Membrane Of Rat Liver Mitochondria Research Articles

Palmitoyl-CoA inhibits the mitochondrial inner membrane anion-conducting channel

Palmitoyl-CoA is shown here to inhibit the pH-dependent anion-conducting channel (IMAC) in the inner membrane of rat liver mitochondria, with half-maximal inhibition at 2.4 μM. It has little effect on the transport of ribose, thiocyanate and glutamate. Palmitic acid and palmitoyl-carnitine stimulate the entry of all the above metabolises. CoASH and carnitine have no effect on chloride uniport. Palmitoyl-CoA and the IMAC may have a role in controlling thermogenesis in liver mitochrondria.

Ca 2+-dependent NAD(P) +-induced alterations of rat liver and hepatoma mitochondrial membrane permeability

Deenergized rat liver or AS-30D hepatoma mitochondria underwent extensive swelling when incubated in the presence of Ca 2+ and an oxidant of mitochondrial pyridine nucleotides. This swelling was stimulated by phosphate and inhibited by ruthenium red, Mg 2+ plus ATP and dibucaine. The degree of inhibition by dibucaine was shown to vary inversely with the Ca 2+ concentration. The inhibition caused by ruthenium red was completely removed by the Ca 2+ ionophore ionomycin indicating that the effect of the cation is mediated by internal binding sites. Since mitochondria were totally deenergized the data definitely rule out the requirement for Ca 2+ cycling across the membrane in this mechanism.

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 A 2. 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 A 2 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 Ca 2+, 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.

The influence of storage temperature on the transition, activation enthalpy, and activity of enzymes associated with inner mitochondrial membranes

The effects of storage at low temperature on the transition in enzyme function, T f ∗ , and the Arrhenius activation energy, E a, were determined for several enzymes associated with the inner membrane of rat liver mitochondria. The enzymes studied were succinate:cytochrome c reductase, cytochrome c oxidase, β-hydroxybutyrate dehydrogenase, and oligomycin-sensitive, Mg 2+-activated ATPase. For freshly isolated mitochondria the T f ∗ , for succinate:cytochrome c reductase and cytochrome c oxidase, occurred at approximately 23 °C and was coincident with a transition in structure, T s ∗ , determined as the change in temperature coefficient of motion for a spin label intercalated with the membrane lipids. This suggests that the change in thermal response of the membraneassociated enzymes is related to a change in molecular ordering of the membrane lipids. When mitochondria were stored at −12 °C, the specific activities of succinate:cytochrome c reductase and cytochrome c oxidase decreased. Concomitant with these changes the E a, above T f ∗ , increased. After 100 days storage at −12 °C, E a above T f ∗ approached the value for E a below T f∗ such that the transition in thermal response could no longer be detected. In contrast, for mitochondria stored at −196 °C, although the specific activity declined over the 100 days storage, no changes in either E a or T f ∗ were evident. The results indicate a need for caution in evaluating comparative studies of T f and E a, for membrane-associated enzymes, using mitochondria which have been frozen and Stored.

Integration of porin synthesized in vitro into outer mitochondrial membranes.

Porin, an intrinsic protein of outer mitochondrial membranes of rat liver, was synthesized in vitro in a cell-free in a cell-free translation system with rat liver RNA. The apparent molecular mass of porin synthesized in vitro was the same as that of its mature form (34 kDa). This porin was post-translationally integrated into the outer membrane of rat liver mitochondria when the cell-free translation products were incubated with mitochondria at 30 degrees C even in the presence of a protonophore (carbonyl cyanide m-chlorophenylhydrazone). Therefore, the integration of porin seemed to proceed energy-independently as reported by Freitag et al. [(1982) Eur. J. Biochem. 126, 197-202]. Its integration seemed, however, to require the participation of the inner membrane, since porin was not integrated when isolated outer mitochondrial membranes alone were incubated with the translation products. Porin in the cell-free translation products bound to the outside of the outer mitochondrial membrane when incubated with intact mitochondria at 0 degrees C for 5 min. When the incubation period at 0 degrees C was prolonged to 60 min, this porin was found in the inner membrane fraction, which contained monoamine oxidase, suggesting that porin might bind to a specific site on the outer membrane in contact or fused with the inner membrane (a so-called OM-IM site). This porin bound to the OM-IM site was integrated into the outer membrane when the membrane fraction was incubated at 30 degrees C for 60 min. These observations suggest that porin bound to the outside of the outer mitochondrial membrane is integrated into the outer membrane at the OM-IM site by some temperature-dependent process(es).

