLUCOSE AS ENERGY SOURCES
The red cell has the capacity to utilize several other substrates in addition to glucose as a source of energy. Among these are adenosine, inosine, fructose, mannose, galactose, dihydroxyacetone, and lactate. Although in the circulation red cells normally rely on glucose as their energy source, the utilization of other substrates, particularly during blood storage (see Chap. 138) and in certain experimental situations, is of interest.
ADENOSINE AND INOSINE
Adenosine has been used as an experimental blood preservative, and it has been suggested that it may also be metabolized by human red cells in vivo.120 Adenosine is deaminated to inosine by the enzyme adenosine deaminase (ADA)121:
It apparently plays a regulatory role in the concentration of purine nucleotides in the red cell. Deficiency of ADA is associated with SCID122,123 (Chap. 86). In this disorder, large quantities of deoxyadenine nucleotides, not normally present in erythrocytes, accumulate. Hereditary increase in activity of erythrocyte ADA results in the depletion of red cell ATP and nonspherocytic hemolytic anemia.124 For reasons that are not understood, ADA activity also increases in the red cells of AIDS patients125,126 and of those with Diamond-Blackfan anemia.127
Inosine formed in the ADA reaction or added directly to red cells may enter the erythrocyte and undergo phosphorolysis to form hypoxanthine and ribose 1-phosphate (R-1-P):
This reaction is of particular interest because it results in the introduction of a phosphorylated sugar, R-1-P, into the erythrocyte without the utilization of ATP.121,128 The R-1-P may then be further metabolized to yield high-energy phosphate. The nucleoside phosphorylase reaction appears to be the only practical means by which ATP may be formed in the cell without first expending ATP to prepare an unphosphorylated substrate for further metabolism. The use of inosine has therefore received much attention in the field of blood banking (see Chap. 138). A deficiency of nucleoside phosphorylase has been associated with immunodeficiency.123
Fructose is readily utilized by the erythrocyte, although at a rate somewhat slower than that of glucose.129 Fructose undergoes phosphorylation at position 6 in the hexokinase reaction:
Fructose 6-phosphate is a normal metabolic intermediate in the anaerobic glycolytic pathway. Thus, the result of fructose phosphorylation is exactly the same as the result of the phosphorylation of glucose.
Fructose may also be metabolized by another red cell enzyme, sorbitol dehydrogenase.130,131 This enzyme reduces fructose to its corresponding polyol, sorbitol, with NADH serving as a hydrogen donor. The reaction is reversible, and a pathway therefore exists for the formation of fructose from glucose through L hexonate dehydrogenase and sorbitol dehydrogenase. An enzyme that facilitates sorbitol permeation through the erythrocyte membrane has been described.
Mannose is also phosphorylated in the hexokinase reaction19:
Mannose 6-phosphate must be isomerized to fructose 6-phosphate before it is further metabolized by erythrocytes. This is accomplished by phosphomannose isomerase132,133:
Phosphomannose isomerase of red cells has very low activity, even at its pH optimum of 5.9.19 The rate of mannose utilization is therefore limited by the activity of phosphomannose isomerase. Young red cells have enhanced phosphomannose isomerase activity and can therefore utilize mannose at a more rapid rate than can mature red cells.
The utilization of galactose by erythrocytes is more complex than that of most other substrates. At low concentrations of galactose, metabolism occurs by way of galactokinase, galactose-1-phosphate uridyl transferase, and phosphoglucomutase.134 Unlike fructose, mannose, and glucose, galactose is phosphorylated at position 1:
The galactose-1-phosphate formed in the galactokinase reaction exchanges with the glucose-1-phosphate moiety of uridine diphosphoglucose (UDPG) in the galactose-1-phosphate uridyl transferase reaction:
The uridine diphosphogalactose (UDPgalactose) formed in this reaction is epimerized to UDPG:
The a-glucose-1-phosphate in the transferase reaction is transformed to a-glucose-6-phosphate in the phosphoglucomutase (PGM) reaction135 with glucose 1,6-diphosphate acting as coenzyme:
The a-glucose-6-phosphate formed may join the direct metabolic stream after conversion by phosphoglucose isomerase to fructose 6-phosphate. It may also undergo anomerization to b-glucose-6-phosphate and enter the hexose monophosphate pathway, if NADP+ is available. Very high concentrations of galactose appear to be metabolized by way of another pathway, as yet poorly delineated. This pathway is known not to involve galactose-1-phosphate uridyl transferase or to have the capacity to reduce NAD+.20
DIHYDROXYACETONE AND GLYCERALDEHYDE
As indicated earlier, glyceraldehyde can be reduced in erythrocytes to glycerol in the L hexonate dehydrogenase reaction. In addition, glyceraldehyde and dihydroxyacetone can each be phosphorylated by ATP in the presence of the enzyme triokinase.136 Like other kinases, this enzyme has a requirement for magnesium. A remarkable feature of this enzyme is its extraordinarily low Km for dihydroxyacetone. It is one-half saturated with this substrate at a concentration of only 0.5 µM. The products of the triokinase reaction, dihydroxyacetone phosphate or glyceraldehyde-3-phosphate, are normal metabolic intermediates and can be metabolized in the usual fashion. Because of its capacity to act as an alternative substrate for red cell energy metabolism and 2,3-BPG formation, dihydroxyacetone had been studied as an experimental additive for blood storage.137,138
Red cells have the capacity to form and to break down glycogen. They contain the enzymes UDPG-glycogen glucosyltransferase and a-1,4-glucan: a-1,4-glucan-6-glycosyltransferase (the brancher enzyme) for the formation of glycogen from glucose 1-phosphate. Red cells also contain the enzymes phosphorylase and amylo-1,6-glucosidase (the debrancher enzyme) for the breakdown of glycogen.139 Only very little glycogen is present in normal red cells,140 and most of what was thought to be in red cells may actually be platelet and leukocyte glycogen.141 The function of glycogen in red cell metabolism is not understood.
GLUTATHIONE METABOLISM OF THE ERYTHROCYTE
The red cell contains a high concentration (approximately 2 mM) of the sulfhydryl-containing tripeptide reduced glutathione.1 Red cell GSH appears to undergo a rapid turnover, with a approximately 4 days.142 Synthesis occurs in two steps:
Both steps are catalyzed by red cell hemolysates.22 The red cell requires a system for the synthesis of GSH because of the active transport of GSSG from the erythrocyte.143 It has also been suggested that a requirement for GSH synthesis comes from the amino acid transporting function of the g-glutamyl cycle.144 However, this pathway is not present in red cells.145,146 and 147
One important function of GSH in the erythrocyte appears to be the detoxification of low levels of hydrogen peroxide that may form spontaneously or as a result of drug administration. In either event, the superoxide radical may be formed first and then be converted to H2O2 by the action of the copper-containing enzyme superoxide dismutase.148 Hydrogen peroxide is reduced to water through the mediation of the enzyme glutathione peroxidase.149,150 Glutathione peroxidase is a selenium-containing enzyme.151 In New Zealand, dietary selenium intake is extremely low, and glutathione peroxidase activities are much lower than are observed elsewhere.152 A polymorphism affecting the activity of the enzyme which is most common in persons of Mediterranean descent153 has also been described. The consequent decreases in enzyme activity are without clinical effect. The genes for several glutathione peroxidases, including that of the erythrocyte, have been cloned.154 The triplet UGA usually acts as a stop codon in this particular message and inserts selenocysteine in the proper location.155 A unique tRNA that has complementary UCA anticodons is aminoacylated with serine. The seryl-tRNA is then converted to selenocysteyl-tRNA and is delivered to the ribosome.156 Recognition elements within the mRNAs are essential for translation of UGA as selenocysteine rather than the usual stop codon.156
GSH also functions in maintaining integrity of the erythrocyte by reducing sulfhydryl groups of hemoglobin, membrane proteins, and enzymes that may become oxidized.157 In the process of reducing peroxides or oxidized protein sulfhydryl groups, GSH is converted to GSSG or may form mixed disulfides. GSSG, like certain other disulfides, has the capacity to inhibit red cell hexokinase,32,158 although greater-than-physiologic levels appear to be needed for this effect. It may also complex with hemoglobin A to form hemoglobin A3.159 Glutathione reductase provides an efficient mechanism for the reduction of GSSG to GSH in the red cell. It is a flavin enzyme, and either NADPH or NADH may serve as a hydrogen donor.160,161 In the intact cell, only the NADPH system appears to function.162 The same enzyme system appears to have the capacity to reduce mixed disulfides of GSH and proteins.13 Although inherited deficiencies of this enzyme exist,163 the activity of red cell glutathione reductase is strongly influenced by the riboflavin content of the diet.164 Red cells also contain thioltransferase that can catalyze GSH-dependent reduction of some disulfides.165
Oxidized glutathione is actively extruded from the erythrocyte166,167 by a system consisting of at least two GSSG-activated ATPases that serve as an enzymatic basis for this transport process.168 In addition to transporting GSSG, the system appears to have the capacity to transport thioether conjugates of GSH and electrophiles formed by the action of glutathione-S-transferase.169,170 Blood cells, specifically including erythrocytes, contain a glutathione-S-transferase that is distinct from the predominant liver forms of the enzyme. This enzyme, designated type III or r to distinguish it from the liver enzymes catalyzes the formation of a thioether bond between GSH and a variety of xenobiotics. The role of glutathione-S-transferase in the erythrocyte has not been established. It may be that it serves to cleanse the blood of xenobiotics to which the red cell membrane is permeable. Glutathione-S-transferase could conjugate such substances to glutathione, and the detoxified product of conjugation would be transported out of the red cell for subsequent disposal. The enzyme has the capacity to reversibly bind heme, and a possible role in heme transport has been postulated.171
Fairly severe deficiency of this enzyme has been associated with hemolytic anemia, but a cause-and-effect relationship has not been established.172
The reduction of methemoglobin in normal red cells is achieved primarily through a NADH-linked system173 (Chap. 48). A methemoglobin reductase (known also as NADH diaphorase or cytochrome b5 reductase) utilizes NADH generated in the glyceraldehyde-phosphate dehydrogenase reaction to reduce cytochrome b5, which in turn reduces the iron of methemoglobin from the trivalent to the divalent form. The gene for this enzyme has been cloned and sequenced.174,175
Red cells also contain a NADPH-linked methemoglobin-reducing system176,177 that functions only in the presence of an artificial electron carrier, such as methylene blue. Nonenzymatic reduction of methemoglobin by GSH and ascorbic acid accounts for only a small portion of the total methemoglobin-reducing rate of red cells.
