AMP deaminase as a cell-age marker in transient erythroblastopenia of childhood and its role in the adenylate economy of erythrocytes.

Erythrocytes from 11 patients with presumptive diagnoses of transient erythroblastopenia of childhood were evaluated retrospectively (six) or prospectively (five) for a possible relationship between erythrocyte adenosine 5'-monophosphate aminohydrolase, adenylic acid deaminase (AMP deaminase) activity and intracellular concentrations of adenine nucleotides. Older red blood cell (RBC) cohorts in these patients consistently exhibited significantly decreased activities of AMP deaminase (approximately 5% to 70% of normal control mean) in association with increased concentrations (up to threefold) of adenosine triphosphate (ATP) and total adenine nucleotides. We postulate that the latter is a direct consequence of the former, since diminishing AMP deaminase activity in aging cells should reduce the drain on the adenine nucleotide pool imposed by irreversible deamination of AMP to inosine 5'-monophosphate. Consistent reductions in AMP deaminase activity indicate that this enzyme should also serve as a reliable marker of mean RBC age useful in diagnostic confirmation of transient erythroblastopenia. The observed increases in ATP and total adenine nucleotides in older RBCs require a reevaluation of the traditional view that age-related losses of these compounds mediate the ultimate demise of senescent erythrocytes. Similar alterations in the balance of degradative and salvage pathways in RBC nucleotide metabolism may also underlie certain cases of so-called "high ATP syndrome."

Inhibition of adenylate kinase by P1,P5-di(adenosine 5') pentaphosphate in assays of erythrocyte enzyme activities requiring adenine nucleotides.

P1,P5-di(adenosine 5')pentaphosphate (Ap5A) is an excellent inhibitor of human hemolysate adenylate kinase at concentrations near 2 microM and above. At ten times this concentration and in hemolysate enzyme assays under conditions described in this paper it appears not to alter reaction data in the case of hexokinase, phosphofructokinase, and phosphoglycerokinase. In the pyruvate kinase assay, very modest reductions in activity are noted, and kinetics with phosphoenolpyruvate, adenosine diphosphate (ADP), and uridine diphosphate (UDP) are unaltered.

Pyruvate kinase Greensboro. A four-generation study of a high K0.5s (phosphoenolpyruvate) variant.

The proband with lifelong hemolytic anemia has a high K0.5s phosphoenolypyruvate (PEP) erythrocyte pyruvate kinase (PK) variant substantially but incompletely normalized by the allosteric modifier fructose-1,6-diphosphate (F-1,6-P2) with conversion of sigmoidal to hyperbolic kinetics. Heterozygotes in four generations express qualitatively identical but less severely abnormal kinetics and lack overt hemolysis. Kinetic abnormalities are closely mimicked by sulfhydryl modification of normal PK. Three distinct clinical and metabolic phenotypes characterize the proband and two sisters: variant PK and hemolytic anemia, variant PK without clinical manifestations or hemolysis, and complete normality. Their mother, whose red cell PK is entirely normal except for a questionably slightly low Vmax, is postulated to express the gene products of nonidentical alleles, one encoding a product with mildly less favorable catalytic characteristics. At low PEP concentrations, the proband and heterozygotes for the PK mutant express only a very small fraction of normal PK activity despite apparent inheritance of one normal allele in the latter. Evidence suggests that disproportionately lowered PK activity may be a property of a heterotetrameric PK. Illusory abnormalities in nucleotide specificity are artifacts of diminished substrate affinity characterizing the mutant PK.

Erythrocyte pyruvate kinase (PK): the variable significance of "nucleotide specificity" in the characterization of mutant variants.

The half-saturation constant (K0.5s) phosphoenolpyruvate (PEP) for red cell pyruvate kinase (PK) with co-factors UDP and GDP is less than one-half that with ADP with or without additions of the allosteric modifier, fructose-1, 6-dephosphate (F-1, 6-P2) to the assay. The Vmax is markedly greater with ADP than with UDP or GDP, but with (PEP) at 0.5 mM, activity with all co-factors is about equal and at lower concentrations greater with UDP and GDP. With high K0.5s (PEP) mutant enzymes, and at the usual test concentration (lmM) for PEP when nucleotide specificity is assessed, the abnormally low saturation of variant enzymes may result in higher activity with UDP and GDP than with ADP--the opposite of the "normal situation." The apparent aberration in nucleotide specificity may thus be illusory and secondary to the abnormal K0.5s (PEP) of the mutant. Example data are recorded. Variations in K0.5s (PEP) may also be introduced during enzyme preparation for assay, particularly when partial purification is employed.

Substrate specificity and pH sensitivity of deoxyribonucleotidase and pyrimidine nucleotidase activities in human hemolysates.

Residual 5'-nucleotidase activities in hemolysates from nine subjects with severe hereditary deficiency of pyrimidine nucleotidase (PyrNase) were compared to those in normal and reticulocyte-rich controls. Dephosphorylation rates of 12 potential ribo- and deoxyribomononucleotide substrates were measured as a function of pH. Data confirmed the existence of at least two isozymes of 5'-nucleotidase, PyrNase, and 2'-deoxy-5'-ribonucleotide phosphohydrolase (dNase) distinguishable by differences in maximal velocities, substrate preferences and restrictions, and pH optima. PyrNase was confirmed to be active principally with pyrimidine substrates (UMP = dCMP greater than CMP much greater than dTMP greater than dUMP) at a pH optimum of 7.5 +/- 0.1. dNase activity occurred with both purine and pyrimidine substrates and was maximal with deoxy analogs (dIMP much greater than dUMP greater than dGMP greater than dTMP = dAMP much greater than dCMP) at a pH optimum of 6.2, but slight cross-reactivity occurred with some nondeoxy substrates (IMP greater than GMP greater than UMP = XMP greater than CMP). PyrNase and dNase may be complementary systems that serve physiologically to clear the cytosol of RNA and DNA degradation products during maturation of erythroid elements by conversion of nucleotide monophosphates to diffusible nucleosides.

