Alvaro Rendon

Subfractionation of the outer membrane of rat brain mitochondria: evidence for the existence of a domain containing the porin-hexokinase complex

Isolated and well-characterized rat brain nonsynaptic mitochondria were subfractionated by digitonin. Antibodies to a uniquely outer membrane protein, porin, have allowed us to use this protein for the first time as an outer membrane marker in brain. Hexokinase, which binds to porin, was also measured. Based upon the sequential release of these and other marker enzymes with increasing concentrations of digitonin, three outer membrane domains have been identified. Two populations of porin were found by this treatment. The most plausible interpretation of our results is that the two porin populations exist in different membrane environments with regard to cholesterol. One of these populations binds most of the hexokinase and appears to be associated with the inner membrane. It is proposed that the porin-hexokinase complex in brain mitochondria is located in a cholesterol-free membrane domain together with inner membrane components. This domain has the features of contact points which have been visualized by electron microscopy.

Purification of non-synaptic and synaptic mitochondria and plasma membranes from rat brain by a rapid Percoll gradient procedure

A method is described for the isolation and purification of non-synaptic and synaptic mitochondria and plasma membranes. The procedure involved differential centrifugation and successively a Ficoll gradient followed by a Percoll one. The method is rapid and maintains constant osmotic conditions. The Percoll-purified fractions show a lower density than those prepared by the conventional methods. The fractions were identified and characterized by enzyme markers and electron microscopy. The method will be useful for routine preparations rendering a good degree of homogeneity and purity.

Intramitochondrial intermembranal large amplitude protein movements: I. A possible novel aspect of membrane fluidity

Summary Large amplitude protein movements between mitochondrial inner membrane and intermembranal space have been studied. o 1 — Effects of exogenous Na+, K+, or NH4+ salts of aspartate, glutamate, acetate, pyruvate, β-hydroxybutyrate, 2-oxoglutarate, succinate, malate, fumarate and phosphate have been tested. 2 — Specificity of the influence of these substances on the protein release phenomenon have been studied by comparing the action of increasing concentrations of these substances and by polyacrylamide gel electrophoresis on the released proteins. 3 — Reversibility of the protein release has been shown to occur by realizing release binding cycles and by polyacrylamide gel analysis. 4 — A discussion excluding possible methodological artifacts and swelling as release vectors is developed. 5 — Possible functional consequences of the transitory presence of certain proteins in two submitochondrial compartments are presented.

Studies on the relationship between the inner and outer membranes of rat liver mitochondria as determined by subfractionation with digitonin.

The present investigation has attempted to define in rat liver mitochondria the distribution of outer membrane proteins in relation to the inner membrane by fractionation with digitonin and phospholipase A2. Porin, the channel-forming protein in the outer membrane, was measured quantitatively by immunological methods. Neither monoamine oxidase nor porin could be released by phospholipase A2 treatment, but both were released by digitonin, at the same detergent concentration. Thus, the release of monoamine oxidase and porin requires the disruption of the cholesterol but not the phospholipid domains of the membrane and the two polypeptides exist in the same, or similar, membrane environment with regard to cholesterol. Changes in the energy state, or binding of brain hexokinase to rat liver mitochondria prior to fractionation with digitonin, did not alter the release patterns of porin and monoamine oxidase. The uptake of Ca2+, however, resulted in the concomitant release of the outer membrane markers together with the matrix marker, malate dehydrogenase. The present findings with liver differ from those obtained recently with brain mitochondria (L. Dorbani et al. (1987) Arch. Biochem. Biophys. 252, 188-196) in which two populations of porin were located in two different cholesterol domains. The significance of these differences in the location of porin in liver and brain mitochondria is discussed.

