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In order to provide a reference range for normal red blood cell enzyme activities in Thai, we analyzed data from 113 healthy non-anemic Thai people (55 males and 58 females) age 1-42 years, who all had a normal pattern of hemoglobin typing (HbA and HbA2 less than 3.5%). Hematological analysis was performed using an automated cell counter and the hemoglobin studies were carried out by low pressure liquid chromatography. Owing to a high frequency of alpha-thalassemia in Thailand, cases with an MCV < 75 fl were excluded from the study since these cases were likely to be heterozygotes for alpha0-thalassemia. Cases with reticulocytes > 2.5% were excluded from the study since reticulocytes have a higher enzyme activity than mature erythrocytes. Cases with abnormal red blood cell morphology, such as spherocytes and ovalocytes, were also excluded. These criteria were applied to select "normal" controls for our analysis. We assayed eight red blood cell enzyme activities in normal subjects: glucose-6-phosphate dehydrogenase (G6PD), 6-phosphogluconate dehydrogenase (6PGD), pyruvate kinase (PK), hexokinase (HK), glucose phosphate isomerase (GPI), phosphofructokinase (PFK), aldolase (ALD) and phosphoglycerate kinase (PGK). The mean normal ranges (+/- SD) for G6PD, 6PGD, PK, HK, GPI, PFK, ALD and PGK were 12.7 (+/-2.2), 10.7 (+/-1.3), 18.5 (+/-4.0), 1.5 (+/-0.4), 80.5 (+/-11.8), 11.8 (+/-2.1), 4.5 (+/-1.6) and 370 (+/-43) IU/gHb, respectively. Age-dependent differences for the reference values for these enzyme activities were summarized. All red blood cell enzyme activities were highest during the early childhood period and slightly lower in the adult period. These values will be of clinically useful for future reference.

This review summarizes currently available information about a crucial part of erythrocyte metabolism, that is, purine nucleotide conversions and their relationships with other conversion pathways. We describe the cellular resynthesis, interconversion, and degradation of purine compounds, and also the regulatory mechanisms in the conversion pathways. We also mention purine metabolism disorders and their clinical consequences. The literature is fragmentary because studies have concentrated only on selected aspects of purine metabolism; hence the need for a synthetic approach.

Two cytoplasmic pyrimidine 5'-nucleotidase have been purified from human erythrocytes to homogeneity and partially characterized. The two enzymes, indicated as PN-I and PN-II, preferentially hydrolyse pyrimidine 5'-monophosphates and 3'-monophosphates, respectively. The kinetic analysis demonstrate that pyrimidine 5'-nucleotidases, in the presence of suitable nucleoside substrates, can operate as phosphotransferases by transferring phosphate to various nucleoside acceptors, including nucleoside analogues known as important drugs widely used in chemotherapy.

We have previously characterized phosphatidylinositol (PtdIns) synthase and PtdIns/myo-inositol-exchange enzyme activities in ghost membranes prepared by hypotonic lysis of turkey erythrocytes [McPhee, Lowe, Vaziri and Downes (1991) Biochem. J. 275, 187-192]. Here we show that PtdIns synthase activity is relatively enriched in plasma-membrane preparations of turkey erythrocytes and that inositol phospholipids labelled by both PtdIns synthase and PtdIns myo-inositol exchange enzymes are susceptible to hydrolysis by the receptor- and G-protein-regulated phospholipase C (PLC), which is present also in ghost preparations. Specific-radioactivity measurements of [3H]PtdIns from ghosts labelled to equilibrium under conditions favouring [3H]inositol incorporation by PtdIns synthase activity indicate that PtdIns synthase can directly access approx. 14% of the total erythrocyte ghost PtdIns. Approx. 16% of the [3H]PtdIns labelled by the PtdIns synthase reaction can be phosphorylated to polyphosphoinositides, which are then hydrolysed by the receptor- and G-protein-stimulated PLC. Since the mass of PtdIns declines to a similar extent as [3H]PtdIns during stimulation in the presence of guanine nucleotides and ATP, it is evident that both the labelled and unlabelled phosphoinositides are susceptible to hydrolysis by the relevant PLC. Phosphoinositides present in nuclei-free plasma membranes were also labelled by [3H]inositol under conditions favouring PtdIns synthase and PtdIns/myo-inositol-exchange enzyme activities respectively. These membranes lack PLC activity [Vaziri and Downes (1992) J. Biol. Chem. 267, 22973-22981], but the labelled lipids were sensitive to purinergic-receptor-stimulated hydrolysis in reconstitution assays using partially purified turkey erythrocyte PLC. The results strongly suggest that at least a portion of the PtdIns synthase in turkey erythrocytes is located in the plasma membrane and has direct access to an agonist-sensitive pool of inositol phospholipids.

