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The main aim of this study was to investigate whether enzyme levels of the malate-aspartate and alpha-glycerophosphate shuttles and of cytochrome b5 reductase in human skeletal muscle are affected by strength training. Muscle biopsy samples from the deltoid muscle of the nondominant arm in untrained (n = 12) and strength-trained (n = 12) subjects were compared. The strength-trained muscles were characterized by a tendency to a higher percentage of type I fibers (67 vs. 59%), a lower percentage of type IIb fibers (12 vs. 18%), 34% larger mean fiber areas, and 19% more capillaries per fiber (P less than 0.1). No difference was noted in levels of enzymes representing the citric acid cycle, fatty acid oxidation, and glycolysis, nor in the number of capillaries per square millimeter. Neither did the levels of malate-aspartate and alpha-glycerophosphate shuttle enzymes nor cytochrome b5 reductase differ. Levels of cytochrome b5 reductase correlated (r = 0.59, P less than 0.01) with levels of the mitochondrial marker enzyme citrate synthase. It is concluded that strength training does not appear to result in increased levels of NADH shuttle enzymes and cytochrome b5 reductase. 相似文献
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Ubiquinol-cytochrome-c oxidoreductase has been isolated from potato (Solanum tuberosum L.) mitochondria by cytochrome-c affinity chromatography and gel-filtration chromatography. The procedure, which up to now only proved applicable to Neurospora, yields a highly pure and active protein complex in monodisperse state. The molecular mass of the purified complex is about 650 kDa, indicating that potato cytochrome c reductase occurs as a dimer. Upon reconstitution into phospholipid membranes, the dimeric enzyme catalyzes electron transfer from a synthetic ubiquinol to equine cytochrome c with a turnover number of 50 s-1. The activity is inhibited by antimycin A and myxothiazol. A myxothiazol-insensitive and antimycin-sensitive transhydrogenation reaction, with a turnover number of 16 s-1, can be demonstrated as well. The protein complex consists of ten subunits, most of which have molecular masses similar to those of the nine-subunit fungal enzyme. Individual subunits were identified immunologically and spectral properties of b and c cytochromes were monitored. Interestingly, an additional 'core' polypeptide which is not present in other cytochrome bc1 complexes forms part of the enzyme from potato. Antibodies raised against individual polypeptides reveal that the core proteins are clearly immuno-distinguishable. The additional subunit may perform a specific function and contribute to the high molecular mass which exceeds those reported for other cytochrome-c-reductase dimers. 相似文献
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The efficacy of chemotherapeutic agents on tumor cells has been shown to be modulated by tumor suppressor gene p53 and its target genes such as Bcl-2 family members (Bax, Noxa, and PUMA). However, various chemotherapeutic agents can induce cell death in tumor cells that do not express the functional p53, suggesting that some chemotherapeutic agents may induce cell death in a p53-independent pathway. Here we showed that etoposide can induce the similar degree of cell death in p53-deficient HCT 116 cells, whereas 5'-FU-mediated cell death is strongly dependent on the existence of functional p53 in HCT 116 cells. Further, we provide the evidence that etoposide can induce the cytochrome c release from isolated mitochondria, and etoposide-induced cytochrome c release is not accompanied with the large amplitude swelling of mitochondria. These data suggest that etoposide can directly induce the mitochondrial dysfunction irrespective of p53 status, and it may, at least in part, account for the p53-independent pathway in cell death induced by chemotherapeutic agents. 相似文献
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Flux control of cytochrome c oxidase in human skeletal muscle 总被引:3,自引:0,他引:3
Kunz WS Kudin A Vielhaber S Elger CE Attardi G Villani G 《The Journal of biological chemistry》2000,275(36):27741-27745
In the present work, by titrating cytochrome c oxidase (COX) with the specific inhibitor KCN, the flux control coefficient and the metabolic reserve capacity of COX have been determined in human saponin-permeabilized muscle fibers. In the presence of the substrates glutamate and malate, a 2.3 +/- 0.2-fold excess capacity of COX was observed in ADP-stimulated human skeletal muscle fibers. This value was found to be dependent on the mitochondrial substrate supply. In the combined presence of glutamate, malate, and succinate, which supported an approximately 1.4-fold higher rate of respiration, only a 1.4 +/- 0.2-fold excess capacity of COX was determined. In agreement with these findings, the flux control of COX increased, in the presence of the three substrates, from 0.27 +/- 0.03 to 0.36 +/- 0.08. These results indicate a tight in vivo control of respiration by COX in human skeletal muscle. This tight control may have significant implications for mitochondrial myopathies. In support of this conclusion, the analysis of skeletal muscle fibers from two patients with chronic progressive external ophthalmoplegia, which carried deletions in 11 and 49% of their mitochondrial DNA, revealed a substantially lowered reserve capacity and increased flux control coefficient of COX, indicating severe rate limitations of oxidative phosphorylation by this enzyme. 相似文献
