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1.
When isolated mitochondria which have been labeled with [3H]leucine are solubilized and treated with anti-serum specific for cytochrome c oxidase, labeled polypeptides which correspond to the three largest polypeptides of this enzyme are immunoprecipitated. This indicates that the three largest polypeptides of cytochrome c oxidase which have Mr of 66,000, 39,000, and 23,000 are synthesized by isolated mitochondria whereas the three smallest ones which have Mr of 14,000, 12,500, and 10,000 are not. The smallest polypeptides are probably synthesized on cytoplasmic ribosomes as has been demonstrated in other systems by in vivo studies. These results are the first demonstration that isolated mammalian mitochondria are capable of synthesizing some of their own polypeptide components. The antiserum used in this study was prepared to highly purified cytochrome c oxidase (12.4 nmol of heme a + a3/mg of protein) from rat liver mitochondria. This antiserum gives a single precipitin line when tested by the Ouchterlony double diffusion technique. Its specificity has been demonstrated by the fact that it: 1) only precipitates heme a + a3, not hemes b, c, or c1, when added to solubilized mitochondria, 2) inhibits cytochrome c oxidase activity at least 85%, and 3) precipitates only those polypeptides found in purified cytochrome c oxidase when added to solubilized mitochondria labeled in vivo. 相似文献
2.
C Montecucco G Schiavo B Bacci R Bisson 《Comparative biochemistry and physiology. B, Comparative biochemistry》1987,87(4):851-856
1. Several bird and fish heart mitochondrial cytochrome c oxidases have been isolated with a rapid and simple method involving hydrophobic and affinity chromatography. 2. Their spectrophotometric and kinetic properties are very similar to those of the mammalian enzymes. 3. These oxidases show a polypeptide composition simpler than the mammalian enzymes being composed of 9-10, instead of 13, different polypeptides. 4. These data suggest that the complexity of the mitochondrial heart oxidase increases with the stage of evolution. 相似文献
3.
Yeast cytochrome c oxidase has been isolated by ion exchange chromatography using lauryl maltoside (n-dodecyl beta-D-maltoside) as the solubilizing detergent. The enzyme prepared in this way has a heme aa3 concentration of 8-9 nmol/mg of protein and a turnover number in the range of 180-210 s-1 at pH 6.2 in 0.01% lauryl maltoside at 20 degrees C. Yeast cytochrome c oxidase prepared by any of several previously published methods which use Triton X-100 contains nine subunits. The enzyme isolated in lauryl maltoside contains these same nine different polypeptides and three others, including homologues of subunits VIa and VIb of the mammalian enzyme. 相似文献
4.
Tissue-specificity overrides species-specificity in cytoplasmic cytochrome c oxidase polypeptides 总被引:4,自引:0,他引:4
With a high-resolving dodecyl sulfate electrophoretic system rat liver cytochrome c oxidase was separated into 13 different polypeptides. An antiserum against rat liver holocytochrome c oxidase immunoreacted with all 13 polypeptides, as demonstrated by immunofluorescence after transfer of the separated Coomassie blue-stained bands on nitrocellulose and coupling with FITC-protein A ("western blot"). Polypeptide-specific antisera reacted only with their corresponding polypeptides indicating that the various protein bands are represented by individual polypeptides. From total proteins of rat liver, kidney, heart, spleen and skeletal muscle mitochondria, only the cytochrome c oxidase polypeptides showed immunofluorescence with an antiserum against the rat liver holoenzyme. In contrast to the polypeptide from liver, polypeptide VIa from heart and skeletal muscle showed little or no reactivity, indicating a tissue-specificity of this polypeptide. Mitochondrial proteins from pig, bovine and blackbird heart were incubated with an antiserum against the rat liver holoenzyme. Immunoreaction was found with most cytochrome c oxidase polypeptides but not with polypeptide VIa. This result demonstrates less immunological relationship between tissue-specific polypeptides (VIa, VIIa and VIII) of the same species than between tissue-unspecific polypeptides of different species. 相似文献
5.
