Two-dimensional isoelectric focussing/sodium dodecyl sulphate polyacrylamide gel electrophoretic mapping and some molecular characteristics of the proteins of the adult guinea-pig small intestinal microvillus membrane

Abstract

(1) The adult guinea-pig small intestinal microvillus membrane was purified approximately 25-fold by both cation-precipitation and differential centrifugation methods. Comparison by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed no substantial differences in polypeptide composition between the two preparations. (2) One-dimensional SDS-PAGE and two-dimensional isoelectric focussing (IEF)/SDS-PAGE, together with Coomassie-blue, silver and lectin-staining, showed three major high molecular weight polypeptides, M r, 108 000, 116 000 and 127 000, as well as a 47 kDa protein (actin), as major constituents of the membrane. The proteins of M r 108 000 and 116 000 were strongly concanavalin A reactive. A detailed two-dimensional IEF/SDS-PAGE map of the membrane was constructed. (3) Sodium carbonate treatment showed the two concanavalin A-reactive glycoproteins, M r 108 000 and 116 000, comprising the sucrase-isomaltase complex, to be loosely-associated ‘extrinsic’ microvillus membrane proteins. Two proteins, M r 127 000 and 135 000, were tightly-associated ‘intrinsic’ microvillus proteins. (4) Despite regional differences in specific activity of some small intestinal microvillar enzymes, most noticeably enterokinase (EC 3.4.21.9) and dipeptidyl peptidase IV (EC 3.4.14.x), no substantial regional differences were seen in microvillus membrane polypeptide composition. In contrast, a substantial increase in the major high molecular weight proteins of M r 108 000 and 116 000 accompanied a 10-fold rise in sucrase-isomaltase activity, and loss of a major protein of M r 131 000 accompanied the complete loss of lactase activity from the membrane during postnatal development.