Chloroplast ATPase in Acetabularia acetabulum: purification and characterization of chloroplast F1-ATPase.
ATPases were isolated from chloroplasts of the unicellular marine alga Acetabularia acetabulum. Two preparations of ATPase, a chloroplast-enriched fraction and an alpha beta gamma-complex were compared. The alpha beta gamma-complex was released into an EDTA solution and purified by anion-exchange chromatography, hydrophobic chromatography, and gel permeation chromatography. The subunit composition of this enzyme appeared to be 52-53 (alpha), 51 (beta), and 40 (gamma) kDa from SDS-PAGE. ATPase activity was enriched about 260-fold to a specific activity of approximate 4.1 U.mg protein-1. The catalytic properties of the alpha beta gamma-complex were as follows: pH optimum at 7.5; substrate specificity, ATP > ITP, GTP > UTP = CTP (Km for ATP 0.2 mM); divalent cation requirement, Mg2+ = Mn2+ = Co2+ > Zn2+ > Ni2+ > Ca2+; ATPase activity was inhibited by monovalent anions (NO3-, SCN-), while monovalent cations had neither inhibitory nor stimulatory effect. Orthovanadate had no inhibitory effect on the enzyme activity of alpha beta gamma-complex. Azide was the most effective inhibitor of the alpha beta gamma-complex. N-Terminal amino acid sequences of the alpha and beta subunits were not obtained and appeared to be blocked. The gamma subunit gave a sequence of AGLKEMKD-XIGSVXNTKKI, which showed 60% similarity to the gamma subunits of spinach and Chlamydomonas reinhardtii CF1-ATPase and EF1-ATPase.
Satoh
S
S
Faculty of Pharmaceutical Sciences, Okayama University, Japan.
Moritani
C
C
Ohhashi
T
T
Konishi
K
K
Ikeda
M
M
D000076:Acetabularia / Q000201:enzymology* / Q000648:ultrastructure; D000595:Amino Acid Sequence; D000838:Anions / Q000378:metabolism; D002384:Catalysis; D002412:Cations / Q000378:metabolism; D002736:Chloroplasts / Q000201:enzymology*; D002850:Chromatography, Gel; D002853:Chromatography, Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D000431:Ethanol / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D000432:Methanol / Q000494:pharmacology; D008969:Molecular Sequence Data; D006180:Proton-Translocating ATPases / Q000032:analysis / Q000037:antagonists & inhibitors / Q000302:isolation & purification*; D013379:Substrate Specificity
D000838:Anions; D002412:Cations; D000431:Ethanol; D006180:Proton-Translocating ATPases; D000432:Methanol