Mouse Liver Mitochondria Research Articles

Acyl group specificity of mitochondrial pools of carnitine acyltransferases

1. 1. Two different pools of carnitine acyltransferases are present in the inner mitochondrial membrane. 2. 2. A new method for the selective assay of the “inner” and the “outer” pools of carnitine acyltransferases has been developed. The acyltransferases are assayed by an isotope exchange method in intact mitochondria in the absence and the presence of external CoASH. Any endogenous external CoASH is removed by oxidation with the disulfide tetrathionate which does not penetrate the inner mitochondrial membrane. 3. 3. This method has been used for the study of the acyl group specificity patterns of the two mitochondrial pools of carnitine acyltransferases in rat, mouse and calf liver. 4. 4. In all three species the greatest activity in the “inner” transferase pool was found with medium-chain acylcarnitines (optimal substrate: C 7). 5. 5. In rat and mouse liver mitochondria the “outer” transferase pool had a specificity pattern different from that of the “inner” pool. These mitochondria had little or no activity with short-chain substrates in the “outer” pool. The optimum medium-chain substrates were C 9 or C 10. 6. 6. The specificity pattern of the “outer” transferase pool was verified by extracting the “outer” transferase by digitonin. 7. 7. In calf liver mitochondria the specificity pattern of the “outer” transferase pool resembled closely that of the “inner” pool. 8. 8. Clofibrate feeding caused increased activities only in the “inner” transferase pool of rat liver mitochondria. The short-chain activity increased 13 times, whereas the medium- and long-chain activities increased only moderately.

Mode of action of garlic oil—Effect on oxidative phosphorylation in hepatic mitochondria of mice

The effect of garlic oil on oxidativc phosphorylation in liver mitochondria of mice has been studied. Garlic oil impairs oxidative phosphorylation in hepatic mitochondria. The coupling activity of sites 1. 2 and 3 appears to be affected by garlic oil. The extent to which respiration is affected varies with the substrates used. For example, garlic oil affects respiration during oxidation of NAD-lmkcd substrates such as glutamate to a greater extent than during oxidation of succinate and ascorbate. The inhibitory effect on respiration is less than that on phosphorylation. Diallyl disulfide, the principal insecticidal component of garlic oil, has a similar inhibitory effect on the mitochondrial oxidative phosphorylation. In contrast, dipropyl disulfide. which is chemically comparable to diallyl disulfide but is devoid of insecticidal activity, has a substantially lesser effect on oxidative phosphorylation.

An x-ray microanalytical study of the ferricyanide reaction for the electron cytochemical demonstration of succinate dehydrogenase activity in isolated mitochondria.

The cytochemical technique for the demonstration of succinate dehydrogenase activity by ferricyanide reduction and simultaneous coupling with Cu2+ has been investigated by the combined techniques of transmission electron microscopy and X-ray microanalysis. The localization of the final reaction product of succinate dehydrogenase activity within isolated mouse liver mitochondria is described, and variations in the composition of the final reaction product are shown to occur, both at various sites within the mitochondrion, as well as after different reaction times.

Action of some insecticides on membranes of mouse liver mitochondria.

Action of some insecticides on membranes of mouse liver mitochondria.

Influence of some insecticides on the ATPase of mouse liver mitochondria.

Effects of insecticides upon both target and non-target organisms often reflect interaction with cellular membranes (Matsumura and O'Brien, 1966). For instance, in the case of vertebrates, these effects include inhibition of plasma membrane ATPase (adenosine triphosphatase) (Davis et al. 1972), increased metabolic transformations by microsomal membranes (Remmer et al. 1967), and disruption of osmotic equilibrium (Janicki and Kinter 1971). Since mitochondrial energy coupling is localized in the inner mitochondrial membrane and since coupling is highly sensitive to assault on the integrity of that membrane, we have undertaken to examine the action of insecticides on processes associated with mitochondrial energy conservation. The present communication presents data indicating that low concentrations of several chemically-unrelated insecticides stimulate mouse mitochondrial ATPase in a manner similar to that of uncouplers of oxidative ATP synthesis.

Phospholipid and protein metabolism in mouse liver mitochondria during riboflavin deficiency and recovery.