OTHER RED CELL ENZYMES
Erythrocytes contain a high concentration of carbonic anhydrase I. In catalyzing the equilibrium between carbon dioxide and carbonic acid, this enzyme aids in oxygen and carbon dioxide transport of the erythrocyte. This enzyme has been obtained from red cells in a highly purified state, and the cDNA has been cloned.178
The red blood cell is a rich source of catalase, the enzyme that decomposes hydrogen peroxide to water and oxygen. Hereditary lack of catalase does not seem to cause any hematologic disorder.179,180 This enzyme functions efficiently only when relatively high concentrations of peroxide are present. Low concentrations of peroxide are detoxified by the enzyme glutathione peroxidase.149,150 There has been an ongoing controversy regarding whether catalase or glutathione peroxidase is the more important means for the protection of the erythrocyte against free radicals15,181,182 and 183; however, it is difficult to understand why one would have to be more important than the other. Either system alone appears to suffice, since deficiencies of either are well tolerated. Superoxide dismutase, a copper-containing enzyme, is also present in erythrocytes.184 It presumably plays an important role in the protection of hemoglobin and other red cell components against a highly reactive superoxide anion. It has been suggested that red cells contain thioredoxin, thioredoxin reductase,185 and glutaredoxin,186 but it is possible that these enzymes might have been present in contaminating leukocytes, since these were inadequately removed. If this system does exist in erythrocytes, it could serve as another defense mechanism against the oxidative damage to which erythrocytes are vulnerable because of their large load of oxygen that is continually being bound by and released by hemoglobin. Erythrocytes are a primary means for the removal not only of nitric oxide but also of nitric oxide synthase-1.187
The red cell membrane contains large amounts of acetylcholinesterase.188 Although the activity of this enzyme is diminished in paroxysmal nocturnal hemoglobinuria, this does not play an etiologic role. It is merely a manifestation of the underlying defect in the phosphotidyl-inositol anchor (see Chap. 35). Hereditary lack of red cell cholinesterase activity is not associated with any clinical hematologic effects.189 AMP-deaminase190 seems to be particularly important because of the regulatory role that it plays in the levels of adenine nucleotides in the red cell. An entirely separate enzyme from ADA, AMP deaminase removes ammonia from the adenine moiety of AMP, converting it to inosine monophosphate. A severe deficiency of this enzyme has been reported,191 and this resulted in elevated red cell ATP levels. However, there were no hematologic consequences.
Red cell membranes also contain protein kinase activities.192 Several such enzymes catalyze the transfer of the terminal phosphate from ATP to various cytoskeletal protein acceptors, primarily band 2 of spectrin and “band 3” (see Chap. 26). One protein kinase is relatively insensitive to stimulation by cAMP and unaffected cGMP. It has the capacity to phosphorylate exogenous protein acceptors, such as casein and histones, as well as endogenous cytoskeletal proteins. In rabbit erythrocytes membrane-associated casein kinase was found to show striking age-dependency.193 The role of this enzyme in the structural properties of the red cell membrane is not yet clear. Several proteolytic systems have been defined in erythrocytes. One of these, calpain, is activated by high concentrations of calcium.194 Reticulocytes contain ubiquitin that, together with ATP and several partially defined enzyme activities, may serve as an important mechanism for the destruction of mitochondrial matrix and unneeded enzymes as the red cell matures from the reticulocytes to the erythrocyte stage.195 The red cell membrane is also believed to contain neutral protease activities.196 Multicatalytic proteinase197,198 and 199 or enzymes that closely resemble it have also been found.200
Several distinct membrane ATPases have been characterized. Each seems to serve a transport function, and each is stimulated to hydrolyze ATP by the substance that is transported. Thus, the Na+- K+-ATPase is a membrane enzyme that functions to move sodium out of the red cell and potassium in at a fixed ratio of two potassium ions for each three sodium ions.201 It appears to have a requirement for bound lipid202,203 and is inhibited by ouabain. A Ca2+ 204,205 serves to extrude calcium from the red cell. It binds calmodulin,204 a regulator of calcium transport that has also been identified in red cells.206 The function of Mg2+-ATPase207 is less clear, since all ATPases require magnesium ions for their function. Guanosine triphosphatase208 and inosine triphosphate209,210 activities have also been characterized in red cells. G-proteins are present.211,212
An aldehyde dehydrogenase of red cells makes it possible for erythrocytes to utilize aldehydes such as formaldehyde as substrates for methemoglobin reduction,213,214 and the enzyme may play a role in drug detoxification.215,216 The presence and usually the characteristics of amino acid-activating enzymes,217 dipeptidases,218 formate-activating enzyme,219 glutamic-oxaloacetic transaminase,1 glyoxalase,220 pyridoxine kinase,221,222 uroporphyrinogen 1 synthetase,223 ribonucleases,224 pyrroline-5-carboxylate reductase,225 acid phosphatase,226 prolidase,227,228 nucleoside diphosphokinase,229 (ADP-ribose)n glycohydrolase,230 ribonuclease,224 ribonuclease inhibitor,231 arylamine-N-acetyltransferase,232 phosphatidylinositol 3-monophosphate-4-kinase,233 phosphatidylinositol 4-kinase,233 protein palmitoyl acyltransferase,234 calpromotin,216,235 D dopachrome tautomerase,236 thiopurine methyltransferase,237 UMP synthetase,238 and numerous other enzymes239 have been reported in erythrocytes.
Most cells achieve the de novo synthesis of purine nucleotides by constructing the heterocyclic purine ring in a series of enzymatic reactions that begin with the synthesis of phosphoribosyl pyrophosphate (PRPP) from ribose 5-phosphate and ATP. Methyl groups are added through the mediation of folate coenzymes, and nitrogens are supplied by glutamine, lysine, and aspartic acid. The initial product of the de novo pathway, inosine 5′-monophosphate (IMP), is then converted to AMP and to guanosine 5′-phosphate through further enzymatic transformations. Pyrimidine nucleotides are synthesized de novo through pathways beginning with the reaction of carbamyl phosphate and aspartic acid to form carbamyl aspartate. Further intermediates include dihydroorotate, orotate, and orotidine 5′-phosphate, which is finally converted to uridine 5′-phosphate.
