Catherine Godinot

Membranes creatine kinase (E. C. 2. 7. 3. 2.) in pig heart mitochondria: Properties and role in phosphate potential regulation

Summary The properties and the role of creatine kinase (CK) in regulating phosphate potential has been studied in pig heart mitochondria, as well as its interactions with adenylate kinase and ATPase. A pH stat procedure has been adjusted to realize the simultaneous determination of creatine kinase and ATPase activities allowing the correction for adenylate kinase contribution. o 1. Creatine and phosphocreatine levels in pig heart mitochondria have been determined and compared to the intact heart levels. 2. The ratio ΔH+/Δ ATP has been measured for all three mitochondrial systems using ATP as a common substrate: creatine kinase, ATPase and adenylate kinase. The meaning of such a ratio is discussed. 3. The intracellular distribution of creatine kinase in pig heart has been measured. Although mitochondria contained only a small percentage of this activity as compared to the intact tissue, the specific activity of creatine kinase was high in mitochondria. 4. Creatine kinase activity in pig heart mitochondria is higher or of the same order as ATP synthesis through oxidative phosphorylation; ATP synthesis being higher than ATPase and adenylate kinase activities. 5. The kinetic parameters of creatine kinase and ATPase were determined: Vm and apparent Km for ATP, Mg2+ and creatine (creatine kinase) as a function of the ratio ATP/Mg2+. The accessibility of intramitochondrial Mg2+ gave informations concerning the mitochondrial location of these two enzymatic systems. 6. The true dissociation constants: Kia, Ka, Kib, Kb of creatine kinase complexes with its substrates have been calculated according to Cleland. 7. The influence of the creatine kinase on the respiratory rate of pig heart mitochondria has been evaluated. The compared contribution of the three enzymatic systems in regulating the phosphate potential in pig heart mitochondria is discussed.

[13] Separation of right-side-out and inside-out submitochondrial particles by affinity chromatography on sepharose-cytochrome c

Publisher Summary When mitochondria are sonicated or treated with a hypotonic solution, they disintegrate into small vesicles, the membrane of which is more or less inverted as compared to normal mitochondria. The chapter describes a method to separate the fight-side-out and inside-out vesicles by free-flow electrophoresis. This method is a very simple way to control the orientation of right-side-out and inside-out vesicles by affinity chromatography in a l0-ml disposable plastic syringe filled with Sepharose-cytochrome c. The technique is based on the fact that the physiological location for cytochrome c is on the membrane surface normally oriented toward the cytoplasm and that the cytochrome c can be easily removed from the membrane by hypotonic and salt treatments. If the endogenous cytochrome c has been removed from the membrane, the right-side-out vesicles should be retained by the column whereas the inside-out ones should be excluded.

Correlations between ATP hydrolysis, ATP synthesis, generation and utilization of ΔpH in mitochondrial ATPase-ATP synthase

Using fluorescence quenching of 9-amino-6-chloro-2- methoxyacridine induced either by ATP hydrolysis in the ATPase-ATP synthase complex or by succinate oxidation in inverted submitochondrial particles, correlation have been established between ATP hydrolysis, ATP synthesis and the generation and utilization of delta pH. The results obtained are best explained in terms of local circuits of protons.

Immunolabelling of mitochondrial superoxide dismutase and of Hsp60 in muscles harbouring a respiratory chain deficiency.

In mitochondrial encephalomyopathies, impairment of the electron transfer chain may lead to overproduction of reduced oxygen species because oxygen consumption is decreased. Whether heat shock proteins (Hsp) are induced or not in mitochondria against oxidative stress is questionable. Muscle ragged-red fibres are the histological hallmark of most respiratory chain deficiencies in humans. They exhibit abnormal mitochondria which accumulate mainly under their sarcolemma. Within these fibres, immunolabelling demonstrated strong expression of mitochondrial manganese-dependent superoxide dismutase and a lack of expression of mitochondrial Hsp60 within the subsarcolemmal spaces. In contrast, Hsp60 was overexpressed within the intermyofibrillar mitochondria. These findings suggest enhanced generation and dismutation of superoxide anions and that processing and integration of imported precursor proteins is impaired within the subsarcolemmal mitochondrial aggregates of ragged-red fibres, whereas protein import and assembly may still be efficient in the intermyofibrillar mitochondria of these fibres.

[73] Monoclonal antibodies to F1-ATPase subunits as probes of structure, conformation, and functions of isolated or membrane-bound F1

Publisher Summary This chapter describes the preparation of monoclonal antibodies (McAb) against pig heart mitochondrial F 1 -ATPase subunits and their use to determine the stoichiometry, the conformations, and the functions of the isolated or membrane-integrated enzyme. The antibodies able to recognize sequences of only a few amino acids serve as tools to define structure-function relationships in enzymes. Conventional antisera are complex mixtures of antibodies of different classes with different affinities for various antigenic determinants; therefore, they cannot provide precise information on the domains or sub-domains of proteins. Because of the complexity of the immune response, no conventional antiserum is exactly like another even if both come from the same species. In contrast, monoclonal antibodies are attractive reagents as once the cell line is established, it provides a permanent source of an antibody. In addition, as monoclonal antibodies recognize a single antigenic determinant, they are suitable as specific probes of protein assembly.