Cytochrome c oxidase isolated from pig liver and heart was incubated with 1-ethyl-3-[3-(dimethylamino) propyl]carbodiimide and [14C]glycine ethyl ester in the presence and absence of cytochrome c. Labelling of individual subunits was determined after separation of the enzyme complexes into 13 polypeptides by SDS-gel electrophoresis. Polypeptide II and additional but different polypeptides were labelled in the liver and in the heart enzyme. Labelling of polypeptide II and of some other polypeptides could be partially or completely suppressed by cytochrome c. From the data two conclusions can be drawn: In addition to polypeptide II, other polypeptides take part in the binding of cytochrome c to cytochrome c oxidase; the binding domain for cytochrome c is different in pig liver and heart cytochrome c oxidase.Cytochrome c oxidase isozymeCytochrome c binding domain1-Ethyl-3-(3-dimethylaminopropyl)carbodiimideTissue specificity 相似文献
6.
Cytochrome c oxidase was isolated from rat liver either by affinity chromatography on cytochrome-c--Sepharose 4B or by chromatography on DEAE-Sepharose. Dodecyl sulfate gel electrophoresis of both preparations showed the same subunit pattern consisting of 13 different polypeptides. Kinetic analysis of the two preparations gave a higher Vmax for the enzyme isolated by chromatography on DEAE-Sephacel. Specific antisera were raised in rabbits against nine of the ten nuclear endoded subunits. A monospecific reaction of each antiserum with its corresponding subunit was obtained by Western blot analysis, thus excluding artificial bands in the gel electrophoretic pattern of the isolated enzyme due to proteolysis, aggregation or conformational modification of subunits. With an antiserum against rat liver holocytochrome c oxidase a different reactivity was found by Western blot analysis for subunits VIa and VIII between isolated cytochrome c oxidases from pig liver or kidney and heart or skeletal muscle. For a quantitative analysis of immunological differences a nitrocellulose enzyme-linked immunosorbent assay was developed. Monospecific antisera against 12 of the 13 subunits of rat liver cytochrome c oxidase were titrated with increasing amounts of total mitochondrial proteins from different rat tissues dissolved in dodecyl sulfate and dotted on nitrocellulose. The absorbance of a soluble dye developed by the second peroxidase-conjugated antibody was measured. From the data the following conclusions were obtained: (a) The mitochondrial encoded catalytic subunits I-III of cytochrome c oxidase are probably identical in all rat tissues. (b) All nine investigated nuclear encoded subunits of cytochrome c oxidase showed immunological differences between two or more tissues. Large immunological differences were found between liver, kidney or brain and heart or skeletal muscle. Minor but significant differences were observed for some subunits between heart and skeletal muscle and between liver, kidney and brain. (c) Between corresponding nuclear encoded subunits of cytochrome c oxidase from fetal and adult tissues of liver, heart and skeletal muscle apparent immunological differences were observed. The data could explain cases of fatal infantile myopathy due to cytochrome c oxidase deficiency. 相似文献
7.
Immunological identification of four different polypeptides in 'subunit VII' of mammalian cytochrome c oxidase 总被引:2,自引:0,他引:2
Rat liver cytochrome c oxidase was separated by SDS-gel electrophoresis into 13 polypeptide bands. Monospecific antisera against the isolated polypeptides VIIa, VIIb and VIIc were raised in rabbits. Cytochrome c oxidase was blotted on nitrocellulose and incubated with the antisera. The antisera reacted only with their corresponding polypeptides, indicating no immunological relationship between polypeptides VIIa, VIIb and VIIc. The data also exclude that these polypeptides are proteolytic breakdown products of larger subunits. 相似文献
8.
R O Poyton E McKemmie C George-Nascimento 《The Journal of biological chemistry》1978,253(18):6303-6306
A two-dimensional electrophoretic method which takes advantage of the "migration anomalies" experienced by some polypeptides on gels of different porosities has been successfully used to resolve the seven subunit polypeptides of yeast cytochrome c oxidase and the nine polypeptides associated with bovine cytochrome c oxidase. The two-dimensional maps provided by this method reveal clear differences between these two cytochrome c oxidases. 相似文献
9.