PtdIns 4-kinase has been purified 83,000-fold from human erythrocyte membranes. The major protein detected by SDS/PAGE is of molecular mass 56 kDa, and enzymic activity can be renatured from this band of the gel. The characteristics of this enzyme are similar to other type II PtdIns kinases previously described: PtdIns presented in Triton X-100 micelles is preferred as a substrate over PtdIns vesicles, the enzyme possesses a relatively low Km for ATP (20 microM), and adenosine is an effective inhibitor. A monoclonal antibody raised against bovine brain type II PtdIns 4-kinase is an effective inhibitor of the purified enzyme. PtdIns(4,5)P2 inhibits by approx. 50% when added in equimolar amounts with PtdIns; PtdIns4P has little effect on activity. A PtdIns3P 4-kinase activity has also been detected in erythrocyte lysates. Approximately two-thirds of this activity is in the cytosolic fraction and one-third in the membrane fraction. No PtdIns3P 4-kinase activity could be detected in the purified type II PtdIns 4-kinase preparation, nor could this activity be detected in a bovine brain type III PtdIns 4-kinase preparation. The monoclonal antibody that inhibits the type II PtdIns 4-kinase does not affect the PtdIns3P 4-kinase activity in the membrane fraction. The cytosolic PtdIns3P 4-kinase can be efficiently recovered from a 60%-satd.-(NH4)2SO4 precipitate that is virtually free of PtdIns 4-kinase activity. We conclude that PtdIns3P 4-kinase is a new enzyme distinct from previously characterized PtdIns 4-kinases, and that this enzyme prefers PtdIns3P over PtdIns as a substrate.

The effect of total body irradiation (5 Gy) on functional mouse erythroid lineage has been studied. The transferrin binding capacity by bone marrow cells and the activity of glycolytic regulatory enzymes and intracellular levels of 2,3 bisphosphoglycerate in peripheral blood erythrocytes have been determined. Results obtained along one year post-irradiation period suggest a complete recovery in the erythroid cell lineage with respect to the biological endpoints investigated.

Adenylate kinase (AK) modulates the interconversion of adenine nucleotides (AMP + adenosine triphosphate----2 ADP). We evaluated the fifth kindred with hereditary erythrocyte (RBC) AK deficiency. The proband had chronic hemolytic anemia. Her RBC had undetectable AK activity when measured spectrophotometrically, whereas those of her parents had half-normal AK activity. AK electrophoresis showed only AK-1 in the parents. The activities of pyruvate kinase and phosphoribosylpyrophosphate synthetase were decreased given the young age of the proband's RBC. Despite the absence of spectrophotometric AK activity, the proband's RBC were able to incorporate 14C-adenine into 14C-adenine nucleotides at 50% of the rate expected for her young RBC population, suggesting the possibility of an alternative pathway for the formation of ADP from AMP. Normal hemolysate had AMP:guanosine triphosphate (GTP) phosphotransferase activity, which produced ADP at 8% to 9% of the rate of AK (6.8 +/- 0.8 IU/mL RBC). AMP:GTP phosphotransferase activity was not detectable in the proband's or parent's hemolysates. These additional biochemical defects in the AK-deficient RBC further support the concept that AK deficiency per se may not cause hemolytic anemia. We propose that defects occur in multiple phosphotransferases in the AK-deficient RBC and that these other biochemical defects may produce deleterious lesions that promote the shortened RBC survival in AK deficiency.

