Mitochondrial Membrane-bound Enzyme Research Articles

Unexpected obesity, rather than tumorigenesis, in a conditional mouse model of mitochondrial complex II deficiency.

Mutations in any of the genes encoding the four subunits of succinate dehydrogenase (SDH), a mitochondrial membrane‐bound enzyme complex that is involved in both the tricarboxylic acid cycle and the electron transport chain, can lead to a variety of disorders. Recognized conditions with such mutations include Leigh syndrome and hereditary tumors such as pheochromocytoma and paraganglioma (PPGL), renal cell carcinoma, and gastrointestinal stromal tumor. Tumors appear in SDH mutation carriers with dominant inheritance due to loss of heterozygosity in susceptible cells. Here, we describe a mouse model intended to reproduce hereditary PPGL through Cre‐mediated loss of SDHC in cells that express tyrosine hydroxylase (TH), a compartment where PPGL is known to originate. We report that while there is modest expansion of TH+ glomus cells in the carotid body upon SDHC loss, PPGL is not observed in such mice, even in the presence of a conditional dominant negative p53 protein and chronic hypoxia. Instead, we report an unexpected phenotype of nondiabetic obesity beginning at about 20 weeks of age. We hypothesize that this obesity is caused by TH+ cell loss or altered phenotype in key compartments of the central nervous system responsible for regulating feeding behavior, coupled with metabolic changes due to loss of peripheral catecholamine production.

Identification and activity of monoamine oxidase in the orb-weaving spider Larinioides cornutus

Monoamine oxidase (MAO) is a mitochondrial membrane-bound enzyme that catalyzes the oxidative deamination of monoamines in a wide array of organisms. While the enzyme monoamine oxidase has been studied extensively in its role in moderating behavior in mammals, there is a paucity of research investigating this role in invertebrates, where the latter utilizes this enzyme in a major pathway to degrade monoamines. There is especially a dismal lack of information on how MAO influences activity in invertebrates, particularly in account of the circadian cycle. Previous studies revealed MAO degrades serotonin and norepinephrine in arachnids, but did not investigate other critically important compounds like octopamine. Larinioides cornutus is a species of orb-weaving spider that exhibits diel fluctuations in behavior, specifically levels of aggression. The monoamines octopamine and serotonin have been shown to influence aggressive behaviors in L. cornutus, thus this species was used to investigate if MAO is a potential site of regulation throughout the day. Not only did gene expression of MAO orthologs and MAO activity fluctuate at different times of day, but the enzymatic activity was substrate-specific producing a higher level of degradation of octopamine as compared to serotonin in vitro. This study further supports evidence that MAO has an active role in monoamine inactivation in invertebrates and provides a first look at how MAO ultimately may be regulating behavior in an invertebrate.

345 - Catecholamine Metabolism via Monoamine Oxidase Disrupts Oxidative Phosphorylation Efficiency in Glutathione Peroxidase-4 Deficient Mouse and Human Myocardium

Glutathione peroxidase-4 (GPx4) is a selenoenzyme expressed in mitochondria and nuclear compartments which exclusively neutralizes lipid peroxides. Monoamine oxidase (MAO), an outer mitochondrial membrane-bound enzyme responsible for catecholamine metabolism, produces a reactive catechol-aldehyde and H2O2 upon substrate deamination. Current findings in human myocardial samples from our lab show that obese/diabetic patients exhibit greater maximal MAO activity and content, but lower GPx4 compared with nondiabetic patients, suggesting that both increased MAO and decreased GPx4 act to compound levels of reactive aldehyde species in diabetic myocardium. Our preliminary experiments using nitro blue tetrazolium staining have revealed protein modification by catechol-aldehydes derived from norepinephrine (NE) and dopamine (DA) in whole heart lysate and isolated cardiac mitochondria from mice and humans, an effect mitigated by MAO inhibitors. We further examined the effects of catecholamine metabolism via MAO on ATP production and O2 consumption (OxPhos) in isolated cardiac mitochondria prepared from WT and GPx4-deficient (GPx4+/-) mice, and in permeabilized myofibers prepared from human atrial myocardium. Both DA and NE significantly decreased ATP production and O2 consumption in GPx4+/- mice, but not WT, by 15% and 13%, respectively (p

Aging-Associated Enzyme Human Clock-1: Substrate-Mediated Reduction of the Diiron Center for 5-Demethoxyubiquinone Hydroxylation

