Chinese hamster purine-nucleoside phosphorylase: purification, structural, and catalytic properties.
Purine-nucleoside phosphorylase (EC 2.4.2.1; purine-nucleosideorthophosphate ribosyltransferase) was purified to apparent homogeneity from Chinese hamster liver and kidneys and from V79 tissue culture cells. The enzymes from both sources appear to have identical structural and catalytic properties. A simple rapid radioisotope assay capable of detecting 0.1 nmol of product for both directions of the purine-nucleoside phosphorylase reaction is described using Bio-Rad Cu2+ Chelex in Pasteur pipet columns. At 37 degrees C the purified enzyme converts 60 mumol of guanine to guanosine per min per mg of protein. Electrophoresis in sodium dodecyl sulfate-polyacrylamide gels indicates that the enzyme is composed of identical subunits of 30 000 molecular weight. The native enzyme behaves as a mixture of dimers of 68 000 molecular weight and trimers of 89 000 molecular weight during Sephadex G-100 chromatography. Sucrose gradient centrifugation indicates that the enzyme had a sedimentation coefficient of 5.4 S, which corresponds to a molecular weight of 94 000 and suggests a trimer structure. The enzyme displays Michaelis-Menten kinetics with apparent Michaelis constants of 20 muM both hypoxanthine and guanine, 35 muM form guanosine, 50 muM for inosine, and 200 muM for both ribose 1-phosphate and phosphate. During isoelectrofocusing, the enzyme forms a single major band at a pI of 5.25.
D000818:Animals; D002460:Cell Line; D006224:Cricetinae; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D046911:Macromolecular Substances; D008970:Molecular Weight; D010430:Pentosyltransferases / Q000378:metabolism; D011683:Purine-Nucleoside Phosphorylase / Q000302:isolation & purification / Q000378:metabolism
D046911:Macromolecular Substances; D010430:Pentosyltransferases; D011683:Purine-Nucleoside Phosphorylase