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1.
Cytochemical and ultrastructural analysis of wild-type cells of Saccharomyces cerevisiac, grown aerobically in a glucose-limited chemostat, shows that cytochrome c peroxidase is localized between the membranes of the cristae, that is, in the intracristal space. This enzyme is thus positioned appropriately within the organelle to act as an alternate terminal oxidase for the respiratory chain. The proximity of the peroxidase to major sites of generation of its two substrates may account for the small leakage of hydrogen peroxide from yeast mitochondria, as compared with the larger outflow from mammalian mitochondria.In the cytoplasmic petite mutant, gross distortion of promitochondrial membrane arrangement is evident. Nevertheless, cytochrome c peroxidase activity is present in the same amounts as is found in wildtype cell, and is localized predominantly within annuli of membrane which constitute the promitochondria in these cells.No unequivocal evidence was obtained for the localization of catalase in microbodies or other organelles in either wild-type or petite cells.  相似文献   

2.
Torulopsis pintolopesii is an indigenous yeast that colonizes the secreting epithelia in the stomachs of mice and rats. A wild-type strain of this microbe was isolated and identified. To attempt to learn characteristics of the yeast that are advantageous to it in colonizing its natural habitat in vivo, we examined some aspects of its nutrition and energy-yielding metabolism and some environmental conditions that influence its growth in vitro. The yeast appeared to be limited in the compounds it can utilize as carbon and nitrogen sources. It grew best at 37 degrees C and did not grow at 23 or 43 degrees C. It grew optimally at neutral pH but could grow aerobically at pH values as low as 2.0 and anaerobically at pH values as low as 3.4. As assessed by measurements of growth rates and yield coefficients, it grew better aerobically than anaerobically. When grown aerobically, it had a cyanide-sensitive system for taking up O(2) and tested positively for cytochrome c oxidase activity. A petite mutant strain isolated from the wild-type strain had a growth rate and yield coefficient when incubated aerobically that were essentially the same as those of the wild-type parent grown anaerobically. Likewise similar to the wild-type parent grown anaerobically, the petite strain, though incubated aerobically, did not take up O(2). Yeast-free mice associated with either the wild-type or the petite mutant strain were colonized at essentially the same rates and to similar final population levels by both strains. The yeast's capacity to respire may be of little advantage to it in its natural environment. By contrast, its abilities to grow best at 37 degrees C and to grow at low pH values are undoubtedly advantageous characteristics in this respect. The limitations in its carbon and nitrogen nutrition are difficult to evaluate as ecological factors in its colonization of the natural habitat.  相似文献   

3.
Light and heavy membrane fractions have been isolated by equilibrium sucrose density centrifugation from Rhodopseudomonas capsulata 938 GCM grown aerobically in the dark (chemotrophically) and anaerobically in the light (phototrophically). The densities of the light and heavy fractions from phototrophic cells were 1.1004 to 1.1006 and 1.1478, respectively, and the densities of the light and heavy fractions from chemotrophic cells were 1.0957 to 1.0958 and 1.1315, respectively. Both fractions were active in photochemical and respiratory functions and in electron transport-coupled phosphorylation. The light membrane fraction isolated from chemotrophic cells contained the reaction center and the light-harvesting pigment-protein complex B 870, but not the variable light-harvesting complex B 800-850. A small amount of the complex B 800-850 was present in the light fraction isolated from phototrophically grown cells, but it was not energetically coupled to the photosynthetic apparatus. From inhibitor studies, difference spectroscopy, and measurement of enzyme activities it was tentatively concluded that the light membrane fraction contains only the reduced nicotinamide adenine dinucleotide-oxidizing electron transport chain having a KCN-insensitive, low-potential cytochrome c oxidase, whereas the heavy fraction contains additionally the succinate dehydrogenase and a high-potential cytochrome b terminal oxidase sensitive to KCN. The light membrane fraction was more labile than the heavy fraction in terms of phosphorylating activity.  相似文献   

