Monamine oxidase in outer membrane of skeletal muscle mitochondria.

(1) Monoamine oxidase (EC 1.4.3.4) is present in rat skeletal muscle mitochondria. (2) A radioassay procedure for the assay of monoamine oxidase in muscle mitochondria is described. It is based on teh procedure using side-chain [2-14C]-tryptamine as substate described by Wurtman, R.J. and Axelrod, J. (1963) Biochem. Pharmacol. 12, 1439--1441 and employs a pH of 8.0 and a substrate concentration of 0.25 mM. (3) The Km of the muscle mitochondrial enzyme at pH 8.0 is 1.34 - 10(-5) M and that of the liver enzyme under the same conditions is 2.5 - 10(-5) M. Muscle mitochondria contain only one quarter of the activity of enzyme present in liver mitochondria. (4) Monoamine oxidase is shown to be in the outer membrane of skeletal muscle mitochondria and thus to be a suitable marker enzyme for use in the fractionation of these mitochondria.     

Monoamine oxidase of rat skeletal muscle: substrate specificity and half-life

The substrate specificity of rat skeletal muscle MAO has been studied. By the use of clorgyline as a MAO A inhibitor, it is found that 5-hydroxytryptamine, tryptamine, and kynuramine are deaminated by MAO A whereas benzylamine is a substrate for both forms of MAO. Phenethylamine displays a concentration-dependent preference for the two forms of MAO. These substrate specificies of the two forms of MAO in skeletal muscle are different from those observed in liver and brain but resemble closely that seen with heart. The half-lives of MAO A and MAO B in muscle estimated by rate of recovery from pargyline inhibition are 6.9 and 6.4 days, respectively.     

Monoamine oxidase and semicarbazide sensitive amine oxidase activities in toad liver mitochondria

1. The deamination of 5-HT and PEA has been assayed by a radiochemical method in mitochondria isolated from toad liver. 2. Time courses of 5-HT and PEA deamination indicate that when PEA is used as the substrate, higher specific activities are obtained. 3. 5-HT is deaminated by MAO A and partially by a SSAO-like enzyme. 4. PEA is deaminated exclusively by SSAO and, MAO B activity, at least under the adopted experimental conditions, is not detectable.     

Nitric oxide inhibits mitochondrial monoamine oxidase activity and decreases outer mitochondria membrane fluidity

The recent finding that mitochondria contain a nitric oxide (NO) synthase suggests that this compound is involved in the regulation of various mitochondrial functions. Monoamine oxidase (MAO) is embedded in the outer mitochondrial membrane. NO modulates membrane fluidity. Thus, the aim of the present work was to study the effect of NO on mitochondrial MAO activity and membrane fluidity. An outer mitochondrial membrane fraction (OMMF) was obtained from rat liver. OMMF was incubated with various concentrations of S-nitroso-N-acetylpenicillamine (SNAP), a NO donor. MAO activity and fluidity were measured by a spectrophotometric assay and by the polarization of fluorescence technique, respectively. It was found that small concentrations of SNAP (0.4-40 microM) were capable of inhibiting MAO activity but unable to decrease fluidity significantly. In contrast, larger amounts of SNAP (40-300 microM) effectively decreased membrane fluidity, but were not able to further decrease MAO activity. This information suggests that mitochondrial MAO and membrane fluidity possess different sensitivity to the effect of NO. Unfortunately, the mechanism by which NO inhibits MAO remains unknown at present. However, it seems likely that the effect of NO on MAO activity is by a direct interaction of the compound or a metabolite to the protein.     

The insertion of monoamine oxidase A into the outer membrane of rat liver mitochondria.

Human monoamine oxidase A that had been synthesized in a reticulocyte lysate translation system was capable of binding to and inserting into either rat liver mitochondria or isolated mitochondrial outer membranes. The inserted form was as resistant to proteinase K as endogenous mitochondrial monoamine oxidase A. The insertion, but not the binding, of monoamine oxidase A was prevented by depleting the reaction mixture of either ATP (with apyrase) or ubiquitin (with purified antibodies against this polypeptide). Addition of ATP or ubiquitin, respectively, to these depleted mixtures restored the insertion of the enzyme. In the absence of mitochondria, in vitro synthesized monoamine oxidase A did not catalyze its own alkylation by the mechanism-based inhibitor, [3H]clorgyline. However, both monoamine oxidase A that had been membrane-inserted in vitro and monoamine oxidase A that had been bound to the mitochondria under conditions of ATP depletion catalyzed adduct formation. Furthermore, reaction of either clorgyline or another mechanism-based inhibitor, pargyline, with the membrane-bound enzyme during ATP depletion inhibited the insertion of monoamine oxidase A when ATP was restored. These observations indicate that monoamine oxidase A acquired a catalytically active conformation on interaction with the mitochondrial outer membranes prior to its ATP and ubiquitin-dependent insertion into the membrane.     