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Considerable thermostable, very probably nonenzymatic, cytochrome c reducing activities are present in all plant extracts studied. These interfere with the assays of NADH- and NADPH-cytochrome c reductases. Since this could be a widespread, but scarcely considered phenomenon, a method was looked for to avoid this problem. Because of the low-molecular-weight nature of the compounds exhibiting the thermostable activity, separation of enzymatic and nonenzymatic activities is achieved by Sephadex G-25 filtration. This separation allows the optimization of the assay of antimycin A-insensitive NADH-cytochrome c reductase of maize root tips. The enzymatic and nonenzymatic activities are shown to differ in many respects (the nonenzymatic activity is not significantly NADH-dependent), but even in the optimized conditions, the nonenzymatic activity is not eliminated totally if homogenates are tested without previous Sephadex filtration. So the separation of the different active compounds on Sephadex G-25 seems to be a prerequisite for obtaining exact data on enzymatic cytochrome c reducing activities in plant extracts. 相似文献
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Basu S Azarova NA Font MD King SB Hogg N Gladwin MT Shiva S Kim-Shapiro DB 《The Journal of biological chemistry》2008,283(47):32590-32597
Small increases in physiological nitrite concentrations have now been shown to mediate a number of biological responses, including hypoxic vasodilation, cytoprotection after ischemia/reperfusion, and regulation of gene and protein expression. Thus, while nitrite was until recently believed to be biologically inert, it is now recognized as a potentially important hypoxic signaling molecule and therapeutic agent. Nitrite mediates signaling through its reduction to nitric oxide, via reactions with several heme-containing proteins. In this report, we show for the first time that the mitochondrial electron carrier cytochrome c can also effectively reduce nitrite to NO. This nitrite reductase activity is highly regulated as it is dependent on pentacoordination of the heme iron in the protein and occurs under anoxic and acidic conditions. Further, we demonstrate that in the presence of nitrite, pentacoordinate cytochrome c generates bioavailable NO that is able to inhibit mitochondrial respiration. These data suggest an additional role for cytochrome c as a nitrite reductase that may play an important role in regulating mitochondrial function and contributing to hypoxic, redox, and apoptotic signaling within the cell. 相似文献
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A monoclonal antibody against cytochrome c oxidase distinguishes cardiac and skeletal muscle mitochondria 总被引:2,自引:0,他引:2
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. 相似文献
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The adenosinetriphosphatase activity of myofibrils isolated from skeletal muscle 总被引:11,自引:7,他引:4 下载免费PDF全文
PERRY SV 《The Biochemical journal》1951,48(3):257-265
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Isolation of the rotenone-sensitive NADH-ubiquinone reductase (complex I) from red beet mitochondria
Allan G. Rasmusson Janet Mendel-Hartvig Jan M. Møller Joseph T. Wiskich 《Physiologia plantarum》1994,90(3):607-615
Complex 1 of the respirator) chain (EC 1.6.531, measured as NADH-duroquinone and NADH-ubiquinone, reductase activities, was isolated from purified red beetroot ( Beta vulgaris L.I mitochondria. The mitochondria were disrupted by freeze-thawing and inner membrane vesicles were pelleted. After solubilization of the vesicles with Triton X-100, the enzyme complex was purified 11-fold (compared to the activity in the inner membrane vesicles) by size-exclusion chromatography on a Sephacryl S-400 HR column and then by ion-exchange chromatography on a DEAE-Sepharose CL-6B column. Triton X-100 was present throughout the purification procedure. Tire purified complex showed approximately 30 bands on SDS-PAGE and about 15 polypeptides including those at 80. 54, 53. 51. 27. 25 and 22 kDa cross-reacted with polyclonal antibodies raised against complex I from Neurospora crassa . This is similar lo the pattern obtained with complex I from Neurospera crassa .
Analysis by nativc-SDS 2-dimensional PAGE revealed the existence of several molecular mass forms of the purified complex.
After reconstitution of the purified complex into phosphatidylcholine vesicles, the NADH-ubiquinone reductase activity had a Km (NADH) of about I μ M and was inhibited by both rotenone and dicyclohexylcarbodiimide. 相似文献
Analysis by nativc-SDS 2-dimensional PAGE revealed the existence of several molecular mass forms of the purified complex.
