Studies on peroxisomal membranes.

The phospholipid/protein ratios of rat liver peroxisomes, mitochondria and microsomes were determined and found to be 257 +/- 26, 232 +/- 20 and 575 +/- 20 nmol.mg-1, respectively. After correction for the loss of soluble protein, a peroxisomal ratio of 153 nmol.mg-1 was calculated. Organelle fractions were treated with sodium carbonate, whereafter membrane fragments containing integral membrane proteins were pelleted. For the membrane fractions of peroxisomes, mitochondria and microsomes phospholipid/protein ratios of 1054 +/- 103, 1180 +/- 90 and 1050 +/- 50 nmol.mg-1 were found, whereas 26 +/- 2, 20 +/- 2 and 49 +/- 2% of the organelle protein was recovered in these membrane fractions, respectively. The phospholipid composition of the different organelle fractions were determined, but no large differences were obtained, except for cardiolipin that was found only in the mitochondrial fraction. After sodium carbonate treatment virtually all enzymatic activity of the enzymes tested was lost. Therefore Triton X-114 phase separation was used to obtain the peroxisomal membrane components. In this fraction 42.9 +/- 3.5% of the protein and 90.2 +/- 3.7% of the phospholipid was found. Enzymatic activity of two integral membrane proteins was recovered for over 90% in the membrane fraction, whereas activity of two matrix proteins was mainly found in the soluble fraction. Urate oxidase, the peroxisomal core protein, behaved differently and was recovered mainly with the membrane components. Recoveries of enzymatic activities after the Triton X-114 phase separation varied from 45 to 116%, and together with the good separation that was obtained between soluble proteins and integral membrane proteins this method provides a useful alternative for the isolation of membrane components.

Immunoquantitative analysis of human carnitine palmitoyltransferase I and II defects.

Carnitine palmitoyltransferase deficiency realizes two distinct clinical forms. We previously showed and confirmed in the present work that CPTII (identified as the carnitine palmitoyltransferase activity assayable in detergent conditions) is decreased in the muscular form whereas it is unaffected and CPTI is decreased in the hepatic form. The antibody previously prepared against human liver mitochondrial CPTII recognizes the same enzyme in muscle, liver, and fibroblasts. Immunoprecipitation experiments were performed in fibroblasts from patients with the muscular and hepatic forms of the defect. As compared with controls, cell lines from two patients with the hepatic form of the defect did not exhibit any qualitative nor quantitative abnormality of cross-reacting material, whereas cell lines from two patients with the muscular form of the defect exhibited a decreased amount of cross-reacting material. These data suggest that CPTII deficiency could result from a decreased production of protein. The amount of cross-reacting material in the two sets of patients only correlates with CPTII activity, which is decreased in the muscular presentation and unaffected in the hepatic form. These results strengthen the hypothesis of distinct proteins supporting CPTI and CPTII activities.

Cholesterol distribution in rat liver and brain mitochondria as determined by stopped-flow kinetics with filipin.

Recently, analysis of protein distribution in rat brain mitochondria suggested the existence of distinct cholesterol domains in the outer membrane (Dorbani et al., 1987, Arch. Biochem. Biophys. 252, 188-196) while such domains were not detected in rat liver mitochondria (Jancsik et al., 1988, Arch. Biochem. Biophys. 264, 295-301). We studied cholesterol distribution in both types of mitochondria by analyzing the kinetics of filipin-cholesterol complex formation, using the stopped-flow technique. In liver mitochondria, the kinetics are characterized by a biphasic curve which presumably corresponds to the two membranes. This was confirmed by the finding that pretreatment with digitonin abolished one of the kinetic components. Sonication of the mitochondria increased the rate of the filipin-cholesterol complex formation and also abolished one of the two components. In the case of brain mitochondria, several distinct cholesterol domains could be revealed: one of them was cholesterol-free and it was directly accessible to filipin. Two other domains were revealed by differences found in the rate of the cholesterol-filipin complex formation. It is noteworthy that only a part of the cholesterol is accessible to filipin. Sonication of mitochondria decreased the proportion of cholesterol molecules accessible to filipin. This suggests specific interactions of cholesterol with other mitochondrial components, which occur only in brain mitochondria.

Isolation of rat liver lysosomes by a single two-phase partition on dextran/polyethylene glycol.

