Location of the carbohydrates present in the HK-ATPase vesicles isolated from hog gastric mucosa.
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.(ABSTRACT TRUNCATED AT 250 WORDS)
Hall
K
K
CURE, Veterans Administration Medical Center, Los Angeles, California 90073.
Perez
G
G
Anderson
D
D
Gutierrez
C
C
Munson
K
K
Hersey
S J
SJ
Kaplan
J H
JH
Sachs
G
G
D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000581:Amidohydrolases / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D050260:Carbohydrate Metabolism; D003208:Concanavalin A; D004591:Electrophoresis, Polyacrylamide Gel; D005700:Galactosyltransferases / Q000378:metabolism; D005753:Gastric Mucosa / Q000201:enzymology*; D006023:Glycoproteins / Q000378:metabolism; D006031:Glycosylation; D017506:H(+)-K(+)-Exchanging ATPase; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D012967:Sodium Dodecyl Sulfate; D013552:Swine; D014316:Tritium; D014531:Uridine Diphosphate Galactose / Q000378:metabolism; D014909:Wheat Germ Agglutinins
D006023:Glycoproteins; D014909:Wheat Germ Agglutinins; D014316:Tritium; D003208:Concanavalin A; D014531:Uridine Diphosphate Galactose; D012967:Sodium Dodecyl Sulfate; D005700:Galactosyltransferases; D000581:Amidohydrolases; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D000251:Adenosine Triphosphatases; D017506:H(+)-K(+)-Exchanging ATPase