Characterization of the mitochondrial carnitine palmitoyltransferase enzyme system. II. Use of detergents and antibodies.

Exposure of rat liver mitochondrial membranes to octyl glucoside, Triton X-100, or Tween 20 solubilized an active and tetradecylglycidyl-CoA (TG-CoA)-insensitive carnitine palmitoyltransferase (presumed to be carnitine palmitoyltransferase II). The residual membranes after octyl glucoside or Triton X-100 treatment were devoid of all transferase activity. By contrast, Tween 20-extracted membranes were still rich in transferase; this was completely blocked by TG-CoA and thus was presumed to be carnitine palmitoyltransferase I. The residual carnitine palmitoyltransferase activity disappeared from the membranes upon subsequent addition of octyl glucoside or Triton X-100 and could not be recovered in the supernatant fraction. Antibody raised against purified rat liver transferase II (Mr 80,000) recognized only this protein in immunoblots from untreated liver mitochondrial membranes containing both transferases I and II. Tween 20-extracted membranes, which contained only transferase I, did not react with the antibody. Purified transferase II from skeletal muscle (also of Mr 80,000) was readily recognized by the antiserum, suggesting antigenic similarity with the liver enzyme. These and other studies on the effects of detergents on the mitochondrial [3H]TG-CoA binding protein provide further support for the model of carnitine palmitoyltransferase proposed in the preceding paper. They suggest that: 1) carnitine palmitoyltransferases I and II in rat liver are immunologically distinct proteins; 2) transferase I is more firmly anchored into its membrane environment than transferase II; 3) association of carnitine palmitoyltransferase I with a membrane component(s) is necessary for catalytic activity. While carnitine palmitoyltransferase I is a different protein in liver and muscle, it seems likely that both tissues share the same transferase II.

Effect of succinate on mitochondrial lipid peroxidation. 2. The protective effect of succinate against functional and structural changes induced by lipid peroxidation.

The damaging effects of ADP/Fe/NADPH-induced lipid peroxidation were studied on the enzymes and membranes of rat liver mitochondria. Succinate, an inhibitor of mitochondrial lipid peroxidation, prevented or delayed most of the damage caused by the peroxidation on different mitochondrial structures and functions. There were marked abnormalities on the electrophoretic pattern of mitochondrial proteins during the course of lipid peroxidation. The disappearance of particular polypeptide bands and the accumulation of high-molecular-weight aggregates could be observed. Succinate was found to delay these effects. As a consequence of lipid peroxidation the succinate oxidase activity of mitochondria was decreased. The succinate dehydrogenase enzyme and the component(s) of the respiratory chain were inactivated. Succinate prevented the inactivation of succinate dehydrogenase but did not protect the other components of terminal oxidation chain. From the matrix enzymes the glutamate dehydrogenase retained its full activity but the NADP-linked isocitrate dehydrogenase was inactivated. The mitochondrial membranes became permeable to large protein molecules. Succinate prevented the inactivation of isocitrate dehydrogenase and delayed the release of protein molecules from mitochondria.