Crude mitochondria from liver rats were added to a two-phase system containing dextran and polyethylene glycol. The polymer and ionic concentration values of the two-phase system were changed in order to separate lysosomes from mitochondria. The best separation of lysosomes and mitochondria was obtained at 6.6-6.6% (w/w) dextran-polyethylene glycol and 5 mmol/kg ammonium chloride as shown by enzyme assays. This procedure showed good reproducibility, and lysosomes were never contaminated with more than 16% mitochondria, as determined by succinate dehydrogenase activity, and beta-D-galactosidase and acid phosphatase activities were enriched five- to sixfold. The lipid composition profile of lysosomes was quite similar to that obtained by means of free carrier electrophoresis, considered a reference method.

[Tocopherol-binding proteins in membranes of rat liver mitochondria]. / Issledovanie tokoferolsviazyvaiushchikh belkov membran mitokhondrii pecheni krys.

The use of the adsorption chromatography on the hydroxyl apatite makes it possible to yield and partly purify the triton X-100-solubilized mitochondrial protein fraction of the rat liver able to bind specifically [3H]-alpha-tocopherol. The method permits removing simultaneously both free detergent and [3H]-alpha-tocopherol from the protein mixture without disturbance of the established equilibrium. When compared with methods used for the removal of free hydrophobic ligands in the in vitro binding experiments, the applied method is the most effective.

The monocarboxylate carrier from rat liver mitochondria. Purification and kinetic characterization in a reconstituted system.

The monocarboxylate (pyruvate) carrier was extracted from rat liver mitochondria with Triton X-100 in the presence of asolectin and partially purified by chromatography on HTP. The HTP eluate reconstituted in liposomes was shown to catalyze active pyruvatein/acetoacetateout and acetoacetatein/pyruvateout counter-exchange. Kinetic characterization of the reconstituted pyruvate carrier was achieved by an original spectrophotometric method consisting of determination of substrate release from proteoliposomes with a coupled enzymatic assay.

Two-dimensional gel electrophoretic resolution of the polypeptides of rat liver mitochondria and the outer membrane.

The proteins of highly purified rat liver mitochondria were resolved by two-dimensional polyacrylamide gel electrophoresis, and detected by staining with either Coomassie blue or silver. Approximately 250 polypeptides were detected with silver staining which is 2- to 3-times that observed with Coomassie blue. Silver staining was especially more effective than Coomassie blue for detecting polypeptides of less than 50 000 daltons. A two-dimensional gel pattern of rat liver microsomes was distinct from that of the mitochondria. The mitochondrial outer membrane was prepared from purified mitochondria either with digitonin or by swelling in a hypotonic medium. As assessed by marker enzymes, the latter method yielded a considerably purer outer membrane preparation (20-fold purification) than the former (2.6-fold purification). Approximately 50 polypeptides were observed in a two-dimensional gel (pH 3-10) of the highly purified outer membrane fraction. Three isoelectric forms of the pore (VDAC) protein were observed with pI values of 8.2, 7.8 and 7.1. Monoamine oxidase was identified as a polypeptide of Mr 60 000. About 50 polypeptides were also resolved in a reverse polarity non-equilibrium pH gradient electrophoresis gel of the outer membrane, pH 3-10, with at least six isoelectric forms of the VDAC protein observed under these conditions. The six isoforms of the VDAC protein were also observed in a non-equilibrium gel with 2 micrograms of the purified protein.

[The temperature dependence of the respiration and phospholipid content of the mitochondria in the organs of heat-adapted rats]. / Temperaturnaia zavisimost' dykhaniia i fosfolipidnyi sostav mitokhondrii organov u adaptirovannykh k teplu krys.

A shift of breaking point to higher temperatures was illustrated on Arrhenius plots in kidney, liver and heart mitochondria of heat-adapted rats on 4.1, 2.7, 2.4 degrees C respectively. Q10 mitochondrion respiration fall in adapted animals in a range of 26-36 degrees C was indicative of temperature compensation. It was shown the elevation of phospholipid plasmalogen form content in heart and kidney mitochondria; as well as saturated fatty acids content in liver mitochondria. Structure and function changes were suggested to be the basic point of elevation of mitochondrion functional ability at high ambient temperature.

Two subcellular locations for peripheral-type benzodiazepine acceptors in rat liver.

