Partial purification and properties of purine nucleoside phosphorylase from rabbit erythrocytes.
1. The partial purification of purine nucleoside phosphorylase from rabbit erythrocytes is described. 2. Analytical and preparative isoelectric focusing gave a pI value for the enzyme of 4.65. 3. Gel-chromatography and sucrose-density-gradient-centrifugation techniques gave estimates of the molecular weight in the range 75000-83000. 4. Lineweaver-Burk plots of kinetic data were non-linear at high inosine concentrations. Extrapolation of the linear part of such plots yielded a Km value for inosine of about 70 micrometer for the rabbit erythrocyte and liver enzymes. 5. A Hill interaction coefficient of 0.75 was obtained, suggesting negative co-operativity with respect to the binding of inosine. 6. Treatment of the enzyme with 5,5'-dithiobis-(2-nitrobenzoic acid) caused partial inactivation, and subsequent Lineweaver-Burk plots with inosine as substrate displayed complete linearity, with an increase in Km value for inosine to 200 micrometer. 7. Starch-gel electrophoresis did not reveal the presence of secondary isoenzymes; all tissue extracts examined gave electrophoretic patterns similar to those obtained with the partially purified enzyme from erythrocytes. 8. Results of hybridization studies with nucleoside phosphorylase from human foetal liver suggest that the rabbit enzyme is also a trimer.
D000818:Animals; D055598:Chemical Phenomena; D002621:Chemistry; D004228:Dithionitrobenzoic Acid / Q000494:pharmacology; D004912:Erythrocytes / Q000201:enzymology*; D007288:Inosine / Q000378:metabolism; D007700:Kinetics; D008970:Molecular Weight; D010430:Pentosyltransferases / Q000097:blood*; D011683:Purine-Nucleoside Phosphorylase / Q000037:antagonists & inhibitors / Q000097:blood* / Q000302:isolation & purification; D011817:Rabbits; D014018:Tissue Distribution
D007288:Inosine; D004228:Dithionitrobenzoic Acid; D010430:Pentosyltransferases; D011683:Purine-Nucleoside Phosphorylase