Papain fragmentation of the gastric (H+ + K+)-ATPase.
Membrane-bound (H+ + K+)-ATPase purified from hog gastric mucosa was exposed to limited papain digestion. Such treatment resulted in a rapid inhibition of the K+-stimulated adenosine triphosphatase and p-nitrophenyl phosphatase activities, with about 90% of these activities lost after 3 min incubation at 37 degrees C with 0.1 units of papain per mg of enzyme protein. Parallel to the inhibition of the enzyme activities, there was a production of a 77 kDa membrane-bound fragment containing the aspartyl phosphate residue of the phospho-intermediate. This fragment accounted for about 45% of the total enzyme protein after the 3 min papain treatment. The digestion barely affected the steady-state level of phosphorylation, allowed the aspartyl phosphate of the 77 kDa fragment to undergo the transition to the E2P form, and did not significantly alter the fraction of ADP-sensitive phosphoenzyme. The presence of KCl, however, depressed the steady-state level of phosphoenzyme formed from [gamma-32P]ATP considerably less than that of the control enzyme. With further exposure to papain the 77 kDa peptide became fragmented into a 28 kDa soluble peptide that retained the phosphorylating site. Binding of fluorescein 5'-isothiocyanate (FITC) to the native enzyme did not affect the sites of papain hydrolysis because the same peptide fragments were obtained. The FITC reaction site was also in the 28 kDa soluble peptide fragment.
D000244:Adenosine Diphosphate / Q000378:metabolism; D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D016650:Fluorescein-5-isothiocyanate; D005452:Fluoresceins; D017506:H(+)-K(+)-Exchanging ATPase; D008565:Membrane Proteins / Q000378:metabolism; D010206:Papain / Q000378:metabolism; D010446:Peptide Fragments / Q000032:analysis; D010766:Phosphorylation; D011188:Potassium / Q000378:metabolism; D013270:Stomach / Q000201:enzymology*; D013329:Structure-Activity Relationship; D013552:Swine; D013861:Thiocyanates
D005452:Fluoresceins; D008565:Membrane Proteins; D010446:Peptide Fragments; D013861:Thiocyanates; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D010206:Papain; D000251:Adenosine Triphosphatases; D017506:H(+)-K(+)-Exchanging ATPase; D016650:Fluorescein-5-isothiocyanate; D011188:Potassium