Chinese hamster purine-nucleoside phosphorylase: purification, structural, and catalytic properties

Abstract

Purine-nucleoside phosphorylase (EC 2.4.2.1; purine-nucleoside:orthophosphate ribosyltransferase) was purified to apparent homogeneity from Chinese hamster liver, kidneys and V79 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.degree. C the purifed enzyme converts 60 .mu.mol of guanine to guanosine/min per mg of protein. Electrophoresis in sodium dodecyl sulfate-polyacrylamide gels indicates that the enzyme is composed of identical subunits of MW 30,000. The native enzyme behaves as a mixture of dimers of MW 68,000 and trimers of MW 89,000 during Sephadex G-100 chromatography. Sucrose gradient centrifugation indicates that the enzyme has a sedimentation coefficient of 5.4S, which corresponds to a MW of 94,000 and suggests a trimer structure. The enzyme displays Michaelis-Menten kinetics with apparent Km of 20 .mu.M for both hypoxanthine and guanine, 35 .mu.M for guanosine, 50 .mu.M for inosine and 200 .mu.M for both ribose 1-phosphate and phosphate. During isoelectrofocusing the enzyme forms a single major band at a pI of 5.25.