Efficient reconstitution of mitochondrial energy-transfer reactions from depleted membranes and F1-ATPase as a function of the amount of bound oligomycin sensitivity-conferring protein (OSCP)

Pénin, François; Deléage, Gilbert; Godinot, Catherine; Gautheron, D

doi:10.1016/0005-2728(86)90056-3

Cited by 25 publications

(11 citation statements)

References 41 publications

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“…Biological Preparations. Previously described procedures were used to prepare pig heart mitochondria (Gautheron et al, 1964;Godinot et al, 1969), ASU particles (Penin et al, 1986), purified Ft (nucleotide depleted; Penin et al, 1979), purified OSCP (Archinard et al, 1986), and purified protein inhibitor (Di Pietro et al, 1988).…”

Section: Methodsmentioning

confidence: 99%

“…Iodinated F¡ (645 cpm/^g of F,) was prepared as previously (Penin et al, 1986): an average value of 0.5 mol of 125I was covalently linked per mole of Fj. It has been checked that iodinated Ft behaved exactly as unlabeled F,: nucleotide binding and release, ATPase activity, reconstitution, and ATP synthesis were not modified.…”

Section: Methodsmentioning

confidence: 99%

“…Reconstitution Procedure. The reconstitution conditions previously described (Penin et al, 1986) were adapted as follows: 530 jug of iodinated Fj preloaded with tritiated adenine nucleotides in the presence or absence of the protein inhibitor was mixed with pure OSCP (30 /ug) and incubated for 30 min at 30 °C with ASU particles (1.6 mg) in 1.1 mL of 50 mM Tris (acetate), pH 7.5, 0.25 M sucrose, 5 mM DTT, and 2.5 mM MgSG4 (final concentrations). The mixture was then centrifuged as 175-gL fractions in a Beckman Airfuge at 25 psi (140000g) for 5 min.…”

Section: Methodsmentioning

confidence: 99%

“…ATPase activity was measured spectrophotometrically in the presence of an ATP regenerating system coupled to NADH oxidation (Pullman et al, 1960) in 50 mM Tris (maléate) buffer, pH 8.0, as detailed previously (Godinot et al, 1979) in the presence of 1.5 mM KCN and with or without 1 pg of oligomycin per assay. Net ATP synthesis was measured at 30 °C (Penin et al, 1986) in the complete ATP synthesis medium described above, in the presence of 2 X 106 cpm of [32P]P¡/Vmol of P¡.…”

Section: Methodsmentioning

confidence: 99%

“…purified from pig heart mitochondria was loaded with labeled nucleotides in the presence or absence of the protein inhibitor. Then F, was reassociated with Frdepleted membranes (ASU particles) to reconstitute efficient ATP synthesis driven by NADH oxidation (Penin et al, 1986). A technique of rapid filtration through nitrocellulose filters was developed to titrate the labeled nucleotides remaining bound to membrane-reassociated F, during the course of ATP hydrolysis or net ATP synthesis.…”

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confidence: 99%

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Fate of nucleotides bound to reconstituted F0-F1 during adenosine 5'-triphosphate synthesis activation or hydrolysis: role of protein inhibitor and hysteretic inhibition

Pénin¹,

Pietro²,

Godinot³

et al. 1988

Biochemistry

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The protein ATPase inhibitor entraps about five nucleotides in pig heart mitochondrial F1, one at least being a triphosphate [Di Pietro, A., Penin, F., Julliard, J.H., Godinot, C., & Gautheron, D.C. (1988) Biochem. Biophys. Res. Commun. 152, 1319-1325]. The fate of these nucleotides was studied during ATP synthesis driven by NADH oxidation in reconstituted inverted submitochondrial particles. Iodinated F1, containing 0.7 mol of endogenous nucleotides/mol, was first loaded with tritiated adenine nucleotides in the presence or absence of the protein inhibitor and then reassociated with F1-depleted submitochondrial particles (ASU particles) to reconstitute an efficient NADH-driven ATP synthesis. In the absence of the protein inhibitor, 1.7 mol of labeled nucleotides remained bound per mole of reassociated F1, 0.8-0.9 mol being rapidly exchangeable against medium ADP or ATP, as measured after rapid filtration through nitrocellulose filters. In the presence of the protein inhibitor, as many as 3.25 mol of labeled nucleotides remained bound per mole of reassociated F1. Under hydrolysis conditions where ATPase activity was highly inhibited, no release of tritiated nucleotide occurred. In contrast, under ATP synthesis conditions where the protonmotive force was generated by NADH oxidation, the progressive reversal of inhibition by the protein inhibitor was correlated to a concomitant release of tritiated nucleotide. When ATP synthesis became fully active, about one nucleotide was completely exchanged whereas more than three nucleotides remained tightly bound and did not appear to be directly involved in ATP synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

