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J. Biol. Chem., Vol. 261, Issue 6, 2542-2547, 02, 1986

Photoaffinity labeling of glyceraldehyde-3-phosphate dehydrogenase by an aryl azide derivative of glucosamine in human erythrocytes

JM May

An aryl azide derivative of glucosamine, N-(4-iodoazidosalicyl)-2-amido- 2-deoxy-D-glucopyranose (GlcNAs), was synthesized as a potential photoaffinity label for the facilitative hexose carrier. The derivative inhibited hexose uptake into intact human erythrocytes half-maximally at 3.5 mM and was itself slowly transported into cells. However, photolysis of iodinated GlcNAs with leaky erythrocyte ghosts produced appreciable labeling on gel electrophoresis only of Band 6, which is glyceraldehyde-3-phosphate dehydrogenase. Band 6 photolabeling in leaky ghosts by GlcNAs was: saturable, due mostly to the aryl azide moiety, inhibited by agents with known affinity for the enzyme including sulfhydryl reagents and the enzyme substrate glyceraldehyde-3- phosphate, and not inhibited by the free-radical scavenger p- aminobenzoic acid. Moreover, GlcNAs also inhibited erythrocyte glyceraldehyde-3-phosphate dehydrogenase activity in a dose-dependent fashion in the dark and more potently following irradiation. In resealed ghosts, Band 6 labeling was decreased by D-glucose, reflecting inhibition of carrier-mediated uptake of the agent. GlcNAs appears to be a specific photoaffinity label for erythrocyte glyceraldehyde-3- phosphate dehydrogenase, and therefore potentially useful for studies of enzyme activity, compartmentation, or membrane association.
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