Studies on (K+ + H+)-ATPase V. Chemical composition and molecular weight of the catalytic subunit.
(1) A (K+ + H+)-ATPase preparation from porcine gastric mucosa is solubilized in sodium dodecyl sulfate, and is subjected to gel filtration. (2) A main subunit fraction is obtained, which is a protein carbohydrate lipid complex, containing 88% protein, 7% carbohydrate and 5% phospholipid. The Detailed composition of the protein and carbohydrate moieties are reported. (3) Sedimentation analysis of the subunit preparation, after detergent removal, reveals no heterogeneity, but the subunits readily undergo aggregation. (4) Acylation of the subunit preparation with citraconic anhydride causes a clear shift of the band obtained after SDS gel electrophoresis, but the absence of broadening and splitting of the band pleads against subunit heterogeneity. (5) Treatment of the subunit preparation with dansyl chloride indicates that the NH2 terminus is blocked, which favors the assumption of homogeneity of the protein. (6) Binding studies with concanavalin A indicate that at least 86% of the subunit preparation is composed of glycoprotein. (7) These findings, taken together, strongly suggest that there is a single subunit which is a glycoprotein and which represents the catalytic subunit of the enzyme. From sedimentation equilibrium analysis a molecular mass value of 119 kDa (S.E. 3, n = 6) is calculated for protein + carbohydrate and of 110 kDa (S.E. 3, N = 6) for protein only. (8) In combination with the molecular mass of 444 kDa (S. E. 10, n = 4) obtained for the intact enzyme by radiation inactivation we conclude that the enzyme appears to be composed of a homo-tetramer of catalytic subunits.
D000251:Adenosine Triphosphatases / Q000302:isolation & purification; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002241:Carbohydrates / Q000032:analysis; D005753:Gastric Mucosa / Q000201:enzymology*; D017506:H(+)-K(+)-Exchanging ATPase; D007425:Intracellular Membranes / Q000201:enzymology; D046911:Macromolecular Substances; D008970:Molecular Weight; D010743:Phospholipids / Q000032:analysis; D013552:Swine
D000596:Amino Acids; D002241:Carbohydrates; D046911:Macromolecular Substances; D010743:Phospholipids; D000251:Adenosine Triphosphatases; D017506:H(+)-K(+)-Exchanging ATPase