Evidence for the presence of a carbohydrate moiety in fluorescein isothiocyanate labeled fragments of rat gastric hydrogen ion-potassium ATPase

Publisher Website
Google Scholar
Abstract
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. These polypeptides also showed Con A binding activity, sensitive to methyl .alpha.-D-mannopyranoside and endoglycosidase H. The association of the FITC label and Con A binding activity throughout the additional tryptic cleavage of the 27-kDa polypeptide indicates the proximity of Lys-517 (FITC labeled) to the residue bearing the N-linked carbohydrate. The amino acid sequence of rat gastric (H+-K+)-ATPase deduced from cDNA clones shows that only one asparagine, Asn-492, meets the minimum specificity requirements for N-linked carbohydrate attachement in this region of the ATPase. These results raise the question whether the N-linked carbohydrate moiety, most likely at Asn-492, is cytoplasmic, as proposed by hydropathy plots, or extracellular as most N-linked carbohydrates are in membrane proteins. Determination of its orientation appears to be crucial in understanding the topology of gastric (H+-K+)-ATPase.
Keywords