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Fate of nucleotides bound to reconstituted F0-F1 during adenosine 5'-triphosphate synthesis activation or hydrolysis: role of protein inhibitor and hysteretic inhibition

Pénin¹,

Pietro²,

Godinot³

et al. 1988

Biochemistry

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Evidence from immunological studies of structure-mechanism relationship of F1 and F1F0

Gautheron¹,

Godinot²

1988

J Bioenerg Biomembr

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Monoclonal and polyclonal antibodies directed against peptides of F1-ATPase of F1F0-ATPase synthase provide new and efficient tools to study structure-function relationships and mechanisms of such complex membrane enzymes. This review summarizes the main results obtained using this approach. Antibodies have permitted the determination of the nature of subunits involved in the complex, their stoichiometry, their organization, neighboring interactions, and vectorial distribution within or on either face of the membrane. Moreover, in a few cases, amino acid sequences exposed on a face of the membrane or buried inside the complex have been identified. Antibodies are very useful for detecting the role of each subunit, especially for those subunits which appear to have no direct involvement in the catalytic mechanism. Concerning the mechanisms, the availability of monoclonal antibodies which inhibit (or activate) ATP hydrolysis or ATP synthesis, which modify nucleotide binding or regulation of activities, which detect specific conformations, etc. brings many new ways of understanding the precise functions. The specific recognition by monoclonal antibodies on the beta subunit of epitopes in the proximity of, or in the catalytic site, gives information on this site. The use of anti-alpha monoclonal antibodies has shown asymmetry of alpha in the complex as already shown for beta. In addition, the involvement of alpha with respect to nucleotide site cooperativity has been detected. Finally, the formation of F1F0-antibody complexes of various masses, seems to exclude the functional rotation of F1 around F0 during catalysis.

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Preparation of highly phosphorylating mitochondria from the yeastSchizosaccharomyces pombe

Jault

Comte

Gautheron

et al. 1994

J Bioenerg Biomembr

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Schizosaccharomyces pombe yeast cells grown on either fermentable or respiratory media were efficiently converted to stable spheroplasts by the alpha-(1-->3)-glucanase Novozym 234 in the presence of 1.2 M sorbitol. Lysis of spheroplasts by gentle homogenization in dilute sorbitol resulted in the preparation of mitochondria with a structure similar to that observed within the starting yeast cells. The isolated mitochondria exhibited high oxidation rates with various respiratory substrates, NADH being the most efficient. The mitochondria appeared well coupled since the second State 4 rate observed after ADP consumption was identical to the initial one. The State 3 rate in the presence of ADP was completely inhibited by low oligomycin concentrations, similarly to the concomitant ATP synthesis of 900 nmol/min x mg protein. These NADH oxidation and dependent ATP-synthesis activities are much higher than those previously described for mitochondria isolated from Schizosaccharomyces pombe, and similar to the highest values reported for Saccharomyces cerevisiae.

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Efficient reconstitution of mitochondrial energy-transfer reactions from depleted membranes and F1-ATPase as a function of the amount of bound oligomycin sensitivity-conferring protein (OSCP)

Cited by 25 publications

References 41 publications

Fate of nucleotides bound to reconstituted F0-F1 during adenosine 5'-triphosphate synthesis activation or hydrolysis: role of protein inhibitor and hysteretic inhibition

Fate of nucleotides bound to reconstituted F0-F1 during adenosine 5'-triphosphate synthesis activation or hydrolysis: role of protein inhibitor and hysteretic inhibition

Evidence from immunological studies of structure-mechanism relationship of F1 and F1F0

Preparation of highly phosphorylating mitochondria from the yeastSchizosaccharomyces pombe

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