Determination of the sidedness of the C-terminal region of the gastric H,K-ATPase alpha subunit.
It cannot be predicted from hydropathy analysis whether the C-terminal end of the alpha subunit of the gastric H,K-ATPase is cytoplasmic or extracytoplasmic. The sideness of the C-terminal amino acids was determined by taking advantage of the two C-terminal tyrosines in the primary sequence of the enzyme. Intact, cytoplasmic side out vesicles derived from hog gastric mucosa or detergent solubilized vesicles were iodinated by the lactoperoxidase method and then the C-terminal amino acids hydrolyzed by carboxypeptidase Y. The alpha and beta subunits were separated by SDS gel electrophoresis. The level of iodination of the alpha subunit following solubilization was about three fold greater than when intact vesicles were iodinated, and the beta subunit was iodinated only when solubilized enzyme was used. Carboxypeptidase Y removed 28 +/- 4% of the radioactivity from the alpha subunit iodinated in intact vesicles. These data are consistent with a cytoplasmic location of the C-terminal amino acids of the alpha subunit and with a mostly extracytoplasmic location of the amino acids of the beta subunit.
Scott
D R
DR
Department of Physiology and Medicine, UCLA.
Munson
K
K
Modyanov
N
N
Sachs
G
G
D000818:Animals; D002268:Carboxypeptidases / Q000378:metabolism; D003600:Cytosol / Q000201:enzymology; D004591:Electrophoresis, Polyacrylamide Gel; D005110:Extracellular Space / Q000201:enzymology; D005753:Gastric Mucosa / Q000201:enzymology*; D017506:H(+)-K(+)-Exchanging ATPase / Q000737:chemistry / Q000378:metabolism*; D007457:Iodine Radioisotopes; D007553:Isotope Labeling; D007784:Lactoperoxidase; D010446:Peptide Fragments / Q000378:metabolism; D012995:Solubility; D013552:Swine; D014443:Tyrosine / Q000378:metabolism
D007457:Iodine Radioisotopes; D010446:Peptide Fragments; D014443:Tyrosine; D007784:Lactoperoxidase; D002268:Carboxypeptidases; C022633:serine carboxypeptidase; D017506:H(+)-K(+)-Exchanging ATPase