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1.
The Cytochromes of Prototheca zopfii   总被引:2,自引:2,他引:0       下载免费PDF全文
The respiratory pigments of Prototheca zopfii include seven cytochromes: two c-type cytochromes, a soluble c(549) and a membrane bound c(551); three b-type cytochromes, b(555), b(559) and b(564); and cytochromes a and a3. Cytochromes a and a3 could be resolved spectrally in the α-band region by reducing the cells in the presence of methanol and cyanide. Methanol shifted the absorption maximum of cytochrome a from 598 to 603 nanometers and permitted dithionite (or substrate) to reduce the cyanide-cytochrome a3 complex to give a well defined 595-nanometer absorption band. Methanol did not interfere with CO binding by cytochrome a3, and CO did not alter the methanol effect on cytochrome a. Azide and cyanide, which partially inhibited exogenous respiration, stimulated endogenous respiration. Frozen steady states of the electron transport chain in the presence of cyanide and azide indicated that the stimulation by these inhibitors was due to an increased autooxidation of one of the b-type cytochromes, possibly b(564).  相似文献   

2.
1. Purified mitochondria have been prepared from wild type Paramecium tetraurelia and from the mutant Cl1 which lacks cytochrome aa3. Both mitochondrial preparations are characterized by cyanide insensitivity. Their spectral properties and their redox potentials have been studied.2. Difference spectra (dithionite reduced minus oxidized) of mitochondria from wild type P. tetraurelia at 77 K revealed the α peaks of b-type cytochrome(s) at 553 and 557 nm, of c-type cytochrome at 549 nm and a-type cytochrome at 608 nm. Two α peaks at 549 and 545 nm could be distinguished in the isolated cytochrome c at 77 K. After cytochrome c extraction from wild type mitochondria, a new peak at 551 nm was unmasked, probably belonging to cytochrome c1. The a-type cytochrome was characterized by a split Soret band with maxima at 441 and 450 nm. The mitochondria of the mutant Cl1 in exponential phase of growth differed from the wild type mitochondria in that cytochrome aa3 was absent while twice the quantity of cytochrome b was present. In stationary phase, mitochondria of the mutant were characterized by a new absorption peak at 590 nm.3. Cytochrome aa3 was present at a concentration of 0.3 nmol/mg protein in wild type mitochondria and ubiquinone at a concentration of 8 nmol/mg protein both in mitochondria of the wild type and the mutant Cl1. Cytochrome aa3 was more susceptible to heat than cytochromes b and c,c1.4. CO difference spectra at 77 K revealed two different Co-cytochrome complexes. The first, found only in wild type mitochondria, was a typical CO-cytochrome a3 complex characterized by peaks at 596 and 435 nm and troughs at 613 and 450 nm. The second, found both in mitochondria of the wild type and the mutant, was a CO-cytochrome b complex with peaks at 567, 539 and 420 nm and a trough at 558-549 nm. Both complexes are photo-dissociable.5. Spectral evidence was obtained for interaction of cyanide with the a-type cytochrome (shift of the α peak at 77 K from 608 to 605 nm), but not with the b-type cytochrome.6. The mid-point potentials of the different cytochromes at neutral pH are as follows: cytochrome aa3 235 and 395 mV, cytochrome c,c1 233 mV, cytochromes b 120 mV.  相似文献   

3.
The cytochrome system in eggs and embryos of the sea urchin, Hemicentrotus pulcherrimus, was investigated. Difference spectra of the mitochondrial fraction demonstrated the presence of a complete cytochrome system in unfertilized eggs. Cytochrome levels and the activities of respiratory enzymes were measured in crude extracts of eggs both before and after fertilization. Unfertilized eggs contained cytochromes aa3, b, and c + c1 in a ratio of 1.0:1.8:0.7. Gastrulae contained almost the same amount of cytochromes aa3and b as unfertilized eggs. However, the amount of cytochrome c + c1 in gastrulae was 1.5 times greater than that in unfertilized eggs. The activity of cytochrome oxidase remained unchanged during development. No cytochrome oxidase inhibitor was found in unfertilized eggs. Both antimycin A-sensitive and insensitive NADH-cytochrome c reductase activities increased during development. The activity of succinate-cytochrome c reductase increased during early development, reached a temporary plateau, and then declined at the pluteus stage. These results are discussed in relation to the increase of respiration during early development.  相似文献   