Reversible intramitochondrial release of protein related to unsaturated fatty acids of membranes

Abstract The influence of temperature on intramitochondrial protein and enzyme release was studied in control and “lipid-deficient” rat liver mitochondria and in synaptosomal and “cell body” mitochondria of rat brain. (i) The fatty acid composition of the phospolipid fraction was shown to be different in control and lipid-deficient preparations. (ii) Arrhenius curves for temperature-dependent release of protein showed breaks. (iii) When comparing control to lipid-free rat liver mitochondria and cell body to synaptosomal rat brain mitochondria, shifts in the breaks in the Arrhenius plots were observed for release of aspartate aminotransferase, protein and malate dehydrogenase. (iv) Intramitochondrial temperature-dependent, succinate-induced protein release was also studied in rat liver mitochondria which had previously undergone a succinate-induced release and rebinding cycle. These mitochondria showed a temperature-dependent protein release identical to that of untreated mitochondria.

Intramitochondrial release and binding of mitochondrial aspartate aminotransferase and malate dehydrogenase in the presence and absence of monovalent and bivalent cations

Abstract Intramitochondrial release and binding of mitochondrial aspartats aminotransferase and malate dehydrogenase was shown to be controlled by a sucrose-cation-sucrose cycle in vitro . The effect of ion concentration on the aspartate aminotransferase release suggests distinct modes of action for bivalent and monovalent cations.

Intramitochondrial release and binding of mitochondrial aspartate aminotransferase and malate dehydrogenase in the presence and absence of exogenous succinate

Abstract Differential shuttling of aspartate aminotransferase and malate dehydrogenase between submitochondrial fractions was shown to occur. This phenomenon is dependent upon the nature of the extramitochondrial compartment.

Microtubule-associated proteins bind to 30 kDa and 60 kDa proteins of rat brain mitochondria: Visualization by ligand blotting

Axonal transport of mitochondria is a microtubule-associated movement. Microtubule-mitochondria interactions were studied in vitro using organelles isolated from rat brain. Thanks to the ligand blotting method we were able to show two mitochondrial membrane proteins with apparent molecular masses of 30 kDa and 60 kDa that bind microtubule-associated proteins. The binding of the 30 kDa protein has an apparent Kd of 8 x 10(-8) M. Digitonin fractionation of mitochondria reveals a bimodal localization of the 30 kDa and the 60 kDa proteins within the outer membrane. The data suggest that these polypeptides could participate to the interactions observed in situ between microtubules and mitochondria.

Seasonal variation of the composition of membrane lipids in liver mitochondria of the hibernator cricetus cricetus relation to intramitochondrial intermembranal protein movement

Abstract 1. 1. European hamster ( Cricetus cricetus ) raised under constant temperature conditions show a seasonal variation in the profiles of the structural fatty acids of the liver mitochondrial membrane. In these animals there are but small differences between sleeping and aroused animals in winter. 2. 2. The most important variation in lipid composition occurs in the prehibernating phase—at the end of summer and the beginning of autumn. 3. 3. However, there is no apparent coordinated relation between the seasonal variation of the composition of the fatty acids and the membrane “fluidity” as expressed by the break in the Arrhenius curve for protein release in intermembranal space of the liver mitochondria. 4. 4. This break occurred at a higher temperature in active animals in winter (arousedl than in summer. 5. 5. No general correlation could be found between the breaks in the Arrhenius curves and the variations of the different fatty acid species during the seasonal cycle, except for the most polyunsaturated fatty acid (docosahexanoic acid) where an excellent inverse correlation was observed. 6. 6. Our results suggest that the more fluid parts of the lipidic leaflet of the mitochondrial membrane are those more specifically involved in such phenomena as succinate induced intramitochondrial protein movement and that the changes in composition of the mitochondrial lipids are a possible adaptative advantages for the hibernator.

Release and binding of proteins and enzymes with isolated inner mitochondrial membranes

Abstract Extramitochondrial substances trigger specific and reversible release of proteins and enzymes from inner membranal compartment towards intermembranal space. This shuttling phenomenon has been shown to occur with isolated inner membranes.