Kinetic and structural differences between cytochrome c oxidases from beef liver and heart 总被引:2,自引:0,他引:2
1. The cytochrome content of beef liver mitochondria differs from that of beef heart mitochondria by an eightfold lower cytochrome aa3 and a twofold lower cytochrome b and c + c1 content. 2. The kinetic properties of cytochrome c oxidases from beef liver and heart were measured with intact cytochrome c-depleted membranes, deoxycholate-dissolved membranes, and with the isolated enzymes at various cytochrome c concentrations with an oxygen electrode. Under all conditions a higher V was found for the liver enzyme, both for the low-affinity and for the high-affinity binding site for cytochrome c. Differences were also found for the Km of the two enzymes. 3. Isolated beef heart mitochondria contained about twice as much cardiolipin than beef liver mitochondria. The isolated enzymes contained one mole cardiolipin per mole of the heart enzyme, but 2 moles cardiolipin per mole of the liver enzyme. 4. By application of a high performance sodium dodecylsulfate gel electrophoretic system the two isolated enzymes could be separated into 13 different protein components, three of which (polypeptides VIa, VIIa and VIII) were found to differ in their apparent molecular weights. The functional meaning of cytochrome c oxidase isoenzymes in liver and heart is discussed. 相似文献
10.
Functional equivalence of monomeric (shark) and dimeric (bovine) cytochrome c oxidase 总被引:2,自引:0,他引:2
D Bickar A Lehninger M Brunori J Bonaventura C Bonaventura 《Journal of inorganic biochemistry》1985,23(3-4):365-372
Cytochrome c oxidase isolated from hammerhead shark red muscle is monomeric in relation to the dimeric form of isolated bovine cytochrome c oxidase but in other ways bears a close resemblance to the enzyme isolated from mammalian tissue [1, 2]. Comparative studies of shark and bovine cytochrome c oxidase were extended to address the degree of functional similarity between the monomeric (shark) and dimeric (bovine) enzymes in the kinetics of peroxide binding and in the extent to which the catalytic action of the enzymes in vesicles can establish a proton gradient. Although the kinetics of peroxide binding and the proton pumping processes are complex, the dimeric and monomeric forms are quite similar with respect to these functional attributes. The kinetic heterogeneity of the process of peroxide binding is expressed in the shark enzyme as well as in the bovine enzyme, and both types of enzymes in vesicles can generate transmembrane proton gradients. On this basis we conclude that the dimeric state of isolated cytochrome c oxidase from mammalian sources is not essential for its function in vitro. 相似文献
11.
K M Sinjorgo T B Hakvoort I Durak J W Draijer J K Post A O Muijsers 《Biochimica et biophysica acta》1987,890(2):144-150
Human cytochrome c oxidase was isolated in an active form from heart and from skeletal muscle by a fast, small-scale isolation method. The procedure involves differential solubilisation of the oxidase from mitochondrial fragments by laurylmaltoside and KCl, followed by size-exclusion high-performance liquid chromatography. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate showed differences between the subunit VI region of cytochrome c oxidases from human heart and skeletal muscle, suggesting different isoenzyme forms in the two organs. This finding might be of importance in explaining mitochondrial myopathy which shows a deficiency of cytochrome c oxidase in skeletal muscle only. In SDS polyacrylamide gel electrophoresis most human cytochrome c oxidase subunits migrated differently from their bovine counterparts. However, the position of subunits III and IV was the same in the human and in the bovine enzymes. The much higher mobility of human cytochrome c oxidase subunit II is explained by a greater hydrophobicity of this polypeptide than of that of the subunit II of the bovine enzyme. 相似文献
12.
The orientation of the thirteen polypeptides of rat-liver cytochrome c oxidase in the inner mitochondrial membrane was studied by proteolytic digestion of mitoplasts and sonicated particles. After separation by sodium dodecylsulfate gel electrophoresis proteins were transferred on nitrocellulose, and individual polypeptides were identified by incubation with polypeptide-specific antisera, followed by fluorescein-isothiocyanate-conjugated protein A. The three catalytic polypeptides I-III and seven nuclear coded polypeptides (IV, Vb, VIa, VIc, VIIa, VIIb and VIII) were found accessible to proteases from the cytoplasmic phase. Polypeptides II, IV, Va, Vb and VIa were accessible from the matrix phase, indicating a transmembraneous orientation of polypeptides II, IV, Vb and VIa. Together with data on cross-linking and on cytochrome-c-protected labeling of polypeptides, a model of the cytochrome c oxidase complex was developed. It is suggested that the cytochrome c binding site on polypeptide II is surrounded by several nuclear-coded polypeptides, which may modulate the affinity of the enzyme towards cytochrome c. 相似文献
13.
