A Cl(-)-translocating adenosinetriphosphatase in Acetabularia acetabulum. 1. Purification and characterization of a novel type of adenosinetriphosphatase that differs from chloroplast F1 adenosinetriphosphatase.
ATPases were solubilized from membranes of Acetabularia acetabulum using nonanoyl-N-methylgluconamide and purified by ion-exchange and gel permeation chromatography. Three fractions of ATPase, Mono Q-I, -II, and -III, were separated. Activity in fraction Mono Q-I was very labile and could not be accurately determined. Fractions Mono Q-II and -III had specific activities of 0.6 and 6 units/mg of protein, respectively. By SDS-polyacrylamide gel electrophoresis, isoelectric focusing, and peptide mapping, it was shown that fractions Mono Q-II and -III consisted of the same polypeptides with molecular masses of 54K (a-subunit) and 50K (b-subunit). Fractions Mono Q-II and -III had the following catalytic properties: pH optimum at 6.0; substrate specificity, ATP = GTP = ITP much greater than UTP = CTP (Km for ATP 0.6 mM); divalent cation requirement, Mn2+ = Mg2+ greater than Co2+ greater than Zn2+ much greater than Ca2+, Ni2+. Both activities were inhibited by monovalent anions, while monovalent cations had neither inhibitory nor stimulatory effects. Orthovanadate inhibited both activities to 50% at 1 mM, and the most effective inhibitor of both was azide (95% inhibition at 100 microM). An enzyme-phosphate complex was formed after incubation of fraction Mono Q-III with [gamma-32P]ATP. The CF1-ATPase subcomplexes were isolated from the same organism and compared with the fraction Mono Q-III. Data supported the difference of fraction Mono Q-III from CF1-ATPase.
Ikeda
M
M
Faculty of Pharmaceutical Sciences, Okayama University, Japan.
Schmid
R
R
Oesterhelt
D
D
D000076:Acetabularia / Q000187:drug effects / Q000201:enzymology*; D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000838:Anions / Q000494:pharmacology; D001692:Biological Transport; D015153:Blotting, Western; D002412:Cations / Q000494:pharmacology; D002712:Chlorides / Q000378:metabolism*; D000460:Chlorophyta / Q000201:enzymology*; D002736:Chloroplasts / Q000187:drug effects / Q000201:enzymology*; D010449:Peptide Mapping; D011487:Protein Conformation; D011489:Protein Denaturation; D012995:Solubility; D013379:Substrate Specificity
D000838:Anions; D002412:Cations; D002712:Chlorides; D000251:Adenosine Triphosphatases