Purification and partial characterization of the (H+,K+)-transporting adenosinetriphosphatase from fundic mucosa.
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 having 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 pig. The N-terminal amino acid was identified to be lysine as the sole residue. Similar 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 approximately P unstable to hydroxylamine and partially (about 25%) sensitive to K+ but having a slow turnover. The levels of E approximately P produced by the pure (H+,K+)-ATPase- and Mg2+-ATPase-rich fractions were 1400 and 178 pmol/mg of protein, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)
Nandi
J
J
Department of Surgery, State University of New York Health Science Center, Syracuse 13210.
Zhou
M A
MA
Ray
T K
TK
D000251:Adenosine Triphosphatases / Q000302:isolation & purification* / Q000378:metabolism; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002458:Cell Fractionation; D002462:Cell Membrane / Q000201:enzymology / Q000648:ultrastructure; D004285:Dogs; D005748:Gastric Fundus / Q000201:enzymology; D005753:Gastric Mucosa / Q000201:enzymology*; D017506:H(+)-K(+)-Exchanging ATPase; D007700:Kinetics; D008861:Microsomes / Q000201:enzymology / Q000648:ultrastructure; D013552:Swine
D000596:Amino Acids; D000251:Adenosine Triphosphatases; D017506:H(+)-K(+)-Exchanging ATPase