Modification of erythrocyte enzyme activities by persulfides and methanethiol: possible regulatory role.

Sulfhydryl modification of 22 human erythrocyte enzymes was achieved by exposing intact erythrocytes, hemolysates, and partially purified enzymes to persulfides (RSSH) generated nonenzymatically from cystine in the presence of pyridoxal phosphate and mercaptopyruvate, which donates its sulfur to suitable acceptors with the mediation of the carrier enzyme, mercaptopyruvate sulfurtransferase (EC 2.8.1.2). The inhibition pattern was qualitatively similar for persulfides and that previously reported by us for the methylthio-group donor, methyl methanethiosulfonate. Thirteen activities were inhibited, and 9 were minimally or not at all affected. Pyruvate kinase was similarly modified by all systems in terms of phosphoenolpyruvate kinetics, thermostability, and interaction with the negative effector ATP. Partial-to-complete reversal of inhibition was documented in a subset of activities inhibited by mercaptopyruvate upon 30-min incubation with 1 mM dithiothreitol. A possible physiologic role for methylthio groups and for persulfides is discussed.

Acute intravascular hemolysis in the black rhinoceros: erythrocyte enzymes and metabolic intermediates.

Enzymes of aerobic and anaerobic glycolysis, glutathione cycling, and nucleotide metabolism were assayed on erythrocytes from 7 healthy rhinoceroses, 2 rhinoceroses during periods of intravascular hemolysis, and 1 rhinoceros without clinical signs of illness, which was the mother of 3 offspring with intravascular hemolytic syndrome. Measurements also were made of erythrocyte concentrations of glycolytic intermediates, adenine nucleotides, and glutathione. Although comparison of results for healthy and affected rhinoceroses did not identify an enzyme abnormality as a cause for the hemolytic syndrome, the data provided information regarding the metabolic characteristics of erythrocytes from healthy rhinoceroses.

Mechanisms of adenosine 5'-monophosphate catabolism in human erythrocytes.

Uncertainties regarding the role of pyrimidine nucleotidase (PyrNase) in AMP catabolism were resolved by studies of erythrocytes from normal controls, controls with young mean cell ages, and patients with hereditary hemolytic anemia due to severe deficiency of PyrNase. Hemolysates from the latter exhibited undiminished capacity to dephosphorylate AMP over a broad range of pH, indicating that PyrNase was not directly involved. In each subject group, the rates of AMP dephosphorylation between pH 5.1 and 8.3 were indistinguishable from those of IMP, suggesting a potential role for AMP-deaminase, an erythrocyte enzyme that was stimulated by coformycin at pH 7.2. Quantitative analysis of catabolites in incubated hemolysates confirmed that AMP degradation preferentially occurred via deamination to IMP with subsequent dephosphorylation by another erythrocyte nucleotidase isozyme, deoxyribonucleotidase. Both AMP-deaminase and deoxyribonucleotidase have acidic pH optima with minimal activities at physiologic pH, suggesting that this pathway of AMP catabolism could accelerate depletion of the adenine nucleotide pool and thereby mediate the demise of senescent erythrocytes sequestered in the spleen.

Identification of thymidine nucleotidase and deoxyribonucleotidase activities among normal isozymes of 5'-nucleotidase in human erythrocytes.

The persistence of normal thymidine nucleotidase (ThyNase) activity in subjects with pyrimidine nucleotidase (PyrNase) deficiency suggested the possible existence of separate isozymes in normal human erythrocytes. This hypothesis was confirmed by studies of PyrNase-deficient individuals from five unrelated families. Erythrocytes deficient in PyrNase retained normal activity of an enzyme system preferentially active at pH 6.2 with a variety of 2'-deoxyribonucleoside 5'-monophosphate substrates, including those of uridine, thymidine, and cytidine. Lesser activities were observed with the corresponding ribonucleotides. Normal control hemolysates were also found capable of effectively dephosphorylating purine nucleotides (dAMP greater than AMP) when pH was lowered sufficiently from the pH 7.4-8.0 region commonly used in conventional assays. Variations in substrate specificity, pH optima, kinetics, and sensitivity to inactivation by Pb2+ indicated the existence of multiple 5'-nucleotidase isozymes in normal erythrocytes: PyrNase and deoxyribonucleotidase(s) that might function physiologically in the conversion of DNA-derived nucleotides to diffusible nucleosides. Evolution of such a unique 5'-nucleotidase suggests that normal erythroblast maturation and nuclear extrusion is accompanied by a degree of karyolysis sufficient to require dephosphorylation and clearance of DNA degradation products.

Pyrimidine nucleotidase deficiency with active dephosphorylation of dTMP: evidence for existence of thymidine nucleotidase in human erythrocytes.

Erythrocytes from a patient with classical pyrimidine nucleotidase (PyN) deficiency had less than 10% residual PyN activity with uridine 5'-monophosphate (UMP) or cytidine 5'-monophosphate (CMP) as substrate, but exhibited brisk nucleotidase activity with thymidine 5'-monophosphate (dTMP). This strongly suggests the existence of separate enzymes or isozymes of PyN in normal human erythrocytes--an hypothesis that should be tested by similar studies in other cases of severe PyN deficiency, whether induced by genetic defects or lead toxicity.