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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.
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
3.
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
4.
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
5.
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
6.
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
7.
The complete amino acid sequences of subunits VII and VIIa from yeast cytochrome c oxidase are reported. Subunits VII and VIIa are 57 residues (Mr = 6603) and 54 residues (Mr = 6303) in length, respectively. Both polypeptides are amphiphilic, have an internal hydrophobic section and hydrophilic NH2 and COOH termini, and terminate at their COOH termini with a basic amino acid. This structural motif is similar to that possessed by subunit VIII of yeast cytochrome c oxidase. All three polypeptides have hydrophobic sections which are long enough to span the inner membrane; all three polypeptides lack methionine at their NH2 termini; and all three polypeptides have COOH termini which could result from proteolysis by a protease with trypsin or cathepsin B-like activity. These observations raise the interesting possibility that subunits VII, VIIa, and VIII are transmembranous polypeptides which are processed at both their NH2 and COOH termini during their biogenesis. 相似文献
8.
Two water-soluble carbodiimides, differing in molecular dimensions, have been used to characterize the cytochrome c binding site of bovine heart cytochrome c oxidase. Several polypeptide components of the enzyme contain acidic residues which are modified by these reagents. Carboxyl groups present in subunit II, VII and polypeptide c, are protected from modification when cytochrome c, equimolar to oxidase, is added and they can cross-link to the substrate once activated by the carbodiimide. Comparison of the modification patterns suggest that the most reactive residues are located on subunit II and VII, the former being also more exposed. The data obtained indicate that even though subunit II plays the major role in binding cytochrome c, at least two other lower Mr polypeptides contribute to the cytochrome c binding domain. 相似文献
9.
The binding of rat liver cytochrome c oxidase to phenyl-Sepharose and various alkyl and omega-aminoalkyl agarose gels has been studied. Deoxycholate-solubilized cytochrome c oxidase was tightly bound to hexyl, octyl, omega-aminohexyl, omega-aminooctyl agarose as well as to phenyl-Sepharose. This hydrophobic interaction was used for the purification of cytochrome c oxidase. The enzyme which was eluted from phenyl-Sepharose was devoid of NADH (NADPH)-acceptor reductase activities. The heme a content was 15.4 nmol per mg of protein. The purified enzyme was resolved into seven polypeptides upon polyacrylamide gel electrophoresis in sodium dodecylsulfate with molecular weights of 40,000, 23,200, 21,500, 14,500, 12,600, 8900, and 4900. Antibodies raised in rabbits against the pure enzyme did not cross-react with cytochrome c oxidases from either beef heart or yeast mitochondria. Cytochrome c oxidase bound to octyl-Sepharose or phenyl-Sepharose exhibited a very low catalytic activity. The possible modes of interaction of cytochrome c oxidase with the hydrophobic ligands are discussed. 相似文献
10.
A protocol has been worked out for separating all thirteen different polypeptides in the beef heart cytochrome c oxidase complex from a single aliquot of enzyme. This involves an initial separation of polypeptides by gel filtration on a Biogel P-60 column in SDS, a step which purifies subunits CIV and CVIII and gives mixtures of CV + CVI, ASA, AED and STA, as well as CVII, CIX and IHQ. These mixtures are then resolved by reverse-phase high-performance liquid chromatography. The separation procedures have been applied to fetal heart cytochrome c oxidase of gestation between 100 and 200 days. No differences were found in the N-terminal sequences of any of the cytoplasmically made subunits or in the entire sequence of CIX between late fetal and adult forms of the enzyme. 相似文献
11.
Separation of mammalian cytochrome c oxidase into 13 polypeptides by a sodium dodecyl sulfate-gel electrophoretic procedure 总被引:20,自引:0,他引:20
A sodium dodecyl sulfate-gel electrophoretic procedure which allows the separation of isolated cytochrome c oxidase from different mammalian sources into 13 different polypeptides is described. Application of the silver-staining procedure results in the same protein pattern as obtained by Coomassie blue staining. From the correlation of the gel bands with 12 isolated polypeptides from which the complete amino acid sequence is known, it is concluded that mammalian cytochrome c oxidase consists of 13 different polypeptides which can all be separated by the described procedure. 相似文献
12.
Church C Goehring B Forsha D Wazny P Poyton RO 《The Journal of biological chemistry》2005,280(3):1854-1863
The biogenesis of multimeric protein complexes of the inner mitochondrial membrane in yeast requires a number of nuclear-coded ancillary proteins. One of these, Pet100p, is required for cytochrome c oxidase. Previous studies have shown that Pet100p is not required for the synthesis, processing, or targeting of cytochrome c oxidase subunits to the mitochondrion nor for heme A biosynthesis. Here, we report that Pet100p does not affect the localization of cytochrome c oxidase subunit polypeptides to the inner mitochondrial membrane but instead functions after they have arrived at the inner membrane. We have also localized Pet100p to the inner mitochondrial membrane in wild type cells, where it is present in a subassembly (Complex A) with cytochrome c oxidase subunits VII, VIIa, and VIII. Pet100p does not interact with the same subunits after they have been assembled into the holoenzyme. In addition, we have identified two subassemblies that are present in pet100 null mutant cells: one subassembly (Complex A') is composed of subunits VII, VIIa, and VIII but not Pet100p, and another subassembly (Complex B) is composed of subunits Va and VI. Because pet100 null mutant cells lack assembled cytochrome c oxidase but accumulate Complexes A' and B it appears likely that these subassemblies of cytochrome c oxidase subunits are intermediates along an assembly pathway for holocytochrome c oxidase and that Pet100p functions in this pathway to facilitate the interaction(s) between Complex A' and other cytochrome c oxidase subassemblies and subunits. 相似文献
13.
