PGK1, a glucose metabolism enzyme, may play an important role in rheumatoid arthritis.

BACKGROUND: Some studies have indicated that glucose metabolism plays an important role in the pathogenesis of rheumatoid arthritis (RA). This study aimed to find the novel genes affecting glucose metabolism in RA. MATERIALS/METHODS: Synovial tissues of collagen-induced arthritis (CIA) were analyzed with Rat Glucose Metabolism RT(2) Profiler™ PCR Array to screen those genes with special expressions in glucose metabolism. Real-time PCR, western blotting, and ELISA were used to confirm the result in synovial tissues and blood of human RA. Culture synovial fibroblast cells (RASF) was treated with siRNA to suppress expressions of the target genes. CCK-8 cell proliferation assay and two-compartment transwell system were performed to examine cell proliferation and cell migration of the treated RASF. RESULTS: Both PCR array and real-time PCR detected the up-regulation of ENO1, HK2, and PGK1 and the down-regulation of PCK1 and PDK4 in synovial tissues of CIA rats. Real-time PCR and western blotting detected the increased expression of ENO1 and PGK1 in RA synovial tissues. ELISA detected a high level of PGK1 in the blood of RA patients. Decreased cell proliferation and cell migration capabilities were significantly detected in RASF following treatment of anti-PGK1 siRNA. IL-1ß and IFN-γ rather than TNF-α and IL-1α levels were significantly declined in supernatants of the treated RASF. CONCLUSIONS: PGK1, a glycolytic enzyme catalyzing the conversion of 3-phosphoglycerate into 2-phosphoglycerate, has increased expression in synovial tissues and blood of RA, which may be involved in pro-inflammation and synovial hyperplasia of the disease.

Long noncoding RNA associated with microvascular invasion in hepatocellular carcinoma promotes angiogenesis and serves as a predictor for hepatocellular carcinoma patients' poor recurrence-free survival after hepatectomy.

UNLABELLED: Survival of patients with hepatocellular carcinoma (HCC) remains poor, which is largely attributed to active angiogenesis. However, the mechanisms underlying angiogenesis in HCC remain to be discovered. In this study, we found that long noncoding RNA associated with microvascular invasion in HCC (lncRNA MVIH) (lncRNA associated with microvascular invasion in HCC) was generally overexpressed in HCC. In a cohort of 215 HCC patients, the overexpression of MVIH was associated with frequent microvascular invasion (P = 0.016) and a higher tumor node metastasis stage (P = 0.009) as well as decreased recurrence-free survival (RFS) (P < 0.001) and overall survival (P = 0.007). Moreover, the up-regulation of MVIH served as an independent risk factor to predict poor RFS. We also found that MVIH could promote tumor growth and intrahepatic metastasis by activating angiogenesis in mouse models. Subsequent investigations indicated that MVIH could activate tumor-inducing angiogenesis by inhibiting the secretion of phosphoglycerate kinase 1 (PGK1). Additionally, in 65 HCC samples, MVIH expression was inversely correlated with the serum level of PGK1 and positively correlated with the microvessel density. CONCLUSION: Deregulation of lncRNA MVIH is a predictor for poor RFS of HCC patients after hepatectomy and could be utilized as a potential target for new adjuvant therapies against active angiogenesis.

Novel serum proteomic signatures in a non-human primate model of retinal injury.