Subcellular fractionation of rat liver by differential centrifugation showed the mitochondrial fractions to have the greatest enrichment of 'peripheral-type' benzodiazepine acceptor. Two peaks of acceptor sites were found on isopycnic density-gradient centrifugation, one peak (rho = 1.19 g/ml) corresponding to the peak of mitochondria as judged by marker enzyme distribution and by transmission electron microscopy, and the other peak (rho = 1.17 g/ml) which is not mitochondrial as judged by the lack of mitochondrial enzyme markers. Whereas the density of the mitochondrial acceptor was sensitive to sonication and was shown to have an outer-membrane location, the density of the non-mitochondrial acceptor was insensitive to sonication. The non-mitochondrial acceptor was shown not to be associated with Golgi, lysosomes, rough endoplasmic reticular microsomes, peroxisomes, or some types of plasma membranes, as judged by differences in the distribution of marker activities. No enrichment of benzodiazepine acceptor was found in the purified nuclear fraction. Both acceptors were shown to be peripheral-type high-affinity acceptors as judged by ligand specificities and by photoaffinity labelling.

Purification and immunohistochemical study of actin in mitochondrial matrix.

Actin was purified to apparent homogeneity from the matrix of ultra-pure mitochondria of rat livers by DNase-I affinity chromatography and HPLC gel filtration. The mitochondrial actin was immunologically identified by an anti-actin antibody, and its apparent molecular weight was 43 KDa, as determined by SDS-polyacrylamide gel electrophoresis. The immunohistochemical study revealed the localization of the mitochondrial actin in the matrix space and on the internal surface of inner membrane. The actin fraction eluted from a DNase-I column by KCl-EGTA solution underwent polymerization and bundling in vitro.

Identification and purification of the tricarboxylate carrier from rat liver mitochondria.

The tricarboxylate carrier from rat liver mitochondria was solubilized with Triton X-100 and purified by chromatography on hydroxyapatite and celite. SDS-gel electrophoresis of the purified fraction showed a single polypeptide band with an apparent Mr of 30,000. When reconstituted into liposomes, the tricarboxylate transport protein catalyzed a 1,2,3-benzenetricarboxylate-sensitive citrate/citrate exchange. We obtained a 1070-fold purification with respect to the mitochondrial extract, the recovery was 22% and the protein yield 0.02%. The properties of the reconstituted carrier, i.e., requirement for a counteranion, substrate specificity and inhibitor sensitivity, were similar to those of the tricarboxylate transport system as characterized in intact mitochondria.

Tianeptine, a new tricyclic antidepressant metabolized by beta-oxidation of its heptanoic side chain, inhibits the mitochondrial oxidation of medium and short chain fatty acids in mice.

Tianeptine is a new tricyclic antidepressant which is metabolized mainly by beta-oxidation of its heptanoic side chain. We determined the effects of tianeptine on the mitochondrial oxidation of natural fatty acids in mice. In vitro, tianeptine (0.5 mM) inhibited by only 32% the formation of beta-oxidation products from [1-14C]palmitic acid by hepatic mitochondria, but inhibited by 71% that from [1-14C]octanoic acid and by 51% that from [1-14C]butyric acid. The activity of the tricarboxylic acid cycle, assessed as the in vitro formation of [14C]CO2 from [1-14C]acetylcoenzyme A was decreased by 51% in the presence of tianeptine (0.5 mM). The inhibition of both beta-oxidation and the tricarboxylic acid cycle appeared reversible in mitochondria from mice exposed to tianeptine in vivo but incubated in vitro without tianeptine. In vivo, administration of tianeptine (0.0625 mmol/kg i.p.), decreased by 53 and 58%, respectively, the formation of [14C]CO2 from [1-14C]octanoic acid and [1-14C]butyric acid, but did not significantly decrease that from [1-14C]palmitic acid. After administration of high doses of tianeptine, however, formation of [14C]CO2 from [1-14C]palmitic acid became inhibited as well, transiently after 0.25 mmol/kg and durably (greater than 24 hr) after 0.75 mmol/kg i.p. Hepatic triglycerides were increased 24 hr after administration of 0.75 mmol/kg i.p. of tianeptine, but not after 0.25 mmol/kg i.p. Microvesicular steatosis of the liver was observed in some mice after 0.75 mmol/kg i.p., but not after 0.5 mmol/kg i.p. We conclude that tianeptine inhibits the oxidation of medium- and short-chain fatty acids in mice. Microvesicular steatosis, however, requires very large doses in mice (0.75 mmol/kg i.p., i.e. 600-times the oral dose in humans), and is therefore unlikely to occur in humans.