A monoclonal antibody against cytochrome c oxidase distinguishes cardiac and skeletal muscle mitochondria

The mitochondrial enzyme cytochrome c oxidase (COX) in eukaryotes consists of at least seven subunits, three of which (I-III) are encoded by mitochondrial DNA (mitDNA) and the others (IV-VII) by the nuclear genome. There is increasing evidence that COX in mammals exists in multiple tissue-specific forms, presumably specified by nuclearly encoded subunits. We performed immunologic studies in human cardiac and skeletal muscle, using a monoclonal antibody raised against subunit IV of COX purified from human cardiac muscle. In immunotitration studies, the antibody bound with high affinity to mitochondria from cardiac muscle, but reacted only weakly with mitochondria from skeletal muscle. Similarly, immunocytochemical studies showed prominent mitochondrial staining in frozen sections of heart, but no staining in sections of mature skeletal muscle. Although this antibody did not stain mitochondria in mature skeletal muscle, it clearly stained mitochondria in myoblasts and immature myotubes of human muscle cultures, suggesting that mitochondria in immature muscle cells are different from those in mature muscle, and similar to heart mitochondria. Immunotitration data using either native or denatured COX protein from heart or skeletal muscle showed similar immunoreactivity. These studies indicate that the epitope for recognition by this antibody is exposed in mitochondria from heart and immature muscle cells, but masked in mitochondria from mature skeletal muscle.     

L-lactate oxidation by skeletal muscle mitochondria

1. Mitochondria isolated from rat skeletal muscle possess lactate dehydrogenase which is involved in direct oxidation of L-lactate in the presence of external NAD. 2. L-lactate oxidation can be stimulated in a reversible manner by ADP. 3. Mitochondrial lactate oxidation is sensitive to oxamate-inhibitor of LDH, alpha-cyano-3-hydroxy-cinnamate-pyruvate translocase inhibitor and respiratory chain inhibitors (rotenone, antimycin A, KCN). 4. In the same conditions the mitochondria did not oxidize pyruvate in the absence of malate, whereas, oxidize pyruvate plus external NADH in an uncoupling manner.     

Changes in membrane fluidity and lipid peroxidation of skeletal muscle mitochondria after exhausting exercise in rats

We studied the effects of exhausting exercise and exercise training on skeletal muscle mitochondrial membrane fluidity and lipid peroxidation in rats. The first part of the study involved 60 untrained rats divided into six equal groups. Of the total number 10 rats were sedentary and acted as controls. The remaining 50 rats exercised to exhaustion and were sacrificed at 0-h, 24-h, 48-h, 72-h, and 96-h post-exercise. The second part of the study involved 40 rats which were divided into four equal groups. Of these 10 rats were sedentary and acted as controls. The remaining 30 rats underwent 8 weeks of exercise training. They were then subjected to a single period of exhausting exercise and were sacrificed at 0-h, 24-h and 48-h post-exercise. Membrane fluidity was measured using the fluorescence polarization method. Lipid peroxidation was estimated by determining the thiobarbituric acid-reactive substances (TBARS) in mitochondria. In the untrained rats, mitochondrial fluorescence polarization and TBARS contents were significantly increased post-exercise compared with the sedentary controls (P < 0.05). They did not return to near control levels until 96 h and 48 h, respectively. In the trained rats, fluorescence polarization was raised compared with the sedentary controls but this was significantly lower than those measured at the same times of the untrained group post-exercise (P < 0.05). Exhausting exercise decreased membrane fluidity and increased lipid peroxidation in rat skeletal muscle mitochondria. These effects were relieved to some extent by exercise training.     

A novel potassium channel in skeletal muscle mitochondria

In this work we provide evidence for the potential presence of a potassium channel in skeletal muscle mitochondria. In isolated rat skeletal muscle mitochondria, Ca(2+) was able to depolarize the mitochondrial inner membrane and stimulate respiration in a strictly potassium-dependent manner. These potassium-specific effects of Ca(2+) were completely abolished by 200 nM charybdotoxin or 50 nM iberiotoxin, which are well-known inhibitors of large conductance, calcium-activated potassium channels (BK(Ca) channel). Furthermore, NS1619, a BK(Ca)-channel opener, mimicked the potassium-specific effects of calcium on respiration and mitochondrial membrane potential. In agreement with these functional data, light and electron microscopy, planar lipid bilayer reconstruction and immunological studies identified the BK(Ca) channel to be preferentially located in the inner mitochondrial membrane of rat skeletal muscle fibers. We propose that activation of mitochondrial K(+) transport by opening of the BK(Ca) channel may be important for myoprotection since the channel opener NS1619 protected the myoblast cell line C2C12 against oxidative injury.     