PURPOSE: To identify candidate protein biomarkers in sera indicative of acute retinal injury. METHODS: We used laser photocoagulation as a model of acute retinal injury in Rhesus macaques. In a paired-control study design, we collected serum from each animal (n=6) at 4 h, 1 day, and 3 days following a mock procedure and then again following retinal laser treatment that produced mild lesions. Samples were fractionated by isoelectric focusing, digested with trypsin, and analyzed by liquid chromatography/tandem mass spectrometry (LC-MS/MS). Spectral counting was used to determine relative protein abundances and identify proteins with statistically significant differences between control and treated sera. RESULTS: Mild retinal injury was confirmed by fundus photography and histological examination. The average number of total proteins detected by LC-MS/MS was 908±82 among samples from all three time points. Following statistical analysis and employing stringent filtering criteria, a total of 19 proteins were identified as being significantly more abundant in sera following laser-induced retinal injury, relative to control sera. Many of the proteins detected were unique to one time point. However, four proteins (phosphoglycerate kinase 1, keratin 18, Lewis alpha-3-fucosyltransferase, and ephrin receptor A2) showed differences that were significant at both 4 h and 1 day after laser treatment, followed by a decrease to baseline levels by day 3. CONCLUSIONS: A serum biomarker response to mild retinal laser injury was demonstrated in a primate model. Among the proteins detected with highest significant differences, most are upregulated within 24 h, and their appearance in the serum is transient. It is conceivable that a panel of these proteins could provide a means for detecting the acute-phase response to retinal injury. Further investigation of these candidate biomarkers and their correlation to retinal damage is warranted.

Impaired age-dependent increases in phosphoglycerate kinase activity in red blood cells of Parkinson's disease patients.

BACKGROUND: Impaired bioenergetics are partially involved in the pathogenesis of Parkinson's disease (PD). Phosphoglycerate kinase (PGK), an essential enzyme for glycolysis, has recently attracted attention due to its pathogenic role in PD and as a target for disease-modifying therapies. This study is aimed to evaluate the profiles of PGK activity in red blood cells (RBCs) of PD patients and controls. METHODS: Sixty-eight PD patients and thirty-four age-matched unrelated controls were enrolled. PGK activities of RBCs were measured by the established colorimetric assay and standardized by the same RBC samples. RESULTS: PGK activity of the PD group was significantly higher than that of the control group in participants aged sixty-five years or younger, whereas it was not significantly different between the two groups at any age. PGK activity was positively correlated with aging in the control group, but this was not noted in the PD group. On multivariable analysis by partial correlation in the PD group, PGK activity was negatively correlated with the specific binding ratio of dopamine transporter scintigraphy in the striatum. The levodopa-equivalent daily dose was not significantly correlated with the enzyme activity. CONCLUSION: The results support the following: 1) elevation of PGK activities in RBCs can be detected in relatively young PD patients and with normal aging; 2) the degree of striatonigral degeneration is associated with elevated PGK activities. These are important considerations when the PGK assay is applied as a diagnostic biomarker of PD and to therapeutically monitor PGK-enhancing treatments.

Development of B lymphocytes in mice heterozygous for the X-linked immunodeficiency (xid) mutation. xid inhibits development of all splenic and lymph node B cells at a stage subsequent to their initial formation in bone marrow.

CBA/N mice were crossed with CBA/Ca-Pgk-1a to produce female F1 hybrids that were heterozygous for both xid and the phosphoglycerate kinase 1 (PGK-1) allozymes. PGK acted as a quantifiable marker for the frequency of cells in which the xid-bearing X chromosome was active in lymphocytic and other cell populations. In adults, such cells (termed xid cells) were virtually absent in FACS-sorted splenic and lymph node B cells, and in all three splenic subpopulations distinguished on the basis of their relative expression of membrane mu and delta chains. Thus, the xid mutation appeared to compromise the development of all B cells. Erythrocytes, thymocytes, T cells, and granulocytes were unaffected. Selection against xid cells was less pronounced in the spleens of 2-6-wk-old mice. In the bone marrow, there was evidence for selection against xid in the production of B cells (except at 2 wk of age), but not at the pre-B cell level. These data suggest that, in competition with normal non-xid cells, newly-formed xid B cells were less likely to be incorporated into the peripheral B cell pool.

A sandwich ELISA for phosphoglycerate kinase.