A rapid and efficient new method of purification of glutamate dehydrogenase by affinity chromatography on GTP-Sepharose

Abstract The very high affinity for GTP of glutamate dehydrogenase was used to purify this enzyme by affinity chromatography. After periodic acid oxidation, GTP was covalently bound to an activated Sepharose. When crude mitochondrial extracts were applied on a column of this GTP-Sepharose, glutamate dehydrogenase was retained with very few other proteins. Glutamate dehydrogenase from rat liver was eluted with a KCl gradient with only one contaminating protein. From a pig heart mitochondrial extract the enzyme was purified 300-fold in one step. A chromatography on hydroxyapatite was sufficient to achieve the purification. This very simple technique avoids the long and troublesome crystallization steps generally involved in glutamate dehydrogenase purification.

Molecular histology of mitochondrial and nuclear transcripts in the muscle of patients harbouring a single mitochondrial DNA deletion

Abstract The distribution of transcripts of mitochondrial and nuclear genes involved in oxidative phosphorylation and of the mitochondrial creatine kinase nuclear gene was examined, using in situ hybridisation, in the skeletal muscle of 11 patients harbouring a heteroplasmic mitochondrial DNA (mtDNA) single deletion. Levels of mRNAs transcribed from genes located within the deletions were not decreased, suggesting that the remaining wild-type mtDNA was still transcribed. Those muscle fibres with characteristic abnormal mitochondrial proliferation always showed overexpression of mRNAs and rRNAs transcribed from mitochondrial genes located outside the deletions. Interestingly, they also showed overexpression of the nuclear-encoded ATP synthase β subunit mRNA, but not of mitochondrial creatine kinase mRNA. These observations lead to three proposals: (1) overexpression of mitochondrial transcripts within fibres harbouring mitochondrial proliferation, together with the apparently normal expression of the remaining wild-type mtDNA, is not related to decreased mitochondrial translation; (2) it is more probably related to an up-regulation mechanism which co-ordinates both mitochondrial and nuclear expression; and (3) this mechanism is restricted to transcripts directly involved in oxidative phosphorylation and to fibres with mitochondrial accumulation.

Reversal of glucose-induced inhibition of newborn rat liver mitochondrial maturation by administration of alkylxanthines at birth

A glucose injection given immediately after birth delays the maturation which normally occurs in rat liver mitochondria and which increases the rate of ATP synthesis coupled to succinate oxidation from a low value at birth to the adult value a few hours after birth [R. Meister, J. Comte, L. Baggetto, C. Godinot and D. C. Gautheron, Biochim. biophys. Acta 722, 36 (1983)]. Alkylxanthine (pentoxifylline, HWA 285) administration at birth has no effect on the maturation of mitochondria prepared from 2-hr-old rat livers while DBcAMP administration increases their RCR and their rate of ATP synthesis. On the contrary, both alkylxanthines and DBcAMP reverse the glucose-induced inhibition of mitochondrial maturation. This DBcAMP effect cannot be mimicked by butyrate and is therefore related to cAMP. The cAMP content of rat liver increases during this postnatal period in both control and glucose-treated rats, although glucose administration tends to decrease the level of cAMP. Alkylxanthine administration restores after 2 hr the cAMP level in glucose-treated animals. The variations of RCR could not be completely correlated with the level of cAMP. The possible involvement of other factors in the mitochondrial maturation and the glucose effect is discussed.

Binding of L-glutamate to glutamate dehydrogenase in the presence of 1,4,5,6-tetrahydronicotinamide adenine dinucleotide

In a previous work with Julliard, a lipid-protein complex showing a high affinity for glutamate was extracted from pig heart mitochondrial membranes; it exhibited many properties expected from a glutamate translocator [ 11. From its protein composition it was suggested...

[43] Use of monoclonal antibodies to purify oligomycin sensitivity-conferring protein and to study its interactions with F0 and F1

Publisher Summary This chapter focuses on the use of monoclonal antibodies to purify oligomycin sensitivity-conferring protein and to study its interactions with F 0 and F 1 . Oligomycin sensitivity-conferring protein (OSCP) is a subunit of the H + -transporting ATP synthase complex in mitochondria. The difficulty in improving the purification is the absence of known intrinsic enzymatic activity of this peptide. To better know the structure and function of OSCP in the complex, anti-OSCP monoclonal antibodies are prepared. These antibodies are useful tools for a new procedure of purification of OSCP that is simple, rapid, and efficient. Anti-OSCP monoclonal antibodies prepared and purified can be used to study the interaction of OSCP with F 0 and F 1 and its function in the whole complex H + -transporting ATP synthase. However, the OSCP-antibody complex cannot be used to reconstitute F 1 with depleted submitochondrial particles (SMP).