Danièle C. Gautheron

Lipid composition and protein profiles of outer and inner membranes from pig heart mitochondria. Comparison with microsomes.

1. Mitochondria, inner and outer mitochondrial membranes and microsomes were isolated and purified from pig heart. Their lipid composition and protein components were studied. 2. The fatty acid distribution in the main phospholipids seemed specific rather of a given phospholipid and not of one type of membrane. 3. Inner mitochondrial membranes were characterized by a high content in cardiolipin and a very low level of triglycerides together with a high degree of unsaturation and C18 acids. Gel electrophoresis revealed 13 different polypeptide subunits of which 5 were major ranging in molecular weights from 10000 to 215000. 4. In outer mitochondrial membranes, total lipid, phosphatidylcholine, phosphatidylinositol, plasmologen and triglyceride contents were much higher than in inner membranes. Fatty acids of phospholipids were mostly saturated and the polypeptide pattern showed 12 components, of which 4 were major of mol. wt 75000, 60000, 20000 and below 10000. 5. Compared to outer membrane, microsomes exhibited a much higher cholesterol content and markedly different protein profiles. They contained significant amounts of cardiolipin and phosphatidylserine, this latter phospholipid being exclusively located in microsomes. However odd similarities were observed in some lipid components of microsomes and inner mitochondrial membranes, but fatty acids were more saturated in microsomes and electrophoretic profiles of protein components appeared very different and revealed components of high mol. wt.

Multipurpose electrode with different enzyme systems bound to collagen films

Abstract Selective, multipurpose electrodes have been developed from the previously described glucose electrode based on amperometric detection of hydrogen peroxide. Several single or multi-enzyme systems, including galactose oxidase, cholesterol oxidase, glucoamylase with glucose oxidase, and invertase with glucose oxidase, can be covalently bound to collagen membranes and attached to a platinum anode for monitoring the hydrogen peroxide generated. The probes allow fast and sensitive measurements of galactose, free cholesterol and maltose. Analogous electrodes are convenient for the assay of sucrose and lactose, with lower sensitivity. For disaccharide measurements, a comparative study of membranes produced by random co-immobilization, stacking of membranes and asymmetric coupling is reported. Asymmetric coupling improved the electrode performances in every case. One enzyme membrane is readily replaced by another in the electrode construction, and the sensors can be used for hundreds of assays.

Pig heart mitochondrial ATPase : properties of purified and membrane-bound enzyme: Effects of flavonoids

Soluble ATPase (F1) has been purified from pig heart mitochondria. The purified enzyme had a high specific activity and was homogeneous as checked by ultracentrifugation and electrofocusing. It could be dissociated into subunits by cold-treatment or sodium dodecyl sulfate denaturation. The molecular weights of the two major and three minor subunits could be estimated by sodium dodecyl sulfate gel electrophoresis. The native enzyme had an isoelectric point of 5.2 while the cold-denatured enzyme showed three main bands focusing at pH 5.0, 5.2, and 5.4. Kinetic properties (Vm and Km (atp) have been compared for the soluble and membrane bound ATPase in presence of various anions. Inhibitory effects of Quercetin and other flavonoids have been tested in order to get an insight on the interaction between ATPase and its natural inhibitor.

Optimization of the purification of mitochondrial F1-adenosine triphosphatase.

A simple technique of purification of the soluble pig heart mitochondrial F1-ATPase is described. It consists of removal of extrinsic proteins from mitochondrial membranes before extraction with chloroform and ammonium sulfate fractionation. A high degree of purity, an excellent stability and a good yield are attained after gel filtration through an Ultrogel ACA 34 column equilibrated in the presence of 50% glycerol. The tested properties of the F1-ATPase prepared by this method are similar to those of the same enzyme extracted by sonication. The enzyme is virtually devoid of tightly bound nucleotides. In addition, some characteristics of the behaviour of the beta subunit are shown.