Changes in physicochemical properties of mitochondrial membranes during the formation process of megamitochondria induced by hydrazine

Changes in some biochemical and physicochemical properties of rat liver mitochondrial membranes during the formation process of megamitochondria induced by hydrazine were analyzed. Hepatic mitochondria obtained from rats placed on a 1% hydrazine diet for 3 days became slightly enlarged and sometimes enlogated, while they became gigantic after 7 days of hydrazine intoxication. Changes were observed in mitochondria from rats treated with hydrazine for 3 days. (1) Total amounts of phospholipids extracted from mitochondria and submitochondrial fractions were increased. Among phospholipid species, relative amounts of acidic phospholipids were increased. (2) Contents of Ca 2+ in mitochondria were increased. (3) Differential scanning calorimetric analysis of mitochondria, especially that of the outer membrane fraction, showed that the thermotropic lipid phase transition temperatures were elevated accompanying the broadening of thermograms and the increase in transition enthalpy. (4) Contents of water in mitochondria were increased significantly with the ratio of freezable water to unfreezable water unchanged. Among the changes observed was that the total amount of phospholipids (except for that of the outer membrane fraction) and the contents of water and Ca 2+ nearly returned to normal in megamitochondria after 7 days of hydrazine intoxication. Relative amounts of phospholipids and thermotropic lipid phase transition temperatures of megamitochondria did not return to normal levels and yet changes were smaller than those obtained from 3 days of hydrazine intoxication. The fluidity of mitochondrial membranes was not affected by hydrazine treatment. These data would suggest that hydrazine-induced megamitochondrial formation is not due simply to the swelling of mitochondria, but might be due to the fusion of adjacent mitochondria by Ca 2+ -acidic phospholipid interactions, and once megamitochondria are formed the mitochondrial membranes are stabilized.

An import factor in reticulocyte lysates which stimulates processing of several precursors destined for the rat liver mitochondrial inner membrane.

An import factor in reticulocyte lysates which stimulates processing of several precursors destined for the rat liver mitochondrial inner membrane.

Effects of Ethanol on the Structure and Function of Rat Liver Mitochondrial and Microsomal Membranes

Effects of Ethanol on the Structure and Function of Rat Liver Mitochondrial and Microsomal Membranes

Effects of ethanol on the structure and function of rat liver mitochondrial and microsomal membranes.

Effects of ethanol on the structure and function of rat liver mitochondrial and microsomal membranes.

Stereochemistry and function of oxaloacetate keto-enol tautomerase.

Oxaloacetate keto-enol tautomerase, partially purified from porcine kidney, catalyzes the conversion of enol- to keto-oxaloacetate by a mechanism in which solvent protons end up equally distributed between the two prochiral positions at C3 of keto-oxaloacetate. This conclusion is based upon the observation that when enzyme catalyzed ketonization is conducted in 3H2O in the presence of excess malate dehydrogenase and NADH, only 50% of the 3H in the isolated (2S)-[3-3H]malate is labilized to solvent upon treatment with fumarase. From a stereochemical perspective, this enzyme is unlike phenylpyruvate keto-enol tautomerase that is known to catalyze stereospecific proton transfer between solvent and the pro-R position of keto-substrate. As a result of an attempt to clarify the physiological importance of oxaloacetate tautomerase activity, keto-oxaloacetate was demonstrated to be directly transported across the inner membrane of rat liver mitochondria on the basis of the results of kinetic and isotope-trapping experiments.

Changes of fatty acid composition of phopholipids and lipid structural order in rat liver mitochondrial membrane subsequent to galactosamine intoxication. Effect of clofibrate

