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Photoaffinity labeling of the human erythrocyte D-glucose transporter.

1982; Elsevier BV; Volume: 257; Issue: 10 Linguagem: Inglês

10.1016/s0021-9258(19)83793-6

1083-351X

Christin Carter‐Su, Jeffrey E. Pessin, René Mora, Wendy Gitomer, M P Czech,

Diet and metabolism studies

We have developed methodology t o label covalently the cytochalasin B-binding component of the D-glucose transport system in the human erythrocyte membrane.This technique takes advantage of an intrinsic property ofcytochalasin B, which upon high intensity ultraviolet irradiation becomes a highly reactive molecule.Irradiation of erythrocyte membranes in the presence of [3H]cytochalasin B resulted in the covalent labeling of a component that migrated as a single broad band on sodium dodecyl sulfate-polyacrylamide gels in the M, range of 45,000-60,000 with a maximum at 54,000.This same labeling pattern was found using freshly prepared whole cells, erythrocyte ghosts, or dimethylmaleic anhydride-extracted ghosts.The photoaffinity labeling of this membrane component by [3H]cytochalasin B in erythrocyte ghosts was inhibited by D-glucose, 3-O-methylglucose, 2-deoxyglucose, and cytochalasin B, but not by D-sorbitol, L-glucose, L-arabinose, o r cytochalasin E. Half-maximal inhibition of binding at 1 p~ [3H]cytochalasin B occurred at 12 n m D-glUCOSe.Maximal labeling of the M, = 54,000 component was achieved with 2 p~ [3HJcytochalasin B while half-maximal labeling occurred at 0.7 PM.Photocatalyzed labeling of bovine serum albumin or rabbit muscle actin could be demonstrated with [3H]cytochalasin B; however, this labeling was not inhibited by the presence of 500 ~l l ~ D-glucose.We conclude that the M, = 54,000photoaffinity-labeled [3Hlcytochalasin B-binding protein exhibits properties identical to those expected for the D-glucose transporter in human erythrocytes.Further, n o evidence was found to support the hypothesis that this cytochalasin B-binding protein is a degradation product of a much larger polypeptide.Over the past several years, extensive efforts have been made to identify and purify the stereospecific D-glucose transporter from human erythrocytes (see Ref. 1 for a recent review).These efforts can be divided into three general types: use of specific reagents that are expected to bind only to the transport protein (2, 3), use of nonspecific reagents that co-