Phosphoglycerate kinase (PGK1) is a key enzyme in glycolysis that can also be released from certain cells. In the extracellular milieu, PGK1 reportedly acts as a disulphide reductase to activate plasmin, resulting in the production of angiostatin, a potent angiogenesis inhibitor. Certain cancer cell lines secrete unusually large amounts of PGK1, raising the possibility that serum PGK1 levels can be used to screen for cancer. To facilitate the characterization of the PGK1 secretory pathway and to monitor serum levels of PGK1, we have developed a sensitive sandwich ELISA using an immuno-affinity-purified chicken polyclonal antibody for capturing PGK1 and an immuno-affinity-purified rabbit polyclonal antibody for detecting it. The assay is about 10-fold more sensitive than other reported PGK1 ELISAs. We used the ELISA to quantify the amount of PGK1 released from HeLa cells and PGK1 serum levels in cancer patients. Of 10 cancer patients whose serum was tested, 3 of 4 with pancreatic cancer had 65-900% higher levels of PGK1 than that found in normal serum.

Level of residual enzyme activity modulates the phenotype in phosphoglycerate kinase deficiency.

OBJECTIVE: To study the variable clinical picture and exercise tolerance of patients with phosphoglycerate kinase (PGK) 1 deficiency and how it relates to residual PGK enzyme activity. METHODS: In this case series study, we evaluated 7 boys and men from 5 families with PGK1 deficiency. Five had pure muscle symptoms, while 2 also had mild intellectual disability with or without anemia. Muscle glycolytic and oxidative capacities were evaluated by an ischemic forearm exercise test and by cycle ergometry. RESULTS: Enzyme levels of PGK were 4% to 9% of normal in red cells and 5% to10% in muscle in pure myopathy patients and 2.6% in both muscle and red cells in the 2 patients with multisystem involvement. Patients with pure myopathy had greater increases in lactate with ischemic exercise (2-3 mmol/L) vs the 2 multisystem-affected patients (<1 mmol/L). Myopathy patients had higher oxidative capacity in cycle exercise vs multisystem affected patients (≈30 vs ≈15 mL/kg per minute). One multisystem-affected patient developed frank myoglobinuria after the short exercise test. CONCLUSIONS: This case series study of PGK1 deficiency suggests that the level of impaired glycolysis in PGK deficiency is a major determinant of phenotype. Lower glycolytic capacity in PGK1 deficiency seems to result in multisystem involvement and increased susceptibility to exertional rhabdomyolysis.

Hereditary hemolytic anemia with human erythrocyte pyrimidine 5'-nucleotidase deficiency.

A severe deficiency of a red cell pyrimidine 5'-nucleotidase was found to be associated with hereditary hemolytic anemia in four members of three kindreds. The syndrome was characterized by marked increases above normal in red cell basophilic stippling, total nucleotides, and GSH and by a fairly severe deficiency of ribosephosphate pyrophosphokinase (EC 2.7.6.1.). Patient erythrocytes uniquely contained large amounts of pyrimidine 5'-ribonucleotides. In earlier studies, these were erroneously considered to be adenosine phosphates, since all previous investigations of the nucleotides of human red cells and reticulocytes have shown 97% or more to contain adenine. Total nucleotides in patient cells were present in amounts 3-6 times greater than normal, and approximately 80% contained pyrimidine. The ultraviolet spectral curves of deproteinized red cell extracts exhibited a shift in maximum absorbance from the usual 256-257 nm to approximately 266-270 nm, and absorbance at 250, 270, 280, and 290 nm, expressed as a ratio of that at 260 nm, differed greatly from normal. The spectral characteristics of extracts provide the basis of a readily performed screening procedure, which does not require enzyme assay. The nucleotidase activity in deficient red cells assayed less than 14%, and usually less than 10%, of normal and much less in terms of reticulocyte-rich blood, where it was consistently found to be increased. The enzyme has a pH optimum of 7.5-8.0, is inhibited by EDTA, and does not utilize purine 5'-ribonucleotides or beta-glycerophosphate as substrates. While comparatively few family members have been available thus far for study, initial data are compatible with an autosomal, recessive mode of transmission of the deficiency. The pyrimidine 5'-ribonucleotides are presumably derived from RNA degradation and, not being diffusible, accumulate when the enzyme catalyzing their dephosphorylation is deficient. It is postulated that the prominent basophilic stippling results from retarded ribosomal RNA degradation secondary to accumulation of degradation products, namely pyrimidine 5'-ribonucleotides. Ribosephosphate pyrophosphokinase deficiency is considered to be an epiphenomenon. The mechanism responsible for increased red cell GSH is unknown.