4.
The respiratory chain components of higher plant mitochondria   总被引:5,自引:25,他引:5       下载免费PDF全文
Tightly coupled mitochondria have been prepared from a variety of plant sources: white potato (Solanum tuberosum), Jerusalem artichoke (Heliantus tuberosus), cauliflower buds (Brassica oleracea), and mung bean hypocotyls (Phaseolus aureus). Mitochondria with no appreciable coupling were also prepared from skunk cabbage spadices (Symplocarpus foetidus).

Room temperature difference spectra show that these mitochondria are very similar in the qualitative and quantitative composition of their electron carriers. The different cytochromes are present in the amounts of 0.1 to 0.3 mμmole per mg of mitochondrial protein. The molar ratios of the different electron carriers are, on the average: 0.7:0.7:1.0:3 to 4:10 to 15 respectively for cytochrome aa3, cytochromes b, cytochromes c, flavoproteins, and pyridine nucleotides.

From low temperature difference spectra carried out under particular experimental conditions, it can be deduced that these mitochondria contain 3 b cytochromes whose α bands are located at 552, 557, and 561 mμ, and 2 c cytochromes, one of which, a c1-like cytochrome, is firmly bound to the mitochondrial membrane. Cytochrome oxidase can be optically resolved into its 2 components a and a3.

For all kinds of mitochondria, the rates of oxidation of succinate are similar as well as the turnover of cytochrome oxidase (50-70 sec−1), regardless of the metabolic activities of the tissues. The number of mitochondria per cell appears to be the controlling factor of the intensity of tissue respiration.

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5.
1. Mitochondria of the obligately aerobic ciliate protozoon, Tetrahymena pyriformis strain ST, are unusual in that they possess a cytochrome oxidase system that does not react with reduced mammalian cytochrome c; the presence of cytochromes a603+a3 is masked in the α-band region of spectra by the broad absorption band of cytochrome a620. 2. Other haemoproteins present include cytochromes b560, b556, c553 and c549. 3. The reaction of reduced cytochrome a3 with CO is reversed by flash photolysis, and in the presence of O2 the subsequent oxidation of this cytochrome is followed by that of cytochrome a603. 4. Cytochromes a620 and b560 also react with CO and with KCN; the latter cytochrome corresponds with that designated cytochrome o by other workers. 5. The contribution of cytochrome a603 to difference spectra is revealed by making use of the fact that it does not react with KCN. 6. Cytochrome a620 is unstable, and its α-absorption band is lost from spectra of mitochondria which have been aged or treated with ultrasound, detergents or organic solvents. 7. Possible pathways of electron transport via the several different terminal oxidases in Tetrahymena mitochondria are proposed.  相似文献   

6.
7.
We have isolated and studied cytochromic-deficient mutants of the amylolytic yeast Schwanniomyces castellii in order to study the possible contribution of cytochromes to alternative pathways. Three mutants were found, lacking cytochrome b, a+a 3,or b and a+a 3.All strains presented two alternative pathways, which were induced in the wild strain when cytochromic respiration was suppressed by growth in the presence of inhibitors, or without copper. If cytochromic respiration was absent, the \({\text{Y}}_{_{\bar S}^x } \) yields in aerobiosis were higher than in anaerobiosis. This shows that the alternative pathways play a part in energy conservation. Cytochrome chrome a+a 3did not appear to be directly involved in the alternative pathways.  相似文献   

