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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.

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.

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.

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.

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.

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.

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 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.

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.

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.

A method for resolving an overlapped polypeptide pattern of sodium dodecyl sulfate-polyacrylamide gel electrophoresis was described. The procedure was essentially a Gaussian fitting using the least squares method, and could resolve more than 20 overlapped components simultaneously. The applicability to overlapped and shouldered patterns was evaluated using practical electrophoretic data with varying amounts of mitochondrial samples. The relative contents of respective polypeptide components gave a good agreement regardless of the loaded amounts.

An N-ethylmaleimide-sensitive phosphate transport protein has been isolated from rat liver mitochondria, substantially purified, and reconstituted into phospholipid vesicles. Purified inner mitochondrial membrane vesicles depleted of F1-ATPase by urea treatment proved to be the most satisfactory starting material. Treatment of these membrane vesicles with Triton X-100 resulted in solubilization of the phosphate transport protein. Further purification was achieved using hydroxylapatite powder. Polyacrylamide gel electrophoresis of the purified fraction in sodium dodecyl sulfate indicated the presence of two Coomassie blue-staining bands with apparent Mr's of 30,000 and 35,000. Labeling of the 35,000 Mr band by the Pi transport inhibitor diazobenzene sulfonate was reduced markedly by prior treatment of the mitochondria with the inhibitor N-ethylmaleimide. The purified fraction containing both proteins could be reconstituted into liposomes prepared from purified asolectin. Phosphate efflux from these vesicles was inhibited by N-ethylmaleimide, by the impermeant mercurial agent, p-chloromercuribenzoate, and by diazobenzene sulfonate. Treatment of the purified fraction with N-ethylmaleimide prior to incorporation into liposomes resulted in a reconstituted system incapable of catalyzing Pi efflux. These studies summarize the first detailed attempt to purify the Pi/H+ transport system from rat liver mitochondria and emphasize the need to commence the purification with purified inner membrane vesicles depleted of F1-ATPase. In addition, these studies show that the final fraction contains a reconstitutively active transport system which when incorporated into phospholipid vesicles has its essential sulfhydryl groups oriented outward. Finally, it is shown that the purified fraction also contains a 30,000 Mr component.

We studied the metabolic variations in grafted livers at different times after transplant by measuring the hepatic energy and redox states. Five groups of rats were studied: control ungrafted Wistar (RT1y) rats (group 1), ungrafted Wistar rats with ligature of the hepatic artery (group 2), isografted Wistar rats (group 3), allografted Wistar rats with livers from ACI (RT1a) donors (group 4, long-term surviving rat strain combination), and allografted Wistar rats with livers from BN (RT1n) rats (group 5, rejector rats). The metabolism of grafted livers was studied for 7 days in groups 2 and 3, for 2 months in group 4, and at the time of rejection in group 5. Adenine nucleotide levels (ATP, ADP, AMP) were significantly impaired at 24 hr and at 48 hr from grafting in isografted and in allografted livers, and the reestablishment of normal values began at the 7th day from grafting. Cytoplasmic NAD+/NADH ratios were lowered at 24 hr from grafting in isografted and in allografted livers. Mitochondrial NAD+/NADH ratios were lowered at 24 hr in isografted livers and at 24 hr and 48 hr from grafting in allografted livers. The metabolic studies performed for 2 months revealed a significant correlation between well-maintained metabolic functions and transplant survival. On the contrary, an important energy loss was evidenced in livers of group 5, at the time of rejection.

Rat-liver mitochondrial DNA (mtDNA) contains 2 cleavage sites of the restriction endonuclease Xbal. The molecular sizes of restriction fragments are 6.6 X 10(6) and 3.7 X 10(6) D. The results of partial cleavage of mtDNA with EcoRI allow the fragment F (0.32 X 10(6) D) to be localized in the sequence ABCEGFHDA. The functional map of mtDNA is constructed for two genes of the ATP-ase mRNAs from rat-liver mitochondria. Molecular hybridization shows that the ATPase genes are located in fragment B and in the GEHD area of mtDNA EcoRI cleavage.