Although all the reactions for synthesis of purine and pyrimidine nucleotides presumably occur in erythroid precursors, the mature erythrocyte depends on the so-called salvage pathway for its supply of purine nucleotides. The adenine phosphoribosyltransferase (APRT) and hypoxanthine-guanine phosphoribosyltransferase (HGPRT) reactions serve to incorporate adenine (in the case of APRT) or hypoxanthine or guanine (in the case of HGPRT) into nucleotides:
The first of these reactions is the basis for the use of adenine in blood preservatives (Chap. 138). Absence of APRT,240 inherited as an autosomal recessive disorder, results in nephrolithiasis, deoxyadenine stones being deposited in the kidneys.241,242 and 243 The function of HGPRT in red cells is unclear, since the role of guanine and inosine nucleotides remains undefined. Absence of this enzyme, inherited as a sex-linked disorder, results in hyperuricemia and a neurologic disorder characterized by self-mutilation, the so-called Lesch-Nyhan syndrome.244,245 Red cells are also able to synthesize adenine nucleotides by phosphorylating adenosine. This reaction is catalyzed by adenosine kinase.246 The bridge between ribonucleotides and deoxyribonucleotides is provided by the enzyme ribonucleotide reductase. All dividing cells require deoxyribonucleotides for DNA synthesis. Ribonucleotides are needed not only for the synthesis of RNA for protein synthesis but also to perform many other functions. For example, ATP and GTP provide the energy for many biochemical processes and serve as precursors of cyclic nucleotides, the regulators of many enzymatic reactions. Uridine nucleotides are sugar carriers that serve as intermediates in various carbohydrate transformations and in the synthesis of glycoproteins and glycolipids.
The mature erythrocyte contains small quantities of pyrimidine nucleotides. Little is known of their function in this cell. The capacity of erythrocytes to metabolize galactose reflects one function of a pyrimidine nucleotide, UDPGlucose, in the erythrocyte. However, since the red cell is a trivial site of galactose metabolism in the body, this function of the pyrimidine nucleotide can hardly be considered of much physiologic importance. The enzyme pyrimidine 5′-nucleotidase247 specifically dephosphorylates pyrimidine mononucleotides and thus presumably plays a role in the catabolism of ribose polynucleotides in the red cell.
The nicotinic acid nucleotides NAD+ and NADP+ are also a vital component of the biochemical machinery of the cell, and pathways for their synthesis exist. NAD+ is synthesized from nicotinic acid as shown in Fig. 26-3. PRPP is attached to the nicotinic acid ring through the mediation of the enzyme desamido-NMN pyrophosphorylase, forming desamido nicotinic acid mononucleotide. After attachment of AMP through a pyrophosphate bond, glutamine provides an amino group for completion of the synthesis of NAD+.248,249 and 250 The only known pathway for the synthesis of NADP+ involves phosphorylation of NAD+ by ATP in the presence of NAD kinase.218,251 Large oral doses of nicotinic acid promote an increase in the concentration of red cell NAD+ but not of NADP+.252 NAD+ is degraded by the enzyme NADase, which hydrolyzes the pyridine nucleotides at the nicotinamide-ribose linkage. The enzymes can catalyze the exchange of free nicotinamide with pyridine nucleotide-bound nicotinamide.218 A deficiency of this activity, apparently without significant effect, has been documented.253
FIGURE 26-3 The nicotinic acid pathway for the biosynthesis of nicotinamide adenine dinucleotide.
The energy metabolism of reticulocytes is more active than that of older erythrocytes. The activity of enzymes that are important in regulating the rate of glycolysis is increased in reticulocytes. Evidence is mounting that there is an abrupt decrease of a number of glycolytic enzymes as reticulocytes mature.27,254,255 and 256 These observations stand in contrast to the earlier held view that a gradual decrease in activities of red cell enzymes occurred throughout their life span.257 This concept was based on the assumption that there was a sufficiently consistent increase in density of erythrocytes during their ageing in the circulation to make possible meaningful separations of red cells into different age groups by density centrifugation. In reality, this does not seem to occur, and the apparent decrease in the activity of many enzymes based on such studies may well be due to decreasing contamination of layers of different density with reticulocytes.
In addition to their capacity to carry out glycolysis at a relatively rapid rate, reticulocytes have mitochondria with a complete complement of mitochondrial enzymes.258 This enables them to metabolize glucose not only through the Embden-Meyerhof pathway and hexose monophosphate shunt but also through the Krebs cycle. The rate of oxygen consumption of reticulocytes is 60 times that of mature red cells; the rate of glucose consumption is 7.5 times as great.259 This metabolic capacity provides these cells with the potential of phosphorylating ADP to ATP at a greatly accelerated rate and of providing succinate for the synthesis of heme.
Beutler E: Red Cell Metabolism: A Manual of Biochemical Methods. Grune & Stratton, New York, 1984.
Baldwin SA, Lienhard GE: Purification and reconstitution of glucose transporter from human erythrocytes. Methods Enzymol 174:39, 1989.
Herzberg V, Boughter JM, Carlisle S, Hill DE: Evidence for two insulin receptor populations on human erythrocytes. Nature 286:279, 1980.
Robinson TJ, Archer JA, Gambhir KK, Hollis VW Jr, Carter L, Bradley C: Erythrocytes: a new cell type for the evaluation of insulin receptor defects in diabetic humans. Science 205:200, 1979.
Eadie GS, MacLeod JJR, Noble EC: Insulin and glycolysis. Am J Physiol 65:462, 1923.
Keitt AS, Bennett DC: Pyruvate kinase deficiency and related disorders of red cell glycolysis. Am J Med 41:762, 1966.
Gerlach E, Duhm J, Deuticke B: Metabolism of 2,3-diphosphoglycerate in red blood cells under various experimental conditions, in Red Cell Metabolism and Function, edited by E Beutler, p 155–174. Plenum, New York, 1970.
Poole RC, Halestrap AP: Identification and partial purification of the erythrocyte L lactate transporter. Biochem J 283:855, 1992.
Heinrich R, Rapoport SM: The utility of mathematical models for the understanding of metabolic systems. Biochem Soc Trans 11:31, 1983.
Joshi A, Palsson BO: Metabolic dynamics in the human red cell. Part II—Interactions with the environment. J Theor Biol 141:529, 1989.
Reddy VN, Liebman MN, Mavrovouniotis ML: Qualitative analysis of biochemical reaction systems. Comput Biol Med 26:9, 1996.
Mulquiney PJ, Kuchel PW: Model of the pH-dependence of the concentrations of complexes involving metabolites, haemoglobin and magnesium ions in the human erythrocyte. Eur J Biochem 245:71, 1997.
Srivastava SK, Beutler E: Glutathione metabolism of the erythrocyte. The enzymic cleavage of glutathione-haemoglobin preparations by glutathione reductase. Biochem J 119:353, 1970.
Scott MD, Wagner TC, Chiu DTY: Decreased catalase activity is the underlying mechanism of oxidant susceptibility in glucose-6-phosphate dehydrogenase-deficient erythrocytes. Biochim Biophys Acta 1181:163, 1993.
Gaetani GF, Ferraris AM, Rolfo M, Mangerini R, Arena S, Kirkman HN: Predominant role of catalase in the disposal of hydrogen peroxide within human erythrocytes. Blood 87:1595, 1996.
Thorburn DR, Kuchel PW: Regulation of the human-erythrocyte hexose-monophosphate shunt under conditions of oxidative stress. Eur J Biochem 150:371, 1985.
Ni TC, Savageau MA: Application of biochemical systems theory to metabolism in human red blood cells—signal propagation and accuracy of representation. J Biol Chem 271:7927, 1996.
Schuster R, Jacobasch G, Holzhütter HG: Mathematical modelling of metabolic pathways affected by an enzyme deficiency—energy and redox metabolism of glucose-6-phosphate-dehydrogenase-deficient erythrocytes. Eur J Biochem 182:605, 1989.
Beutler E, Teeple L: Mannose metabolism in the human erythrocyte. J Clin Invest 48:461, 1969.
Beutler E, Mathai CK: Genetic variation in red cell galactose-1-phosphate uridyl transferase, in Hereditary Disorders of Erythrocyte Metabolism, edited by E Beutler, p 66–86. Grune & Stratton, New York, 1968.
Beutler E, Kuhl W: Unpublished 1991.
Beutler E, Gelbart T: Improved assay of the enzymes of glutathione synthesis: gamma-glutamylcysteine synthetase and glutathione synthetase. Clin Chim Acta 158:115, 1986.
Oski FA, Sugerman HJ, Miller LD: Experimentally induced alterations in the affinity of Hb for oxygen: I. In vitro restoration of erythrocyte 2,3-DPG and its relationship to erythrocyte purine nucleoside phosphorylase activity in a variety of species. Blood 39:522, 1972.
Beutler E, Kuhl W, Gelbart T: Blood cell phosphogluconolactonase: assay and properties. Br J Haematol 62:577, 1986.
Lachant NA, Gottlieb AJ, DiFino SM, Landaw SA, Tanaka KR: Increased Heinz body formation and impaired erythrocyte pentose phosphate shunt function during pregnancy. Am J Hematol 18:131, 1985.
Magnani M, Stocchi V, Chiarantini L, Serafini G, Dacha M, Fornaini G: Rabbit red blood cell hexokinase. Decay mechanism during reticulocyte maturation. J Biol Chem 261:8327, 1986.