4.
Torulopsis pintolopesii is an indigenous yeast that colonizes the secreting epithelia in the stomachs of mice and rats. A wild-type strain of this microbe was isolated and identified. To attempt to learn characteristics of the yeast that are advantageous to it in colonizing its natural habitat in vivo, we examined some aspects of its nutrition and energy-yielding metabolism and some environmental conditions that influence its growth in vitro. The yeast appeared to be limited in the compounds it can utilize as carbon and nitrogen sources. It grew best at 37°C and did not grow at 23 or 43°C. It grew optimally at neutral pH but could grow aerobically at pH values as low as 2.0 and anaerobically at pH values as low as 3.4. As assessed by measurements of growth rates and yield coefficients, it grew better aerobically than anaerobically. When grown aerobically, it had a cyanide-sensitive system for taking up O2 and tested positively for cytochrome c oxidase activity. A petite mutant strain isolated from the wild-type strain had a growth rate and yield coefficient when incubated aerobically that were essentially the same as those of the wild-type parent grown anaerobically. Likewise similar to the wild-type parent grown anaerobically, the petite strain, though incubated aerobically, did not take up O2. Yeast-free mice associated with either the wild-type or the petite mutant strain were colonized at essentially the same rates and to similar final population levels by both strains. The yeast's capacity to respire may be of little advantage to it in its natural environment. By contrast, its abilities to grow best at 37°C and to grow at low pH values are undoubtedly advantageous characteristics in this respect. The limitations in its carbon and nitrogen nutrition are difficult to evaluate as ecological factors in its colonization of the natural habitat.  相似文献   

5.
1. During anaerobic glucose de-repression the respiration rate of whole cells of Saccharomyces carlsbergensis remained constant and was insensitive to antimycin A but was inhibited by 30% by KCN. Aeration of cells for 1 h led to increased respiration rate which was inhibited by 80% by antimycin A or KCN. 2. Homogenates were prepared from sphaeroplasts of anaerobically grown, glucose de-repressed cells and the distribution of marker enzymes was investigated after zonal centrifugation on sucrose gradients containing MgCl(2). These homogenates contained no detectable cytochrome c oxidase or catalase activity. The complex density distributions of NADH- and NADPH-cytochrome c oxidoreductases and adenosine triphosphatase(s) [ATPase(s)] were very different from those of anaerobically grown, glucose-repressed cells. 3. The specific activity of total ATPase was lowered and sensitivity to oligomycin decreased from 58 to 7% during de-repression. 4. Cytochrome c oxidase and catalase activities were detectable in homogenates of cells after 10min aeration. Zonal centrifugation indicated complex, broad sedimentable distributions of all enzyme activities assayed; the peaks of activity were at 1.27g/ml. 5. Centrifugation of homogenates of cells adapted for 30min and 3 h indicated a shift of density of the major sedimentable peak from 1.25g/ml (30min) to 1.235g/ml (3 h). After 30min adaptation a minor zone of oligomycin-sensitive ATPase and 15% of the total cytochrome c oxidase activities were detected at rho=1.12g/l; these particles together with those of higher density containing cytochrome c oxidase, ATPase and NADH-cytochrome c oxidoreductase activities were all sedimented at 10(5)g-min. 6. Electron microscopy indicated that the mitochondria-like structures of anaerobically grown, glucose-de-repressed cells were similar to those of repressed cells. After 10min of respiratory adaptation highly organized mitochondria were evident which resembled the condensed forms of mitochondria of aerobically grown, glucose-de-repressed cells. High-density zonal fractions of homogenates of cells after adaptation also contained numerous electron-dense vesicles 0.05-0.2mum in diameter. 7. The possibility that the ;promitochondria' of anaerobically grown cells may not be the direct structural precursors of fully functional mitochondria is discussed.  相似文献   

