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 alphabetagamma-complex were compared. The alphabetagamma-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 alphabetagamma-complex were as follows: pH optimum at 7.5; substrate specificity, ATP > ITP, GTP > UTP = CTP (K(m) 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 effects. Orthovanadate had no inhibitory effect on the enzyme activity of alphabetagamma-complex. Azide was the most effective inhibitor of the alphabetagamma-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.