48 PARTIAL PURIFICATION OF THE NUCLEOSIDE TRANSPORTER FROM HUMAN ERYTHROCYTES

Abstract

The nucleoside transporter of human erythrocytes can be identified by photolabelling with radiolabelled nitrobenzyl-thioinosine, a potent high affinity inhibitor of nucleoside transport in human erythrocytes and some somatic cells. The nucleoside transporter migrates as a broad band on SDS potyacrylamide gels with a molecular weight between 45,000 and 65,000 daltons. This band width probably reflects heterogeneous glycosylation. The nucleoside transporter from human erythrocytes has been partially purified by a combination of high pH washes to remove extrinsic proteins followed by ion exchange chromotography on DEAE-cellulose. In this fashion, the nucleoside transporter has been purified approximately 10 to 20 fold from human erythrocyte ghosts. However, the nucleoside transporter preparation was contaminated by the glucose transporter. Efforts are currently underway to generate proteolytic fragments of the nucleoside transporter that are distinct and separable from the glucose transporter and to separate the glucose transporter from the nucleoside transporter by immunoaffinity chromatographic techniques. The focus of these studies is to generate a sufficiently pure preparation of nucleoside transporter that can be used to obtain amino acid sequence or to generate antibodies that can be subsequently exploited for the isolation of molecular clones encoding the human nucleoside transport system.