Rubella Virus Maturation and Production in Two Host Cell Systems

When inoculated at the same MOI, Vero cells released a larger amount of infectious rubella virus into the culture medium than did BHK21 cells, However, BHK21 cells (in monolayer or in suspension) produced more intracellular infectious virus than Vero cells when tested 24 h after infection. Maturation of the virus in BHK21 cells occurred at the plasma membrane and, in a larger quantity, in the cytoplasm (Golgi apparatus and vacuoles). Viral particles consisted of an electron-dense core (32 nm) surrounded by a capsid and enveloped by a single membrane (8-10 nm). Aberrant forms (elongated and twisted) in the vacuole and double virions in the plasma membrane were observed as early as 65 h after infection.

ANTHEPROT: a package for protein sequence analysis using a microcomputer.

A simple microcomputer package is described to make the theoretical analysis of protein sequences. Several methods designed to compare two sequences, to model proteolytic reactions and to predict the secondary structure, the hydrophobic/hydrophilic regions and the potential antigenic sites of proteins have been included in an Apple II microcomputer software. The package comprises 21 programs as well as the secondary structure database of Kabsch and Sander (1983).

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.

Two-dimensional gel electrophoresis of membrane proteins using anionic and cationic detergents. Application to the study of mitochondrial F0-F1-ATPase

Polyacrylamide gel electrophoresis in the presence of a cationic detergent, tetradecyltrimethylammonium bromide (TDAB) has been compared to electrophoresis in the presence of an anionic detergent, sodium dodecyl sulfate (SDS). Although, in both systems, the peptides generally migrated as a function of their molecular weight, the TDAB electrophoresis permitted us to obtain a much better resolution of several peptides of the mitochondrial F0-F1-ATPase, especially for the alpha and beta subunits and for the oligomycin sensitivity conferring protein (OSCP). The differences between the two electrophoretic profiles have been used to devise a new technique of two-dimensional electrophoresis using successively anionic and cationic detergents. This method could be very useful in the case of membrane proteins, which are generally soluble only in the presence of powerful ionic detergents. It has been particularly successful in resolving the small peptides of the F0-F1-ATPase which were difficult to differentiate by other techniques in one- or two-dimensional polyacrylamide gel electrophoresis.

Enzyme Electrode with Collagen-Immobilized Cholesterol Oxidase for the Microdetermination of Free Cholesterol

Abstract Cholesterol oxidase has been immobililzed on collagen films associated to an electrochemical sensor to form a “cholesterol electrode”. The electrode poised at a potential of + 650 my vs Ag/AgCl detects the hydrogen peroxide produced in the enzymatic reaction. This device presents a very high sensitivity and a wide range of linearity (10−7 M - 0.8.10−4 M). The use of a non enzymatic electrode associated with the enzymatic one allowed the detection and correction of electrochemical interferences when applied to human sera for free cholesterol determination.

Effects of ATP on various steps controlling the rate of oxidative phosphorylation in newborn rat liver mitochondria

Preincubation of newborn rat liver mitochondria with ATP increases their state 3 respiration rate [J. K. Pollak (1975) Biochem. J. 150, 477-488; J. R. Aprille, and G. K. Asimakis (1980) Arch. Biochem. Biophys. 201, 564-575]. To determine which reactions contribute to control the rate of succinate oxidation with and without prior exposure to ATP, the effects of inhibitors specific for various reactions were studied. The adenine nucleotide translocator does not control the respiration in newborn more than in the adult mitochondria. The supply of reducing equivalents to the respiratory chain is an important step controlling the rate of oxidative phosphorylation by mitochondria from newborn rat liver, especially after preincubation with ATP. On the contrary, titrations with oligomycin show that the preincubation with ATP markedly decreases the control exerted by the ATPase-ATP synthase complex. That the rate of ATP synthesis is one of the steps controlling the rate of oxidative phosphorylation in newborn rat liver mitochondria is in striking contrast to the behavior of adult rat liver mitochondria. Other differences include a greater permeability to protons and a marked increase in sensitivity to mersalyl, indicating an easier accessibility of the proteins involved in oxidative phosphorylation to the thiol reagent.