8.
The enzymatic activities and the cytochrome components of the respiratory chain were investigated with membrane fractions from chemoheterotrophically grown Rhodopseudomonas palustris. Whereas the level of electron transfer carriers was not distinctly affected by a change of the culture conditions, the potential activities of the enzymes were clearly increased when the cells were grown aerobically. Reduced-minus oxidized difference spectra of the membrane fractions prepared from dark aerobically grown cells revealed the presence of three b-type cytochromes b 561, b 560 and b 558, and at least two c-type cytochromes c 556 and c 2 as electron carriers in the electron transfer chain. Cytochrome of a-type could not be detected in these membranes. Reduced plus CO minus reduced difference spectra of the membrane fractions were indicative of cytochrome o, which may be equivalent to cytochrome b 560, appearing in substrate-reduced minus oxidized difference spectra. Cytochrome o was found to be the functional terminal oxidase. CO difference spectra of the high speed supernatant fraction indicated the presence of cytochrome c′. Succinate and NADH reduced the same types of cytochromes. However, a considerable amount of cytochrome b 561 with associated β and γ bands at 531 and 429 nm, respectively, was reducible by succinate, but not by NADH. A substantial fraction of the membrane-bound b-type cytochrome was non-substrate reducible and was found in dithionite-reduced minus substrate-reduced spectra. Cytochrome c 2 may be localized in a branch of the electron transport system, with the branch-point at the level of ubiquinone. The separate pathways rejoined at a common terminal oxidase. Two terminal oxidases with different KCN sensitivity were present in the respiratory chain, one of which was sensitive to low concentrations of KCN and was connected with the cytochrome chain. The other terminal oxidase which was inhibited only by high concentrations of cyanide was located in a branched pathway, through which the electrons could flow from ubiquinone to oxygen bypassing the cytochrome chain.  相似文献   

9.
T.J. Kula  M.I.H. Aleem  David F. Wilson 《BBA》1982,680(2):142-151
(1) Cells of ThiobacillusA2 grown chemoautotrophically on thiosulfate or heterotrophically on succinate with oxygen contained b-, c-, o-, a- and a3-type cytochromes. The amount of cytochrome per mg of cell protein was much greater in thiosulfate-grown cells and differences in the relative concentrations of cytochromes were observed for the different growth conditions. (2) The half-reduction potentials at pH 7.0 (Em,7.0) and spectral maxima of c-, b-, a- and a3-type cytochromes were similar in cells grown aerobically with thiosulfate or with succinate as the growth substrate. (3) The half-reduction potential of the ‘invisible’, or high-potential copper, as determined from the potentiometric behavior of the carbon monoxide-reduced cytochrome a3 complex at pH 8.0, was 365 mV. (4) Reducing equivalents from thiosulfate appear to enter the respiratory chain at the cytochrome c level; however, studies in cell-free extracts were limited due to a loss in respiratory activity with thiosulfate as a substrate upon cell disruption.  相似文献   

10.
Cytochrome P-450 cannot be detected spectrophotometrically in testis mitochondria of untreated rats because of the high cytochrome a3 to Cytochrome P-450 ratio. Injection of human chorionic gonadotrophin (HCG) causes a large increase in mitochondrial cytochrome P-450. After 14 days injection, mitochondrial cytochrome P-450 levels are increased 15- to 30-fold (from 0.007 to 0.134 nmoles/mg protein) over control levels. Levels of cytochrome a + a3 are not altered by this treatment. Mitochondrial cytochrome P-450 can also be demonstrated by injection of HCG into rats which were hypophysectomized 24 days previously. During hypophysectorny the mitochondrial cytochromes c + ci, a + a3 and mitochondrial protein decay with halflives of 14, 16, and 15.5 days, respectively. HCG treatment for 8 days increases mitochondrial cytochrome P-450 (from < 0.003 to 0.24 nmoles/mg protein) without altering the levels of the other mitochondrial cytochromes. The control of cytochrome P-450 levels in the mitochondria by HCG suggests that the level of this key component of cholesterol side-chain cleavage enzyme may be of importance in the regulation of steroidogenesis in the testis.  相似文献   