Since it has been earlier reported that d-galactosamine induces an inhibition of palmitoylcarnitine transferase I and a depletion of mitochondrial phospholipids which were both prevented by clofibrate, an evaluation of the effects of these drugs on mitochondrial fatty acid composition was made. Galactosamine does not alter the fatty acid pattern of these fatty acids whereas clofibrate induces a 2-fold increase in monounsaturated / saturated fatty acids ratio and a 10-fold decrease of the 20:4 ( n − 6)/20:3 ( n − 6) ratio in phosphatidylcholine. These alterations suggest an increase of Δ 9-desaturation and decrease of Δ 5-desaturation. To determine whether the drug-induced changes in mitochondrial phospholipids has an effect on the physical properties of the membrane, the lipid structural order of mitochondrial preparations was studied using the lipophilic probes DPH and TMA-DPH. Mitochondria isolated either from galactosamine- or clofibrate-treated rats showed a decrease in fluorescence polarization, indicating an overall decrease in lipid structural order. This alteration is more drastic when both drugs are administered. This phenomenon suggests drastic changes in the bulk phase of inner mitochondrial membrane lipids after treatments and could explain the altered kinetic properties of palmitoylcarnitine transferase I.

Consequences of defective vitamin A transportation on mitochondrial membrane integrity during protein depletion.

The relationships between the structural integrity and functionality of rat liver mitochondrial membranes, and different levels of dietary protein and vitamin A transportation during protein depletion in animals have been investigated. Although the vitamin A content of the protein-depleted diet was 1680 +/- 35 IU/kg diet, and that of the control diet was 1,650 +/- 30 IU/kg diet, the vitamin A content of the liver of depleted rats was reduced to 16.7% of controls. The hepatic mitochondria of rats fed a protein-depleted diet showed excessive passive swelling (about 3-fold of controls) in isotonic solutions. Whereas a seemingly inverse relationship existed between the vitamin A content of the liver and the osmotic behaviour of hepatic mitochondria of rats fed a protein-depleted diet, there is a direct relationship between their hepatic mitochondrial vitamin A and the respiratory control ratio. The implications of these observations are discussed.

Monoamine oxidase A and monoamine oxidase B activities are catalyzed by different proteins

Monoamine oxidases A and B (amino: oxygen oxidoreductase (deaminating) (flavin-containing), EC 1.4.3.4) have been identified in the outer membranes of rat liver mitochondria by their covalent reaction with the inhibitor, [ 3H]pargyline. On analysis by polyacrylamide gel electrophoresis under denaturing conditions. Monoamine oxidase A was found to migrate more slowly that monoamine oxidase B. Proteins which correspond to monoamine oxidases A and B (as identified by the electrophoretic distribution of covalently bound [ 3H]pargyline) were excised from the gels. Subsequent analysis showed that both monoamine oxidase A and monoamine B had been highly purified by this procedure. Electrophoretic analysis of the peptides produced by limited proteolysis with bovine trypsin, α-chymotrypsin, Staphylococcus aureus V8 proteinase and cyanogen bromide indicate that monoamine oxidases A and B have different amino acid sequences.

The phosphate potential maintained by mitochondria in State 4 is proportional to the proton-motive force

Evidence is presented for a proportional relationship between the extramitochondrial phosphate potential (Δ G ex p) and the proton-motive force (Δ \\ ̃ gm H + ) across the mitochondrial membrane in rat-liver mitochondria oxidising succinate in State 4, when Δ \\ ̃ gm H + is varied by addition of uncouplers or malonate. This relationship was found when precautions were taken to minimise interference with the determination of Δ G p ex and Δ \\ ̃ gm H + by intramitochondrial nucleotides, adenylate kinase activity, the quenching method, and Δ \\ ̃ gm H + -dependent changes in matrix volume. A non-proportional Δ G p ex/Δ \\ ̃ gm H + relationship was obtained when these precautions were omitted. Our results do not support mosaic protonic coupling, but are not necessarily in conflict with other localised coupling schemes.

