Evidence for the presence of a carbohydrate moiety in fluorescein isothiocyanate labeled fragments of rat gastric (H+-K+)-ATPase.
Limited tryptic digestion of fluorescein isothiocyanate (FITC)-labeled (H+-K+)-ATPase from rat resting light gastric membranes produced a soluble 27-kDa polypeptide which retained the fluorescence of the parent enzyme. Its production was markedly enhanced in the presence of an amphiphilic detergent, Zwittergent 3-14, which potently inhibits the ATPase activity. This increase is probably due to protection of certain tryptic cleavage sites through conformational changes of the membrane enzyme by the detergent. The NH2-terminal sequence of the 27-kDa polypeptide corresponded exactly to that beginning at Asn-369 of the cDNA-deduced primary structure of the rat ATPase. The presence of the phosphorylation site, Asp-385, and FITC-labeled Lys-517, which is known to be a part of the ATP-binding site, indicates that the 27-kDa polypeptide contains a major cytoplasmic portion of (H+-K+)-ATPase. Interestingly, the polypeptide was stained with periodate-Schiff's base, indicating its glycoprotein nature. The carbohydrate group attached to the polypeptide seems to include at least an N-linked high-mannose moiety, since the polypeptide showed Con A binding activity as detected with a Con A-biotin/avidin-peroxidase assay on nitrocellulose transblots. Also, its Con A binding activity was inhibited by excess methyl alpha-D-mannopyranoside and disappeared upon treatment of the polypeptide with endoglycosidase H and N-glycanase. Further tryptic action converted the 27-kDa polypeptide to 2 smaller FITC-labeled polypeptides of 25 and 15 kDa, which lost 18 and 96 amino acid residues, respectively, from the NH2 terminus of the parent polypeptide.(ABSTRACT TRUNCATED AT 250 WORDS)
Tai
M M
MM
Diabetes and GI Diseases Research, Upjohn Company, Kalamazoo, Michigan 49001.
Im
W B
WB
Davis
J P
JP
Blakeman
D P
DP
Zurcher-Neely
H A
HA
Heinrikson
R L
RL
D000251:Adenosine Triphosphatases / Q000302:isolation & purification*; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002241:Carbohydrates / Q000302:isolation & purification*; D003208:Concanavalin A; D016650:Fluorescein-5-isothiocyanate; D005452:Fluoresceins; D017506:H(+)-K(+)-Exchanging ATPase; D066298:In Vitro Techniques; D008297:Male; D008358:Mannose / Q000302:isolation & purification; D008566:Membranes / Q000201:enzymology; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000302:isolation & purification; D051381:Rats; D011919:Rats, Inbred Strains; D013270:Stomach / Q000201:enzymology; D013861:Thiocyanates; D014357:Trypsin
D002241:Carbohydrates; D005452:Fluoresceins; D010446:Peptide Fragments; D013861:Thiocyanates; D003208:Concanavalin A; D014357:Trypsin; D000251:Adenosine Triphosphatases; D017506:H(+)-K(+)-Exchanging ATPase; D016650:Fluorescein-5-isothiocyanate; D008358:Mannose