Characterization of the subunits of purine nucleoside phosphorylase from cultured normal human fibroblasts

✍ Scribed by Vassilis I. Zannis; Lorraine J. Gudas; David W. Martin

• Publisher: Springer
• Year: 1979
• Tongue: English
• Weight: 434 KB
• Volume: 17
• Category: Article
• ISSN: 0006-2928
• DOI: 10.1007/bf00502122

✦ Synopsis

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 ( Ip 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 isoelectrie 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 eleetrophoretie 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 eleetrophoretic patterns of the erythrocyte and fibroblast enzymes. The purine nueleoside phosphorylase subunit 1 is detectable in the autoradiogram from a two-dimensional electrophoretic analysis of a crude,