11.
A minor form of hepatic microsomal cytochrome P-450 has been purified to apparent homogeneity from rats treated with the polychlorinated biphenyl mixture, Aroclor 1254. This newly isolated hemoprotein, cytochrome P-450e, is inducible in rat liver by Aroclor 1254 and phenobarbital, but not by 3-methylcholanthrene. Two other hemoproteins, cytochromes P-450b and P-450c, have also been highly purified during the isolation of cytochrome P-450e based on chromatographic differences among these proteins. By Ouchterlony double-diffusion analysis with antibody to cytochrome P-450b, highly purified cytochrome P-450e is immunochemically identical to cytochrome P-450b but does not cross-react with antibodies prepared against other rat liver cytochromes P-450 (P-450a, P-450c, P-450d) or epoxide hydrolase. Purified cytochrome P-450e is a single protein-staining band in sodium dodecyl sulfate-polyacrylamide gels with a minimum molecular weight (52,500) slightly greater than cytochromes P-450b or P-450d (52,000) but clearly distinct from cytochromes P-450a (48,000) and P-450c (56,000). The carbon monoxide-reduced difference spectral peak of cytochrome P-450e is at 450.6 nm, whereas the peak of cytochrome P-450b is at 450 nm. Ethyl isocyanide binds to ferrous cytochromes P-450e and P-450b to yield two spectral maxima at 455 and 430 nm. At pH 7.4, the 455:430 ratio is 0.7 and 1.4 for cytochromes P-450b and P-450e, respectively. Metyrapone binds to reduced cytochromes P-450e and P-450b (absorption maximum at 445–446 nm) but not cytochromes P-450a, P-450c, or P-450d. Metabolism of several substrates catalyzed by cytochrome P-450e or P-450b reconstituted with NADPH-cytochrome c reductase and dilauroylphosphatidylcholine was compared. The substrate specificity of cytochrome P-450e usually paralleled that of cytochrome P-450b except that the rate of metabolism of benzphetamine, benzo[a]pyrene, 7-ethoxycoumarin, hexobarbital, and testosterone at the 16α-position catalyzed by cytochrome P-450e was only 15–25% that of cytochrome P-450b. In contrast, cytochrome P-450e catalyzed the 2-hydroxylation of estradiol-17β more efficiently (threefold) than cytochrome P-450b. Cytochrome P-450d, however, catalyzed the metabolism of estradiol-17β at the greatest rate compared to cytochromes P-450a, P-450b, P-450c, or P-450e. The peptide fragments of cytochromes P-450e and P-450b, generated by either proteolytic or chemical digestion of the hemoproteins, were very similar but not identical, indicating that these two proteins show minor structural differences.  相似文献   

12.
K.S. Cheah 《BBA》1975,387(1):107-114
1. The cytochrome system in Ascaris muscle mitochondria was further characterized using purer preparations.2. Difference spectra (at 22 °C and ?196 °C) of the mitochondrial preparations using succinate and ascorbate plus N,N,N′,N′-tetramethyl-p-phenylenediamine show that Ascaris muscle mitochondria contain cytochromes c1, c and aa3, and also at least three b-type cytochromes. The b-type cytochrome is the predominant component.3. Cytochrome c and Ascaris cytochrome b-560 can be extracted from the mitochondrial preparations with 150 mM KCl, leaving the membrane-bound cytochromes c1, b and aa3 in the KCl residue.  相似文献   

13.
The rate of incorporation of [14C]aminolevulinic acid (ALA) into cytochrome hemes was used to measure mitochondrial cytochrome synthesis in the fat body of adult male Blaberus discoidalis cockroaches. The hemes of cytochromes aa3+b and c+c1, were chemically separated to observe differential rates in their synthesis and regulation. [14C]ALA was linearly incorporated into cytochrome hemes for at least 8 h. No significant pool of endogenous ALA was detected relative to the amount of administered [14C]ALA. Peak cytochrome synthesis occurred 4 to 6 days after adult emergence. Endocrine disruption by corpora cardiaca-corpora allata extirpation or cervical ligation eliminated the 4-day developmentally related increase in the rate of cytochrome aa3+b synthesis but had no effect on the production of cytochromes c+c1. Injections of corpora cardiaca extracts into cervically ligated animals stimulated the rate of production of cytochromes aa3+b by 2.5 times but did not affect cytochromes c+c1. By comparison, juvenile hormone injections did not affect the rate of synthesis of either cytochrome fraction. These findings indicate that a neurohormone regulates the rate of synthesis of cytochromes a+b in insect fat body mitochondria.  相似文献   

