Studies on the mechanism of oxidative phosphorylation. Positive cooperativity in ATP synthesis.

A Matsuno-Yagi,Y Hatefi

doi:10.1016/s0021-9258(19)40198-1

Abstract

Kinetic and nucleotide binding studies have shown that submitochondrial particles from bovine heart possess three exchangeable binding sites for ADP or GDP. In order of decreasing affinity at neutral pH, these sites will be referred to as sites I, II, and III, and their respective dissociation constants as KI, KII, and KIII. In oxidative phosphorylation experiments in the presence of saturating amounts of inorganic phosphate, rapid ATP (or GTP) synthesis occurred only upon ADP (or GDP) binding to site III. The Eadie-Hofstee plots (v/[S] on the ordinate versus v on the abscissa) of the kinetics of ATP (or GTP) synthesis at variable ADP (or GDP) were, therefore, composed of an initial upward phase, indicating positive cooperativity with respect to substrate concentration, followed by a downward phase where rapid product formation took place. These data allowed calculation of KII from the upward phase and KIII (equivalent to apparent Km) from the downward phase. KI was estimated from Scatchard plots of binding data with radiolabeled ADP or GDP. Thus, together with our previous results, these findings have allowed characterization of the process of ATP or GTP synthesis by bovine-heart submitochondrial particles in terms of KI, KII, KIII, and kcat.

Highlights

Kinetic and nucleotide binding studies have shown that submitochondrial particles from bovine heart possess three exchangeable binding sites for ADP or GDP
The data were analyzed with the assumption that at least 2 GDP molecules must bind before rapid GTP synthesis can be measured
The results presented above can be summarized in the two schemes in Fig. 8 for ATP and GTP synthesis

Summary

MATERIALSAND METHODS

Unlabeled nucleotides were obtained from Pharmacia LKB Biotechnology Inc. Luciferin and luciferase were from Boehringer Mannheim. The assay mixtures at pH 7.5 contained 0.25 M sucrose, 50 mM Tris acetate, 0.5 mM EDTA, 25 mM glucose, 5 mM MgC&, 20 mM potassium phosphate containing 5-15 x lo cpm of “P, 70 pg of hexokinaselml, 50 pg of SMP/ml, nucleotide at the concentrations indicated, and 0.5 mM NADH or 6.7 mM potassium succinate as the respiratory substrate. The assay mixture at pH 7.5, contained, in a final volume of 1.5 ml, 50 mM Tris-SOI, 5 mM MgSO,, 0.5 mM EDTA, 1 mM phosphoenolpyruvate, 0.1 mM luciferin, and 7.5 rg of luciferase To this mixture was added an aliquot (lo-20 ~1) of the extract, and light emission was continuously recorded in an SLM. For measurement of the ATP-nonchaseable fraction of bound VH]ADP, MgATP was added at a final concentration of 2 mM after the initial.30-s incubation with. . the mixture was further incubated for 1 min at 30 “C before applyingit to a Sephadex centrifuge column

RESULTS

DISCUSSION

A Matsuno-Yagi and Y Hatefi