The interaction of adriamycin with cardiolipin in model and rat liver mitochondrial membranes

The interaction of adriamycin with cardiolipin in model membranes and in various membrane preparations derived from rat liver mitochondria was studied and the results are analyzed in the light of a possible specific interaction between adriamycin and cardiolipin. It was found that adriamycin binds to cardiolipin-containing model membranes with a fixed stoichiometry of two drug molecules per cardiolipin. Furthermore, the extent of drug complexation by mitochondria and mitoplasts (inner membrane plus matrix) is in reasonable agreement with their cardiolipin content. In contrast, adriamycin-binding curves of inner membrane ghosts and submitochondrial particles reveal considerable association to an additional site, presumably RNA. The evidence for the potential importance of RNA as a target comes from experiments on outer membranes and microsomes which both appear to bind substantial amounts of adriamycin. Removal of the major part of the RNA associated with these fractions by EDTA treatment is accompanied by a dramatic reduction of binding capacity. We propose that endogenous RNA present in mitochondria and mitoplasts is not accessible for adriamycin at low concentrations of the drug due to the presence of an intact lipid barrier. This potential site comes to expression in ghosts and submitochondrial particles, due to the absence of an intact lipid bilayer and due to the inside-out orientation of the limiting membrane, respectively. Electron microscopical studies show that adriamycin induces dramatic changes in mitochondrial morphology, similar to the uncoupler-induced effects described by Knoll and Brdiezka (Biochim. Biophys. Acta 733, 102–110 (1983)). Adriamycin has an uncoupling effect on mitochondrial respiration and oxidative phosphorylation. The concentration dependence of this effect correlates with the adriamycin-binding curve for mitochondria which implies that only bound adriamycin actively inhibits respiration.

Discrimination between the binding sites of modulators of the H +-translocating ATPase activity in rat liver mitochondrial membranes

The properties of the components of the mitochondrial ATPase which interact with modulators of energy transduction have been examined. The chromatographic behavior and the size of the components which bind trialkyl tins, carbodiimides and uncouplers, have been shown to be different. However, they all appear to be proteolipids with apparent molecular weights around 10,000. On this basis it is proposed that these inhibitors act at different sites in the membrane sector of the ATP synthase of rat liver mitochondria.

Binding of citrate synthase and malate dehydrogenase to mitochondrial inner membranes: Tissue distribution and metabolite effects

Citrate synthase and malate dehydrogenase bind to mitochondrial membrane preparations obtained from various species of animals and lemon fruit. The amount of enzyme bound per mg mitochondrial protein was comparable in all tissues studied. The effect of various substrates, products, and substrate analogs on citrate synthase binding to rat liver mitochondrial inner membrane was examined. OAA was the most effective inhibitor of binding followed by AcCoA, CoA, citrate, ATP, and MgATP. Neither D- nor L- malate were effective in blocking binding. The wide distribution of binding of citrate synthase and malate dehydrogenase to the inner membrane and specificity of substrate effects on the binding of citrate synthase are discussed in relation to the possible physiologic nature of these phenomena.

Degradation of proteins in rat liver mitochondrial outer membrane transplanted into different cell types. Evidence for alternative processing.

The degradation of proteins in reductively [3H]methylated mitochondrial outer membrane (MOM) transplanted into cells by a poly(ethylene glycol)-mediated process has been studied. The average rate of degradation (t1/2 24-28 h) of MOM proteins transplanted into HTC cells was not the same as for endogenous MOM proteins (t1/2 56 h), mitoplast proteins (t1/2 120 h), plasma membrane proteins (t1/2 approx. 90 h) or cytosol proteins (t1/2 75 h). The degradation of transplanted MOM proteins was inhibited to the same extent (30-45%) as that of endogenous mitochondrial and plasma membrane proteins by leupeptin and NH4Cl. No inhibition of HTC cell cytosol protein degradation by NH4Cl was observed. NH4Cl differentially inhibited the degradation of endogenous MOM and mitoplast protein subunits as shown after sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Proteins in MOM transplanted into tissue culture cells were degraded either with t1/2 24-28 h (MRC-5, B82 and A549 cells) or with t1/2 55-70 h (CHO-K1 and 3T3-L1 cells) similar to that of proteins in MOM transplanted into rat hepatocytes [Evans & Mayer (1983) Biochem. J. 216, 151-161]. The data suggest that membrane protein destruction is but the end part of a fundamental intracellular membrane recognition process.