Hydrogen ion-ATP synthase from rat liver mitochondria. A simple, rapid purification method of the functional complex and its characterization

Abstract

A novel, simple, and rapid preparative method for purification of rat liver H(+)-ATP synthase by anion-exchange HPLC was developed. The H(+)-ATP synthase purified had higher ATPase activity in the absence of added phospholipids than any preparation reported previously, and this activity was completely inhibited by oligomycin. When reconstituted into proteoliposomes, the H(+)-ATP synthase showed an ATP-dependent 8-anilinonaphthalene-1-sulfonate response and ATP-Pi exchange activity, both of which were also completely inhibited by oligomycin and an uncoupler, indicating the intactness of the H(+)-ATP synthase. An immunochemical study and a labeling experiment with N,N'-[14C]dicyclohexylcarbodiimide ([14C]DCCD) demonstrated the presence of chargerin II ( a product of mitochondrial A6L DNA) and DCCD-binding protein (subunit c) in the complex. The subunits of the complex were separated into 11 main fractions by reverse-phase HPLC, and 3 of them and the delta subunit in F1 were partially sequenced. A search for sequence homologies indicated that these components were subunit b, coupling factor 6, subunit delta, and subunit epsilon. This is the first report of the existence of subunit b, factor 6, and chargerin II in H(+)-ATP synthase purified from rat liver mitochondria.

Mitochondrial disease associated with the T8993G mutation of the mitochondrial ATPase 6 gene: a clinical, biochemical, and molecular study in six families

“…Table 2 shows the activities of ATPase, measured as a reaction linked to pyruvate kinase and lactate dehydrogenase15 and normalised to that of citrate synthase of several samples from different members.…”

“…Rotenone sensitive NADH coenzyme Q1 reductase (complex I), succinate dehydrogenase (part of complex II),12 cytochrome c oxidase (complex IV),13 antimycin sensitive ubiquinol cytochrome c reductase (complex III),14 oligomycin sensitive ATPase (complex V),15 and citrate synthase16 were measured in duplicate in muscle homogenates, as well as in cultured fibroblasts.…”

Gene Expression of Subunit c(P1), Subunit c(P2), and Oligomycin Sensitivity-conferring Protein May Play a Key Role in Biogenesis of H+-ATP Synthase in Various Rat Tissues

“…To clarify the coordinate transcriptional regulatory system(s) of the mammalian H ϩ -ATP synthase, we have developed a simple and rapid purification method of H ϩ -ATP synthase from rat (8,9) that can carry out the physiological experiments. We then determined the primary sequences of the synthase subunits by the protein sequence and cDNA cloning techniques (13)(14)(15)(16)(17).…”

Functional Disorders of the Oxidative Phosphorylation System in the Heart Mitochondria of Mice with Juvenile Visceral Steatosis.

“…Reagents used for detemination of ATPase activity, ATP-Pi exchange activity, SDS-PAGE, and Western blotting were obtained as described previously. [20][21][22] …”

“…The mammalian H ϩ -ATP synthase is a supramolecule composed of at least 14 subunits 21,[24][25][26][27] that have a constant stoichiometry. 28,29) Six of them construct the catalytic site of ATP synthesis called Fig.…”