Sequence divergence of the mitochondrial leucytl-tRNA cistron in several eukaryotes has been examined by RNA-DNA hybridized. Rat mitochondria Leucyl-tRNA was hybridized with rat, mouse, guinea pig, monkey, chicken, and yeast mitochondrial DNAs (mtDNA) immobilized onfilters. Hybridization was carried out in 50% formamide (Tm -12degrees) or in 20% fromamide (Tm -21degrees). melting profiles of the hybrids were obtained for evaluation of the extent of base sequence micmatching. Under the more stringent hybridization conditions (50% formamide, Tm -12degrees), only mouse and quines pig mtDNAs hybridized with rat mitochondrial leucyl-tRNA. The Tm's of the heterohybrids were depressed by 2 and 9 degrees, respectively. Under less stringent hybridization conditions (Tm-21 degrees), monkey mtDNA also hybridized, and the Tm was depressed by about 15 degrees. Chicken and yeast mtDNAs did not form specific hybrids with rat mibochondrial leucyl-tRNA under these hybridization conditions. Mitochondrial leucyl-tRNA sequences in different eukaryotes appear to be conserved to a less extent than cytoplasmic rRNA, 5S RNA, or hemoglobin mRNA sequences.

The in vivo administration of [1-14C]pantothenic acid, which is the precursor of coenzyme A, resulted in the radioactive labelling of several mitochondrial proteins in rat liver. The incorporated radioactivity could be released by glutathione or 2-mercaptoethanol. Two mitochondrial matrix proteins acetyl-CoA acetyltransferase (liver and heart), an enzyme involved in the biosynthesis or degradation of ketone bodies, and 3-oxoacyl-CoA thiolase (liver), a protein participating in fatty acid oxidation were identified as modified proteins. The radioactivity was localized exclusively in forms A1 and A2 indicating that these forms represent the modified states of the acetyl-CoA acetyltransferase protein. Kinetics of incorporation of radioactivity revealed an accumulation of the modified forms. The ratio of specific radioactivities of A2 compared to A1 was 2.41 +/- 0.15 (n = 10). After in vivo labelling with [14C]leucine, the specific radioactivity of acetyl-CoA acetyltransferase depended on the state of the enzyme protein. The unmodified enzyme exhibited a lower specific radioactivity than its modified forms suggesting different turnover rates of these proteins.

A low citrullinogenesis (less than 60 per cent of the adult value) was observed throughout the suckling period when mitochondria isolated from newborn rat liver were incubated in vitro with L-glutamate or succinate as oxidizable substrates. The adult value was reached after weaning. From birth to weaning, intact mitochondria synthesized more citrulline when supplemented with L-glutamate than with succinate. The low citrullinogenesis could not be explained by low carbamoylphosphate synthetase-I and ornithine transcarbamoylase activities that reached adult values at birth. The decreased citrullinogenesis seen for the first three days of life seemed to be related to the low intramitochondrial concentration of N-acetylglutamate, an activator of the carbamoylphosphate synthetase-I. The concentration of this activator did not differ from that reported for adult rat liver mitochondria after the fourth day of life. The discrepancy between the normal value of N-acetylglutamate concentration and the low activity of the N-acetylglutamate synthetase (15 to 30 per cent of the adult activity) is discussed on the basis of acetyl-CoA or L-glutamate availability in mitochondria isolated from newborn or young rats.

Biotin clearance, its distribution in liver and liver fractions after intravenous administration of 5 muCi/100 g body weight (21.55 microgram) of biotin-14COOH in normal and biotin-deficient rats are reported. In the biotin deficient animal there is a more rapid disappearance of the labeled biotin from the blood stream. Biotin-14COOH incorporation in the liver of the deficient rat is more rapid and larger than the incorporation in normal rat liver. Almost all the biotin recovered from liver homogenate is found in the mitochondria and in the pH 5.2 cytosol fraction; whereas in the microsomes only a very small amount is present. The intracellular distribution of biotin is in agreement with its known metabolic roles.

