Interaction of a permeant maleimide derivative of cysteine with the erythrocyte glucose carrier. Differential labelling of an exofacial carrier thiol group and its role in the transport mechanism.

May JM
Biochem J. 1989 263 (3): 875-81

PMID: 2489029 · PMCID: PMC1133512 · DOI:10.1042/bj2630875

S-(Bismaleimidomethyl ether)cysteine (Cys-Mal) was synthesized as a probe for reactive thiol groups on the erythrocyte glucose carrier. Although Cys-Mal entered cells, its reaction with intracellular GSH prevented alkylation of endofacial membrane proteins, limiting its effect to the cell surface at concentrations below 5 mM. Cys-Mal irreversibly inhibited hexose transport half-maximally at 1.5 mM by decreasing the maximal rate of transport, with no effect on the affinity of substrate for the carrier. Reaction occurred with the outward-facing form of the carrier, but did not affect the ability of the carrier to change orientation. In intact cells, several exofacial proteins were labelled by [35S]Cys-Mal, including the band-4.5 glucose carrier, the labelling of which occurred on a single site sensitive to transport inhibitors. The reactive exofacial group was a thiol group, since both transport inhibition and band-4.5 labelling by Cys-Mal were abolished by the thiol-specific and impermeant compound 5,5'-dithiobis(2-nitrobenzoic acid). Selectivity for carrier labelling in cells was increased by a double differential procedure, which in turn allowed localization of the exofacial thiol group to the Mr 18,000-20,000 membrane-bound tryptic carrier fragment. In protein-depleted ghosts the exofacial thiol group was preferentially labelled at low concentrations of [35S]Cys-Mal, whereas with the reagent at 10 mM the Mr 26,000-45,000 tryptic carrier fragment was also labelled. Cys-Mal should be useful in the study of carrier thiol-group location and function.

MeSH Terms (13)

Biological Transport Cell Membrane Permeability Cysteine Erythrocyte Membrane Humans In Vitro Techniques Maleimides Monosaccharide Transport Proteins Peptide Mapping Structure-Activity Relationship Sulfhydryl Compounds Sulfhydryl Reagents Trypsin

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