title: Synthesis of medium pyrophosphate by soluble mitochondrial F1 through dimethyl sulfoxide-water transitions.
abstract: Soluble F1 from heart mitochondria incubated in mixtures that have Mg2+, inorganic phosphate, and dimethyl sulfoxide (40% (v/v)) catalyzes the spontaneous synthesis of ATP and pyrophosphate (Tuena de Gómez-Puyou, M., García, J. J., and Gómez-Puyou, A. (1993) Biochemistry 32, 2213-2218). By filtration techniques, it was determined that synthesized ATP and pyrophosphate are enzyme bound, albeit the affinity for pyrophosphate was lower than that of ATP. After ATP and pyrophosphate were formed 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 experiments 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, ATP underwent hydrolysis. Studies on the effect of arsenate on the synthesis and hydrolysis of ATP and PPi in F1 showed that hydrolysis of synthesized PPi at its site of synthesis was slower than that of ATP. Thus, the question of whether differences in the rates of hydrolysis accounted for the dilution-induced release of PPi but not of ATP was addressed. Synthesis and hydrolysis of ATP and pyrophosphate were examined in preparations of soluble F1 in complex with its inhibitor protein; the complex had an ATPase activity about 100 times lower than that of free F1. In mixtures that contained dimethyl sulfoxide, the complex synthesized ATP and pyrophosphate at nearly the same rates; upon dilution, hydrolysis of both compounds occurred also at similar rates, yet only pyrophosphate was released. The same phenomenon was observed in F1 that had been depleted of adenine nucleotides. Hence, dilution-induced 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 of adenine nucleotides, it is likely that the failure of ATP to be released is due to the high affinity that F1 exhibits for the synthesized ATP. Nevertheless, the results illustrate that a complete catalytic cycle that starts with medium Pi and ends with medium pyrophosphate may be reproduced in soluble mitochondrial F1.
authors.affiliation: Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, México, D. F., México.
authors.forename: M
authors.initials: M
authors.lastname: Tuena de Gómez-Puyou
authors.forename: F
authors.initials: F
authors.lastname: Sandoval
authors.forename: A
authors.initials: A
authors.lastname: Gómez-Puyou
mesh_terms: D000255:Adenosine Triphosphate / Q000096:biosynthesis; D004121:Dimethyl Sulfoxide; D011756:Diphosphates / Q000378:metabolism*; D008928:Mitochondria / Q000201:enzymology; D006180:Proton-Translocating ATPases / Q000502:physiology*
chemical_list: D011756:Diphosphates; D000255:Adenosine Triphosphate; D006180:Proton-Translocating ATPases; D004121:Dimethyl Sulfoxide