The mitochondrial membrane-bound enzyme Clock-1 (CLK-1) extends the average longevity of mice and Caenorhabditis elegans, as demonstrated for Δclk-1 constructs for both organisms. Such an apparent impact on aging and the presence of a carboxylate-bridged diiron center in the enzyme inspired this work. We expressed a soluble human CLK-1 (hCLK-1) fusion protein with an N-terminal immunoglobulin binding domain of protein G (GB1). Inclusion of the solubility tag allowed for thorough characterization of the carboxylate-bridged diiron active site of the resulting GB1-hCLK-1 by spectroscopic and kinetic methods. Both UV-visible and Mössbauer experiments provide unambiguous evidence that GB1-hCLK-1 functions as a 5-demethoxyubiquinone-hydroxylase, utilizing its carboxylate-bridged diiron center. The binding of DMQn (n = 0 or 2) to GB1-hCLK-1 mediates reduction of the diiron center by nicotinamide adenine dinucleotide (NADH) and initiates O2 activation for subsequent DMQ hydroxylation. Deployment of DMQ to mediate reduction of the diiron center in GB1-hCLK-1 improves substrate specificity and diminishes consumption of NADH that is uncoupled from substrate oxidation. Both Vmax and kcat/KM for DMQ hydroxylation increase when DMQ0 is replaced by DMQ2 as the substrate, which demonstrates that an isoprenoid side chain enhances enzymatic hydroxylation and improves catalytic efficiency.

Transcriptional regulation of the rat steroidogenic acute regulatory protein gene by steroidogenic factor 1.

Steroidogenic acute regulatory (StAR) protein is synthesized in response to tropic hormones to facilitate cholesterol transport to the inner mitochondrial membrane-bound P450 side-chain cleavage enzyme (P450scc), the first enzymatic step in the steroid hormone biosynthetic pathway. Gonadotropins activate expression of their target genes via the cAMP second messenger system. We have demonstrated that cAMP administration to rat luteal cells stimulates expression of both StAR messenger RNA and protein. Because cholesterol delivery is the first regulated step in steroidogenesis, and because StAR messenger RNA levels are increased in response to tropic hormone and cAMP stimulation, the mechanism by which tropic hormones/cAMP stimulate transcription needs to be elucidated. To this end, approximately 2.7 kb of the rat StAR promoter was isolated and sequenced. Sequence analysis revealed the presence of a TATA-like element as well as multiple regulatory motifs including steroidogenic factor 1 (SF-1) binding sites, an estrogen receptor half-site, and two AP-1 sites within the promoter region. 5'-RACE experiments determined the transcription start site to be located 82 bp upstream of the ATG translation start codon. Electrophoretic mobility shift assays and supershift analysis demonstrated SF-1 binding to three SF-1 binding sites in the rat StAR promoter with high affinity and two SF-1 binding sites with low affinity. Transfection of mouse Y1 adrenal tumor cells and human bladder carcinoma cells (HTB9s) with the rat StAR promoter demonstrated that SF-1 was able to activate transcription of the luciferase reporter gene and that the rat StAR promoter was responsive to cAMP. Nested deletions of the rat StAR promoter (1.9 kb) identified a region between -1413 and -998 that is essential for maximal activation of the rat StAR gene in HTB9 cells; however, deletion of this region does not affect responsiveness to cAMP. 5'-Deletion and site-directed mutagenesis experiments demonstrated that the SF-1 motifs identified within the rat StAR promoter (located at positions -764, -455, and -106) were sufficient to activate transcription as well as confer cAMP responsiveness to the rat StAR gene. Site-directed mutagenesis studies using the smallest promoter fragment demonstrated that the two proximal SF-1 binding sites are crucial for StAR gene transcription, both at a basal level and in response to cAMP stimulation. These studies provide novel insights into the regulation of the rat StAR gene at the transcriptional level by SF-1.

