SYNTHESIS OF MEDIUM PYROPHOSPHATE BY SOLUBLE MITOCHONDRIAL F1 THROUGHDIMETHYL SULFOXIDE-WATER TRANSITIONS

Soluble F-1 from heart mitochondria incubated in mixtures that have Mg 2+, inorganic phosphate, and dimethyl sulfoxide (40% (v/v)) catalyzes the spontaneous synthesis of ATP and pyrophosphate (Tuena de Gomez-Puy ou, M., Garcia, J. J,, and Gomez-Puyou, A. (1993) Biochemistry 32, 221 3-2218). By filtration techniques, it was determined that synthesized ATP and pyrophosphate are enzyme bound, albeit the affinity for pyroph osphate was lower than that of ATP. After ATP and pyrophosphate were f ormed in dimethyl sulfoxide mixtures, dilution with aqueous buffer to a dimethyl sulfoxide concentration of 6.0% brought about the partition of pyrophosphate into the media. This was evidenced by filtration exp eriments as well as by the accessibility of synthesized pyrophosphate to soluble inorganic pyrophosphatase. Release of pyrophosphate induced by dilution occurred in less than 15 s. Under conditions that produce release of pyrophosphate, no release of ATP was observed; instead, AT P underwent hydrolysis. Studies on the effect of arsenate on the synth esis and hydrolysis of ATP and PPi in F-1 showed that hydrolysis of sy nthesized PPi at its site of synthesis was slower than that of ATP. Th us, the question of whether differences in the rates of hydrolysis acc ounted for the dilution-induced release of PPi but not of ATP was addr essed, Synthesis and hydrolysis of ATP and pyrophosphate were examined in preparations of soluble F-1 in complex with its inhibitor protein; the complex had an ATPase activity about 100 times lower than that of free F-1. In mixtures that contained dimethyl sulfoxide, the complex synthesized ATP and pyrophosphate at nearly the same rates; upon dilut ion, hydrolysis of both compounds occurred also at similar rates, yet only pyrophosphate was released. The same phenomenon was observed in F -1 that had been depleted of adenine nucleotides. Hence, dilution-indu ced release of PPi was independent of the overall catalytic properties of the enzyme or its content of adenine nucleotides. Since synthesis of ATP occurs at the expense of the ADP that remains after depletion o f adenine nucleotides, it is likely that the failure of ATP to be rele ased is due to the high affinity that F-1 exhibits for the synthesized ATP. Nevertheless, the results illustrate that a complete catalytic c ycle that starts with medium P-i and ends with medium pyrophosphate ma y be reproduced in soluble mitochondrial F-1.