6.
In the preceding paper (Ross, E., and Schatz, G. (1976) J. Biol. Chem. 251, 1991-1996) yeast cytochrome c1 was characterized as a 31,000 dalton polypeptide with a covalently bound heme group. In order to determine the site of translation of this heme-carrying polypeptide, yeast cells were labeled with [H]leu(be under the following conditions: (a) in the absence of inhibitors, (b) in the presence of acriflavin (an inhibitor of mitochondrial translation), or (c) in the presence of cycloheximide (an inhibitor of cytoplasmic translation). The incorporation of radioactivity into the hemeprotein was measured by immunoprecipitating it from mitochondrial extracts and analyzing it by dodecyl sulfate-polyacrylamide gel electrophoresis. Label was incorporated into the cytochrome c1 apoprotein only in the presence of acriflavin or in the absence of inhibitor, but not in the presence of cycloheximide. Cytochrome c1 is thus a cytoplasmic translation product. This conclusion was further supported by the demonstration that a cytolasmic petite mutant lacking mitochondrial protein synthesis still contained holocytochrome c1 that was indistinguishable from cytochrome c1 of wild type yeast with respect to molecular weight, absorption spectru, the presence of a covalently bound heme group, and antigenic properties. Cytochrome c1 in the mitochondria of the cytoplasmic petite mutant is firmly bound to the membrane, and its concentration approaches that typical of wild type mitochondria. However, its lability to proteolysis appeared to be increased. A mitochondrial translation product may thus be necessary for the correct conformation or orientation of cytochrome c1 in the mitochondrial inner membrane. Accumulation of cytochrome c1 protein in mitochondria is dependent on the abailability of heme. This was shown with a delta-aminolevulinic acid synthetase-deficient yeast mutant which lacks heme and any light-absorbing peaks attributable to cytochromes. Mitochondria from mutant cells grown without added delta-aminolevulinic acid contained at least 20 times less protein immunoprecipitable by cytochrome c1-antisera than mitochondria from cells grown in the presence of the heme precursor. Similarly, the respiration-deficient promitochondria of anaerobically grown wild type cells are almost completely devoid of material cross-reacting with cytochrome c1-antisera. A 105,000 X g supernatant of aerobically grown wild type cells contains a 29,000 dalton polypeptide that is precipitated by cytochrome c1-antiserum but not by nonimmune serum. This polypeptide is also present in high speed supernatants from the heme-deficient mutant or from anaerobically gorwn wild type cells. The possible identity of this polypeptide with soluble apocytochrome c1 is being investigated.  相似文献   

7.
It has been a long-standing hypothesis that the endosymbiotic rhizobia (bacteroids) cope with a concentration of 10 to 20 nM free O2 in legume root nodules by the use of a specialized respiratory electron transport chain terminating with an oxidase that ought to have a high affinity for O2. Previously, we suggested that the microaerobically and anaerobically induced fixNOQP operon of Bradyrhizobium japonicum might code for such a special oxidase. Here we report the biochemical characteristics of this terminal oxidase after a 27-fold enrichment from membranes of anaerobically grown B. japonicum wild-type cells. The purified oxidase has TMPD (N,N,N',N'-tetramethyl-p-phenylenediamine) oxidase activity as well as cytochrome c oxidase activity. N-terminal amino acid sequencing of its major constituent subunits confirmed that presence of the fixN,fixO, and fixP gene products. FixN is a highly hydrophobic, heme B-binding protein. FixO and FixP are membrane-anchored c-type cytochromes (apparent Mrs of 29,000 and 31,000, respectively), as shown by their peroxidase activities in sodium dodecyl sulfate-polyacrylamide gels. All oxidase properties are diagnostic for it to be a member of the cbb3-type subfamily of heme-copper oxidases. The FixP protein was immunologically detectable in membranes isolated from root nodule bacteroids, and 85% of the total cytochrome c oxidase activity in bacteroid membranes was contributed by the cbb3-type oxidase. The Km values for O2 of the purified enzyme and of membranes from different B. japonicum wild-type and mutant strains were determined by a spectrophotometric method with oxygenated soybean leghemoglobin as the sole O2 delivery system. The derived Km value for O2 of the cbb3-type oxidase in membranes was 7 nM, which is six- to eightfold lower than that determined for the aerobic aa3-type cytochrome c oxidase. We conclude that the cbb3-type oxidase supports microaerobic respiration in endosymbiotic bacteroids.  相似文献   

