Nicotinamide riboside phosphorylase from beef liver: purification and characterization.
Nicotinamide riboside phosphorylase (NR phosphorylase) from beef liver has been purified to apparent homogeneity at 300-fold purification with a 35% yield. Kinetic constants for the enzyme-catalyzed phosphorolysis were as follows Knicotinamide riboside, 2.5 +/- 0.4 mM; Kinorganic phosphate, 0.50 +/- 0.12 mM; Vmax, 410 +/- 30 X 10(-6) mol min-1 mg protein-1, respectively. The molecular weights of the native enzyme and subunit structure were determined to be 131,000 and 32,000, respectively, suggesting the beef liver NR phosphorylase to be tetrameric in structure and consistent with the presence of identical subunits. The amino acid composition was shown to be very similar to that reported for human erythrocyte purine-nucleoside phosphorylase but differing considerably from that found for rat liver purine-nucleoside phosphorylase. In addition to catalytic activity with nicotinamide riboside, the beef liver enzyme catalyzed a phosphorolytic reaction with inosine and guanosine exhibiting activity ratios, nicotinamide riboside:inosine: guanosine of 1.00:0.35:0.29, respectively. These ratios of activity remained constant throughout purification of the beef liver enzyme and no separation of these activities was detected. Phosphorolysis of nicotinamide riboside was inhibited competitively by inosine (Ki = 75 microM) and guanosine (Ki = 75 microM). Identical rates of thermal denaturation of the beef liver enzyme were observed when determined for the phosphorolysis of either nicotinamide riboside or inosine. These observations coupled with studies of pH and specific buffer effects indicate the phosphorolysis of nicotinamide riboside, inosine, and guanosine to be catalyzed by the same enzyme.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002417:Cattle; D006151:Guanosine; D007288:Inosine; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008970:Molecular Weight; D009536:Niacinamide / Q000031:analogs & derivatives; D010430:Pentosyltransferases / Q000378:metabolism*; D011487:Protein Conformation; D011683:Purine-Nucleoside Phosphorylase / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D011726:Pyridinium Compounds; D013379:Substrate Specificity
D000596:Amino Acids; D011726:Pyridinium Compounds; C018613:nicotinamide-beta-riboside; D006151:Guanosine; D009536:Niacinamide; D007288:Inosine; D010430:Pentosyltransferases; D011683:Purine-Nucleoside Phosphorylase