Jansen G, Koenderman L, Rijksen G, Punt K, Dekker AW, Staal GEJ: Age dependent behaviour of red cell glycolytic enzymes in haematological disorders. Br J Haematol 61:51, 1985.
Zimran A, Torem S, Beutler E: The in vivo ageing of red cell enzymes: direct evidence of biphasic decay from polycythemic rabbits with reticulocytosis. Br J Haematol 69:67, 1988.
Rose IA, Warms JVB, O’Connell EL: Role of inorganic phosphate in stimulating the glucose utilization of human red blood cells. Biochem Biophys Res Commun 15:33, 1964.
Fujii S, Beutler E: High glucose concentrations partially release hexokinase from inhibition by glucose-6-phosphate. Proc Natl Acad Sci USA 82:1552, 1985.
Gerber G, Kloppick E, Rapoport S: über den Einfluss des Anorganischen Phosphats auf die Glykolyse; seine Unwirksamkeit auf die Hexokinase des Menschenerythrozyten. Acta Biol Med Ger 18:305, 1967.
Beutler E, Teeple L: The effect of oxidized glutathione (GSSG) on human erythrocyte hexokinase activity. Acta Biol Med Ger 22:707, 1969.
Dische Z: The pentose phosphate metabolism in red cells, in The Red Blood Cell, edited by C Bishop, DM Surgenor, pp 189–209. Academic, New York, 1964.
Beutler E: 2,3-Diphosphoglycerate affects enzymes of glucose metabolism in red blood cells. Nature (New Biol) 232:20, 1971.
Kaplan JC, Beutler E: Hexokinase isoenzymes in human erythrocytes. Science 159:215, 1968.
Altay C, Alper CA, Nathan DG: Normal and variant isoenzymes of human blood cell hexokinase and the isoenzyme patterns in hemolytic anemia. Blood 36:219, 1970.
Murakami K, Blei F, Tilton W, Seaman C, Piomelli S: An isozyme of hexokinase specific for the human red blood cell (HKR). Blood 75:770, 1990.
Ruzzo A, Andreoni F, Magnani M: Structure of the human hexokinase type I gene and nucleotide sequence of the 5′ flanking region. Biochem J 331:607, 1998.
Murakami K, Piomelli S: Identification of the cDNA for human red blood cell-specific hexokinase isozyme. Blood 89:762, 1997.
Magnani M, Stocchi V, Dacha M, Fornaini G: Rabbit red blood cell hexokinase: Intracellular distribution during reticulocytes maturation. Mol Cell Biochem 63:59, 1984.
Stocchi V, Magnani M, Piccoli G, Fornaini G: Hexokinase microheterogeneity in rabbit red blood cells and its behaviour during reticulocytes maturation. Mol Cell Biochem 79:133, 1988.
Thorburn DR, Beutler E: Ubiquitin-dependent proteolysis of reticulocyte hexokinase. Blood 74:218A, 1989.
Rijksen G, Akkerman JWN, van den Wall Bake AWL, Pott Hofstede O, Staal GEJ: Generalized hexokinase deficiency in the blood cells of a patient with nonspherocytic hemolytic anemia. Blood 61:12, 1983.
Hirono A, Forman L, Beutler E: Enzymatic diagnosis in non-spherocytic hemolytic anemia. Medicine (Baltimore) 67:110, 1988.
Detter JC, Ways PO, Giblett ER, et al: Inherited variations in human phosphohexose isomerase. Ann Hum Genet 31:329, 1968.
Xu WM, Lee P, Beutler E: Human glucose phosphate isomerase: exon mapping and gene structure. Genomics 29:732, 1995.
Walker JIH, Morgan MJ, Faik P: Structure and organization of the human glucose phosphate isomerase gene (GPI). Genomics 29:261, 1995.
Fujii S, Matsuda M, Okuya S, Yoshizaki Y, Miura-Kora Y, Kaneko T: Fructose-6-phosphate, 2-kinase activity in human erythrocytes. Blood 70:1211, 1987.
Bosca L, Aragon JJ, Sols A: Modulation of muscle phosphofructokinase at physiological concentration of enzyme. J Biol Chem 260:2100, 1985.
Vora S: Isozymes of human phosphofructokinase: Biochemical and genetic aspects, in Isozymes: Current Topics in Biological and Medical Research, edited by MC Rattazzi, JG Scandalios, GS Whitt, pp 3–23. Liss, New York, 1983.
Nakajima H, Noguchi T, Yamasaki T, Kono N, Tanaka T, Tarui S: Cloning of human muscle phosphofructokinase cDNA. FEBS Lett 223:113, 1987.
Levanon D, Danciger E, Dafni N, Groner Y: Construction of a cDNA clone containing the entire coding region of the human liver-type phosphofructokinase. Biochem Biophys Res Commun 147:1182, 1987.
Darville MI, Crepin KM, Vandekerckhove J, et al: Complete nucleotide sequence coding for rat liver 6-phosphofructo-2-kinase/fructose-2,6- bisphosphatase derived from a cDNA clone. FEBS Lett 224:317, 1987.
Beutler E, Scott S, Bishop A, Margolis N, Matsumoto F, Kuhl W: Red cell aldolase deficiency and hemolytic anemia: a new syndrome. Trans Assoc Am Phys 86:154, 1974.
Kishi H, Mukai T, Hirono A, Fujii H, Miwa S, Hori K: Human aldolase A deficiency associated with a hemolytic anemia: thermolabile aldolase due to a single base mutation. Proc Natl Acad Sci USA 84:8623, 1987.
Izzo P, Costanzo P, Lupo A, et al: A new human species of aldolase A mRNA from fibroblasts. Eur J Biochem 164:9, 1987.
Maquat LE, Chilcote R, Ryan PM: Human triosephosphate isomerase cDNA and protein structure. J Biol Chem 260:3748, 1989.
Watanabe M, Zingg BC, Mohrenweiser HW: Molecular analysis of a series of alleles in humans with reduced activity at the triosephosphate isomerase locus. Am J Hum Genet 58:308, 1996.
Schneider A, Forman L, Westwood B, et al: New insights into the interrelationships of the -5, -8, and -24 mutations with triosephosphate isomerase (TPI) deficiency. Blood 90 (Suppl 1):273a, 1997.
Schrier SL: Organization of enzymes in human erythrocyte membranes. Am J Physiol 210:139, 1966.
Brookes PS, Land JM, Clark JB, Heales SJR: Stimulation of glyceraldehyde-3-phosphate dehydrogenase by oxyhemoglobin. FEBS Lett 416:90, 1997.
Chen S-H, Malcolm LA, Yoshida A, Giblett ER: Phosphoglycerate kinase: an X-linked polymorphism in man. Am J Hum Genet 23:87, 1971.
Yoshida A, Watanabe S, Chen S-H, Giblet ER, Malcolm LA: Human phosphoglycerate kinase II. Structure of a variant enzyme. J Biol Chem 247:446, 1972.
Huang IY, Rubinfien E, Yoshida A: Complete amino acid sequence of human phosphoglycerate kinase. Isolation and amino acid sequence of tryptic peptides. J Biol Chem 255:6408, 1980.
Michelson AM, Markham AF, Orkin SH: Isolation and DNA sequence of a full-length cDNA clone for human X chromosome-encoded phosphoglycerate kinase. Proc Natl Acad Sci USA 80:472, 1983.
Raskind WH, Wijsman E, Pagon RA, et al: X-linked sideroblastic anemia and ataxia: linkage to phosphoglycerate kinase at Xq13. Am J Hum Genet 48:335, 1991.
Rosa R, Gaillardon J, Rosa J: Diphosphoglycerate mutase and 2,3-diphosphoglycerate phosphatase activities of red cells: Comparative electrophoretic study. Biochem Biophys Res Commun 51:536, 1973.
Ikura K, Sasaki R, Narita H, Sugimoto E, Chiba H: Multifunctional enzyme, bisphosphoglyceromutase/2,3-bisphosphoglycerate phosphatase/phosphoglyceromutase from human erythrocytes. Eur J Biochem 66:515, 1976.
Rose ZB: The enzymology of 2,3-bisphosphoglycerate. Adv Enzymol 51:211, 1980.
Joulin V, Peduzzi J, Romeo PH, et al: Molecular cloning and sequencing of the human erythrocyte 2,3-bisphosphoglycerate mutase cDNA: revised amino acid sequence. EMBO J 5:2275, 1986.
Yanagawa S, Hitomi K, Sasaki R, Chiba H: Isolation and characterization of cDNA encoding rabbit reticulocyte 2,3-bisphosphoglycerate synthase. Gene 44:185, 1986.