A study was made of the effect of riboflavin deficiency and recovery upon the structure and function of mitochondrial membranes in mouse liver. In the deficient animals the mitochondria were enlarged and contained reduced numbers of inner membrane profiles. The activity of enzymes of the inner membranes (succinate cytochrome c reductase, cytochrome oxidase) was reduced in the deficient mitochondria, as was the content of diphosphatidylglycerol, an inner membrane phospholipid; monoamine oxidase, a mitochondrial outer membrane enzyme, and the coupling reactions of oxidative phosphorylation did not appear to be affected. Incorporation in vivo of 32Pi into phospholipids and of 3H-leucine into proteins of deficient mitochondria was normal or somewhat enhanced. Following the administration of riboflavin to the deficient mice, liver mitochondria appeared to undergo division, and inner membrane profiles seemed more numerous. Riboflavin administration increased the 3H labelling of tightly bound mitochondrial proteins, and decreased the 32P labelling of the phosphatidylcholine fraction.

Quantitative Ultrastructural Study of Mitochondrial Form After Partial Hepatectomy

Induced swelling of isolated residual rat and mouse liver mitochondria following partial removal has been shown to be diminished. This observation can be explained by two possible mechanisms; (a) the conformation of organelles in residual liver is altered and already swollen prior to isolation or, (b) the isolated mitochondria are rendered unreactive to certain swelling inducing systems because of specific changes that have occurred in the membrane.In order to evaluate the former of these proposed mechanisms, quantitative morphometric analysis of the heterogeneity of mitochondria has been attempted in situ by ultrastructural techniques after the manner of Bahr.

Antagonistic effect of two forms of clofibrate on mitochondrial oxidation of succinate

Clofibrate can both inhibit and stimulate the oxidation of succinate by mouse liver mitochondria, depending on whether the salt or the esterified form of the drug is used. Sodium clofibrate is a strong inhibitor whereas clofibrate ethyl ester is an active stimulator of succinate oxidation by mouse liver mitochondria.

Release of respiratory control by uncouplers: The question of stoichiometry

An alternative kinetic model of uncoupler action is envisaged in which the observed stoichiometry of uncoupler and titratable factor do not reflect chemical binding reactions. This model assumes (a) that highly effective uncouplers dissolve in the membrane, whereas less effective uncouplers remain largely in aqueous solution; (b) that a high affinity exists between respiratory chains and “low energy states”; (c) that maximal respiration requires the presence of only a small number of low-energy states and forms high-energy from low-energy states; (d) that uncouplers act by a bimolecular “collision” process without binding. In this model, respiratory chains must be capable of driving energy equivalents into a storage mode, with no effect on respiration rate until the store is almost filled. Results obtained with 2,4-dinitrophenol and S-13 show that the shapes of curves obtained when uncoupler concentration is plotted against respiration rate are the same for both “stoichiometric” and classical uncouplers. Mouse liver mitochondria, in the absence of added cations, showed a “titration” response to both dinitrophenol and S-13. Differences are: (1) the dinitrophenol titration point (approximately 35 μ m) is less dependent on mitochondrial concentration than that of S-13 (approximately 0.09 μ m) (for 1 mg of protein/ml); (2) a “lag” phase of some 30 sec occurs with S-13 at concentrations (approximately 0.1 μ m) giving the same uncoupling effect as 50 μ m-dinitrophenol (which shows no lag phase). Cytochrome b reduction level continues to fall upon DNP addition, and the rapid decline in per cent reduction of cytochrome b by a given increment of DNP ceases at approximately 35 μ m DNP, the concentration required for maximal respiration rate. Under some circumstances the release of respiration by uncoupler in the presence of azide shows an apparent titration independent of the final rate of respiration. This is true for DNP as well as for the apparent “stoichiometric” uncouplers. It is suggested that this does not necessarily identify a more specific uncoupling reaction than that observed in the absence of azide. The apparent release of azide inhibition by uucouplers is attributed to changes in the steady state level of cytochrome reduction on passing from state 3 to the uncoupled state, and to an oligomycin-like effect of azide on the mitochondrial ATPase.

Mitochondrial Protein and RNA Synthesis of X-Irradiated Mouse Livers

The effects of 2000 R on protein and RNA synthesis of mouse liver mitochondria were determined. After 40-minute injection in vivo of14 C-leucine or3 H-uridine the uptake of both isotopes by the irradiated liver mitochondria was 80% and 36% higher respectively than control. Mitochondria from x-irradiated livers also incorporated 40% more14 C-leucine and 12% more ${}^{3}{\rm H}\text{-}{\rm UTP}$ during 40 minutes incubation in vitro at 37°C than control. The distribution of nascent protein and RNA in submitochondrial fractions after in vitro incorporation of14 C-leucine or ${}^{3}{\rm H}\text{-}{\rm UTP}$ indicated that the mitochondrial membrane fraction from x-irradiated livers incorporated 12.5% more ${}^{3}{\rm H}\text{-}{\rm UTP}$ and 34% more14 C-leucine. The supernatant (excluding mito-ribosomal particles) incorporated 58.5% more ${}^{3}{\rm H}\text{-}{\rm UTP}$ and 103% more14 C-leucine than control. The sedimentation profile of mito-ribosomes of control showed a higher proportion of particles in th...

