Sulfhydryl modification of 22 human erythrocyte enzymes was achieved by exposing intact erythrocytes, hemolysates, and partially purified enzymes to (i) persulfides (RSSH) generated nonenzymatically from cystine in the presence of pyridoxal phosphate and (ii) 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.In previous studies (1, 2) we have reported the effects of methyl methanethiosulfonate, CH3SO2SCH3, a CH3S donor developed by Smith et al. (3), on certain erythrocyte enzymes. CH3SO2SCH3 is one of a family of alkyl alkanethiosulfonates and delivers its CH3S group under mild conditions to accessible cysteinyl residues of enzyme proteins. It readily traverses the erythrocyte (RBC) membrane and produces profound alterations in certain enzyme properties, including catalytic activity and thermostability (1, 2).Persulfides are intermediates in sulfur compound transformations in which -SH is bonded to another sulfur atom to form RSSH. Accumulating evidence of a potential physiological role for persulfide sulfur in biological systems (partially reviewed in ref 4) prompted the present study comparing persuffide effects with those of CH3SO2SCH3 on RBC enzymes. Since low molecular weight persulfides are unstable near pH 7, it was necessary to use incubation systems in which persulfide was continuously generated. The first of these was initially described by Cavallini et al. (5), who showed that pyridoxal with a metal causes the P-elimination reaction of cystine, with the nonenzymatic formation of cysteine persulfide, ammonia, and pyruvate. The second incubation system utilized mercaptopyruvate as the sulfur donor (4). This sulfhydryl compound is the substrate for sulfurtransferase (EC 2.8.1.2) (6), which accepts, transports, and transfers its sulfur to form persulfides with suitable acceptors. This enzyme is present in human RBC (7). METHODS RBC and Hemolysates. Blood treated with the anticoagulant heparin was washed and freed of leukocytes by passage with saline over a column of microcellulose (8). After washing, centrifugation, and resuspension in saline, the RBC were adjusted to =4-4.5 x 106 per A.l. Where indicated, cells were lysed as described in text.Partially Purified Lysates. Hemolysates were largely freed of Hb precipitated by ZnSO4 at pH 8.0 in 0.1 M Tris HCl ...

No abstract

The persistence of normal thymidine nucleotidase (ThyNase) activity in subjects with pyrimidine nucleotidase (PyrNase) Recognition of the existence of pyrimidine-specific nucleotidase (PyrNase) evolved from studies of patients who shared an unusual hemolytic syndrome in which affected erythrocytes (i) retained 10% or less of normal PyrNase activity, (ii) accumulated enormous quantities of cytidine and uridine nucleotides, and (iii) exhibited prominent basophilic stippling with Wright's stain (1). None of these alterations occurs in heterozygous relatives, who have 40-60% of con-

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.