Rose ZB, Salon J: The identification of glycolate-2-P as a constituent of normal red blood cells. Biochem Biophys Res Commun 87:869, 1979.
Vora S, Spear D: Demonstration and quantitation of phosphoglycolate in human red cells. Clin Res 34:664A, 1986.
Fujii S, Beutler E: Glycolate kinase activity in human red cells. Blood 65:480, 1985.
Fujii S, Beutler E: Where does phosphoglycolate come from in red cells? Acta Haematol (Basel) 73:26, 1985.
Sasaki H, Fujii S, Yoshizaki Y, Nakashima K, Kaneko T: Phosphoglycolate synthesis by human erythrocyte pyruvate kinase. Acta Haematol (Basel) 77:83, 1987.
Badwey JA: Phosphoglycolate phosphatase in human erythrocytes. J Biol Chem 252:2441, 1977.
Beutler E, West C: An improved assay and some properties of phosphoglycolate phosphatase. Anal Biochem 106:163, 1980.
Hass LF, Kappel WK, Muller KB, Engle RL: Evidence for structural homology between human red cell phosphoglycerate mutase and 2,3- bisphosphoglycerate synthase. J Biol Chem 253:77, 1978.
Chen S-H, Anderson JE, Giblett ER: Human red cell 2,3-diphosphoglycerate mutase and monophosphoglycerate mutase: genetic evidence for two separate loci. Am J Hum Genet 29:405, 1977.
Hoorn RKJ, Filkweert JP, Staal GEJ: Purification and properties of enolase of human erythrocytes. Int J Biochem 5:845, 1974.
Chen S-H, Giblett ER: Enolase: Human tissue distribution and evidence for three different loci. Ann Hum Genet 39:277, 1976.
Valentine WN, Tanaka KR, Paglia DE: Hemolytic anemias and erythrocyte enzymopathies. Ann Intern Med 103:245, 1985.
Noguchi T, Yamada K, Inoue H, Matsuda T, Tanaka T: The L- and R-type isozymes of rat pyruvate kinase are produced from a single gene by use of different promoters. J Biol Chem 262:14366, 1987.
Blume KG, Hoffbauer RW, Busch D, Arnold H, Löhr GW: Purification and properties of pyruvate kinase in normal and in pyruvate kinase deficient human red blood cells. Biochim Biophys Acta 227:364, 1971.
Kahn A, Marie J, Garreau H, Sprengers ED: The genetic system of the L-type pyruvate kinase forms in man. Subunit structure, interrelation and kinetic characteristics of the pyruvate kinase enzymes from erythrocytes and liver. Biochim Biophys Acta 523:59, 1978.
Tani K, Yoshida MC, Satoh H, et al: Human M2-type pyruvate kinase: cDNA cloning, chromosomal assignment and expression in hepatoma. Gene 73:509, 1988.
Tani K, Fujii H, Tsutsumi H, et al: Human liver type-pyruvate kinase: cDNA cloning and chromosomal assignment. Biochem Biophys Res Commun 143:431, 1987.
Takayasu S, Fujiwara S, Waki T: Hereditary lactate dehydrogenase M-subunit deficiency: lactate dehydrogenase activity in skin lesions and in hair follicles. J Am Acad Dermatol 24:339, 1991.
Kitamura M, Iijima N, Hashimoto F, Hiratsuka A: Hereditary deficiency of subunit H of lactate dehydrogenase. Clin Chim Acta 34:419, 1971.
Miwa S, Nishina T, Kakehashi Y, Kitamura M, Hiratsuka A, Shizumo K: Studies on erythrocyte metabolism in a case with hereditary deficiency of H-subunit of lactate dehydrogenase. Acta Haematol Jpn 34:2, 1971.
Joukyuu R, Mizuno S, Amakawa T, Tsukada T, Nishina T, Kitamura M: Hereditary complete deficiency of lactate dehydrogenase H-subunit. Clin Chem 35:687, 1989.
Wakabayashi H, Tsuchiya M, Yoshino K, Kaku K, Shigei H: Hereditary deficiency of lactate dehydrogenase H-subunit. Intern Med 35:550, 1996.
Beutler E: G6PD: Population genetics and clinical manifestations. Blood Rev 10:45, 1996.
Maekawa M, Sudo K, Nagura K, Li SS, Kanno T: Population screening of lactate dehydrogenase deficiencies in Fukuoka Prefecture in Japan and molecular characterization of three independent mutations in the lactate dehydrogenase-B(H) gene. Hum Genet 93:74, 1994.
Yoshida A, Beutler E (eds): Glucose-6-Phosphate Dehydrogenase. Academic, Orlando, FL, 1986.
Yoshida A, Stamatoyannopoulos G, Motulsky A: Negro variant of glucose-6-phosphate dehydrogenase deficiency (A-) in man. Science 155:97, 1967.
Chen EY, Cheng A, Lee A, et al: Sequence of human glucose-6-phosphate dehydrogenase cloned in plasmids and a yeast artificial chromosome (YAC). Genomics 10:792, 1991.
Martini G, Toniolo D, Vulliamy T, et al: Structural analysis of the X-linked gene encoding human glucose 6-phosphate dehydrogenase. EMBO J 5:1849, 1986.
Persico MG, Viglietto G, Martino G, et al: Isolation of human glucose- 6-phosphate dehydrogenase (G6PD) cDNA clones: primary structure of the protein and unusual 5′ non-coding region. Nucl Acids Res 14:2511, 1986.
Takizawa T, Huang IY, Ikuta T, Yoshida A: Human glucose-6-phosphate dehydrogenase: primary structure and cDNA cloning. Proc Natl Acad Sci USA 83:4157, 1986.
Naylor CE, Rowland P, Basak AK, et al: Glucose 6-phosphate dehydrogenase mutations causing enzyme deficiency in a model of the tertiary structure of the human enzyme. Blood 87:2974, 1996.
Rattazzi MC: Glucose-6-phosphate dehydrogenase from human erythrocytes: Molecular weight determination by gel filtration. Biochem Biophys Res Commun 31:16, 1968.
Kirkman HN, Hendrickson EM: Glucose-6-phosphate dehydrogenase from human erythrocytes. II. Subactive states of the enzyme from normal persons. J Biol Chem 237:2371, 1962.
Yoshida A: Hemolytic anemia and G-6-PD deficiency. Science 179:532, 1973.
Avigad G: Inhibition of glucose-6-phosphate dehydrogenase by adenosine-5-triphosphate. Proc Natl Acad Sci USA 56:1543, 1966.
Ben-Bassat I, Beutler E: Inhibition by ATP of erythrocyte glucose-6- phosphate dehydrogenase variants. Proc Soc Exp Biol Med 142:410, 1973.
Beutler E, Kuhl W: Limiting role of 6-phosphogluconolactonase in erythrocyte hexose monophosphate pathway metabolism. J Lab Clin Med 106:573, 1985.
Rakitzis ET, Papandreou P: Kinetic analysis of 6-phosphogluconolactone hydrolysis in hemolysates. Biochem Mol Biol Int 37:747, 1995.
Beutler E, Kuhl W, Gelbart T: 6-Phosphogluconolactonase deficiency, a hereditary erythrocyte enzyme deficiency: Possible interaction with glucose-6-phosphate dehydrogenase deficiency. Proc Natl Acad Sci USA 82:3876, 1985.
Thorburn DR, Kuchel PW: Computer simulation of the metabolic consequences of the combined deficiency of 6-phosphogluconolactonase and glucose-6-phosphate dehydrogenase in human erythrocytes. J Lab Clin Med 110:70, 1987.
Shih L, Justice P, Hsia DY: Purification and characterization of genetic variants of 6-phosphogluconate dehydrogenase. Biochem Genet 1:359, 1968.
Parr CW, Fitch LI: Inherited quantitative variations of human phosphogluconate dehydrogenase. Ann Hum Genet 30:339, 1967.
Bruns FH, Noltmann E, Vahlhaus E: über den Stoffwechsel von Ribose-5-phosphat in Hämolysaten. I. Aktivitäts-messung und Eigenschaften der Phosphoribose-isomerase. II. Der Pentosephosphate- Cyclus in roten Blutzellen. Biochem Z 330:483, 1958.
Brownstone YS, Denstedt OF: The pentose phosphate metabolic pathway in the human erythrocyte. II. The transketolase and transaldolase activity of the human erythrocyte. Can J Biochem 39:533, 1961.
Nakasaki H, Ohta M, Soeda J, et al: Clinical and biochemical aspects of thiamine treatment for metabolic acidosis during total parenteral nutrition. Nutrition 13:110, 1997.
Wolfe SJ, Brin M, Davidson CS: The effect of thiamine deficiency on human erythrocyte metabolism. J Clin Invest 37:1476, 1958.