Enzymatic synthesis of acetylglutamate by mammalian liver preparations and its stimulation by arginine

An enzymatic activity to synthesize N-acetyl-L-glutamate, known as an essential allosteric activator of the ammonia-dependent carbamyl phosphate synthetase, from L-glutamate and acetyl-CoA was demonstrated in the extracts of rat and mouse liver mitochondria. A partially purified enzyme had a strict substrate specificity: various amino acids and acyl-CoA analogues could not substitute for the both substrates. A notable finding is that the enzyme activity was markedly stimulated by L-arginine. The effect of arginine was specific, and all other amino acids tested, including usual components of proteins and intermediates of urea cycle as well as several guanidino compounds were totally ineffective. Arginine also stimulated the acetylglutamate synthesis in intact mitochondria. The occurrence of an enzyme specific for acetylglutamate synthesis and control of its activity by arginine may provide an important regulatory mechanism for the production of carbamyl phosphate as the first step of urea biosynthesis.

A comparison of ultrastructural localization of carnitine acetyltransferase activity in mouse liver mitochondria with that in cardiac muscle.

En microscopie electronique l'etuee de la carnitine acetyltransferase a montre une meme localisation dans les mitochondries du foie et du muscle cardiaque de souris. La grande difference d'activite que decelent les methodes biochimiques entre ces deux types de mitochondries peut donc etre attribuee simplement a la presence 'und nombre beaucoup plus grand de cretes dans les mitochondries du muscle cardiaque.

Ultrastructural localization of succinate dehydrogenase in mouse liver mitochondria; a cytochemical study.

Ultrastructural localization of succinate dehydrogenase, employed as a marker of the electron transfer chain, was studied in mouse liver mitochondria either in orthodox or condensed conformation. Activity of succinate dehydrogenase in the orthodox form mitochondria was localized primarily on the cristal membrane facing the matrix. A prolonged incubation time resulted in reaction product filling the intracristal space, whereas a short incubation time resulted also in a product which appeared mainly at opposite points on both sides of the crista. No such arrangement of the reaction product was noted in mitochondria in the condensed conformation. These findings, supported by studies of serial sections, suggest that the reaction product in orthodox form mitochondria appears in bands or coils surrounding the whole cristal membrane.

Site II-specific inhibition of mitochondrial oxidative phosphorylation by trehalose-6,6′-dimycolate (cord factor) of Mycobacterium tuberculosis

Trehalose-6,6′-dimycolate (cord factor), a toxic glycolipid of Mycobacterium tuberculosis, inhibited the phosphorylation coupled to the oxidation of either succinate or NADH-linked substrates and caused a loss of respiratory control in mouse liver mitochondria. Preincubation of the mitochondrial suspension with cord factor was necessary for this inhibitory action. Liver mitochondria from a variety of animal species were susceptible to the action of cord factor. Phosphorylation coupled to the oxidation of succinate by ferricyanide (site II phosphorylation) was completely damaged, while that associated with the oxidation of ascorbate-TMPD (site III phosphorylation) was not affected by cord factor. Mycolic acid, either alone or with trehalose, acetylated cord factor, and a nontoxic synthetic cord factor analog, 6, x-di-(3′-acetoxy, x-methoxymycolanoyl)- N-acetyl- d-glucosamine, were without effect, while two toxic cord factor analogs, methyl 6-(3′, x-sulphitomycolanoyl)-α- d-glucoside and 6-(3′-acetoxy, x-methoxymycolanoyl)- N-acetyl- d-glucosamine, affected the mitochondrial oxidative phosphorylation as did cord factor. Cord factor stimulated slightly the mitochondrial ATPase. The effect of cord factor and DNP to induce the mitochondrial ATPase was additive. The 14C-labeled cord factor was not firmly bound to mitochondria.

Action of a toxic glycolipid of Corynebacterium diphtheriae on mitochondrial structure and function.