Heterogeneity of human platelets. V. Differences in glycolytic and related enzymes with possible relation to platelet age.

Human platelets were separated by desity-centrifugation into heavy and light populations. Heavy platelets have an average volume approximately twofold greater than light platelets, and have previously been shown to be young platelets. All 11 enzymes of the Embden-Meyerhof pathway plus the five related enzymes: phosphoglucomutase, glucose-6-P dehydrogenase, 6-P-gluconic dehydrogenase, alpha-glycerol-P dehydrogenase, and glutathione reductase (TPNH) were examined in cell lysates from total, heavy, and light platelet populations. Apparent Km for individual enzymes were measured in a total platelet population. Empirical V(max) of the individual enzymes were measured in total, heavy, and light platelet populations. The three apparent rate-limiting enzymes for glycolysis were hexokinase, phosphofructokinase, and glyceraldehyde-3-P dehydrogenase. Heavy platelets contained approximately twofold greater enzyme activity (per gram wet weight) than light platelets for 7 of the 16 enzymes measured: hexokinase, phosphohexoisomerase, phosphofructokinase, glyceraldehyde-3-P dehydrogenase, phosphoglycerokinase, lactic dehydrogenase, and phosphoglucomutase. Heavy platelets also contained 1.9-fold greater reduced glutathione (GSH), 1.7-fold greater DPNH, and 1.2-fold greater TPNH than light platelets. Heavy platelets contained 1.8-fold less lipid peroxidation products (malonyl aldehyde equivalents) than light platelets and were 2.4-fold more resistant to lipid peroxidation catalyzed by 0.1 mM FeCl(3). Sterile incubation of heavy platelets, in vitro for 17 hr, resulted in a significant loss of enzyme activity for the "elevated" seven enzymes when compared with the remainder. Reducing agents such as GSH (0.1 mM), ascorbic acid (0.1 mM), and dithiothreitol (0.01 mM), when added to the incubation mixture, significantly reduced the in vitro loss of activity. In vitro incubation was also associated with a significant loss of GSH and DPNH and a 1.8-fold increase in lipid peroxidation products.

The effect of copper on red cell enzyme activities.

Previous studies have shown a marked effect of very high levels of copper on red cell glucose-6-phosphate dehydrogenase and glutathione. When the effect of more nearly physiological levels of copper were studied, red cell hexokinase, phosphofructokinase, phosphoglyceric kinase, pyruvate kinase, and 6-phosphogluconate dehydrogenase were found to be inhibited. Inhibition was observed both when copper was added directly to hemolysates or when hemolysates were prepared from red cells from whole blood which had been incubated with copper and washed. The inhibition of red cell enzymes by copper was completely reversed by the addition of EDTA.

A study of phosphoglycerate kinase in human erythrocytes. II. Kinetic properties.