We have investigated the levels of fructose 2,6-bisphosphate and its synthesizing enzyme 6-phosphofructo-2-kinase in red blood cells from different congenital anemias. Fructose 2,6-bisphosphate concentration and 6-phosphofructo-2-kinase activity are markedly influenced by the number of reticulocytes in all the cases studied with the exception of homozygous pyruvate kinase deficiency, where no correlation was observed with the percentage of reticulocytes.

A human erythrocyte cytosolic phosphatidylinositol-4-phosphate 5-kinase (PIP kinase) and a membrane-bound PIP kinase have been purified by phosphocellulose chromatography. Fractionation of the membrane-bound PIP kinase activities by phosphocellulose separated activity into two peaks, which eluted at 0.6 M NaCl (type I PIP kinase) and 1.0 M NaCl (type II PIP kinase). The cytosolic PIP kinase and the membrane-bound type II PIP kinase are 53 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, have indistinguishable 125I-peptide maps, and are immunochemically indistinguishable, suggesting that they are sequence identical. Antibodies raised to the cytosolic PIP kinase inhibit activity of both the membrane-bound type II and the cytosolic PIP kinases. The type I PIP kinase appears to be distinct from the cytosolic and membrane-bound type II PIP kinase; it is not immunocross-reactive, and antibodies toward type II PIP kinases do not inhibit type I PIP kinase. Further, membrane-bound type II PIP kinase can be removed from type I PIP kinase without loss of activity. Functional characterization of the PIP kinases demonstrates that the type I kinase has a 10-fold lower Km for PIP and a 5-fold higher Km for ATP compared with the type II enzymes. The type I and type II (membrane-bound or cytosolic) PIP kinases are modulated differentially by spermine and heparin. Finally, the type I PIP kinase phosphorylates intrinsic PIP on isolated erythrocyte membranes, whereas the type II PIP kinases have no activity toward native membranes.

It has been suggested that the erythrocytes of myotonic dystrophy (MyD) patients have a decreased calcium-stimulated phosphatidic acid (PA) accumulation. This could be the result of a defect in the calcium-stimulated hydrolysis of the polyphosphoinositides (calcium-dependent phosphodiesterase) or in the subsequent formation of PA from its precursors (diacylglycerol kinase). In vitro assays were established for both enzymes in erythrocyte membranes. Calcium-dependent phosphodiesterase activity was assayed with both endogenous 32P-labeled erythrocyte diphosphoinositide and triphosphoinositide and with the same phospholipids isolated from rat brain. No significant differences in activity were found between MyD patients and normal controls with either method of substrate preparation. No difference in diglyceride kinase activity was found between ghosts prepared from MyD patients and normal controls. Thus, there were no differences in either of the membrane-associated enzymes of phosphatidic acid metabolism.

1,2-Diacylglycerol kinase activity was measured in human erythrocyte membranes using an assay procedure in which the substrate was generated endogenously, either by treatment with a bacterial phospholipase C or by incubation with Ca24, which activates a membrane-bound polyphosphoinositide phosphodiesterase. The properties of 1,2-diacylglycerol kinase were broadly similar to those described previously, except that in the present work maximum activities were higher and there was evidence for a double pH optimum.

Purine metabolism has been much investigated in human erythrocytes, both for the interest focused on this peculiar cell biochemistry, and four purine pathological implications in man. Most mammalian red blood cells lack de novo purine synthesis, and are completely dependent on base salvage pathway for their purine nucleotide requirement. Besides, human erythrocyte is devoid of some purine interconversion enzymes, thus becoming dependent on external supply of bases and nucleosides, particularly the adenylic ones. Red blood cell purine metabolism is very active, owing to its great need for nucleotides, and seems to aim greatly at the preservation of synthesized nucleotides against catabolic events. Available data on purine enzyme kinetic parameters and regulation, substrate and product cellular concentration and uptake are reviewed.

