Characterization of the subunits of purine nucleoside phosphorylase from cultured normal human fibroblasts.
In previous communications we have demonstrated that the subunits of normal human erythrocyte purine nucleoside phosphorylase can be resolved into four major (1-4) and two minor (1p and 2p) components with the same molecular weight but different apparent isoelectric points (and net ionic charge). The existence of subunits with different charge results in a complex isoelectric focusing pattern of the native erythrocytic enzyme. In contrast, the isoelectric focusing pattern of the native enzyme obtained from cultured human fibroblasts is simpler. The multiple native isoenzymes obtained from human erythrocytes and human brain have isoelectric points ranging from 5.0 to 6.4 and from 5.2 to 5.8 respectively, whereas cultured human fibroblasts have two major native isoenzymes with apparent isoelectric points of 5.1 and 5.6. Purine nucleoside phosphorylase has been purified at least a hundredfold from 35S-labeled cultured human fibroblasts. A two-dimensional electrophoretic analysis of the denatured purified normal fibroblast enzyme revealed that it consists mainly of subunit 1 (90%) with small amounts of subunits 2 (10%) and 3 (1%). This accounts for the observed differences between the native isoelectric focusing and the electrophoretic patterns of the erythrocyte and fibroblast enzymes. The purine nucleoside phosphorylase subunit 1 is detectable in the autoradiogram from a two-dimensional electrophoretic analysis of a crude, unpurified extract of 35S-labeled cultured normal human fibroblasts. The fibroblast phosphorylase coincides with the erythrocytic subunit 1 of the same enzyme, and the cultured fibroblasts of a purine nucleoside phosphorylase deficient patient (patient I) lack this protein component, genetically confirming the identity of the purine nucleoside phosphorylase subunit in cultured fibroblasts.
D001051:Apoenzymes; D001059:Apoproteins; D004591:Electrophoresis, Polyacrylamide Gel; D004912:Erythrocytes / Q000201:enzymology; D005347:Fibroblasts / Q000201:enzymology; D006801:Humans; D007525:Isoelectric Focusing; D010430:Pentosyltransferases; D011683:Purine-Nucleoside Phosphorylase
D001051:Apoenzymes; D001059:Apoproteins; D010430:Pentosyltransferases; D011683:Purine-Nucleoside Phosphorylase