Location of the carbohydrates present in the hydrogen ion-potassium ATPase vesicles isolated from hog gastric mucosa

Abstract

The glycosylation of H+K+-ATPase vesicles isolated from hog gastric mucosa was investigated by various methods. Following protein separation on sodium dodecyl sulfate reducing gels and transfer to poly(vinyl difluoride) membranes, binding of concanavalin A was confined to the 94-kDa band which corresponds to the catalytic subunit. In contrast, wheat germ agglutinin binding occurred in a region below the 94-kDa subunit, corresponding to the 60-85-kDa region, and also to protein just above the catalytic subunit. Treatment with glycopeptidase F removed most of the concanavalin A staining and also the wheat germ agglutinin staining found below the 94-kDa region, but spared the higher molecular weight wheat germ agglutinin reactive material. During the deglycosylation experiments a protein of 35-kDa was produced. Sequencing analysis of V8 protease generated peptide fragments of the 35-kDa protein show at least 30% homology with the Na+K+-ATPase .beta.-subunits. Labeling of the carbohydrates by galactosyltransferase and [3H]uridine diphosphate-galactose showed that the sites of labeling were extracellular and were confined to the wheat germ agglutinin staining regions. Two molecular weight regions, below the 94-kDa region, of 60 and 85 kDa were identified. Electron microscopy using postembedding staining techniques showed that both concanavalin A and wheat germ agglutinin staining occurred on the extracellular face of the gastric vesicles. It is concluded that there are three classes of glycosylated proteins in hog gastric vesicles: (1) the 94-kDa protein containing a core (simple) oligosaccharide, with no or few N-acetylglucosamine residues available for either wheat germ agglutinin binding or galactose transfer; (2) the 60-85-kDa region containing two protein bands that have complex N-linked oligosaccharides; and (3) a protein of higher molecular weight than the catalytic unit which contains O-linked complex oligosaccharides. All of these glycoproteins appear to be located on the extracellular face of the gastric vesicles.