Beutler E, Guinto E: The reduction of glyceraldehyde by human erythrocytes. L hexonate dehydrogenase activity. J Clin Invest 53:1258, 1974.
Das B, Srivastava SK: Purification and properties of aldose reductase and aldehyde reductase II from human erythrocyte. Arch Biochem Biophys 238:670, 1985.
Kim HD: Is adenosine a second metabolic substrate for human red blood cells? Biochim Biophys Acta 1036:113, 1990.
Gabrio BW, Finch CA, Huennekens FM: Erythrocyte preservation: a topic in molecular biochemistry. Blood 11:103, 1956.
Resta R, Thompson LF: SCID: the role of adenosine deaminase deficiency. Immunol Today 18:371, 1997.
Mitchell BS, Kelley WN: Purinogenic immunodeficiency diseases: clinical features and molecular mechanisms. Ann Intern Med 92:826, 1980.
Valentine WN, Paglia DE, Tartaglia AP, Gilsanz G: Hereditary hemolytic anemia with increased red cell adenosine deaminase (45- to 70-fold) and decreased adenosine triphosphate. Science 195:783, 1977.
Casoli C, Lisa A, Magnani G, et al: Prognostic value of adenosine deaminase compared to other markers for progression to acquired immunodeficiency syndrome among intravenous drug users. J Med Virol 45:203, 1995.
Palomba E, David O, Boltri A, Gabiano C, Tevo P-A: Increased erythrocyte adenosine deaminase activity in children with perinatal human immunodeficiency virus infection. Pediatr Infect Dis J 8:862, 1989.
Glader BE, Backer K, Diamond LK: Elevated erythrocyte adenosine deaminase activity in congenital hypoplastic anemia. N Engl J Med 309:1486, 1983.
Accorsi A, Piacentini MP, Piatti E, Fazi A: Purine nucleoside phosphorylase from human erythrocytes: a kinetic study of the fully separated isoenzymes. Biochem Int 24:23, 1991.
Valentine WN, Oski FA, Paglia DE, Baughan MA, Schneider AS, Naiman NL: Erythrocyte hexokinase and hereditary hemolytic anemia, in Hereditary Disorders of Erythrocyte Metabolism, edited by E Beutler, p 288. Grune & Stratton, New York, 1968.
Morsches B, Holzmann H, Bettingen C: Zum Nachweis der Sorbit-dehydrogenase in menschlichen Erythrocyten. Klin Wochenschr 47:672, 1969.
Barretto OCO, Beutler E: The sorbitol oxidizing enzyme of red blood cells. J Lab Clin Med 85:645, 1975.
Bruns FH, Noltmann E: Phosphomannoisomerase, an SH-dependent metal-enzyme complex. Nature 181:1467, 1958.
Bruns FH, Noltmann E, Willemsen A: Phosphomannose-isomerase. I. über die Aktivitätsmessung und die Sulfhydryl-sowie die Metallabhängigkeit der Enzymwirkung in einigen tierischen Geweben. Biochem Z 330:411, 1958.
Beutler E: Galactosemia: screening and diagnosis. Clin Biochem 24:293, 1991.
Noltmann E, Bruns FH: über die Phosphoglucomutase der Erythrocyten und des Serums. Hoppe-Seyler’s Z Physiol Chem 313:194, 1959.
Beutler E, Guinto E: Dihydroxyacetone metabolism by human erythrocytes: Demonstration of triokinase activity and its characterization. Blood 41:559, 1973.
Brake JM, Deindoerfer FH: Preservation of red blood cell 2,3-diphosphoglycerate in stored blood containing dihydroxyacetone. Transfusion 13:84, 1973.
Wood L, Beutler E: The effect of ascorbate and dihydroxyacetone on the 2,3-diphosphoglycerate and ATP levels of stored human red cells. Transfusion 14:272, 1974.
Moses SW, Chayoth R, Levin S, Lazarovitz E, Rubinstein D: Glucose and glycogen metabolism in erythrocytes from normal and glycogen storage disease type III subjects. J Clin Invest 47:1343, 1968.
Sidbury JB Jr, Cornblath M, Fisher J, House E: Glycogen in erythrocytes of patients with glycogen storage disease. Pediatrics 27:103, 1961.
Bartels H: Untersuchungen zur Frage des Glykogen-Gehaltes von Erythrocyten, in Metabolism and Membrane Permeability of Erythrocytes and Thrombocytes, edited by E Deutsch, E Gerlach, K Moser, p 132. Georg Thieme Verlag, Stuttgart, 1968.
Dimant E, Landberg E, London IM: The metabolic behavior of reduced glutathione in human and avian erythrocytes. J Biol Chem 213:769, 1955.
Lunn G, Dale GL, Beutler E: Transport accounts for glutathione turnover in human erythrocytes. Blood 54:238, 1979.
Viña JR, Palacin M, Puertes IR, Hernandez R, Viña J: Role of the gamma-glutamyl cycle in the regulation of amino acid translocation. Am J Physiol 257:E916, 1989.
Board PG, Smith JE: Erythrocyte gamma-glutamyl transpeptidase. Blood 49:667, 1977.
Srivastava SK, Awasthi YC, Miller SP, Yoshida A, Beutler E: Studies on gamma-glutamyl transpeptidase in human and rabbit erythrocytes. Blood 47:645, 1976.
Young JD, Ellory JC, Wright PC: Evidence against the participation of the gamma-glutamyltransferase-gamma-glutamylcyclotransferase pathway in amino acid transport by rabbit erythrocytes. Biochem J 152:713, 1975.
Winterbourn CC, Hawkins RE, Brian M, Carrell RW: The estimation of red cell superoxide dismutase activity. J Lab Clin Med 85:337, 1975.
Mills GC, Randall HP: Hemoglobin catabolism II. The protection of hemoglobin from oxidative breakdown in the intact erythrocyte. J Biol Chem 232:589, 1958.
Cohen G, Hochstein P: Glutathione peroxidase: the primary agent for the elimination of hydrogen peroxide in erythrocytes. Biochemistry 2:1420, 1963.
Rotruck JT, Pope AL, Ganther HE, Swanson AB, Hafeman DG, Hoekstra WG: Selenium: biochemical role as a component of glutathione peroxidase. Science 179:588, 1973.
Thomson CD, Rea HM, Doesburg VM, Robinson MF: Selenium concentrations and glutathione peroxidase activities in whole blood of New Zealand residents. Br J Nutr 37:457, 1977.
Beutler E, Matsumoto G: Ethnic variation in red cell glutathione peroxidase activity. Blood 46:103, 1975.
Burk RF: Molecular biology of selenium with implications for its metabolism. FASEB J 5:2274, 1991.
Chambers I, Harrison PR: A new puzzle in selenoprotein biosynthesis: selenocysteine seems to be encoded by the “stop” codon, UGA. TIBS Rev 12:255, 1987.
Stadtman TC: Selenocysteine. Ann Rev Biochem 65:83, 1996.
Jacob HS, Jandl JH: Effects of sulfhydryl inhibition on red blood cells. I. Mechanism of hemolysis. J Clin Invest 41:779, 1962.
Magnani M, Stocchi V, Ninfali P, Dacha M, Fornaini G: Action of oxidized and reduced glutathione on rabbit red blood cell hexokinase. Biochim Biophys Acta 615:113, 1980.
Huisman THJ, Dozy AM: Studies on the heterogeneity of hemoglobin. V. Binding of hemoglobin with oxidized glutathione. J Lab Clin Med 60:302, 1962.
Wong KK, Blanchard JS: Human erythrocyte glutathione reductase: pH dependence of kinetic parameters. Biochemistry 28:3586, 1989.
Icen A: Glutathione reductase of human erythrocytes purification and properties. Scand J Clin Lab Invest 96:1, 1967.
Beutler E, Yeh MKY: Erythrocyte glutathione reductase. Blood 21:573, 1963.
Loos H, Roos D, Weening R, Houwerzijl J: Familial deficiency of glutathione reductase in human blood cells. Blood 48:53, 1976.
Beutler E: Glutathione reductase: Stimulation in normal subjects by riboflavin supplementation. Science 165:613, 1969.
Mieyal JJ, Starke DW, Gravina SA, Hocevar BA: Thioltransferase in human red blood cells: kinetics and equilibrium. Biochemistry 30:8883, 1991.
Srivastava SK, Beutler E: The transport of oxidized glutathione from human erythrocytes. J Biol Chem 244:9, 1969.
Kondo T, Dale GL, Beutler E: Glutathione transport by inside-out vesicles from human erythrocytes. Proc Natl Acad Sci USA 77:6359, 1980.
Kondo T, Kawakami Y, Taniguchi N, Beutler E: Glutathione disulfide- stimulated Mg2+-ATPase of human erythrocyte membranes. Proc Natl Acad Sci USA 84:7373, 1987.
