Isolation, purification, and characterization of coupling factor 1 from Chlamydomonas reinhardi.
Chloroplast thylakoid particles were prepared from wild-type Chlamydomonas reinhardi by gentle sonication. These particles catalyzed phenazine methosulfate dependent photophosphorylation with rates ranging from 300 to 700 mumol of adenosine 5'-triphosphate (ATP) formed (mg of chlorophyll)-1h-1. Photophosphorylation was not sensitive to tentoxin but was sensitive to an anticoupling factor 1 (CF1) antiserum preparation made against spinach CF1. The C. reinhardi chloroplast CF1 was isolated from thylakoid particles by either chloroform or ethylenediaminetraacetic acid extraction. The former enzyme appeared to be missing the gamma subunit and did not reconstitute with partially resolved thylakoid particles. The latter enzyme reconstituted with partially resolved particles and had a specific activity at 37 degrees C of 2-5 umol of ATP hydrolyzed (mg of protein)-1 min-1. The enzyme utilized both MnATP and MgATP. CaATP was a poor substrate, and SrATP was not hydrolyzed. The enzyme was not activated by heat or proteolysis but was stimulated approximately 2-fold by 50 mM dithiothreitol. Alcohols reversibly stimulated the ATPase activity of the enzyme 5-25-fold. Ethanol, 20%, dramatically lowered the temperature optimum from approximately 75 to approximately 45 degrees C and slightly lowered the pH optimum from 8.5 to 8.2. Ethanol had no effect on the activation energy of the ATPase reaction (17 +/- 1.7 kcal/mol). The kinetics of the ATPase reaction catalyzed by the C. reinhardi enzyme are complex. Both free divalent cations and divalent cation ATP inhibited the activity of the enzyme. The apparent Km for MgTAP (55 uM free Mg2+) was approximately 0.2 mM.
D000251:Adenosine Triphosphatases / Q000302:isolation & purification* / Q000378:metabolism; D000936:Antigen-Antibody Complex; D002413:Cations, Divalent; D002696:Chlamydomonas / Q000201:enzymology*; D004492:Edetic Acid / Q000494:pharmacology; D004789:Enzyme Activation; D000431:Ethanol / Q000494:pharmacology; D007106:Immune Sera; D007700:Kinetics; D008970:Molecular Weight; D010086:Oxidative Phosphorylation Coupling Factors / Q000302:isolation & purification* / Q000378:metabolism; D006180:Proton-Translocating ATPases
D000936:Antigen-Antibody Complex; D002413:Cations, Divalent; D007106:Immune Sera; D010086:Oxidative Phosphorylation Coupling Factors; D000431:Ethanol; D004492:Edetic Acid; D000251:Adenosine Triphosphatases; D006180:Proton-Translocating ATPases