The quantitative determination of protein by means of the biuret method frequently yields erroneous values, especially when applied to turbid, lipid- and hemoglobin-containing materials. These errors can only partially be abolished either by the addition of detergents or by destroying the Cu-protein complex by KCN addition. It was found that most disturbances were almost completely eliminated after prior precipitation of protein by the addition of Triton X-100 to the solubilizing biuret reagent and absorbance measurements being performed at a wavelength of 572 nm before and after the addition of KCN. Values of protein determinations according to the proposed assay and protein concentrations calculated from Kjeldahl nitrogen determinations have been shown to agree fairly satisfactory. The proposed assay represents a relatively simple and versatile approach for the evaluation of protein concentrations in a variety of materials containing lipids, hemoglobin and of some other turbidities as well.

The ratio of ATP content/ADP content in the mitochondrial matrix was found to be 2.07 +/- 0.21 and 2.26 +/- 0.22 as determined with six different preparations of isolated hepatocytes subfractionated with the digitonin and non-aqueous-fractionation procedures, respectively. In contrast, the mitochondrial matrix ATP/ADP determined with isolated haemoglobin-free perfused liver by using the non-aqueous-fractionation procedure was about 0.2, whereas the cytosolic values obtained with isolated cells and with the intact organ were similar. It is concluded that the relatively higher ATP/ADP ratio in the mitochondrial matrix of isolated hepatocytes represents a biochemical difference due to properties of the model rather than a methodological artifact.

Hepatocyte morphology of the three zones of the liver lobule was evaluated by stereological analysis in 3-month-old and 35-month-old virgin female WAG/Rij rats. In the old rats, the volume of the nucleus and cytoplasm, and volume densities of smooth endoplasmic reticulum and lysosomes were increased in all areas of the liver lobule, while the volume density of glycogen was decreased. In addition, mitochondrial and vacuolar densities decreased in midzonal hepatocytes. These results are discussed in comparison with other (conflicting) literature data, and the relevance of a more uniform choice of ages, breeding conditions, sex, strain and other secondary criteria is emphasized. The importance of a subdivision of the liver lobule into zones is stressed, as it allows the consideration of environmental factors in the interpretation of morphological changes with age.

Administration of alpha-p-chlorophenoxyisobutyrate (0.25% in the diet) to rats increased the liver weight, hepatic contents of ubiquinone and mitochondrial protein with no effect on the sterols. The increase was progressive with the period of drug treatment and was potentiated by simultaneous cold exposure. Withdrawal of the drug treatment as well as the cold stress resulted in a return of the liver weight and mitochondrial content to normal levels but this was not so for the ubiquinone content. Treatment with alpha-p-chlorophenoxyisobutyrate with or without cold exposure also resulted in a small but significant increase in the mitochondrial lipids which could be accounted for completely by an increase in the phospholipids with no change in the neutral lipid content. Analysis of the individual phospholipids showed that the drug treatment per se resulted in a specific increase in phosphatidylethanolamine content whereas simultaneous cold exposure or cold per se showed an increase in phosphatidylcholine. Cardiolipin content was unaffected. Mitochondria isolated from drug-treated animals maintained at an ambient or low environmental temperature showed a small but significant decrease in the respiratory control index for the oxidation of glutamate and malate whereas the coupled oxidation rates and ADP/O ratios were normal. Such a feature was also observed in the animals exposed to short periods of cold stress without the drug treatment. In all the cases the oxidation of succinate was unaffected. The role of accumulated phospholipids in the mitochondrial membranes in drug treatment and cold exposure is discussed in relation to the possible involvement in increased thermogenesis.

