Location of the carbohydrates present in the HK-ATPase vesicles isolated from hog gastric mucosa

• Published in: Biochemistry (Easton) Vol. 29; no. 3; pp. 701 - 706
• Main Authors: HALL, K, PEREZ, G, ANDERSON, D, GUTIERREZ, C, MUNSON, K, HERSEY, S. J, KAPLAN, J. H, SACHS, G
• Format: Journal Article
• Language: English
• Published: Washington, DC American Chemical Society 23.01.1990
• Subjects: ACID ANHYDRASES; Adenosine Triphosphatases - metabolism; AGGLUTININS; Amidohydrolases - metabolism; AMINO ACID SEQUENCE; Analytical, structural and metabolic biochemistry; ANIMALS; ANTIBODIES; ATP-ASE; BASIC BIOLOGICAL SCIENCES; BIOCHEMICAL REACTION KINETICS; Biological and medical sciences; BIOLOGICAL LOCALIZATION; Carbohydrate Metabolism; CARBOHYDRATES; CONCANAVALIN; Concanavalin A; DIGESTIVE SYSTEM; DOMESTIC ANIMALS; ELECTRON MICROSCOPY; ELECTROPHORESIS; Electrophoresis, Polyacrylamide Gel; ENZYMES; Fundamental and applied biological sciences. Psychology; Galactosyltransferases - metabolism; Gastric Mucosa - enzymology; GASTROINTESTINAL TRACT; GLYCOPROTEINS; Glycoproteins - metabolism; Glycosylation; H super(+)/K super(+)-transporting ATPase; H(+)-K(+)-Exchanging ATPase; HYDROGEN COMPOUNDS; HYDROLASES; ISOTOPE APPLICATIONS; KINETICS; MAMMALS; MEMBRANES; MICROSCOPY; Microscopy, Electron; Molecular Sequence Data; MOLECULAR STRUCTURE; MOLECULAR WEIGHT; mucosa; MUCOUS MEMBRANES; ORGANIC COMPOUNDS; Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; PHOSPHOHYDROLASES; PROTEINS; REACTION KINETICS; Sodium Dodecyl Sulfate; SWINE; TRACER TECHNIQUES; Tritium; TRITIUM COMPOUNDS; Uridine Diphosphate Galactose - metabolism; VERTEBRATES 550201 > Biochemistry > Tracer Techniques; Wheat Germ Agglutinins; Vertebrata; Mammalia; Agglutinin; Carbohydrate; Glycoproteins; Artificial membrane; Artiodactyla; Concanavalin A; Localization; Electron microscopy; Ungulata; Pig
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi00455a016

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.