The amino acid sequence of an active site peptide from the H,K-ATPase of gastric mucosa.
The gastric H,K-ATPase is an active transport protein that is responsible for the maintenance of a large pH gradient across the secretory canaliculus of the mammalian parietal cell. Acid secretion across these epithelial cell membranes is coupled to the potassium-stimulated hydrolysis of ATP catalyzed by H,K-ATPase, but the mechanism of coupling between ion transport and ATP hydrolysis is unknown. In order to investigate the enzymatic mechanism of this coupling, a peptide derived from the ATP binding site of H,K-ATPase has been purified and its amino acid sequence has been determined. The peptide was identified by the incorporation of a fluorescent probe, fluorescein 5'-isothiocyanate (FITC), into the active site before trypsin digestion of the protein. The labeling of the enzyme by FITC was associated with the irreversible inhibition of enzymatic activity, and both the labeling of the tryptic peptide and inhibition of activity were prevented when the reaction was performed in the presence of ATP. At 100% inhibition of activity, 3.5 +/- 1.6 nmol of FITC were incorporated per mg of protein. The amino acid sequence of the active site peptide is His-Val-Leu-Val-Met-Lys-Gly-Ala-Pro-Glu-Gln-Leu-Ser-Ile-Arg, and FITC reacts with the lysine. This sequence is very similar to sequences of fluorescein-labeled peptides from the ATP binding sites of Na,K-ATPase and Ca2+-ATPase, and suggests that the active site structures of these ion transport ATPases are similar.
Farley
R A
RA
Faller
L D
LD
D000251:Adenosine Triphosphatases / Q000032:analysis*; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D000252:Calcium-Transporting ATPases / Q000032:analysis; D002851:Chromatography, High Pressure Liquid; D016650:Fluorescein-5-isothiocyanate; D005452:Fluoresceins; D005753:Gastric Mucosa / Q000201:enzymology*; D017506:H(+)-K(+)-Exchanging ATPase; D008297:Male; D000254:Sodium-Potassium-Exchanging ATPase / Q000032:analysis; D013552:Swine; D013861:Thiocyanates; D014357:Trypsin / Q000378:metabolism
D005452:Fluoresceins; D013861:Thiocyanates; D014357:Trypsin; D000251:Adenosine Triphosphatases; D017506:H(+)-K(+)-Exchanging ATPase; D000252:Calcium-Transporting ATPases; D000254:Sodium-Potassium-Exchanging ATPase; D016650:Fluorescein-5-isothiocyanate