The thyroid hormone receptor of the inner membrane of rat liver mitochondria was purified by osmotic and freeze-thaw lyses followed by partial purification on Sephadex G-200, and then by affinity chromatography with T3-Sepharose 4B. A single predominant protein band demonstrable on sodium dodecylsulphate (SDS) polyacrylamide gel electrophoresis was present in the first 4 mM NaOH elution peak of affinity chromatography. This was collected from affinity peaks from about 30 rat livers followed by preparative polyacrylamide gel electrophoresis. A single absorbance peak was observed by high pressure liquid chromatography (HPLC). The purified protein was analyzed for binding constants, amino acid composition, and characterized by analytical ultracentrifugation. The association constant (KA) exceeded 10(11) M-1. The sedimentation coefficient (S20,W) was 2.2S, partial specific volume (v) 0.72, frictional coefficient (f/fo)s M 1.68 and the molecular weight was estimated at 28 000. The amino acid composition was obtained.

In order to recognize the significance of elevated liver calcium level in the early phase of carbon tetrachloride intoxication, the subcellular distribution of calcium in liver was investigated one hour after administration of CCl4 intraperitoneally. During preparation of subcellular fractions attempts were made to prevent redistribution of calcium by adding Ruthenium Red and EGTA or LaCl3 to homogenization medium, and by shortening of differential centrifugation to a minimum. The latter caused a loss of purity in sub cellular fractions which was overcome by correction of calcium values from atom absorption spectrometry by means of marker enzyme activity. The calcium levels in normal rat liver were found to be 70 nmoles/g liver wet wt in cytosol, 310 nmoles/g liver in microsomes and about 500 nmoles/g liver in mitochondria. A minor part of the latter fraction may belong to nuclei and plasma membranes. One hour after CCl4 administration calcium levels in cytoplasma were not altered, in microsomes were decreased to 200 nmoles/g liver and in mitochondria were elevated to 2.5 mumoles/g liver. In rats pretreated with vitamin D the whole additional calcium, after carbon tetrachloride application in the range of 9 mumoles/g liver, was sequestrated in mitochondria. In the early phase of carbon tetrachloride intoxication all border membranes of liver cells have to participate in bringing about the reversible increase of liver cell calcium.

The presence and localization of lectin receptor sites on rat liver cell nuclear and other endomembranes was studied by light and electron microscopy using fluorescein and ferritin-coupled lectin conjugates. Isolated nuclei labelled with fluorescein-conjugated Concanavalin A (Con A) or wheat germ agglutinin (WGA) often showed membrane staining, which sometimes was especially bright on small stretches of the nuclear surface. Unlabelled nuclei and nuclei with a complete ring fluorescence were also seen. The nuclear fluorescence corresponded in intensity to that seen on the surface of isolated rat liver cells. Con A-ferritin particles were seldom detected on the cytoplasmic surface of the intact nuclear envelope. However, at places where the 2 leaflets of the envelope were widely separated or where the outer nuclear membrane was partly torn away, heavy labelling was seen on the cisternal surface of both the inner and outer nuclear membranes. Labelling with Con A-ferritin was also found on the cisternal side of rough endoplasmic reticulum present in the specimens. No labelling was seen on the cytoplasmic surface of mitochondrial outer membrane. The results demonstrate the presence of binding sites for Con A and WGA in nuclei and an asymmetric localization of these sites on the cisternal side of ribosome-carrying endomembranes in rat liver cells.

The presence or absence of autoantibodies in acute hepatitis B was investigated longitudinally in a prospective study of 38 patients, 37 of whom recovered completely. Antibodies to nuclei, bile canaliculi or mitochondria were not found in any of the 354 investigated sera. Smooth muscle antibody (SMA) was present in 23 patients for median 4 weeks and from (median) -1 to +3 weeks from peak SGPT. Titers reached from 1:10 to 1:400, with a median of 1:50. In the patient with persistent HBs-antigenemia, SMA - present in low titer (1:10) - persisted as well. Besides smooth muscle cells, other localisations were: glomerular (15 patients), around hepatocytes ('polygonal' 11 pts), around renal tubuli (10) and in the gastric mucosal layer (8). These fluorescence patterns, the presence of which was not correlated to the SMA titer height, disappeared either earlier than or simultaneously with smooth muscle cellular fluorescence. A maximal titer greater than 1:50 was associated with a thymol turbidity ten times the upper limit of normal or more (i.e. greater than 25 S-H U) and with the peak serumgammaglobulin about simultaneously with peak SGPT. The presence of SMA was not correlated with extrahepatic manifestations nor with the peak values attained for ESR, SGPT, alkaline phosphatase or bilirubin.