The toxicity for mice of the cord factor of Cornebacterium diphtheriae, trehalose-6,6'-dicorynomycolate, was studied. The diphtherial cord factor showed a delayed lethal toxicity for mice; the median lethal dose was 120 mug. Mouse liver mitochondria were disrupted in vivo under the toxic action of the diphtherial cord factor into fragments deficient in both respiration and phosphorylation. The site of metabolic defect in the mitochondrial electron transport system was located at the region prior to the level of cytochrome c in the livers of cord factor-intoxicated mice. These effects of the diphtherial cord factor on the host-cell mitochondria were essentially similar to those of the cord factor of Mycobacterium tuberculosis, trehalose-6,6'-dimycolate.

Studies of a biochemical lesion in experimental tuberculosis in mice. 8. Effect of derivatives and chemical analogues of cord factor on structure and function of mouse liver mitochondria.

Studies of a biochemical lesion in experimental tuberculosis in mice. 8. Effect of derivatives and chemical analogues of cord factor on structure and function of mouse liver mitochondria.

A Hexokinase-initiated Inhibition of Oxygen Uptake in Tomato Fruit Mitochondria Uncoupled by Dinitrophenol

The stimulation of oxygen uptake in plant mitochondria uncoupled by 2,4-dinitrophenol (DNP) depends on adenine nucleotide in the absence of hexokinase (EC 2.7.1.1). Laties (9) observed this dependence with cauliflower bud mitochondria oxidizing malate, while WViskich and Bonner (13) reported a doubling of the DNP-rate of oxygen uptake by adenine nucleotide in sweet potato mitochondria oxidizing succinate. 'Tomato fruit mitochondria also showed a dependence on adenine nucleotide in the presence of DNP and succinate and the absence of hexokinase (5). Heytler (7) reported a graduated suppression of oxygen uptake with increasing concentrations of the uncoupler, in-Cl-carbonyl cyanide phenylhydrazone, in the presence of hexokinase, in mouse liver mitochondria oxidizing succinate. Inhibition of succinate oxidation in rat liver and potato mitochondria in the absence of hexokinase depended on the concentration of uncoupler -(12, 13). The inhibition of succinate oxidation may be due to an inhibition of succinate dehyodrogenase (EC 1.3.99.1) by oxaloacetate (13). Wiskich and Bonner (13) suggested that ATP, which could be formed

Isolation and characterization of a rapidly labelled RNA from mouse liver mitochondria

1. 1. The isolation and characterization of a rapidly labelled RNA from mouse liver mitochondria is described. This RNA fraction was shown to have the characteristics of messenger RNA (mRNA). 2. 2. The rapidly labelled RNA fraction exhibits a molecular weight of 1.5 · 10 5 and is continuously synthesized both in vivo and in vitro for 120 min. 3. 3. The amino acid incorporation into mitochondria in vitro is slightly stimulated by this RNA fraction. It also prevents the inhibitory effects of chloramphenicol. 4. 4. Hybridization of the rapidly labelled RNA with mitochondria DNA suggests the mitochondrial origin of this RNA.

H+ Changes Associated with Divalent Cation Uptake by Mouse Liver Mitochondria

The effect of concentration of Ca++ and mitochondria on the stoichiometry of H+ release associated with Ca++ uptake supported by succinate oxidation was examined. Ratios less than unity were observed with low concentrations of Ca++, and higher ratios were obtained with intermediate concentrations of Ca++. The highest values (greater than unity but less than 2) could be obtained with increased mitochondrial concentrations. The concentrations of Ca++ at which the highest ratios were obtained were shifted to higher levels as the mitochondrial concentration was raised. The low ratios observed with low levels of divalent cations are attributed in part to the metabolism-independent binding of Ca++, since 45Ca++ was observed to be bound by mitochondria in the presence of antimycin and oligomycin. This energy-independent uptake of Ca++ was also dependent on the concentration of Ca++ and mitochondrial protein. In contrast to the energy-dependent process, the release of H+ was small or absent when antimycin was present. Added NaCl or KCl, but not further additions of mannitol or sucrose, completed for this Ca++-accessible space. These findings suggested that H+:Ca++ ratios of unity may be underestimated, and those ratios which are based on the assumption that all of the added calcium ions are taken up by an energy-linked mechanism may have to be corrected. Under conditions where the ratio was corrected for the energy-independent binding of Ca++, the (K+ + H+):Ca++ ratio approached 2.

Growth hormone stimulation of protein synthesis in isolated mouse liver mitochondria.

Mouse liver mitochondria from animals of various ages incorporated amino acids into their protein at approximately the same rate. Treating mice with growth hormone produced an increase in the rate of in vitro protein synthesis by mitochondria from all animals. Our studies indicated that isolated mouse liver mitochondria have the ability to incorporate amino acids into their contractile protein. This capability was enhanced by growth hormone treatment. (Endocrinology 80: 772, 1967)