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Soluble F
Volume 270,
Number 28,
Issue of July 14, pp. 16820-16825, 1995
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
through
Dimethyl Sulfoxide-Water Transitions
from heart mitochondria incubated in
mixtures that have Mg, inorganic phosphate, and
dimethyl sulfoxide (40% (v/v)) catalyzes the spontaneous synthesis of
ATP and pyrophosphate (Tuena de Gómez-Puyou, M., Garca, 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 PP
in
F showed that hydrolysis of synthesized PP 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 PP but not of ATP was
addressed. Synthesis and hydrolysis of ATP and pyrophosphate were
examined in preparations of soluble F in complex with its
inhibitor protein; the complex had an ATPase activity about 100 times
lower than that of free F. 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 F that had been
depleted of adenine nucleotides. Hence, dilution-induced release of
PP 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 F exhibits for
the synthesized ATP. Nevertheless, the results illustrate that a
complete catalytic cycle that starts with medium P and ends
with medium pyrophosphate may be reproduced in soluble mitochondrial
F.
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