Concentration of cytochroms in the liver of adult male rats has been studied during the acclimation to cold. One group of rats was kept at about 24 degrees, another at 2--4 degrees. After a month of acclimation to cold the cytochroms concentration in the liver increased, whereas the mitochondrial concentration of cytochroms remained unchanged. It is possible to distinguish three steps of cytochroms concentration changes; 1. on the 9th day of acclimation to cold cytochroms concentration increased 1.5--2 times; 2. by the 14th day the cytochroms concentration decreased (to the control level and lower); 3. after about one month of acclimation to cold the cytochroms concentration increased again. In the skeletal muscle it increased on the 14th (but not on the 9th) day.

A sensitive and precise method for measuring endogenous phylloquinone (K1) and menaquinone (MK-n) in human liver was developed, based on gradient elution high-performance liquid chromatography using platinum-black catalyst reduction and fluorimetric detection. Subnanogram levels of vitamin K compounds in 1 g of liver specimen were detectable. We measured vitamin K concentrations in 38 human resected livers. K1 and MK-4 to MK-13 were detected. The concentrations of MK-10 to MK-12 in livers with chronic hepatitis (n = 10) and cirrhosis (n = 22) were significantly lower than in normal livers (n = 6). It is suggested that the decreased concentrations indicate functional damage of the hepatocytes.

Sperm mtDNA exhibits unique physicochemical properties compared with mtDNA from somatic tissues in the ram. The buoyant density of sperm mtDNA was 1,6983 g/cm3 while the brain, heart and liver mtDNAs was about 1.7005 g/cm3. The Tm of the liver and sperm mtDNA was 71.0 degrees C and 69.5 degrees C, respectively. The G + C content of sperm mtDNA was 3.0 moles % lower than the liver mtDNA. The contour length of the circular sperm mtDNA was 5.01 micrometers compared with the liver mtDNA with contour length of 5.33 micrometers.

Various aspects of membrane solubilization by the Triton X-series of nonionic detergents were examined in pig liver mitochondrial membranes. Binding of Triton X-100 to nonsolubilized membranes was saturable with increased concentrations of the detergent. Maximum binding occurred at concentrations exceeding 0.5% Triton X-100 (w/v). Solubilization of both protein and phospholipid increased with increasing Triton X-100 to a plateau which was dependent on the initial membrane protein concentration used. At low detergent concentrations (less than 0.087% Triton X-100, w/v), proteins were preferentially solubilized over phospholipids. At higher Triton X-100 concentrations the opposite was true. Using the well-defined Triton X-series of detergents, the optimal hydrophile-lipophile balance number (HLB) for solubilization of phosphatidylglycerophosphate synthase (EC 2.7.8.5) was 13.5, corresponding to Triton X-100. Activity was solubilized optimally at detergent concentrations between 0.1 and 0.2% (w/v). The optimal protein-to-detergent ratio for solubilization was 3 mg protein/mg Triton X-100. Solubilization of phosphatidylglycerophosphate synthase was generally better at low ionic strength, though total protein solubilization increased at high ionic strength. Solubilization was also dependent on pH. Significantly higher protein solubilization was observed at high pH (i.e., 8.5), as was phosphatidylglycerophosphate synthase solubilization. The manipulation of these variables in improving the recovery and specificity of membrane protein solubilization by detergents was examined.