Human liver long-chain 3-hydroxyacyl-coenzyme a dehydrogenase is a multifunctional membrane-bound beta-oxidation enzyme of mitochondria

We have purified to homogeneity the long-chain specific 3-hydroxyacyl-CoA dehydrogenase from mitochondrial membranes of human infant liver. The enzyme is composed of non-identical subunits of 71kDa and 47kDa within a native structure of 230kDa. The pure enzyme is active with 3-ketohexanoyl-CoA and gives maximum activity with 3-ketoacyl-CoA substrates of C 10 to C 16 acyl-chain length but is inactive with acetoacetyl-CoA. In addition to 3-hydroxyacyl-CoA dehydrogenase activity, the enzyme possesses 2-enoyl-CoA hydratase and 3-ketoacyl-CoA thiolase activities which cannot be separated from the dehydrogenase. None of these enzymes show activity with C 4 substrates but all are active with C 6 and longer acyl-chain length substrates. They are thus distinct from any described previously. This human liver mitochondrial membrane-bound enzyme catalyses the conversion of medium- and long-chain 2-enoyl-CoA compounds to: 1) 3-ketoacyl-CoA in the presence of NAD alone and 2) to acetyl-CoA (plus the corresponding acyl-CoA derivatives) in the presence of NAD and CoASH. It is therefore a multifunctional enzyme, resembling the beta-oxidation enzyme of E. coli, but unique in its membrane location and substrate specificity. We propose that its existence explains the repeated failure to detect any intermediates of mitochondrial beta-oxidation.

Altered kinetic properties of liver mitochondrial membrane-bound enzyme activities following paracetamol hepatotoxicity in the rat

The effects of treatment with subtoxic (375 mg/kg) and toxic (750 mg/kg) doses of paracetamol on NADH oxidase, succinoxidase and Mg2+-ATPase activities in rat liver submitochondrial particles were examined. In the NADH oxidase system, treatment with subtoxic doses of paracetamol resulted in a 37% increase in activation energy in the high temperature range (E1) while the phase transition temperature (Tt) for this system decreased by 9‡C. Subtoxic doses caused a 43% decrease in E1. For the succinoxidase system, Tt decreased by 2.4 to 3.4‡C after paracetamol administration. E2 increased by 42% only in the subtoxic-treatment group while E1 remained unaltered in both paracetamol-treated groups. For the Mg2+-ATPase system, subtoxic doses of paracetamol treatment did not change the values of E1 E2 and Tt whereas toxic dose treatment resulted in a 29% decrease in E2 with a concomitant increase in Tt by 2.4‡C without any change in the value of E1 The results thus suggest that treatment with toxic and subtoxic doses of paracetamol results in possible differential alterations in the membrane lipid milieu.

Purification and Characterization of the Soluble F1-ATPase of Oat Root Mitochondria

The properties of the soluble moiety (F(1)) of the mitochondrial H(+)-ATPase from oat roots were examined and compared to those of the native mitochondrial membrane-bound enzyme. The chloroform soluble preparation was purified by Sephadex G-200 and DEAE-cellulose chromatography. The purified F(1) preparation contained major polypeptides corresponding to alpha, beta, gamma, delta, and epsilon of apparent molecular mass 58, 55, 35, 22, and 14 kilodaltons, respectively. The purified F(1)-ATPase, like the native enzyme, was inhibited by azide (I(50) = 10 micromolar), nitrate (I(50) = 7-10 millimolar), 4,4'-diisothiocyano-2,2'-stilbene disulfonic acid (I(50) = 1-3 micromolar), and 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (I(50) = 3 micromolar). F(1)-ATPase activity was stimulated by bicarbonate but not by chloride. In both the native and the F(1)-form of the ATPase, ATP was hydrolyzed in preference to GTP. The results indicate that these properties of the native membrane-bound mitochondrial ATPase have been conserved in the purified F(1). In contrast to the membrane-bound enzyme, the F(1)-ATPase was not inhibited by oligomycin or by N,N'-dicyclohexylcarbodiimide. The mitochondrial F(1)-ATPase from oat roots is analogous to other known F(1)F(0)-ATPases.

Role of phospholipid in the low affinity reactions between cytochrome c and cytochrome oxidase

The steady-state oxidation of ferrocytochrome c by cytochrome oxidase monitored spectrophotometrically showed that: (1) the kinetics were strictly biphasic with purified enzyme, while mitochondrial membrane-bound enzyme exhibited multiphasic kinetics with extended low affinity phases; (2) the TN max for the highest affinity phase was as slow as 5–10 electron · s −1 for both preparations, while for the low affinity phases it was about 45 electron · s −1 for the purified enzyme and 150 electron · s −1 for the mitochondrial membrane-bound enzyme; (3) reconstitution of purified enzyme into acidic phospholipid vesicles partially repleted the extended low affinity phases, while reconstitution into uncharged vesicles had no effect.