Isolation, subunit composition, and site of synthesis of human cytochrome c oxidase 总被引:13,自引:0,他引:13
Cytochrome c oxidase (ferrocytochrome c:oxygen oxidoreductase, EC 1.9.3.1), the terminal oxidase of the respiratory chain in eucaryotic cells, has been purified from human placenta mitochondria. Seven polypeptides have been identified reproducibly by high-resolution electrophoresis of the enzyme complex through sodium dodecyl sulfate (Na-DodSO4)--urea polyacrylamide gels; these correspond closely in size to the subunits of beef heart cytochrome c oxidase. When HeLa cells, grown in suspension culture, were pulse-labeled with [35S]methionine in the presence of cycloheximide to inhibit cytoplasmic protein synthesis and chased with an excess of unlabeled methionine in the absence of the drug, the mitochondrially synthesized polypeptides were resolved into at least 17 components by NaDodSO4--urea polyacrylamide gel electrophoresis. After labeled HeLa mitochondria were mixed with human placenta mitochondria and the cytochrome c oxidase was isolated, three of the labeled components were found to copurify with the three largest subunits of the complex. We conclude that human cytochrome c oxidase contains seven subunits, the three largest of which are synthesized on mitochondrial ribosomes, while the other four are synthesized in the cytoplasm. 相似文献
14.
R Rizzuto H Nakase B Darras U Francke G M Fabrizi T Mengel F Walsh B Kadenbach S DiMauro E A Schon 《The Journal of biological chemistry》1989,264(18):10595-10600
15.
Isolation of ubiquinol oxidase from Paracoccus denitrificans and resolution into cytochrome bc1 and cytochrome c-aa3 complexes 总被引:17,自引:0,他引:17
An enzyme complex with ubiquinol-cytochrome c oxidoreductase, cytochrome c oxidase, and ubiquinol oxidase activities was purified from a detergent extract of the plasma membrane of aerobically grown Paracoccus denitrificans. This ubiquinol oxidase consists of seven polypeptides and contains two b cytochromes, cytochrome c1, cytochrome aa3, and a previously unreported c-type cytochrome. This c-type cytochrome has an apparent Mr of 22,000 and an alpha absorption maximum at 552 nm. Retention of this c cytochrome through purification presumably accounts for the independence of ubiquinol oxidase activity on added cytochrome c. Ubiquinol oxidase can be separated into a 3-subunit bc1 complex, a 3-subunit c-aa3 complex, and a 57-kDa polypeptide. This, together with detection of covalently bound heme and published molecular weights of cytochrome c1 and the subunits of cytochrome c oxidase, allows tentative identification of most of the subunits of ubiquinol oxidase with the prosthetic groups present. Ubiquinol oxidase contains cytochromes corresponding to those of the mitochondrial bc1 complex, cytochrome c oxidase complex, and a bound cytochrome c. Ubiquinol-cytochrome c oxidoreductase activity of the complex is inhibited by inhibitors of the mitochondrial bc1 complex. Thus it seems likely that the pathway of electron transfer through the bc1 complex of ubiquinol oxidase is similar to that through the mitochondrial bc1 complex. The number of polypeptides present is less than half the number in the corresponding mitochondrial complexes. This structural simplicity may make ubiquinol oxidase from P. denitrificans a useful system with which to study the mechanisms of electron transfer and energy transduction in the bc1 and cytochrome c oxidase sections of the respiratory chain. 相似文献
16.
The mitochondrial encephalomyopathies in man are characterized by heterogeneous defects leading to an impairment in the pathway of aerobic energy production. As a means of investigating the molecular and genetic mechanisms underlying these disorders we have developed a procedure for selecting mammalian cell lines with features resembling the human pathological phenotypes. The principle of the selection is the use of a fluorescent amphiphilic dye, 2,4-(dimethylamino)-1-styrylmethylpyridiniumiodine, a cation showing two main features. Firstly, it is accumulated by mitochondria to an extent correlated with the magnitude of the electrochemical gradient of protons across the mitochondrial inner membrane. Secondly, upon irradiation with UV light, it gives rise to formation of free radicals, which inflict damage to the cell. Mutant cells with an impairment in oxidative phosphorylation will have more chance to survive than wild type cells. The selection procedure was applied to a stock of mutagenized Chinese hamster ovary cells. After subcloning of the cells which survived the selection procedure, twenty-six independent clones were isolated. Eighteen of the clones had a partial deficiency of cytochrome c oxidase ranging from 30 to 60% of the activity in control cells. The properties of two of the clones are described. One clone has been cultured under non-selective conditions for at least 12 months with retention of the partial deficiency of cytochrome c oxidase. 相似文献
17.