14.
D.L. Knook  J.Van&#x;t Riet  R.J. Planta 《BBA》1973,292(1):237-245
1. The participation of cytochromes in the membrane-bound, nitrate and oxygen respiratory systems of Klebsiella (Aerobacter) aerogenes has been investigated. The membrane preparations contained the NADH, succinate, lactate and formate oxidase systems, and in addition a high respiratory nitrate reductase activity.2. Difference spectra indicated the presence of cytochromes b, a1, d, and o. Cytochromes of the c-type could not be detected in these membranes. Both cytochrome b content and respiratory nitrate reductase activity were the highest in bacteria grown anaerobically in the presence of nitrate.3. Cytochrome b was the only cytochrome which, after being reduced by NADH, could be partially reoxidized anaerobically in the presence of nitrate. Furthermore, nitrate caused a lower aerobic steady state reduction only of cytochrome b.4. NADH oxidase and NADH-linked respiratory nitrate reductase activities were both inhibited by antimycin A, 2-n-heptyl-4-hydroxyquinoline-N-oxide and KCN. NADH oxidase activity was selectively inhibited by CO, while azide was found to inhibit only the respiratory nitrate reductase. In the presence of azide, nitrate did not affect the level of reduction of cytochrome b.5. The evidence presented suggests that cytochrome b is a carrier in the electron transport systems to both nitrate and oxygen; from cytochrome b branching occurs, with one branch linked to the respiratory nitrate reductase and one branch linked to oxidase systems, containing the cytochromes a1, d and o.  相似文献   

15.
Storey BT 《Plant physiology》1972,49(3):314-322
The cytochromes c of mung bean (Phaseolus aureus) mitochondria become reduced when sulfide, a cytochrome oxidase inhibitor free from uncoupling side effects, is added to the aerobic mitochondrial suspension in the absence of added substrate. The cytochromes b remain largely oxidized. Subsequent addition of ATP results in partial oxidation of the cytochromes c and partial reduction of the cytochromes b due to ATP-driven reverse electron transport through the second site of energy conservation, or coupling site, of the respiratory chain. Cytochrome a is also oxidized under these conditions, but there is no concomitant reduction of the flavoprotein components, of ubiquinone, or of endogenous pyridine nucleotide. The reaction is abolished by oligomycin. The reducing equivalents transported from the cytochromes c and a in ATP-driven reverse electron transport are about 2-fold greater than those which appear in the cytochromes b. It is suggested that the equivalents not accounted for are present in a coupling site enzyme at the second site of energy conservation which interacts with the respiratory chain carriers by means of the dithiol-disulfide couple; this couple would not show absorbance changes with redox state over the wavelength range examined. With succinate present, reverse electron transport can be demonstrated at both coupling sites in both the aerobic steady state and in anaerobiosis. ATP-driven reverse electron transport in anaerobiosis maintains cytochrome a 30% oxidized while endogenous pyridine nucleotide is 50% reduced.  相似文献   

16.
The midgut of Hyalophora cecropia actively transports potassium from hemolymph to lumen and the energy for this process appears to be intimately linked to oxidative metabolism. In the present investigation, we monitored concurrently the rate of active transport and the redox levels of the components of the respiratory chain in the intact tissue under a variety of experimental conditions. Approximately equal concentrations of cytochromes a3, a, c and b-557 were found. Other investigators (Pappenheimer, Jr, A. M. and Williams, C. M. (1954) J. Biol. Chem. 209, 915, Shappirio, D. G. and Williams, C. M. (1957) Proc. R. Soc. Lond. Ser. B 147, 233 and Chance, B. and Pappenheimer, Jr, A. M. (1954) J. Biol. Chem. 209, 931) have identified cytochrome b-557 with b5 and found that it exists primarily in an extramitochondrial location.Steady-state experiments demonstrated that all these cytochromes were approximately 50% reduced while active transport proceeded at a high rate in regular cecropia Ringer containing 32 mM KCl. When the potassium concentration was reduced, the active transport decreased and all the cytochromes became more oxidized. Addition of 1 mM cyanide inhibited active transport by 90% and caused a 100% reduction of all cytochromes. Redox state and short circuit current (Isc) kinetics measured as the tissue was made anoxic showed that all the respiratory enzymes, except cytochrome b-557, became fully reduced at a faster rate than the rate of inhibition of the Isc. The rate of cytochrome b-557 reduction followed kinetically the Isc.These observations are interpreted in a scheme where cytochrome b-557 (possibly b5) branches off cytochrome c from the conventional respiratory chain, utilizing cytochrome a3 as the terminal oxidase for both branches. Cytochrome b-557 may be involved in providing a direct link between oxidative metabolism and active transport in the midgut of the silkworm.  相似文献   

