ATP dependent reactions catalyzed by inner membrane vesicles of rat liver mitochondria. Kinetics, substrate specificity, and bicarbonate sensitivity

P. L. Pedersen

Research output: Contribution to journalArticlepeer-review

Abstract

Three ATP dependent reactions catalyzed by the inner membrane of rat liver mitochondria, and the ATPase reaction catalyzed by purified mitochondrial ATPase (F1), were studied with respect to kinetic properties, substrate specificity, and sensitivity to bicarbonate. The ATP dependent transhydrogenase reaction (reduction of NADP+ by NADH) catalyzed by inner membrane vesicles displays typical Michaelis Menten kinetics in both Tris Cl and Tris bicarbonate buffers, with K(m) (ATP) values of 0.035 mM and 0.054 mM, respectively. The V(max) of transhydrogenase activity (25 nmol min-1mg-1) is the same in Tris bicarbonate or Tris Cl buffer. ITP and GTP readily substitute for ATP in the transhydrogenase reaction. The ATP P(i) exchange reaction catalyzed by inner membrane vesicles displays typical Michaelis Menten kinetics in both Tris Cl and Tris bicarbonate buffers, with K(m) (ATP) values of 1.0 mM and 1.4 mM, respectively. The V(max) of exchange (200 nmol min-1mg-1) is the same in either buffer. ITP and GTP do not effectively replace ATP in the exchange reaction. The ATPase reaction catalyzed by inner membrane vesicles deviates significantly from typical Michaelis Menten kinetics in Tris Cl buffer but not in Tris bicarbonate. Eadie Hofstee plots for ATP hydrolysis are biphasic in Tris Cl (K(m) = 0.068 mM, 0.21 mM) and monophasic in Tris bicarbonate (K(m) = 0.076 mM). Relative to the maximal velocity observed in Tris Cl (3000 nmol min-1 mg-1), the V(max) in Tris bicarbonate is 2 fold higher. ITP and GTP are hydrolyzed readily by the membrane bound ATPase also, however, kinetic plots are monophasic in both buffer systems tested. Relative to the maximal velocity observed in Tris Cl buffer, neither the V(max) for ITP hydrolysis nor the V(max) for GTP hydrolysis is altered significantly in Tris bicarbonate. The ATPase reaction catalyzed by the purified ATPase (F1) exhibits similar but not identical properties to the membrane bound enzyme. Differences are noted in nucleoside specificity and in the K(m) values for nucleoside triphosphates when assays are carried out in Tris Cl buffer. However, the purified and membrane bound ATPase appear to have essentially identical properties when assays are carried out in Tris bicarbonate buffer. These studies clearly show that the three ATP dependent reactions examined differ significantly in their kinetic properties, substrate specificity, and sensitivity to bicarbonate. In order to interpret these findings, it is suggested that in the inner mitochondrial membrane of rat liver, the oligomycin sensitive ATPase contains at least two, and perhaps as many as four, functionally distinct ATP binding sites. It is suggested also that the membrane bound and purified ATPase (F1) may have similar conformations in Tris bicarbonate and different conformations in Tris Cl.

Original languageEnglish (US)
Pages (from-to)934-940
Number of pages7
JournalJournal of Biological Chemistry
Volume251
Issue number4
StatePublished - 1976

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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