UNISITE HYDROLYSIS OF [GAMMA-P-32]ATP BY SOLUBLE MITOCHONDRIAL F1-ATPASE AND ITS RELEASE BY EXCESS ADP AND ATP - EFFECT OF TRIFLUOPERAZINE

Some of the characteristics of unisite hydrolysis of [gamma(32)P]ATP a s well as the changes that occur on the transition to multisite cataly sis were further studied. It was found that a fraction of [gamma(32)P] ATP bound at the catalytic sites of F-1 under unisite conditions under goes both hydrolysis and release induced by medium nucleotides upon ad dition of millimolar concentrations of ADP or ATP. The fraction of [ga mma(32)P]ATP that undergoes release is similar to the fraction that un dergoes hydrolytic cleavage, indicating that the rates of the release and hydrolytic reactions of bound [gamma(32)P]ATP are in the same rang e. As part of studies on the mechanisms through which trifluoperazine inhibits ATP hydrolysis, its effect on unisite hydrolysis of [gamma(32 )P]ATP was also studied. Trifluoperazine diminishes the rate of unisit e hydrolysis by 30-40%. The inhibition is accompanied by a nearly tenf old increase in the ratio of [gamma(32)P]ATP/(32)Pi bound at the catal ytic site and a 50% diminution in the rate of (32)Pi release from the enzyme into the media. Trifluoperazine also induces heterogeneity of t he three catalytic sites of F-1 in the sense that in a fraction of F-1 molecules, the high-affinity catalytic site has a turnover rate lower than the other two. Trifluoperazine does not modify the release of pr eviously bound [gamma(32)P]ATP induced by medium nucleotides. The latt er indicates that hindrances in the release of Pi do not necesarily ac company alterations in the release of ATP even though both species lie in the same site.