The alterations of three erythrocyte purine enzymes were studied in 12 patients with diseases associated with reticulocytosis, two patients with a partial deficiency of hypoxanthine-guanine phosphoribosyltransferase, seven patients with severe megaloblastic anemia, and 14 normal subjects. The specific activity of adenine phosphoribosyltransferase was positively correlated (r = 0.81) with the reticulocyte percentate in ten patients with a normal hypoxanthine-guanine phosphoribosyltransferase. Two apparent types of alterations of this enzyme were distinguished: (1) increased specific activity with a normal half life as in megaloblastic anemia, and (2) a prolonged half life with or without an elevation of specific activity as in the deficiency of hypoxanthine-guanine phosphoribosyltransferase. Hypoxanthine-guanine phosphoribosyltransferase and phosphoribosylpyrophosphate synthetase were increased in megaloblastic anemia, but were not correlated with the reticulocyte percentage and did not have a consistent change in the half life in the other disorders studied. The data show that acquired disorders associated with reticulocytosis may cause an elevation of the specific activity of purine enzymes in peripheral circulating erythrocytes. Therefore, these factors must be carefully considered in the interpretation of an elevated level of enzyme activity.

Normal duck erythrocytes and erythrocytes infected with Plasmodium lopharae have all of the enzymes for coenzyme A biosynthesis, whereas parasites freed from their host cells have non. Since erythrocytefree cultivation of P. lophurae requires an exogenous source of coenzyme A, this parasite must obtain its coenzyme A entirely from the host cell during infection.

An assay suitable for initial velocity studies was developed to measure adenosine kinase (ATP: adenosine 5'-phosphotransferase, EC 2.7.1.20) activity in crude lystaes of human erthrocytes. The pH optimum for the reaction was dependent on the ratio of Mg2+/ATP used in the assay. With a Mg2+/ATP ratio of 5.0, the pH optimum was 5.1 and only 10% of the maximal activity was retained at pH 7.3. When a Mg2+/ATP ratio of 0.50 was used, the pH optimum was 6.2, but 70% of the maximal activity was retained at pH 7.3 For assays performed at pH 7.3, the optimal Mg2+/ATP ratio was 0.50 for ATP concentrations between 0.50 and 2.0 mmol/l. Mg2+ was required for reaction, presumably to form the physiological substrate MgATP2-. However, excess free (uncomplexed) Mg2+ was a strong inhibitor of the enzyme at pH 7.3. At pH 7.3, the Km values for adenosine and MgATP2- were 0.66 mu mol/l and 82 mu mol/l respectively.

Rare genetic variants of human red cell 2,3-diphosphoglycerate mutase (DPGM) and monophosphoglycerate mutase (MPGM) were compared by starch gel electrophoresis. The isozyme patterns showed that genetic variation of the enzymes were independent from each other, thus DPGM and MPGM must be controlled by two separate loci.

The effects of two sulfhydryl reagents on the three activities of bisphosphoglycerate mutase have been compared. Under N-ethylmaleimide treatment all the activities were inhibited except for 60% of the non-stimulated phosphatase. With iodoacetamide the mutase and the stimulated-phosphatase activities were completely inhibited whereas the non-stimulated phosphatase and 60% of the synthase activities were unaffected. 2,3-bisphosphoglycerate protected all the activities of the enzyme against inactivation by the two sulfhydryl reagents whereas 3-phosphoglycerate protected them only against iodoacetamide. 2-phosphoglycolate had an identical effect to that of 3-phosphoglycerate except for its effect on the non-stimulated phosphatase activity, which was slightly enhanced under N-ethylmaleimide treatment.

Blood samples wer collected from three populations of full-blood Aborigines, and twelve different enzymes were assayed in red blood cells. These results were compared with those of Caucasian controls. In the majority of enzymes studied significant differences were observed between the Aboriginal populations and also between individual Aboriginal populations and the Caucasian controls.

