Purification and partial characterization of proton-potassium-transporting adenosine triphosphatase from fundic mucosa

Abstract

The microsomal (H+,K+)-ATPase systems from dog and pig fundic mucosa were purified to homogeneity and partially characterized. The method involves sodium dodecyl sulfate (SDS) (0.033%) w/v) extraction of the microsomal non-ATPase proteins under appropriate conditions followed by sucrose density gradient centrifugation. Two distinct membrane bands of low (buoyant density = 1.08 g/mL) and high (buoyant density = 1.114 g/mL) densities have distinct enzymatic and chemical composition were harvested. The low-density membrane was highly enriched in Mg2+- or Ca2+-stimulated ATPase and 5''-nucleotidase activities but totally devoid of (H+,K+)-ATPase and K+-p-nitrophenylphosphatase activities. The latter two activities were found exclusively in the high-density membrane. SDS-polyacrylamide gel electrophoresis revealed the high-density membranes to consist primarily of a major 100-kilodalton (kDa) protein and a minor 85-kDa glycoprotein, the former being the catalytic subunit of the (H+,K+)-ATPase. The amino acid composition of the pure dog (H+,K+)-ATPase revealed close similarities with that from the pig. The N-terminal amino acid was identified to be lysine as the sole residue. Simlar to the high-density membrane-associated pure (H+,K+)-ATPase, the low-density membranes containing high Mg2+-ATPase activity also contained a 100-kDa peptide and a 85-kDa glycopeptide in addition to numerous low molecular weight peptides. Also, similar to the pure (H+,K+)-ATPase, the Mg2+-ATPase-rich fraction produced an E.apprx.P unstable to hydroxylamine and partially (about 25%) sensitive to K+ but having a slow turnover. The levels of E.apprx.P produced by the pure (H+,K+)-ATPase- and Mg2+-ATPase-rich fractions were 1400 and 178 pmol/mg of protein, respectively. The possibility of the low-density membrane-associated Mg2+-ATPase to be a modified form of the (H+,K+)-ATPase has been discussed.