Kinetic studies on phosphoglycerate kinase (EC 2.7.2.3) were performed in the forward reaction leading from 1,3-diphosphoglycerate to 3-phosphoglycerate. Substrate activation was observed at fixed levels of ADP or Mg2+ and varying concentrations of 1,3-diphosphoglycerate. A biphasic curve was obtained in both linear and double reciprocal plots demonstrating two Km values (Km1 1.9 - 10(-6) and Km2 9.8 - 10(6) M). Michaelis-Menten-type kinetics were observed in both the linear and double reciprocal plots at fixed levels of 1,3-diphosphoglycerate of ADP and varying concentrations of Mg2+. Apparent Michaelis-Menten kinetics were observed in linear plots when conditions of fixed concentrations of 1,3-diphosphoglycerate or Mg2+ were maintained with varying concentrations of ADP. However, the double-reciprocal plots demonstrated biphasic curves with two Km values (Km1 1.7 - 10(-5) and Km2 1.0 - 10(-4)M). Apparent negative cooperativity was observed with respect to 1,3-diphosphoglycerate and ADP. Phosphoglycerate kinase activity was found to be inhibited by AMP and 2,3-diphosphoglycerate. Substrate activation by 1,3-diphosphoglycerate was maintained in the presence of AMP or 2,3-diphosphoglycerate but at a reduced level of enzyme activity. AMP was found to inhibit enzyme activity non-competitively with respect to 1,3-diphosphoglycerate, ADP and Mg2+. 2,3-Diphosphoglycerate inhibits phosphoglycerate kinase activity with respect to 1,3-diphosphoglycerate, ADP and Mg2+. 2,3-Diphosphoglycerate inhibits phosphoglycerate kinase activity non-competitively with respect of 1,3-diphosphoglycerate.

A study of phosphoglycerate kinase in human erythrocytes. I. Enzyme isolation, purification and assay.

The enzyme ATP-3-phospho-D-glycerate-1-phosphotransferase (EC 2.7.2.3) (phosphoglycerate kinase) has been isolated from human red cells in crystalline form by a modification of the method of Yoshida and Watanabe (1972) J. Biol. Chem. 247, 440-445). The crystalline enzyme was further purified by electrofocusing using carrier ampholytes (pH 7-9). The isoelectric point of phosphoglycerate kinase was estimated to be 8.75. The specific activity of purified phosphoglycerate kinase from electrofocusing was 2200 units per mg of protein at pH 8.3 (37 degrees C). Enzyme activity was assayed in the forward direction leading from 1,3-diphosphoglycerate to a 3-phosphoglycerate using a fluorimetric procedure for NAD-coupled enzymes for the measurement of the reaction rate at very low substrate concentrations. The auxiliary indicator enzymes were added in excess to yield true initial velocity kinetics, i.e. with no time lag upon addition of substrate (1,3-diphosphoglycerate). This was established theoretically using a mathematical model and confirmed experimentally. Further phosphoglycerate kinase was shown to be the rate-limiting step when the assay conditions were varied.

Organization of enzymes of glycolysis and of glutathione metabolism in human red cell membranes.

1) The activities of 16 enzymes of glycolysis and of glutathione metabolism were determined in intact human red cell membranes (ghosts) which were prepared by hypotonic hemolysis. 2) Enzymes and hemoglobin of the ghosts were resolved by two toluene extractions. Only the four enzymes hexokinase, fructose-bisphosphate aldolase, glyceraldehyde-phosphate dehydrogenase and pyruvate kinase could not be released completely from the ghosts. 3) The residual membrane fraction, which was obtained after the toluene extraction of ghosts prepared at 30 imOsM, contained 0.02% of the original hemoglobin content of the red cell. Between 6.5 and 23% of the hemolysate activities of glyceraldehyde-phosphate dehydrogenase, phosphoglycerate kinase, pyruvate kinase and fructose-bisphosphate aldolase were detected in this fraction after mechanical disruption. 4) Sonication of intact ghosts increased the activities of fructose-bisphosphate aldolase, pyruvate kinase and phosphoglycerate kinase. 5) In "white" ghosts prepared at 5 imOsM phosphate buffer which contained 0.5% of the original hemoglobin the activities of fructose-bisphosphate aldolase and glyceraldehyde-phosphate dehydrogenase were detected at high levels. The activities of pyruvate kinase and phosphoglycerate kinase were low in these preparations. 6) The results indicate that one part of all enzymes is loosely attached to the inner surface of the membrane as is hemoglobin. A second part, the "cryptic enzyme activity", is available after resolving by toluene. A residual part of four enzymes is firmly bound to the membrane. Two of them (fructose-bisphosphate aldolase and glyceraldehyde-phosphate dehydrogenase) are oriented toward the inner surface of the membrane, whereas pyruvate kinase and phosphoglycerate kinase are hidden in the lipid core of the membrane.

