ATPase activity of a highly stable alpha(3)beta(3)gamma subcomplex of thermophilic F(1) can be regulated by the introduced regulatory region of gamma subunit of chloroplast F(1).
A mutant F(1)-ATPase alpha(3)beta(3)gamma subcomplex from the thermophilic Bacillus PS3 was constructed, in which 111 amino acid residues (Val(92) to Phe(202)) from the central region of the gamma subunit were replaced by the 148 amino acid residues of the homologous region from spinach chloroplast F(1)-ATPase gamma subunit, including the regulatory stretch, and were designated as alpha(3)beta(3)gamma((TCT)) (Thermophilic-Chloroplast-Thermophilic). By the insertion of this regulatory region into the gamma subunit of thermophilic F(1), we could confer the thiol modulation property to the thermophilic alpha(3)beta(3)gamma subcomplex. The overexpressed alpha(3)beta(3)gamma((TCT)) was easily purified in large scale, and the ATP hydrolyzing activity of the obtained complex was shown to increase up to 3-fold upon treatment with chloroplast thioredoxin-f and dithiothreitol. No loss of thermostability compared with the wild type subcomplex was found, and activation by dithiothreitol was functional at temperatures up to 80 degrees C. alpha(3)beta(3)gamma((TCT)) was inhibited by the epsilon subunit from chloroplast F(1)-ATPase but not by the one from the thermophilic F(1)-ATPase, indicating that the introduced amino acid residues from chloroplast F(1)-gamma subunit are important for functional interaction with the epsilon subunit.
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Chemical Resources Laboratory, Tokyo Institute of Technology, Nagatsuta 4259, Midori-ku, Yokohama 226-8503, Japan.
Noji
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Stumpp
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Yoshida
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Hisabori
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D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry; D001407:Bacillus / Q000737:chemistry; D002736:Chloroplasts / Q000737:chemistry*; D004229:Dithiothreitol / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D006868:Hydrolysis; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010084:Oxidation-Reduction; D010957:Plasmids; D011485:Protein Binding; D006180:Proton-Translocating ATPases / Q000737:chemistry* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013696:Temperature; D013879:Thioredoxins / Q000494:pharmacology; D013997:Time Factors
D000596:Amino Acids; D011994:Recombinant Proteins; D013879:Thioredoxins; D000255:Adenosine Triphosphate; D006180:Proton-Translocating ATPases; D004229:Dithiothreitol