[Isolation of intact mitochondria from the rat liver using vibration]. / Vydelenie intaktnykh mitokhondrii iz pecheni krys s ispol'zovaniem vibratsii.

A method for isolating mitochondria from the rat liver is described. In this method the homogenization step is replaced by vibration with the frequency of 50 Hz realized by a simple device. Mitochondria isolated by vibration demonstrate higher indices of the oxidative phosphorylation with succinate and glutamate + malate used as substrates than those isolated by homogenization do. The method described permits decreasing considerably the isolation medium expenditure remaining the mitochondria yield per gram of the liver unchanged.

Intracellular distribution of copper in the liver of copper-loaded sheep--a subcellular fractionation study.

Eighteen ewes in two groups were dosed orally with CuSO4 to induce chronic Cu toxicity. Copper dosing was stopped at the first rise of acid AP activity in the serum in group 1 sheep and on the first day of haemolysis in group 2 sheep. Liver samples were obtained 1 week prior to the start of Cu dosing, at the first rise of acid phosphatase (AP) activity in serum and on the first day of haemolysis. These liver samples were homogenized and were separated into nuclear (N), heavy mitochondrial (MH), light mitochondrial (ML), microsomal (MI) and cytosolic (CY) fractions by centrifugation. The Cu concentration and specific activities of AP were determined in the liver, LH and subcellular fractions. The composition of the fractions was studied by light and electron microscopy. In the predosing biopsies, the concentration and percentage of Cu and the total specific activity of AP were highest in the ML fractions. With increasing Cu loading, the concentration of Cu in all fractions increased; the percentage of Cu increased in the N and MH fractions, decreased in the ML and MI fractions and was maintained at a constant level in the CY fractions. The total specific activities of AP in LH, N, MH, MI and CY fractions were increased and the activity was highest in the MH fraction. The results indicate that the increase in the concentration of Cu in liver cells was predominantly in lysosomes and cytosol. Furthermore, it is suggested that the necrosis of isolated hepatocytes observed in chronic Cu-poisoned sheep may be due to a saturation of the uptake of Cu into the lysosomal system of the cell, leading to the accumulation of toxic levels of Cu in the cytosol.

Intravenously administered tetra-thiomolybdate and the removal of copper from the liver of copper-loaded sheep.

Eighteen ewes divided into two groups were dosed orally with CuSO4 in order to induce chronic Cu toxicity. Copper dosing was stopped at the first rise of serum acid phosphatase activity in sheep of group 1 and on the first day of haemolysis in sheep of group 2. Tetra-thiomolybdate was administered intravenously to five group 1 sheep (group 1B) and to group 2 from the cessation of Cu dosing. Following thiomolybdate administration, in groups 1B and 2, there was a reduction in the concentration of Cu in the liver and liver fractions, the number and size of electron-dense lysosomes in particulate liver fractions, the volume density and the mean volume of electron-dense lysosomes in hepatocytes and the number of necrotic cells in the liver. Thiomolybdate appeared to remove Cu from the lysosomes and the cytosol of Cu-loaded liver cells. However, neither the total specific activity of acid phosphatase in liver homogenate and liver fractions nor the numerical density of electron-dense lysosomes in hepatocytes decreased significantly. This may be due to the production of new lysosomes in the liver cells. Furthermore, following thiomolybdate administration, Mo concentration in the liver and liver fractions increased indicating that Mo of thiomolybdate was entering liver cells. The percentage distribution of Cu and Mo in the liver fractions was similar. This may suggest that Mo is bound to Cu and that they remain together with each fraction. The decrease in Cu concentration may indicate that the liver retains its ability to excrete copper via bile.

Purification of the mitochondrial carnitine carrier by chromatography on hydroxyapatite and celite.

The carnitine carrier from rat liver mitochondria has been extracted with Triton X-100 ad partially purified by chromatography on hydroxyapatite and celite. During purification the activity of the carrier was monitored by functional reconstitution into liposomes. The purified fraction is 250-fold enriched with respect to the N-ethylmaleimide-sensitive carnitine/carnitine transport activity. The substrate specificity and the inhibitor sensitivity of carnitine transport in liposomes resemble closely those described for the transport of carnitine in mitochondria.