Biosynthesis of intestinal microvillar proteins. Pulse-chase labelling studies on aminopeptidase N and sucrase-isomaltase

Publisher Website
Google Scholar
Abstract
The biogenesis of 2 microvillar enzymes, aminopeptidase N (EC 3.4.11.2) and sucrase (EC 3.2.1.48)-isomaltase (EC 3.2.1.10), was studied by pulse-chase labeling of pig small intestinal explants kept in organ culture. Both enzymes became inserted into the membrane during or immediately after polypeptide synthesis, indicating that translation takes place on ribosomes attached to the rough endoplasmic reticulum. The earliest detectable forms of aminopeptidase and sucrase-isomaltase were polypeptides of MW 140,000 and 240,000, respectively. These polypeptides were susceptible to treatment with endo-.beta.-N-acetylglucosaminidase H (EC 3.2.1.96), suggesting that the microvillar enzymes during or immediately after completion of protein synthesis become glycosylated with a high-mannose oligosaccharide structure similarly to other plasma membrane and secretory proteins. After 20-40 or 60-90 min of chase, respectively, aminopeptidase N and sucrase-isomaltase were reglycosylated to give the polypeptides of MW 166,000 (aminopeptidase N) and 265,000 (sucrase-isomaltase). These were expressed at the microvillar membrane after 60-90 min. During the entire process of synthesis and transport to the microvillar membrane the enzymes were bound to membranes, indicating that the biogenesis of aminopeptidase N and sucrase-isomaltase occurs in accordance with the membrane flow hypothesis.