Membrane-bound ATP fuels the Na/K pump. Studies on membrane-bound glycolytic enzymes on inside-out vesicles from human red cell membranes.

ATP stimulates Na transport into inside-out vesicles (IOVs) made from human red cell membranes; strophanthidin inhibits the ATP-stimulated transport. The substrates for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and phosphoglycerate kinase (PGK) (glycolytic enzymes bound to the cytoplasmic surface of the red cell membrane) also stimulate Na transport into IOVs without added ATP. The elution of GAPDH from the membranes prevents the stimulation by the substrates, but not by exogenous ATP. Hexokinase plus glucose (agents that promote breakdown of ATP) prevent stimulation of Na transport by exogenous ATP but not by the substrates for GAPDH and PGK. [32P]orthophosphate is incorporated into a membrane-bound organic phosphate compound shown chromatographically to be ATP. The level of membrane-bound ATP is decreased when Na is added, and this decrease is inhibited by strophanthidin. When further synthesis of [32P]ATP is blocked by the addition of unlabeled orthophosphate, all of the membrane-bound [32P]ATP is dissipated by the addition of Na. From these observations it was concluded that membrane-bound glycolytic enzymes synthesize ATP and deposit it in a membrane-associated compartment from which it is used by the Na/K pump.

X-linked gene expression in the Virginia opossum: differences between the paternally derived Gpd and Pgk-A loci.

Expression of X-linked glucose-6-phosphate dehydrogenase (G6PD) and phosphoglycerate kinase-A (PGK-A) in the Virginia opossum (Didelphis virginiana) was studied electrophoretically in animals from natural populations and those produced through controlled laboratory crosses. Blood from most of the wild animals exhibited a common single-banded phenotype for both enzymes. Rare variant animals, regardless of sex, exhibited single-banded phenotypes different in mobility from the common mobility class of the respective enzyme. The laboratory crosses confirmed the allelic basis for the common and rare phenotypes. Transmission of PGK-A phenotypes followed the pattern of determinate (nonrandom) inactivation of the paternally derived Pgk-A allele, and transmission of G6PD also was consistent with this pattern. A survey of tissue-specific expression of G6PD phenotypes of heterozygous females revealed, in almost all tissues, three-banded patterns skewed in favor of the allele that was expressed in blood cells. Three-banded patterns were never observed in males or in putatively homozygous females. These patterns suggest simultaneous, but unequal, expression of the maternally and paternally derived Gpd alleles within individual cells (i.e., partial paternal allele expression). The absence of such partial expression was noted in a parallel survey of females heterozygous at the Pgk-A locus. Thus, it appears that Gpd and Pgk-A are X-linked in D. virginiana and subject to preferential paternal allele inactivation, but that dosage compensation may not be complete for all paternally derived X-linked genes. The data establish the similarity between the American and Australian marsupial patterns of X-linked gene regulation and, thus, support the hypothesis that this form of dosage compensation was present in the early marsupial lineage that gave rise to these modern marsupial divisions. In addition, the data provide the first documentation of the differential expression of two X-linked genes in a single marsupial species. Because of its combination of X-linked variation, high fecundity, and short generation time, D. virginiana is a unique model for pursuing questions about marsupial gene regulation that have been difficult to approach through studies of Australian species.

Metabolic correlates of immune dysfunction in malnourished children.

