SYNTHESIS AND RELEASE OF ATP BY SOLUBLE MITOCHONDRIAL F1 IN COMPLEX WITH ITS INHIBITOR PROTEIN DURING DIMETHYLSULFOXIDE-WATER TRANSITIONS

Soluble mitochondrial Fl and FI in complex with the natural ATPase inh ibitor protein (F1-IP) catalyze the spontaneous synthesis of [gamma-P- 32]ATP from medium [P-32]phosphate and enzyme-bound ADP when incubated in media with dimethylsulfoxide (Me2SO); under these conditions, the synthesized [gamma-P-32]ATP is not released into the media, it remains tightly bound to the enzymes [Gomez-Puyou, A., Tuena de Gomez-Puyou, M. & de Meis, L. (1986) Eur. J. Biochem. 159, 133-140]. Some of the ch aracteristics of the synthesized [gamma-P-32]ATP were studied in F1 an d F1-IP (ATPase activities of 70 and 1-3 mu mol.min(-1).mg(-1), respec tively). In Me2SO media, gamma-phosphate of synthesized ATP in FI or F 1-IP exchanges with medium phosphate. From the rates of the exchange r eaction, the half-times for hydrolysis of the synthesized ATP in F1 an d FI-TP were calculated: 45 min and 58 min for F1 and F1-IP, respectiv ely. The course that synthesized [gamma-P-32]ATP follows after dilutio n of the Me2SO synthetic mixture with aqueous buffer was determined. A fter dilution, the half-life of synthesized ATP in F1 was less than 1 min. In FI-IP, ATP was also hydrolyzed, but at significantly lower rat es. In FZ-LP, dilution also produced release of the synthesized [gamma -P-32]ATP. This was assayed by the accessibility of [gamma-P-32]ATP to hexokinase. About 25% of [gamma-P-32]ATP synthesized in FI-IP, but no t in F1, was released into the media after dilution with aqueous buffe r that contained 20 mM phosphate. Release of tightly bound ATP require d the binding energy of phosphate and solvation of F1-IP however, the particular kinetics of F1-IP were also central for medium ATP synthesi s in the absence of electrochemical H+ gradients.