Purine Nucleoside Phosphorylase from Human Erythrocytes

Evidence has been obtained on the content and essential role of sulfhydryl groups in crystalline human erythrocytic purine nucleoside phosphorylase.The present report is concerned with the total number of -SH groups of purine nucleoside phosphorylase and their behavior.'.Sulfhydryl groups of purine nucleoside phosphorylase differ greatly in their reactivity with sulfhydryl reagents, 5,5'-dithiobis(Znitrobenzoic acid) (DTNB) and p-chloromercuribenzoic acid (PCMB).One mole (81,000 g) of native purine nucleoside phosphorylase binds with 11 to 12 moles of 14C-labeled PCMB, and there is no increase in number upon denaturation with sodium dodecyl sulfate (SDS).DTNB reacts with four to five -SH groups in the native enzyme, but upon denaturation with SDS 11 to 12 -SH groups react with DTNB.The reaction of the first four or five -SH groups of purine nucleoside phosphorylase with PCMB inactivates the enzyme completely, but activity can be restored by addition of an excess of dithiothreitol.However, the reaction of DTNB with four -SH groups causes only a partial loss of activity (60%) which is not reactivated completely by treatment with dithiothreitol.Guanine, hypoxanthine, adenine, inosine, and Formycin B partially protect the -SH groups from the reaction with DTNB whereas ribose l-phosphate and phosphate are ineffective.However,