Board PG: Transport of glutathione S-conjugate from human erythrocytes. FEBS Lett 124:163, 1981.
Kondo T, Murao M, Taniguchi N: Glutathione S-conjugate transport using inside-out vesicles from human erythrocytes. Eur J Biochem 125:551, 1982.
Harvey JW, Beutler E: Binding of heme by glutathione S-transferase: a possible role of the erythrocyte enzyme. Blood 60:1227, 1982.
Beutler E, Dunning D, Dabe IB, Forman L: Erythrocyte glutathione S-transferase deficiency and hemolytic anemia. Blood 72:73, 1988.
Prchal JT, Borgese N, Moore MR, Moreno H, Hegesh E, Hall MK: Congenital methemoglobinemia due to methemoglobin reductase deficiency in two unrelated American black families. Am J Med 89:516, 1990.
Katsube T, Sakamoto N, Kobayashi Y, et al: Exonic point mutations in NADH-cytochrome b5 reductase genes of homozygotes for hereditary methemoglobinemia, types I and III: putative mechanisms of tissue-dependent enzyme deficiency. Am J Hum Genet 48:799, 1991.
Tomatsu S, Kobayashi Y, Fukumaki Y, Yubisui T, Orii T, Sakaki Y: The organization and the complete nucleotide sequence of the human NADH-cytochrome b5 reductase gene. Gene 80:353, 1989.
Sass MD: Observations on the role of TPNH dehydrogenase in human red cells. Clin Chim Acta 21:101, 1968.
Xu F, Quandt KS, Hultquist DE: Characterization of NADPH-dependent methemoglobin reductase as a heme-binding protein present in erythrocytes and liver. Proc Natl Acad Sci USA 89:2130, 1992.
Fraser PJ, Curtis PJ: Molecular evolution of the carbonic anhydrase genes: calculation of divergence time for mouse carbonic anhydrase I and II. J Mol Evol 23:294, 1986.
Takahara S: Acatalasemia in Japan, in Hereditary Disorders of Erythrocyte Metabolism, edited by E Beutler, p 21. Grune & Stratton, New York, 1968.
Wen J-K, Osumi T, Hashimoto T, Ogata M: Molecular analysis of human acatalasemia. Identification of a splicing mutation. J Mol Biol 211:383, 1990.
Scott MD, Lubin BH, Zuo L, Kuypers FA: Erythrocyte defense against hydrogen peroxide: preeminent importance of catalase. J Lab Clin Med 118:7, 1991.
Scott MD, Zuo L, Lubin BH, Chiu DTY: NADPH, not glutathione, status modulates oxidant sensitivity in normal and glucose-6-phosphate dehydrogenase-deficient erythrocytes. Blood 77:2059, 1991.
Mueller S, Riedel HD, Stremmel W: Direct evidence for catalase as the predominant H2O2-removing enzyme in human erythrocytes. Blood 90:4973, 1997.
Roth EF Jr, Gilbert HS: The pyrogallol assay for superoxide dismutase: Absence of a glutathione artifact. Anal Biochem 137:50, 1984.
Cha MK, Kim IH: Thioredoxin-linked peroxidase from human red blood cell: evidence for the existence of thioredoxin and thioredoxin reductase in human red blood cell. Biochem Biophys Res Commun 217:900, 1995.
Papov VV, Gravina SA, Mieyal JJ, Biemann K: The primary structure and properties of thioltransferase (glutaredoxin) from human red blood cells. Protein Sci 3:428, 1994.
Jubelin BC, Gierman JL: Erythrocytes may synthesize their own nitric oxide. Am J Hypertens 9:1214, 1996.
Lawson AA, Barr RD: Acetylcholinesterase in red blood cells. Am J Hematol 26:101, 1987.
Johns RJ: Familial reduction in red-cell cholinesterase. N Engl J Med 267:1344, 1962.
Sasaki R, Ikura K, Katsura S, Chiba H: Regulation of human erythrocyte AMP deaminase by ATP and 2,3-biphosphoglycerate. Agricult Biol Chem 40:1797, 1976.
Ogasawara N, Goto H, Yamada Y, et al: Deficiency of AMP deaminase in erythrocytes. Hum Genet 75:15, 1987.
Tuy FPD, Henry J, Rosenfield C, Kahn A: Protein kinases in normal human blood cells. Am J Hematol 15:105, 1983.
Jindal HK, Ai Z, Gascard P, Horton C, Cohen CM: Specific loss of protein kinase activities in senescent erythrocytes. Blood 88:1479, 1996.
Michetti M, Salamino F, Tedesco I, et al: Autolysis of human erythrocyte calpain produces two active enzyme forms with different cell localization. FEBS Lett 392:11, 1996.
Raviv O, Heller H, Hershko A: Alterations in components of the ubiquitin-protein ligase system following maturation of reticulocytes to erythrocytes. Biochem Biophys Res Commun 145:658, 1987.
Vettore L, De Matteis MC, DiIorio EE, Winterhalter KH: Erythrocytic proteases: Preferential degradation of alpha hemoglobin chains. Acta Haematol (Basel) 70:35, 1983.
Kinoshita M, Hamakubo T, Fukui I, Murachi T, Toyohara H: Significant amount of multicatalytic proteinase identified on membrane from human erythrocyte. J Biochem (Tokyo) 107:440, 1990.
Sacchetta P, Battista P, Santarone S, Di Cola D: Purification of human erythrocyte proteolytic enzyme responsible for degradation of oxidant- damaged hemoglobin. Evidence for identifying as a member of the multicatalytic proteinase family. Biochim Biophys Acta 1037:337, 1990.
Ohkubo I, Gasa S, Namikawa C, Makita A, Sasaki M: Human erythrocyte multicatalytic proteinase: activation and binding to sulfated galacto- and lactosylce ramides. Biochem Biophys Res Commun 174:1133, 1991.
Fagan JM, Waxman L: Purification of a protease in red blood cells that degrades oxidatively damaged haemoglobin. Biochem J 277:779, 1991.
Glynn IM, Karlish SJ: The sodium pump. Annu Rev Physiol 37:13, 1975.
Giraud F, Claret M, Bruckdorfer KR, Chailley B: The effects of membrane lipid order and cholesterol on the internal and external cationic sites of the Na+-K+ pump in erythrocytes. Biochim Biophys Acta 647:249, 1981.
Roelofsen B: The (non)specificity in the lipid-requirement of calcium- and (sodium plus potassium)-transporting adenosine triphosphatases. Life Sci 29:2335, 1981.
James P, Maeda M, Fischer R, et al: Identification and primary structure of a calmodulin binding domain of the Ca2+ pump of human erythrocytes. J Biol Chem 263:2905, 1988.
Smallwood JI, Gugi B, Rasmussen H: Regulation of erythrocyte Ca2+ pump activity by protein kinase C. J Biol Chem 263:2195, 1988.
Larsen FL, Katz S, Roufogalis BD: Calmodulin regulation of Ca2+ transport in human erythrocytes. Biochem J 200:185, 1981.
Auland ME, Morris MB, Roufogalis BD: Separation and characterization of two Mg2+-ATPase activities from the human erythrocyte membrane. Arch Biochem Biophys 312:272, 1994.
Beutler E, Kuhl W: Guanosine triphosphatase activity in human erythrocyte membranes. Biochim Biophys Acta 601:372, 1980.
Vanderheiden BS: Genetic studies of human erythrocyte inosine triphosphatase. Biochem Genet 3:289, 1969.
Zachara B, Kopff M: Activity of inosine triphosphate pyrophosphohydrolase in fresh and stored human erythrocytes. Haematologia (Budap) 14:277, 1981.
Carty DJ, Iyengar R: A 43 kDa form of the GTP-binding protein Gi3 in human erythrocytes. FEBS Lett 262:101, 1990.
Damonte G, Sdraffa A, Zocchi E, et al: Multiple small molecular weight guanine nucleotide-binding proteins in human erythrocyte membranes. Biochem Biophys Res Commun 166:1398, 1990.
Metzenthin M, Meier-Tackmann D, Agarwal DP, Zschaber R, Weh HJ: Aldehyde dehydrogenase-mediated metabolism of acetaldehyde and mafosfamide in blood of healthy subjects and patients with malignant lymphoma. Adv Exp Med Biol 414:147, 1997.
Matthies H: Untersuchungen über eine Aldehyd-dehydrogenase in kernlosen Erythrocyten. Biochem Z 329:421, 1957.
Johnson RD, Bahnisch J, Stewart B, Shearman DJC, Edwards JB: Optimized spectrophotometric determination of aldehyde dehydrogenase activity in erythrocytes. Clin Chem 38:584, 1992.