Purification of rat liver monoamine oxidase by octyl glucoside extraction and reconstitution

Catalytically active isoenzymes of rat liver monoamine oxidase have been copurified from the outer mitochondrial membrane by a novel method involving repetitive solubilization with octyl-β- d-glucopyranoside followed by reconstitution into lipid vesicles. As analyzed using sodium dodecyl sulfate-gel electrophoresis, the purified enzyme migrates as a single band of protein of molecular weight 60,000. The preparation is capable of metabolizing 576 nmol serotonin and 777 nmol β-phenylethylamine/min/mg protein. Apparent K m values and sensitivity to the inhibitor clorgyline are very similar for the purified and outer mitochondrial membrane-bound enzyme when determined with the substrates β-phenylethylamine, serotonin, and tyramine.

Corresponding changes in kynurenine hydroxylase activity, membrane fluidity, and sterol composition in Saccharomyces cerevisiae mitochondria.

The effect of sterol composition on the properties of the mitochondrial membrane of Saccharomyces cerevisiae was investigated. The physical state of mitochondrial membranes from wild-type strains and sterol mutants was compared, using a fluorescence polarization technique with 1,6-diphenyl-1,3-5-hexatriene. Changes in the rate of depolarization of the probe molecule as a function of temperature suggest the occurrence of a phase transition in the mitochondrial membranes isolated from the sterol mutants but not in the membranes isolated from the wild types. Arrhenius kinetics of the mitochondrial membrane-bound enzyme L-kynurenine-3-hydroxylase exhibited changes in activation energy at temperatures similar to those observed in the fluorescence polarization study. The ratio of mitochondrial sterol to phospholipid and the phospholipid fatty acid composition of the organisms were characterized.

Membrane-bound glutathione peroxidase-like activity in mitochondria

A membrane-bound glutathione peroxidase-like activity has been detected in liver and cardiac mitochondrial membrane. This enzyme activity differs from the cytosol and mitochondrial matrix selenium-dependent glutathione peroxidase in that it is membrane bound, sensitive to sonication and triton-X-100, and is unaffected by prolonged feeding of a selenium-free diet. This mitochondrial membrane-bound enzyme activity differs from the glutathione-S-transferases which exhibit glutathione peroxidase activity in that it is capable of utilizing both cumene hydroperoxide and hydrogen peroxide as substrates. Digitonin fractionation studies indicate that this enzyme is not located in either inner or outer mitochondrial membrane but rather within inter-membrane space. This newly described membrane-bound enzyme activity may play an important role in the maintenance of cardiac mitochondrial integrity in that mitochondrial matrix does not contain glutathione peroxidase.

Discontinuities in the arrhenius plots of mitochondrial membrane-bound enzyme systems from a poikilotherm: Acclimation temperature of carp affects transition temperatures

1. Evidence is presented for ‘breaks’ in the Arrhenius plots of succinate and cytochrome oxidase systems of mitochondria from carp liver and red epaxial muscle. 2. Arrhenius activation energies (Ea) of succinate and cytochrome oxidase systems above and below the transition temperatures (T*) are about half those reported for homoiotherms (Table 3). Succinate oxidase and cytochrome oxidase obey Michaelis-Menten kinetics. The Km-values for succinate and tetramethyl-p-phenylenediamine (TMPD) (25°C) are similar to those in homoiotherms; they are not affected by acclimation temperature (Table 2). 3. The change inEa of carp liver mitochondria (C.L.M.) occurs at low (high) experimental temperatures if carps are acclimated to low (high) ambient temperatures for at least 4 (3) weeks prior to the experiments (Fig. 1). 4. TheT* shift in Arrhenius plots following a change in acclimation temperature has been studied for succinate oxidation by carp liver mitochondria (Fig. 2). The time constant ofT* change was calculated as taccl.1/2=4.3 days (Fig. 3). Based on the concept of lipid protein interaction the acclimation temperature dependency ofT* is discussed as a possible consequence of a lipid adaptation. 5. Carp acclimated to 26°C and 10°C do not differ significantly with respect to specific activity of liver succinate oxidase determined at 25°C (Table 1). The same is true for the liver-somatic index and the amount of mitochondrial protein per gram of liver (Table 1). Changes, however, are observed in these parameters if carp are in a transitory state of temperature acclimation (early after changing from 26°C to 10°C; Table 1). 6. A bimodal action of temperature change during the process of acclimation is discussed combining the results in a model of succinate oxidase system of carp liver (Fig. 4).