Partial purification and properties of purine nucleoside phosphorylase from rabbit erythrocytes

Abstract

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.

Stability of Carbovir, a Potent Inhibitor of HIV, to Phosphorolysis by Human Purine Nucleoside Phosphorylase

“…PNP catalyses the reversible phosphorolysis of ribose and 2'-deoxyribose purine nucleosides of guanine and hypoxanthine. PNP has been purified from a variety of mammalian tissues including human erythrocytes (Kim et aI., 1968;Stoeckler et al, 1978), human granulocytes (Wiginton et al, 1980), rabbit erythrocytes (Savage and Spencer, 1977), and bovine erythrocytes and spleen (Agarwal et al, 1975).The kinetic data of PNP from these mammalian tissues indicate that the enzymes have similar substrate characteristics. Interest in the enzyme arises as there is a remarkably high level of PNP in human erythrocytes (Stoeckler et al, 1982),therefore various nucleoside analogues of chemotherapeutic potential may be degraded during their transportation through the blood to the target tissues.…”

Rabbit erythrocyte purine nucleoside phosphorylase. Differential-inactivation studies

“…5). Spencer, 1977). When similar experiments were per-Diflerential inactivation ofcalfspleenpurine nucleoside formed with the enzyme in 50mM-Tris/HCl buffer, phosphorylase pH 7.5, instead ofphosphate buffer, a biphasic pattern Differential-inactivation studies on calf spleen of inactivation was observed (Fig.…”

“…This was measured by using the xanthine oxidaselinked method previously described (Savage & Spencer, 1977), except that xanthine oxidase was extensively dialysed against 5OmM-Tris/HCI buffer, pH7.5, before use in the assay mixture.…”

“…It has been proposed by Zyk et al (1969) that the binding of such ligands may cause a change in the conformation of the enzyme structure that is reflected in stabilization towards proteolysis and thermal inactivation, and the term 'conformative response' has been suggested by Citri & Zyk (1967) to describe conformational changes that accompany specific ligand binding. In the preceding paper (Savage & Spencer, 1979) and elsewhere (Savage & Spencer, 1977) it was proposed that negative homotropic interactions may occur between the active sites of rabbit erythrocyte purine nucleoside phosphorylase when the concentration of either substrate is varied at a fixed saturating concentration of the second substrate. The kinetic data reported in both studies (Savage & Spencer, 1977, 1979 are consistent with a model in which the binding of each substrate molecule causes conformational changes in the enzyme structure that lead to a decrease in affinity between the substrate and vacant sites on neighbouring subunits.…”

“…In the preceding paper (Savage & Spencer, 1979) and elsewhere (Savage & Spencer, 1977) it was proposed that negative homotropic interactions may occur between the active sites of rabbit erythrocyte purine nucleoside phosphorylase when the concentration of either substrate is varied at a fixed saturating concentration of the second substrate. The kinetic data reported in both studies (Savage & Spencer, 1977, 1979 are consistent with a model in which the binding of each substrate molecule causes conformational changes in the enzyme structure that lead to a decrease in affinity between the substrate and vacant sites on neighbouring subunits.…”