Abstract
Abstract Treatment of erythrocytes or their isolated membranes with radioactive iodoacetate results in the specific modification of one of the membrane proteins, as determined by acrylamide gel electrophoresis in sodium dodecyl sulfate. The reactive groups of this protein are almost completely blocked before any of the other major membrane proteins react.The reaction with N-ethylmaleimide does not show a similar specificity of labeling, indicating a considerable degree of reagent specificity for the reaction. The specifically labeled protein was extracted from the membrane with sodium chloride and further purified by chromatography on Sephadex G-200 in sodium dodecyl sulfate. This preparation shows a single band on acrylamide gel electrophoresis in sodium dodecyl sulfate with a molecular weight of 35,000. The labeled amino acid was identified as cysteine from its elution position on ion exchange chromatography.
Highlights
The reactive groups of this protein are almost completely blocked before any of the other major membrane proteins react
The isolated ghosts TTere washed with Krebs-Ringer buffer, with 7 m;M phosphate before being prepared for electrophoresis
The molecular weight of the isolated membrane protein was estimated from its elution position on G-200 in comparison to a standard curve prepared using bovine serum albumin, ovalbumin, and cytochrome c
Summary
Treatment of erythrocytes or their isolated membranes with radioactive iodoacetate results in the specific modification of one of the membrane proteins, as determined by acrylamide gel electrophoresis in sodium dodecyl sulfate. The labeled protein was extracted from the membrane with sodium chloride and further purified by chromatography on Sephadex G-200 in sodium dodecyl sulfate This preparation shows a single band on acrylamide gel electrophoresis in sodium dodecyl sulfate with a molecular weight of 35,000. Ln enhanced specificity of reaction with membrane sulfhydryl groups can be achieved with low concentrations of iodoacetic acid, limiting reaction almost exclusively to a single protein. This observation, coupled with the isolation and partial characterization of this protein, offer the possibility of a dual approach to understanding some of the problems of membrane protein chemistry
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