Purified phosphoglyceromutase was hybridized in vitro with pure biphosphoglyceromutase . The hybrid showed an electrophoretic mobility identical to that of the intermediate band of red cell phosphoglyceromutase activity in the hemolysate patterns. Electrophoretic tests showed that the partially purified hybrid displayed both bisphosphoglyceromutase and phosphoglyceromutase activities and that the sample contained also a small portion of non-hybrid bisphosphoglyceromutase . By constrast to a non-hybrid mixture of the two purified mutases the hybrid exhibited heat instability of bisphosphoglyceromutase activity and neutralization of phosphoglyceromutase activity by anti- bisphosphoglyceromutase antibody.

Guanylate kinase was purified from human erythrocytes by affinity chromatography using GMP-agarose, and the four isozymes which are present were separated by chromatofocusing. The kinetic properties of each isozyme were analyzed with respect to the natural substrates GMP and dGMP, and the 5'-monophosphate derivatives of the antiviral nucleoside analogs 9-(1,3-dihydroxy-2-propoxymethyl)guanine (DHPG) and 9-(2-hydroxyethoxymethyl)guanine (ACV, Acyclovir). The analysis of substrate kinetics yielded Km values for DHPG 5'-monophosphate which were similar with all isozymes (42-54 microM), and about 3-fold higher than the Km values obtained for GMP. Km values obtained with ACV 5'-monophosphate were 10-20-fold higher than the GMP values and varied nearly 4-fold among isozymes (209-753 microM). GMP produced the highest enzyme velocities with all isozymes, followed by dGMP, DHPG 5'-monophosphate, and ACV 5'-monophosphate, in that order. Differences in maximal velocities among isozymes were generally small. DHPG 5'-monophosphate inhibited the isozymes by a simple competitive mechanism with respect to GMP. In contrast, ACV 5'-monophosphate acted as an apparent hyperbolic mixed-type inhibitor. Similar patterns of inhibition were obtained with all isozymes. It is probable that differences is the reactivity of DHPG 5'-monophosphate and ACV 5'-monophosphate with individual guanylate kinase isozymes do not contribute significantly to differences in their antiviral effects.

The frequency of variant forms of 6 red cell enzymes, adenylate kinase, adenosine deaminase, phosphoglucomutase, acid phosphatase, 6-phosphogluconate dehydrogenase and glutathione reductase, were determined in 9 Greek populations. The frequencies of the variants in these populations were similar to those previously reported in most other European populations. However, several differences, particularly in the 6-phosphogluconate dehydrogenase, phosphoglucomutase and acid phosphatase alleles, were found in a comparison of Greeks and Bulgarians, in accordance with their separate ethnic origins. The Macedonians resembled the other Greeks and differed from the Bulgarians.

5 serum protein polymorphic systems (haptoglobin, alkaline phosphatase, group-specific (Gc) proteins, beta2-glycoprotein 1 and leucine aminopeptidase) and 6 red-cell polymorphisms (adenosine deaminase, adenylate kinase, phosphoglucomutase, glutamic-pyruvic transaminase, phosphogluconate dehydrogenase and acid phosphatase) have been investigated in 54 subjects with tuberous sclerosis. The frequencies of all systems were compared with those of a control sample drawn from a similar mentally retarded population and abnormal distributions were detected in the haptoglobin and Gc system. Quantitative estimation of the serum levels of the Gc protein failed to detect any inter-group differences. Data on the deviations from the Hardy-Weinberg equlibrium, Haldane's Log ratio test between groups, and gene frequencies of both test and control groups are given. It is suggested that selection by mortality is the possible causation for the abnormal distribution of the Gc phenotypes, but the haptoglobin phenotype distribution requires further investigation with care being taken in the selection of control subjects.

The concentration of fructose 2,6-bisphosphate found in freshly isolated erythrocytes was below the limit of detection (20 pmol/ml of packed cells). However, it increased to about 250 pmol/ml of cells when erythrocytes were incubated with glucose at pH 6.9, but not at pH 7.4 or 8.2. This could be explained by variations in the content of glycerate 2,3-bisphosphate, which was found to inhibit 6-phosphofructo-2-kinase, the enzyme responsible for fructose 2,6-bisphosphate synthesis. Glycerate 2,3-bisphosphate was also found to inhibit the potato enzyme (pyrophosphate:fructose-6-phosphate 1-phosphotransferase) used for the measurement of fructose 2,6-bisphosphate.