17.
The role of cytochromes in photosynthetic electron transfer system has been studied using the pale green mutant of Chlamydomonas reinhardi (ATCC 18302). The existence of cytochromes b563 and f is confirmed, while no significant amount of ascorbate-reducible cytochrome b559 is detected in this mutant. The presence of cytochrome c and a small amount of a-type cytochrome is determined in these cells.  相似文献   

18.
A comparative study of the interaction of various cytochromes c with phospholipid vesicles and with mitochondrial membranes was undertaken. Both mammalian and yeast types of cytochrome c bind preferentially in the oxidized form as evidenced by the midpoint redox potential (Em 7.0) becoming more negative upon binding. Cytochrome c which is reincorporated into cytochrome c-depleted mitochondria is kinetically comparable with the native cytochrome c component; rate of cytochrome b oxidation is maximally restored at ratios of c1:c:a of 1:1:1. Comparison between the electron paramagnetic spectrum of cytochrome c labeled at methionine 65 or cysteine 103 reveals that upon binding to the mitochondrial membrane, the former is immobilized and not the latter. This result suggests that cytochrome c binds to the membrane at the side at which methionine 65 is located.  相似文献   

19.
The cytochrome complement of Methylophilus methylotrophus and its respiratory properties were determined during batch culture and in continuous culture under conditions of methanol-, nitrogen- and O2-limitation. About 35% of the cytochrome c produced by the bacteria was released into the growth medium, and of the remaining cytochrome c about half was membrane-bound and half was soluble. Two cytochromes c were always present on membranes (redox potentials 375mV and 310mV), and these probably correspond to the soluble cytochromes c described previously [Cross & Anthony (1980) Biochem. J. 192, 421–427]. A third minor component of cytochrome c (midpoint potential 356mV) was only detected on membranes of methanol-limited bacteria. M. methylotrophus always contained two membrane-bound cytochromes b with α-band absorption maxima of about 556 and 563nm (measured at room temperature) and midpoint potentials of 110 and 60mV respectively. There appeared to be relatively more of the cytochrome b563 in methanol-limited bacteria. A third b-type cytochrome with an α-band absorption maximum at 558 (at 77K) reacted with CO and had a high midpoint redox potential (260mV); it is thus a potential oxidase and hence is called cytochrome o. The roles of these cytochromes in electron transport were confirmed by investigating the patterns of respiratory inhibition. It is proposed that two cytochromes are physiological oxidases: cytochrome a+a3, which is present only in methanol-limited conditions, and the cytochrome o, which is induced 10-fold in conditions of methanol excess. Schemes for electron transport from methanol and NAD(P)H to O2 in M. methylotrophus under various limitations are proposed. Spectra and potentiometric titrations of cytochromes in whole cells and membranes of M. methylotrophus grown under various nutrient limitations have been deposited as Supplementary Publication SUP 50111 (10 pages) at the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1978) 169, 5.  相似文献   

20.
Cytochrome oxidase was purified twentyfold from mitochondria of seedlings of wheat genotypes 28, 31 MS, and 31 MS/28. The enzyme of the hybrid exceeded in activity the parental enzymes. Mixtures of cytochrome oxidase of the parents exhibited complementation in that they approached the activity of the hybrid cytochrome oxidase. Hybrid mitochondria also exhibited heterosis in NADH: cytochrome c reductase activity. Complementation by parent mitochondria was observed for this enzyme also. The Michaelis constant of cytochrome oxidase and NADH: cytochrome reductase was markedly less in the hybrid and the mixture than in the parents. Difference spectra revealed the following: strain 28 had cytochromes a and b but was deficient in cytochrome c; strain 31 MS had cytochromes b and c but no a; the hybrid had all three cytochromes, as did the mixture. The relationship of cytochromes to heterosis and complementation is considered.This work was supported by DeKalb AgResearch, Inc.  相似文献   

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