A chemical procedure for determining the sidedness of the NH2 terminus in a membrane protein. The small intestinal sucrase-isomaltase.

Abstract

We have developed a chemical procedure for determining the sidedness of the NH2 termini of sucrase-isomaltase complex in the small intestinal brush-border membrane. The methodology involves amidination of sealed right-side-out brush-border membrane vesicles with the impermeant imidoester 3-[dimethyl-2-[( 3H]acetimidoxyethyl)ammonio]propanesulfonate with subsequent quantitation of this reaction at the NH2 termini of sucrase-isomaltase. It was found that amidination yields were every similar for the reaction of 3-[dimethyl-2-[( 3H]acetimidoxyethyl)-ammonio] propanesulfonate with intact membrane vesicles and with leaky, deoxycholate-extracted membrane fragments. This demonstrates that the NH2 termini were equally accessible to the impermeant reagent in both systems and, hence, are exposed on the outside of the sealed membrane vesicles. The methodology developed does not involve proteolysis and should be of wide applicability.