Characterization of purine nucleoside phosphorylase from human granulocytes and its metabolism of deoxyribonucleosides.
Purine nucleoside phosphorylase (EC 2.4.2.1) has been purified 715-fold from human granulocytes by formycin B affinity chromatography. The purified enzyme has a subunit molecular weight of 32,800 +/- 1,300 and an estimated native molecular weight of 102,000, which indicates a trimeric subunit structure. The purified enzyme migrates as a single band upon denaturing and nondenaturing polyacrylamide gel electrophoresis. Its amino acid composition has been determined. Isoelectric focusing of the purified enzyme produces three protein bands with isoelectric pH values of 5.8 to 6.1. The purified enzyme catalyzes the phosphorolysis of inosine, deoxyinosine, guanosine, and deoxyguanosine with apparent Km values of 0.21 mM, 0.32 mM, 0.21 mM, and 0.24 mM, respectively. The ribonucleosides and deoxyribonucleosides of adenine and the pyrimidines are not substrates. There are no major differences in metabolism of ribonucleosides and deoxyribonucleosides. Purine nucleoside phosphorylase activity is inhibited by formycin B in a competitive manner and by adenine in a mixed noncompetitive manner. Antiserum to the purified protein has been prepared and is useful in the immunochemical assay of phosphorylase over the range 10 to 100 ng of protein.
D000596:Amino Acids / Q000032:analysis; D003853:Deoxyribonucleosides / Q000378:metabolism*; D006098:Granulocytes / Q000201:enzymology*; D006801:Humans; D007122:Immunoelectrophoresis; D007700:Kinetics; D046911:Macromolecular Substances; D008970:Molecular Weight; D010430:Pentosyltransferases / Q000097:blood*; D011683:Purine-Nucleoside Phosphorylase / Q000097:blood* / Q000302:isolation & purification
D000596:Amino Acids; D003853:Deoxyribonucleosides; D046911:Macromolecular Substances; D010430:Pentosyltransferases; D011683:Purine-Nucleoside Phosphorylase