8.
M C Liu  W J Payne  H D Peck  Jr    J LeGall 《Journal of bacteriology》1983,154(1):278-286
Pseudomonas perfectomarinus (ATCC 14405) is a facultative anaerobe capable of either oxygen respiration or anaerobic nitrate respiration, i.e., denitrification. A comparative study of the electron transfer components of cells revealed five c-type cytochromes and cytochrome cd in the soluble fraction from anaerobically grown cells and four c-type cytochromes in the soluble fraction from aerobically grown cells. Purification procedures yielded three c-type cytochromes (designated c-551, c-554, and acidic c-type) from both kinds of cells as indicated by similarities in absorption spectra, molecular weight, and electrophoretic mobility. Cytochrome cd, a diheme c-type cytochrome (cytochrome c-552), and a split-alpha c-type cytochrome were recovered only from anaerobically grown cells. A c-type cytochrome with a low ratio of alpha to beta absorption peak heights was uniquely present in the aerobically grown cells. Liquid N2 temperature absorption spectroscopy on the membrane fraction from anaerobically grown cells revealed residual cytochrome cd as well as differences in the relative amounts of c-type and b-type cytochromes in membranes prepared from cells grown under the two different conditions.  相似文献   

9.
Levels of thiosulfate-oxidizing enzyme (TSO) and tetrathionate reductase (TTR) were measured in washed cell suspensions of a heterotrophic marine thiosulfate-oxidizing bacterium, strain 16B. TSO activity remained virtually constant in aerobically and anaerobically grown cells and was unaffected by the presence or absence of thiosulfate and tetrathionate in the growth medium. TTR was also present in cells grown aerobically and anaerobically, but its activity was threefold greater in cells cultured in media containing tetrathionate or thiosulfate. Tetrathionate appears to be the inducer of increased TTR activity in both aerobically and anaerobically grown cells. TTR (constitutive or induced) and TSO have different pH and temperature optima. Both TTR activities were unaffected by 10 mM KCN, which reversed oxygen inhibition of tetrathionate reduction. TSO was partially inhibited by 5 μM KCN and completely inhibited by 90 μM KCN. These findings and results of experiments to determine the influence of several inorganic electron donors and acceptors on TSO and TTR activities suggest that constitutive TSO and TTR represent reverse activities of the same enzyme, whereas inducible TTR is a separate enzyme used by strain 16B only for anaerobic respiration of tetrathionate. The bacterium appears well adapted to growth in environments characterized by low oxygen tension, dilute organic carbon concentrations, and the presence of a variety of reduced, inorganic sulfur compounds.  相似文献   

10.
Diaminobenzidine, DAB, was applied to segments of aerobically and anaerobically grown coleoptiles of rice, Oryza sativa L., with the object of studying the location of cytochrome oxidase at the electron-microscope level. A specific staining of mitochondrial cristae and inner membrane was obtained, with no reaction in other organelles; with extended periods of incubation, the reaction product filled the mitochondria completely. In anaerobically grown coleoptiles, the reaction was much slower and the difference was particularly marked in vascular bundle companion cells and parenchyma, which gave the strongest reaction in aerobic tissue, but in the anaerobic stained even less than the cortical parenchyma. The reaction was inhibited by boiling and slowed very much by lowering of the incubation temperature from 27 to 4 degrees C. This indicated the involvement of an enzymic reaction and cyanide inhibition indicated that a haem enzyme was involved. The catalase inhibitor aminotriazole did not inhibit DAB oxidation. Nevertheless the specificity of the reaction for cytochrome oxidase must be questioned, because preheating of the tissue to 60 degrees C before incubation, which would be expected to destroy cytochrome oxidase activity, failed to decrease the oxidation, at least in aerobically grown coleoptiles. It is concluded that DAB is oxidized in the rice coleoptile tissue by a cytochrome system, and the development of this system is inhibited by anaerobiosis, but the oxidation cannot be claimed to represent cytochrome oxidase activity exclusively. Perhaps other autoxidizable, more heat-stable cytochromes participate in the reaction.  相似文献   