Red cell adenylate kinase (AK) phenotypes were studies in 195 patients with affective disorders (41 with the bipolar and 81 with the unipolar form of the disease) and 418 controls. No significant differences were found between patients and controls and between patients with different types of affective disorders. Thus the previous observation by Rundle et al. (1977) showing an increased frequency of the AK2 allele in the unipolar group was not confirmed.

Adenylate kinase exists in the human erythrocyte in a number of molecular forms with two allels at a single polymorphic locus coding for most of the enzyme forms. The predominant enzyme form, AK a, was purified to constant specific activity in excess of 3000 and appeared homogeneous by chromatography, electrophoresis, and ultracentrifugation. Sedimentation velocity and partial specific volume measurments of AK a yielded values of s20,w = 2.1 S and 0.722 cm-3per g. The molecular weight of the native enzyme was estimated to be 22,500 by sedimentation equilibrium and gel filtration analyses. The molecular weight of the denatured enzyme did not differ, indicating an absence of subunit structure in confirmation of genetic evidence of a single locus coding for the enzyme. The isolated enzyme demonstrated remarkable stability to denaturants (heat, guanidine HCl, urea) in the presence of appropriate stabilizing agents and could not be distinguished from rabbit muscle enzyme on this basis (as well as by a number of other kinetic and physicochemical parameters). The erythrocyte adenylate kinases have a common molecular size but differ in their charge properties. They demonstrate anomalous electrophoretic behavior, migrating anionic to hemoglobin in starch gel, yet exhibit isoelectric points considerably alkaline to hemoglobin (e.g. AK a, pI = 9.0) by isoelectric focusing.

Properties of phosphatidylinositol kinase activities in rabbit erythrocyte membranes were studied by measuring 32P incorporation into di- and triphosphoinositide from Mg-[gamma-32P]ATP. The Km's for 32P incorporation into di- and triphosphoinositide were 110 and 48 microM ATP, respectively. The optimal temperature for 32P incorporation into diphosphoinositide was at 32 degrees C, whereas the optimum for triphosphoinositide labeling occurred at 43 degrees C. Differences in the effects of pH on the rate of 32P incorporation into di- and triphosphoinositide were also found. At 37 degrees C but not at 25 degrees C 32P-labeled diphosphoinositide was phosphorylated to triphosphoinositide in the presence of Mg-ATP. Triton X-100 partially inhibited 32P incorporation into diphosphoinositide but completely inhibited the synthesis of triphosphoinositide. At physiological concentrations, 0.4 mM MgCl2 half-maximally activated di- and triphosphoinositide synthesis. Higher concentrations of MgCl2 (5 to 50 mM) decreased 32P incorporation into diphosphoinositide and greatly enhanced 32P incorporation into triphosphoinositide. NaCl or KCl (less than or equal to 100 mM) did not have any effects on polyphosphoinositide synthesis, whereas 150 to 300 mM NaCl or KCl decreased synthesis of diphosphoinositide and increased synthesis of triphosphoinositide. Further studies showed that 50 mM MgCl2 and 200 mM NaCl or KCl stimulate kinase-mediated phosphorylation of diphosphoinositide to triphosphoinositide. Triton X-100 inhibited the ability of 50 mM MgCl2 and neomycin to stimulate phosphorylation of diphosphoinositide to triphosphoinositide. The pathways for synthesis of di- and triphosphoinositides in erythrocyte membranes are discussed.

The red-cell glucose-6-phosphate dehydrogenase (G.-6-P.D.) activity and red-cell pyridoxal-kinase (P.L.K.) activity of 27 Nigerian children with severe Plasmodium falciparum parasitaemia were compared with those of 26 healthy Nigerian children and 6 White adults. The mean P.L.K. activity of the malaria patients was similar to that of the Whites but significantly higher than that of the Nigerian controls. Correction for reduced mean red-cell age in patients was made by comparing the P.L.K.: G.-6-P.D. ratio for those subjects with stable G.-6-P.D. phenotypes. The mean P.L.K.:G.-6-P.D. ratio was the same for malaria patients and adult White but significantly higher than that for the Nigerian controls. These results suggest that the relatively high frequency of low red-cell P.L.K. activity among Blacks may have been selected for by falciparum malaria.