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
14.
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
15.
The structure and the orientation of cytochrome c oxidase molecules in crystalline cytochrome c oxidase membranes (Vanderkooi, G., Senior, A.E., Capaldi, R.A., and Hayashi, H. (1972) Biochim. Biophys. Acta 274, 38-48) were studied by image analysis of electron micrographs and by reacting the crystalline preparations with immune gamma-globulins against individual cytochrome c oxidase subunits. Binding of gamma-globulins to the membranes was detected by the following two methods: (a) electrophoretic identification of gamma-globulin polypeptides in the washed membranes; (b) electron microscopic examination of the negatively stained membranes. The membranes bound immune gamma-globulins against subunit IV (which faces the matrix side in intact mitochondria) but failed to bind immune gamma-globulins against subunits II + III (which face the outer side of the inner membrane in intact mitochondria). In contrast, solubilized cytochrome c oxidase bound either of the two immune gamma-globulins. All cytochrome c oxidase molecules in the crystalline membranes are thus asymmetrically arranged so that subunit IV faces outward and subunits II + III face toward the interior. This orientation is opposite to that found with intact mitochondria. The data also suggest that the crystalline membranes form closed vesicles which are impermeable to externally added gamma-globulins. 相似文献
16.
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
17.
S D Power M A Lochrie T E Patterson R O Poyton 《The Journal of biological chemistry》1984,259(10):6571-6574
The amino acid sequence of subunit VIII from yeast cytochrome c oxidase is reported. This 47-residue (Mr = 5364) amphiphilic polypeptide has a polar NH2 terminus, a hydrophobic central section, and a dilysine COOH terminus. An analysis of local hydrophobicity and predicted secondary structure along the peptide chain predicts that the hydrophobic central region is likely to be transmembranous. Subunit VIII from yeast cytochrome c oxidase exhibits 40.4% homology to bovine heart cytochrome c oxidase subunit VIIc , at the level of primary structure. Secondary structures and hydrophobic domains predicted from the sequences of both polypeptides are also highly conserved. From the location of hydrophobic domains and the positions of charged amino acid residues we have formulated a topological model for subunit VIII in the inner mitochondrial membrane. 相似文献
18.
The syntheses of two new radioactive probes derived from cardiolipin and phosphatidylcholine are reported. These probes are derivatives of natural lipids and contain an amine-specific benzaldehyde in the head-group region. This functional group allows a choice of timing of the reaction (e.g., after equilibration and detergent removal) because an irreversible covalent bond is formed only upon the addition of reducing agent. These probes, as well as a benzaldehyde analogue of phosphatidic acid, and a water-soluble benzaldehyde reagent were covalently attached to bovine heart cytochrome c oxidase. After reconstitution into vesicles, the lipid-benzaldehyde probes selectively incorporated into the smaller polypeptides of the enzyme, while the remaining subunits (I-IV) exhibited little incorporation of label. The accessibility of amine groups labeled under the conditions used here was independent of the structural and charge differences between the benzaldehyde probes. This suggests that all three lipid probes react with polypeptides of the cytochrome c oxidase complex at general contact sites for membrane phospholipids. A water-soluble benzaldehyde reagent predominantly labeled subunits IV, Va, and Vb and polypeptides of VII-VIII. A comparison of these results facilitates a more refined view of the disposition of polypeptides of cytochrome c oxidase in respect to the lipid and aqueous phases. 相似文献
19.
Cloning and sequencing of the Paracoccus denitrificans ccmG gene indicates that it codes for a periplasmic protein–disulphide oxidoreductase; the presence of the sequence Cys-Pro-Pro-Cys at the CcmG active site suggests that it may act in vivo to reduce disulphide bonds rather than to form them. A CcmG–PhoA fusion confirmed the periplasmic location. Disruption of the ccmG gene resulted in not only the expected phenotype of pleiotropic deficiency in c -type cytochromes, but also loss of spectroscopically detectable cytochrome aa 3 , cytochrome c oxidase and ascorbate/TMPD oxidase activities; there was also an enhanced sensitivity to growth inhibition by some component of rich media and by oxidized thiol compounds. Dithiothreitol promoted the growth of the ccmG mutant on rich media and substantially restored spectroscopically detectable cytochrome aa 3 and cytochrome c oxidase activity, although it did not restore c -type cytochrome biogenesis. Assembly of the disulphide-bridged proteins methanol dehydrogenase and Escherichia coli alkaline phosphatase was unaffected in the ccmG mutant. It is proposed that P. denitrificans CcmG acts in vivo to reduce protein–disulphide bonds in certain protein substrates including c -type cytochrome polypeptides and/or polypeptides involved in c -type cytochrome biogenesis. 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