Sequence homologies and structural similarities between the polypeptides of yeast and beef heart cytochrome c oxidase 总被引:5,自引:0,他引:5
The homologous polypeptides in yeast and beef heart cytochrome c oxidase have been identified by sequence comparisons and structural similarities. The properties of individual polypeptides have been used to specify which components are extrinsic, and which intrinsic and bilayer spanning, in the cytochrome c oxidase complex. 相似文献
18.
We have studied the mobility of yeast mitochondrial translation products during electrophoresis on polyacrylamide gels of different composition and found that these polypeptides can be divided into two groups. One, to which subunit II of cytochrome c oxidase belongs, behaves normal as all water-soluble reference proteins. The other, to which cytochrome b and subunits I and III of cytochrome c oxidase belong, shows a free electrophoretic mobility about twice as fast as the first group. Conditions have been found to separate cytochrome c1 from cytochrome b. 相似文献
19.
Cytochrome c release from mitochondria induces caspase activation in cytosols; however, it is unclear whether the redox state of cytosolic cytochrome c can regulate caspase activation. By using cytosol isolated from mammalian cells, we find that oxidation of cytochrome c by added cytochrome oxidase stimulates caspase activation, whereas reduction of cytochrome c by added tetramethylphenylenediamine (TMPD) or yeast lactate dehydrogenase/cytochrome c reductase blocks caspase activation. Scrape-loading of cells with this reductase inhibited caspase activation induced by staurosporine. Similarly, incubating intact cells with ascorbate plus TMPD to reduce intracellular cytochrome c strongly inhibited staurosporine-induced cell death, apoptosis, and caspase activation but not cytochrome c release, indicating that cytochrome c redox state can regulate caspase activation. In homogenates from healthy cells cytochrome c was rapidly reduced, whereas in homogenates from apoptotic cells added cytochrome c was rapidly oxidized by some endogenous process. This oxidation was prevented if mitochondria were removed from the homogenate or if cytochrome oxidase was inhibited by azide. This suggests that permeabilization of the outer mitochondrial membrane during apoptosis functions not just to release cytochrome c but also to maintain it oxidized via cytochrome oxidase, thus maximizing caspase activation. However, this activation can be blocked by adding TMPD, which may have some therapeutic potential. 相似文献
20.
In order to obtain information on the role of subunit III in the function and aggregation state of cytochrome c oxidase, the kinetics of ferrocytochrome c oxidation by the bovine cytochrome c oxidase depleted of its subunit III were studied and compared with those of the oxidase isolated from P. denitrificans which contains only two subunits. The aggregation state of both enzymes dispersed in dodecyl maltoside was also compared. The two-subunit oxidase from P. denitrificans gave linear Eadie-Hofstee plots and the enzyme resulted to be monomeric (Mr = 82 000) both, in gel filtration and sucrose gradient centrifugation studies. The bovine heart subunit III depleted enzyme, under conditions when the P. denitrificans cytochrome c oxidase was in the form of monomers, was found to be dimeric by sucrose gradient centrifugation analysis. At lower enzyme concentrations monomers were, however, detected by gel filtration. Depletion of subunit III was accompanied by the loss of small polypeptides (VIa, VIb and VIIa) and of almost all phospholipid (1-2 molecules were left per molecule of enzyme). The electron-transfer activity of the subunit III-depleted enzyme showed a monophasic Eadie-Hofstee plot, which upon addition of phospholipids became non-linear, similar to that of the control bovine cytochrome c oxidase. One of the roles of subunit III may be that of stabilising the dimers of cytochrome c oxidase. Lack of this subunit and loss of phospholipid is accompanied by a change in the kinetics of electron transfer, which might be the consequence of enzyme monomerisation. 相似文献