Carbonic anhydrase in guinea pig skeletal muscle mitochondria

The presence of carbonic anhydrase activity was demonstrated in guinea pig skeletal muscle mitochondria purified by Percoll gradient centrifugation such that contamination by sarcoplasmic reticulum vesicles was less than 5%. Assay of purified heavy sarcoplasmic reticulum vesicles for carbonic anhydrase activity showed these to have somewhat less activity than the mitochondria, so that any contribution by sarcoplasmic reticulum vesicles to mitochondrial activity would be negligible. In agreement with this observation, rabbit skeletal muscle mitochondria prepared by the Percoll method had no detectable activity. Assay of the guinea pig muscle mitochondrial enzyme activity in the presence of Triton X-100 showed a sixfold greater activity than in its absence, indicating a matrix location for the carbonic anhydrase. The enzyme is highly sensitive to the sulfonamide inhibitor ethoxzolamide, with Ki = 8.7 nM. The activation energy obtained from the rate constant for CO2 hydration, kenz with units (mg/ml)-1 s-1, over the range 4 to 37 degrees C was 12.8 kcal/mol. These properties are those expected for a carbonic anhydrase of the CA II class of isozymes, rather than for CA I, CA III, and the liver mitochondrial enzyme CA V.     

Probing the outer mitochondrial membrane in cardiac mitochondria with nanoparticles

The outer mitochondrial membrane (OMM) is the last barrier between the mitochondrion and the cytoplasm. Breaches of OMM integrity result in the release of cytochrome c oxidase, triggering apoptosis. In this study, we used calibrated gold nanoparticles to probe the OMM in rat permeabilized ventricular cells and in isolated cardiac mitochondria under quasi-physiological ionic conditions and during permeability transition. Our experiments showed that under control conditions, the OMM is not permeable to 6-nm particles. However, 3-nm particles could enter the mitochondrial intermembrane space in mitochondria of permeabilized cells and isolated cardiac mitochondria. Known inhibitors of the voltage-dependent anion channel (VDAC), K?nig polyanion, and 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid inhibited this entrance. Thus, 3-nm particles must have entered the mitochondrial intermembrane space through the VDAC. The permeation of the isolated cardiac mitochondria OMM for 3-nm particles was approximately 20 times that in permeabilized cells, suggesting low availability of VDAC pores within the cell. Experiments with expressed green fluorescent protein showed the existence of intracellular barriers restricting the VDAC pore availability in vivo. Thus, our data showed that 1), the physical diameter of VDAC pores in cardiac mitochondria is >or=3 nm but 相似文献   

Properties of creatine kinase from skeletal muscle mitochondria

Creatine kinase from pigeon breast muscle was obtained in a homogeneous (as evidenced from polyacrylamide gel SDS electrophoresis) state. The molecular mass of the enzyme monomer is 43,000. Ultracentrifugation in a sucrose density gradient and gel filtration revealed that the enzyme is present in solution as a mixture of two major forms, i.e., octamer and dimer, which differ in their activity. The decrease of ionic strength from 0.25 to 0.02 results in reversible dissociation of the octameric form. A temperature rise from 5 degrees to 20 degrees C or the nature of monovalent anions (e.g., Cl-, CH3COO-, NO3-) and cations (K+, Na+) present in the medium do not influence the distribution of oligomeric forms. At pH 6.0 the major form is represented by the octamer; its dissociation is caused by an increase of pH. The octamer dissociation occurs in a mixture of substrates of the creatine kinase reaction in the presence of Mg2+; no such dissociation is observed in the absence of Mg2+ and in the presence of each of the reaction substrates. The non-interacting pair of substrates--ADP and creatine--causes the dissociation of the octamer in the presence of nitrate ions but not acetate. It is concluded that the dissociating effect of substrates is due to the conformational changes of subunits during catalysis. At physiological concentrations of nucleotide substrates the degree of octamer dissociation depends on the ratio of creatine phosphate and creatine concentrations, as well as on the presence of chlorine and phosphate ions. A qualitative estimation of the rate of pH- and substrate-dependent dissociation of creatine kinase octamer revealed that under the given experimental conditions the pH-dependent dissociation is completed within hours, whereas the substrate-dependent one--within seconds or minutes. According to its properties, mitochondrial creatine kinase from pigeon breast muscle is close to its bovine heart counterpart; the observed differences were found to be quantitative.