Propyl alcohol and butyl alcohol had similar effects to ethyl alcohol on ultrastructure of liver mitochondria. Rats were given 32% ethyl alcohol, 32% n-propyl alcohol, and 6.9% n-butyl alcohol in drinking water for up to three months. After one month, mitochondria in hepatocytes obtained from the experimental animals became elongated, constricted or cup-shaped with scanty cristae. After two months, mitochondria in some hepatocytes became gigantic. In extreme cases, the megamitochondria exceeded 10 micron in diameter. Coupling efficiencies of hepatic mitochondria obtained from alcohol-fed animals were well preserved despite their drastic morphologic changes.

The respiratory parameters and the membrane potential of liver mitochondria from rats treated with either hexachlorobenzene, iron or hexachlorobenzene plus iron, to induce experimental porphyria, have been studied. Partial uncoupling of oxidative phosphorylation has been observed in mitochondria from hexachlorobenzene- and hexachlorobenzene plus iron-treated rats. Direct evidence has been presented that this uncoupling is due to the action of pentachlorophenol endogenously formed by metabolism of hexachlorobenzene. No irreversible damage of mitochondria membrane has been revealed under both these conditions. Normal oxidative phosphorylation has been found in mitochondria from rats treated with iron alone. In contrast, they presented an anomalous membrane potential, fully restored by oligomycin. A possible involvement of lipid peroxidation process, induced by iron, in causing these abnormalities has been suggested.

The comparison of fluorescein polarization spectra in living cells and in isolated subcellular structures identified the mitochondria as the cytoplasmic domain in which on excitation at 470 nm the sharp fluorescein emission polarization peak at 510 nm is formed. Changes in the emission polarization peak during the cell cycle or those induced by growth stimulators and inhibitors reflect structural changes in the mitochondria on their transition from the resting, orthodox into the active, ATP-generating, condensed conformation and vice versa. Possible mechanisms for the formation of the sharp emission polarization peak are discussed.

A commonly used model for the interaction of the motional narrowing of ESR lines is shown to be qualitatively misleading. An analysis of lipid extracts of mitochondrial preparations labeled with 12-nitroxide stearic acid produced linear plots of the logarithm of the correlation time versus the reciprocal of the absolute temperature when analyzed with stochastic computer simulations. However, when the data were analyzed with isotropic Lorentzian line shape approximations, nonlinear plots were obtained.

Quantitative estimation of free fatty acids was carried out in liver mitochondria of normal and thyroidectomized rabbits by means of two procedures--spectrophotometry using rhodamine 6Zh and a fluorescent method with 1,8-aniline naphthalene sulfonate /ANS/ as a dye. The use of ANS enabled to estimate quantitatively the free fatty acids beginning from 25 nmol in a sample /with eggor of about 17%/ by visual evaluation of the spot areas. As compared with the rhodamine procedure, use of ANS permitted to increase 5-fold the sensitivity of free fatty acid estimation, to elevate 6-fold the accuracy of the analysis, to overcome the step of fatty acid elution from chromatogramms and to decrease the time required for analysis. The data obtained suggest that the hyperthyroid state in rabbits was accompanied by an increase in the phospholipase A2 activity in liver mitochondria.

The rate at which isolated rat liver mitochondria synthesized citrulline with NH4C1 as nitrogen source was markedly dependent on the protein content of the diet. 2. Citrulline synthesis was not rate-limited by substrate concentration, substrate transport or ornithine transcarbamoylase activity under the conditions used. 3. The intramitochondrial content of an activator of carbamoyl phosphate synthase, assumed to be N-acetyl-glutamate, varied markedly with dietary protein content. The variation in the concentration of this activator was sufficient to account for the observed variation in the rates of citrulline synthesis if this synthesis were rate-limited by the activity of carbamoyl phosphate synthase. 4. The rates of urea formation from NH4Cl as nitrogen source in isolated liver cells showed variations in response to diet that closely paralleled the variations in the rates of citrulline synthesis observed in isolated mitochondria. 5. These results are consistent with the postulate that when NH4Cl plus ornithine are present in an excess, the rate of urea synthesis is regulated at the level of carbamoyl phosphate synthase activity.