Dockham PA, Sreerama L, Sladek NE: Relative contribution of human erythrocyte aldehyde dehydrogenase to the systemic detoxification of the oxazaphosphorines. Drug Metab Dispos 25:1436, 1997.
Izak G, Wilner T, Mager J: Amino acid activating enzymes in red blood cells of normal anemic and polycythemic subjects. J Clin Invest 39:1763, 1960.
Kaplan NO: Metabolic pathways involving niacin and its derivatives, in Metabolic Pathways, edited by DM Greenberg, p 627. Academic Press, New York, 1961.
Bertino JR, Simmons B, Donohue DM: Purification and properties of the formate-activating enzyme from erythrocytes. J Biol Chem 237:1314, 1962.
Valentine WN, Tanaka KR: The glyoxalase content of human erythrocytes and leukocytes. Acta Haematol (Basel) 26:303, 1961.
Anderson BB, Mollin DL: Red-cell metabolism of pyridoxine in sideroblastic anaemias and related anaemias. Br J Haematol 23:159, 1972.
Chern CJ, Beutler E: Pyridoxal kinase: Decreased activity in red blood cells of Afro-Americans. Science 187:1084, 1975.
Rimington C: A review of the enzymic errors in the various porphyrias. Scand J Clin Lab Invest 45:291, 1985.
Yasuda T, Mizuta K, Kishi K: Purification and characterization of two ribonucleases from human erythrocytes: immunological and enzymological comparison with ribonucleases from human urine. Arch Biochem Biophys 279:130, 1990.
Micheli V, Simmonds HA, Sestini S, Ricci C: Importance of nicotinamide as an NAD precursor in the human erythrocyte. Arch Biochem Biophys 283:40, 1990.
Dissing J, Johnsen AH, Sensabaugh GF: Human red cell acid phosphatase (ACP1). The amino acid sequence of the two isozymes Bf and Bs encoded by the ACP1*B allele. J Biol Chem 266:20619, 1991.
Endo F, Tanoue A, Nakai H, et al: Primary structure and gene localization of human prolidase. J Biol Chem 264:4476, 1989.
Holmgren A, Luthman M: Tissue distribution and subcellular localization of Bovine thioredoxin determined by radioimmunoassay. Biochemistry 17:4071, 1978.
Presecan E, Vonica A, Lascu I: Nucleoside diphosphate kinase from human erythrocytes: purification, molecular mass and subunit structure. FEBS Lett 250:629, 1989.
Tanuma S, Endo H: Purification and characterization of an (ADP- ribose)n glycohydrolase from human erythrocytes. Eur J Biochem 191:57, 1990.
Moenner M, Vosoghi M, Ryazantsev S, Glitz DG: Ribonuclease inhibitor protein of human erythrocytes: characterization, loss of activity in response to oxidative stress, and association with Heinz bodies. Blood Cells Mol Dis 24:149, 1998.
Ward A, Hickman D, Gordon JW, Sim E: Arylamine N-acetyltransferase in human red blood cells. Biochem Pharmacol 44:1099, 1992.
Graziani A, Ling LE, Endemann G, Carpenter CL, Cantley LC: Purification and characterization of human erythrocyte phosphatidylinositol 4-kinase. Phosphatidylinositol 4-kinase and phosphatidylinositol 3-monophosphate 4-kinase are distinct enzymes. Biochem J 284:39, 1992.
Das AK, Dasgupta B, et al: Purification and biochemical characterization of a protein-palmitoyl acyltransferase from human erythrocytes. J Biol Chem 272:11021, 1997.
Moore RB, Shriver SK, Jenkins LD, Mankad VN, Shah AK, Plishker GA: Calpromotin, a cytoplasmic protein, is associated with the formation of dense cells in sickle cell anemia. Am J Hematol 56:100, 1997.
Bjoerk P, Aman P, Hindemith A, et al: A new enzyme activity in human blood cells and isolation of the responsible protein (D-dopachrome tautomerase) from erythrocytes. Eur J Haematol 57:254, 1996.
Kröplin T, Weyer N, Iven H: Determination of thiopurine methyltransferase activity in erythrocytes using 6-thioguanine as the substrate. Adv Exp Med Biol 431:741, 1998.
Micheli V, Jacomelli G, Zammarchi E, Pompucci G: Erythrocyte UMP synthetase activity—an HPLC-linked non-radiochemical assay in normal subjects and in one case of oroticaciduria. Adv Exp Med Biol 431:161, 1998.
Friedemann H, Rapoport SM: Enzymes of the red cell; a critical catalogue, in Cellular and Molecular Biology of Erythrocytes, edited by H Yoshikawa, SM Rapoport, p 181. University Park Press, Baltimore, 1974.
Hidaka Y, Palella TD, O’Toole TE, Tarle SA, Kelley WN: Human adenine phosphoribosyltransferase. Identification of allelic mutations at the nucleotide level as a cause of complete deficiency of the enzyme. J Clin Invest 80:1409, 1987.
Cartier P, Hamet M: Une nouvelle maladie metabolique: Le deficit complet en adenine-phosphoribosyltransferase avec lithiase de 2,8- dihydroxyadenine. C R Acad Sci (Paris) 279:883, 1974.
Van Acker KJ, Simmonds A, Potter C, Cameron JS: Complete deficiency of adenine phosphoribosyltransferase. Report of a family. N Engl J Med 297:127, 1977.
Kamatani N, Hakoda M, Otsuka S, Yoshikawa H, Kashiwazaki S: Only three mutations account for almost all defective alleles causing adenine phosphoribosyltransferase deficiency in Japanese patients. J Clin Invest 90:130, 1992.
Nyhan WL: The recognition of Lesch-Nyhan syndrome as an inborn error of purine metabolism. J Inherit Metab Dis 20:171, 1997.
Seegmiller JE, Rosenbloom FM, Kelley WN: Enzyme defect associated with a sex-linked human neurological disorder and excessive purine synthesis. Science 155:1682, 1967.
Kyd JM, Bagnara AS: Adenosine kinase from human erythrocytes: determination of the conditions required for assay in crude hemolysates. Clin Chim Acta 103:145, 1980.
Amici A, Emanuelli M, Ferretti E, Raffaelli N, Ruggieri S, Magni G: Homogeneous pyrimidine nucleotidase from human erythrocytes: enzymic and molecular properties. Biochem J 304:987, 1994.
Rocchigiani M, Sestini S, Micheli V, Bari M, Simmonds HA: NAD synthesis in human erythrocytes: determination of the activities of some enzymes. Adv Exp Med Biol 309B:337, 1991.
Sestini S, Ricci C, Micheli V, Pompucci G: Nicotinamide mononucleotide adenylyltransferase activity in human erythrocytes. Arch Biochem Biophys 302:206, 1993.
Preiss J, Handler P: Biosynthesis of diphosphopyridine nucleotide II. Enzymatic aspects. J Biol Chem 233:493, 1958.
Pescarmona GP, Bracone A, David O, Sartori ML, Bosia A, et al: Regulation of NAD and NADP synthesis in human red cell. Acta Biol Med Ger 36:759, 1977.
Beutler E: Red cell metabolism. A. Defects not causing hemolytic disease. B. Environmental modification. Biochim Biophys Acta 54:759, 1972.
Ng WG, Donnell GN, Bergren WR: Deficiency of erythrocyte nicotinamide adenine dinucleotide nucleosidase (NADase) activity in the negro. Nature 217:64, 1968.
Kostic MM, Rapoport SM: Maturation-dependent changes of the rabbit reticulocyte energy metabolism. FEBS Lett 250:40, 1989.
Thorburn DR, Beutler E: The loss of enzyme activity from erythroid cells during maturation, in Red Blood Cell Aging, edited by M Magnani, A DeFlora, p 15. Plenum, New York, 1991.
Zimran A, Forman L, Suzuki T, Dale GL, Beutler E: In vivo aging of red cell enzymes: study of biotinylated red blood cells in rabbits. Am J Hematol 33:249, 1990.
Seaman C, Wyss S, Piomelli S: The decline in energetic metabolism with aging of the erythrocyte and its relationship to cell death. Am J Hematol 3:31, 1980.
Rapoport S: Reifung und Alterungsvorgaenge in Erythrozyten. Folia Haematol (Leipz ) 78:364, 1961.
Rapoport S, Rosenthal S, Schewe T, Schultze M, Miller N: The metabolism of the reticulocyte, in Cellular and Molecular Biology of the Erythrocytes, edited by H Yoshikawa, SM Rapoport, pp 93–141. University Park Press, Baltimore, 1974.
Copyright © 2001 McGraw-Hill
Ernest Beutler, Marshall A. Lichtman, Barry S. Coller, Thomas J. Kipps, and Uri Seligsohn