The mean pyridoxal kinase activity in red blood cells of American blacks was approximately 50 percent lower than that of American whites. Lymphocytes, granulocytes, and cultured skin fibroblasts from black and white donors contained identical pyridoxal kinase activity. The pyridoxal kinase of blacks was indistinguishable from that of whites with respect to heat stability, chromatographic mobility on microgranular diethylaminoethyl cellulose, Michaelis-Menten constant for pyridoxine, and susceptibility to inhibition by 4-deoxypyridoxine. The difference of the activity of this enzyme in whites and in blacks is much greater than any previously observed biochemical difference between the races.

Bisphosphoglyceromutase and 2,3-bisphosphoglycerate phosphatase activities responsible for 2,3-bisphosphoglycerate metabolsim in human red cells are displayed by the same enzyme protein which has phosphoglyceromutase activity [Sasaki, R., et al. (1975) Eur J. Biochem. 50, 581-593]. This enzyme was subjected to chemical modification by trinitrobenzenesulfonate. The three enzyme activities were inactivated by trinitrobenzenesulfonate at the same rate. The sulfhydryl content of the enzyme was unchanged during trinitrophenylation, indicating that derivatization was through the amino group. Trinitrophenylation of about one amino group per mole of the enzyme resulted in complete loss of the three activities. Both 2,3-bisphosphoglycerate and 1,3-bisphosphoglycerate inhibited trinitrophenylation and effectively protected the enzyme from inactivation. Although monophosphoglycerates did not show any protective effect at concentrations which should be adequate based upon their kinetic constants, they were protective at higher concentrations. Inactivation by trinitrophenylation was an apparent first-order reaction. The dissociation constant of the enzyme - 2,3-bisphosphoglycerate complex was determined by analyzing the first-order reaction on the assumption that the protective effect of 2,3-bisphosphoglycerate was due to competition with trinitrobenzenesulfonate. The dissociation constant was in good agreement with kinetic constants of 2,3-bisphosphoglycerate in the enzyme reactions, which indicated that 2,3-bisphosphoglycerate did indeed exert its protective effect through competition with trinitrobenzenesulfonate for an amino group of the enzyme. The protective effect of monophosphoglycerates could be rationalized with kinetic evidence that 2-phosphoglycerate at high concentrations interacts with the 2,3-bisphosphoglycerate binding site. These results indicate that the enzyme exhibits the three enzyme activities at a common active site at which one amino group essential for binding of bisphosphoglycerates is located. Based on the multifunctional properties of this enzyme, a possible mechanism was discussed for regulation of 2,3-bisphosphoglycerate metabolism in human red cells.

The specific activity of bisphosphoglycerate synthase (EC 2.7.5.4) is significantly higher in rat mature erythrocytes than in reticulocytes from phenylhydrazine treated animals, while 2,3-bisphosphoglycerate levels do not differ between them. The enzyme specific activity and the concentration of 2,3-bisphosphoglycerate are both negligible in the erythroid cell pool from the bone marrow of anaemic animals. Thus, a cellular specialization in the last stages of erythropoiesis is confirmed. Human bisphosphoglycerate synthase shows a lower specific activity than that of rat without a parallel decrease in 2,3-bisphosphoglycerate levels.

Content of phosphoribosyl pyrophosphate and the activity of ribose phosphate pyrophosphokinase and AMP-pyrophosphorylase were studied in erythrocytes of healthy persons and of patients with various types of anemia. Deficiency of phosphoribosyl pyrophosphate and a decrease in the activity of enzymes studied were observed in erythrocytes under microspherocytic and hypoplastic anemia. The alterations correlated with impairments in energy metabolism. At the same time an activation of phosphoribosyl pyrophosphate enzymatic system and an increase of its content in erythrocytes did not depend on energy metabolism in Marchiafava--Micheli disease.