After reconstitution of the purified complex into phosphatidylcholine vesicles, the NADH-ubiquinone reductase activity had a K
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《Biochemical medicine》1978,19(3):366-373
Mitochondria were isolated from small muscle biopsy specimens, and the cytochrome content was calculated from the reduced minus oxidized difference spectrum recorded at room temperature.From the difference spectra obtained after reduction of the cytochromes with dithionite it is concluded that human mitochondrial suspensions are contaminated with hemoglobin or myoglobin.The cytochrome content calculated after reduction of the cytochromes with succinate plus KCN is lower than that obtained after reduction with dithionite, indicating incomplete reduction of the cytochromes by the former method.Storage of muscle tissue at −70°C before isolation of mitochondria results in a loss of cytochrome c + c1 from these mitochondria. 相似文献
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Mitochondrial translation of subunits of the rotenone-sensitive NADH:ubiquinone reductase in Neurospora crassa. 总被引:3,自引:0,他引:3 下载免费PDF全文
The rotenone sensitive NADH:ubiquinone was isolated from mitochondria of Neurospora crassa as a monodisperse preparation with the apparent mol. wt. in Triton solution of 0.9 X 10(6). The enzyme is composed of at least 22 subunits with apparent mol. wts. in SDS between 70 and 11 kd. Six of the subunits with the mol. wts. 70, 48, 37, 25, 22 and 18 kd were radioactively labelled in the enzyme isolated from cells which had incorporated [35S]methionine in the presence of cycloheximide. These subunits are synthesized in the mitochondria. Eleven subunits were radioactively labelled in the enzyme from cells which had incorporated [35S]methionine in the presence of chloramphenicol. These subunits are synthesized in the cytoplasm. The site of translation of the other subunits could not be established by the pulse-labelling technique. The assignment of the mitochondrially synthesized subunits to unidentified reading frames on the mitochondrial DNA is discussed. 相似文献
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Human skeletal muscle carnitine palmitoyltransferase I activity determined in isolated intact mitochondria 总被引:2,自引:0,他引:2
Berthon Phanelie M.; Howlett Richard A.; Heigenhauser George J.F.; Spriet Lawrence L. 《Journal of applied physiology》1998,85(1):148-153
This study was designed to compare theactivity of skeletal muscle carnitine palmitoyltransferase I (CPT I) intrained and inactive men (n = 14) andwomen (n = 12). CPT Iactivity was measured in intact mitochondria, isolated from needlebiopsy vastus lateralis muscle samples (~60 mg). The variability ofCPT I activity determined on two biopsy samples from the same leg onthe same day was 4.4, whereas it was 7.0% on two biopsy samples fromthe same leg on different days. The method was sensitive to the CPT Iinhibitor malonyl-CoA (88% inhibition) and therefore specific for CPTI activity. The mean CPT I activity for all 26 subjects was 141.1 ± 10.6 µmol · min1 · kgwet muscle (wm)1 and wasnot different when all men vs. all women (140.5 ± 15.7 and 142.2 ± 14.5 µmol · min1 · kgwm1, respectively) were compared. However, CPT Iactivity was significantly higher in trained vs. inactive subjects forboth men (176.2 ± 21.1 vs. 104.1 ± 13.6 µmol · min1 · kgwm1) and women (167.6 ± 14.1 vs. 91.2 ± 9.5 µmol · min1 · kgwm1). CPT I activity was also significantly correlatedwith citrate synthase activity (all subjects,r = 0.76) and maximal oxygen consumption expressed in milliliters per kilogram per minute (all subjects, r = 0.69). Theresults of this study suggest that CPT I activity can be accurately andreliably measured in intact mitochondria isolated from human musclebiopsy samples. CPT I activity was not affected by gender, and higheractivities in aerobically trained subjects appeared to be the result ofincreased mitochondrial content in both men and women. 相似文献
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The cross-reactivity of human placental microsomal NADPH-cytochrome c reductase antiserum, REDFBIV, against the endometrial reductase alone and as a component of the endometrial aromatase was investigated. Human endometrial particulate fractions were incubated with various amounts of REDFBIV for 1 h at 4 degrees C and both enzyme activities were measured at the end of incubation. The extent of inhibition of these endometrial enzymes was compared with the ability of this antiserum to inhibit the placental microsomal reductase and aromatase activities. The antiserum effectively inhibited the activities of both enzymes in both tissues in a dose dependent manner with aromatase activity inhibited to a greater extent than reductase activity. These results indicate the antiserum to the placental microsomal NADPH-cytochrome c reductase component of aromatase recognizes the reductase component of the aromatase enzyme system in endometrium. 相似文献