title: Effect of free fatty acids and detergents on H,K-ATPase. The steady-state ATP phosphorylation level and the orientation of the enzyme in membrane preparations.
abstract: The effects of detergents and free fatty acids on the K(+)-activated ATPase activity and on the steady-state phosphorylation level of pig gastric H,K-ATPase were studied. Unsaturated free fatty acids inhibited the K(+)-activated ATPase activity, due to inactivation of the enzyme (long-term effects) and to a decrease in the K(+)-sensitive dephosphorylation rate (short-term effects). The degree of inhibition depended on the reaction conditions: the protein concentration, the temperature and the ligands used. No effect was observed when saturated- or methylated unsaturated fatty acids were tested. Free fatty acids and the detergent C12E8 increased the steady-state ATP phosphorylation level, indicating the presence of vesicular structures in the H,K-ATPase preparations. At higher concentrations these compounds inactivated H,K-ATPase, which was measured as a decrease in phosphorylation capacity. By combining the data from the ATP phosphorylation level in the absence and presence of C12E8 (without inactivation) and the data from the K(+)-activated ATPase activity with and without ionophore the tightness of vesicular preparations and the orientation of H,K-ATPase was determined. A rather simple method for the isolation of H,K-ATPase is reported, which yields highly purified H,K-ATPase preparations with a ATP phosphorylation capacity of 3.9 nmol P per mg protein or 0.57 mol P per mol alpha beta protomer. This number suggests that each alpha-subunit H,K-ATPase can be phosphorylated at the same time.
authors.affiliation: Department of Biochemistry, University of Nijmegen, The Netherlands.
authors.forename: H G
authors.initials: HG
authors.lastname: Swarts
authors.forename: T J
authors.initials: TJ
authors.lastname: Van Uem
authors.forename: S
authors.initials: S
authors.lastname: Hoving
authors.forename: J A
authors.initials: JA
authors.lastname: Fransen
authors.forename: J J
authors.initials: JJ
authors.lastname: De Pont
mesh_terms: D000251:Adenosine Triphosphatases / Q000037:antagonists & inhibitors / Q000378:metabolism* / Q000648:ultrastructure; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D002462:Cell Membrane / Q000201:enzymology / Q000648:ultrastructure; D019826:Cholic Acid; D002793:Cholic Acids / Q000494:pharmacology; D003902:Detergents / Q000494:pharmacology*; D005230:Fatty Acids, Nonesterified / Q000494:pharmacology*; D005753:Gastric Mucosa / Q000201:enzymology; D017506:H(+)-K(+)-Exchanging ATPase; D007700:Kinetics; D008051:Lipid Bilayers; D008854:Microscopy, Electron; D009550:Nigericin / Q000494:pharmacology; D019301:Oleic Acid; D009829:Oleic Acids / Q000494:pharmacology; D010766:Phosphorylation; D012967:Sodium Dodecyl Sulfate / Q000494:pharmacology; D013552:Swine
chemical_list: D002793:Cholic Acids; D003902:Detergents; D005230:Fatty Acids, Nonesterified; D008051:Lipid Bilayers; D009829:Oleic Acids; D019301:Oleic Acid; D012967:Sodium Dodecyl Sulfate; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D017506:H(+)-K(+)-Exchanging ATPase; D019826:Cholic Acid; D009550:Nigericin