Mononuclear cells of malnourished children contain diminished activity of phosphoglycerate kinase and pyruvate kinase (PK). The PK activity of these cells correlates well with the percentage of circulating thymus-derived lymphocytes (T-cells). Phytohemagglutinin causes an immediate increase in PK activity of mononuclear cells of malnourished patients. The correlation of PK activity with T-cells and the response of PK activity to phytohemagglutinin are in distinct contrast to observed perturbations of neonatal mononuclear cell metabolism. The relationship of the metabolic alterations to the pathophysiology of the immune system in malnutrition has not yet been defined.

Growth hormone deficiency and empty sella syndrome in a boy with dup(X) (q13.3----q21.2).

A 2 8/12-year-old boy with severe growth failure and mental retardation was found to have a maternally derived tandem duplication of the long arm of X chromosome, dup(X) (q13.3----q21.2). Karyotypic interpretation was further confirmed in this patient by a double gene dose for red blood cell phosphoglycerate kinase. DNA replication study showed that the duplicated X chromosome was always late replicating in peripheral blood lymphocytes as well as in skin fibroblasts from the mother. Endocrine studies in the patient demonstrated growth hormone deficiency. Magnetic resonance imaging of the head then disclosed the empty sella syndrome. This appears to be the first report of a dup(Xq) patient associated with a growth hormone deficiency and the empty sella syndrome. We emphasize that duplication of the proximal Xq in males represents another microduplication syndrome (Thode-Leonard syndrome).

Tissue levels of glycolytic enzymes in phosphoglycerate kinase deficiency.

Phosphoglycerate kinase deficiency is a rate, X-linked disorder associated with a severe haemolytic anaemia. In general the deficiency has been demonstrated only in erythrocytes and leucocytes. However, in a subject with this condition, the activity of phosphoglycerate kinase in lymphocytes and platelets was also shown to be less than 5% of the normal value. Following the death of this subject in 1979, the deficiency was also found to occur in tissue samples of brain, skeletal muscle, liver and cardiac muscle, obtained at the autopsy. Values for phosphoglycerate kinase were of the order of 0.5-5% of normal controls. Other glycolytic enzymes which were tested were hexokinase, pyruvate kinase, enolase and 2-phosphoglyceromutase. In general, values for these enzymes were either normal or slightly raised.

Human erythrocyte phosphoglycerate kinase deficiency: presence in a deficient patient of a stable variant with lowered catalytic activity.

The phosphoglycerate kinase deficiency found in one boy with hemolytic anemia was associated with an almost normal concentration of phosphoglycerate kinase-immunologically related material in the patient's erythrocytes. Consequently the catalytic activity of the mutant enzyme was drastically lowered. Besides, the abnormal phosphoglycerate kinase was more stable to heat and to urea than normal phosphoglycerate kinase and its isoelectric point was slightly increased.

Hemolytic anemia in hereditary pyrimidine 5'-nucleotidase deficiency. II. Effect of pyrimidine nucleotides and their derivatives on glycolytic and pentose phosphate shunt enzyme activity.

We evaluated the glycolytic intermediate concentrations from the erythrocytes of a patient with hereditary pyrimidine 5'-nucleotidase (P5'N) deficiency. Conclusive evidence for a metabolic block was not found. We evaluated the effects of the pyrimidine (cytidine and uridine) tri- and diphosphate nucleotides (CTP, CDP, UTP, UDP) and the choline and ethanolamine derivatives of CDP (CDP-choline, CDP-ethanolamine) on the activities of key enzymes of the Embden-Meyerhof pathway. CTP and UTP inhibited fructose-6-phosphate competitively for phosphofructokinase and phosphoenolpyruvate competitively for pyruvate kinase. In both cases, the Ki of the pyrimidine nucleotide and Km of the glycolytic substrate were above their intraerythrocytic concentrations. CTP was a competitive inhibitor of ADP for pyruvate kinase with a Ki near its intraerythrocytic concentration. CDP-choline and CDP-ethanolamine had no effect on the activities of Embden-Meyerhof or pentose phosphate shunt enzymes. Thus, the nature of the hemolytic anemia in hereditary P5'N deficiency remains enigmatic.