11.
Growth, bacteriochlorophyll a content, electron transport chain (ETC), and activities of the tricarboxylic acid (TCA) cycle enzymes were studied in R and M phase variants of Rhodobacter sphaeroides cells grown anaerobically in the light and aerobically in the dark. Under all cultivation conditions tested, bacteriochlorophyll a content was 2–3 times lower in the cells of the M variant compared to the R variant, which therefore was predominant in the cultures grown in the light. In both variants, activity of all TCA cycle enzymes was higher for the cells grown in the dark under aerobic conditions. When grown aerobically in the dark, the R variant, unlike the M variant, did not contain cytochrome aa 3, acting as cytochrome c oxidase, in its ETC. An additional point of coupling the electron transfer to the generation of the proton gradient at the cytochrome aa 3 level provided for more efficient oxidation of organic substrates, resulting in predominance of the M variant in the cultures grown in the dark under aerobic conditions.  相似文献   

12.
Anaerobically grown yeast cells lack cytochrome c peroxidase activity but rapidly acquire it upon aeration. In order to study the oxygen-induced formation of this hemoprotein, extracts of anaerobic and aerobic yeast cells were resolved by one- and two-dimensional acrylamide gel electrophoresis and the separated polypeptides were then checked for comigration with radiolabeled purified cytochrome c peroxidase from aerobic cells or for reaction with cytochrome c peroxidase antiserum. Both types of extracts contained roughly equal amounts of a polypeptide which was indistinguishable from apocytochrome c peroxidase with respect to antigenicity, isoelectric point, and apparent molecular weight in three different gel systems. In confirmation of an earlier report by Sels. A.A., and Cocriamont, C. (1968) (Biochem. Biophus. Res. Commun. 32, 192-198) the oxygen-induced formation of cytochrome c peroxidase was insensitive to inhibitors of protein synthesis and could be mimicked by the addition of heme to extracts of anaerobic cells. We conclude that the oxygen-induced formation of yeast cytochrome c peroxidase involves the addition of heme to the apoenzyme which is already present in the anaerobically grown cells.  相似文献   

13.
A respiratory-competent wild-type strain and a nuclear isogenic, mitochondrial DNA-less, petite mutant strain of Saccharomyces cerevisiae were grown under conditions of catabolite repression in batch cultures and under conditions of catabolite derepression in chemostat cultures. Subcellular fractions were isolated and the capacity of these fractions to incorporate sn-[2-3H]glycerol 3-phosphate into phospholipids was studied. Neither catabolite repression nor loss of mitochondrial DNA appreciably altered the total in vitro lipid synthesized by mitochondrial fractions during the incubation. Mitochondria isolated from catabolite-derepressed wild-type and petite cells had approximately the same specific activity in vitro for the synthesis of phosphatidylinositol. phosphatidic acid, phosphatidylethanolamine, phosphatidylserine, and neutral lipids. Mitochondria isolated from the petite cells retained the capacity to synthesize phosphatidylglycerol and diphosphatidylglycerol, although the synthesis of these phospholipids was far less extensive than that by the mitochondria isolated from the wild-type cells. In both cases, mitochondria prepared from catabolite-repressed cells synthesized a greater proportion of phosphatidylserine than did mitochondria from catabolite-derepressed cells. The proportions of phospholipid species synthesized in vitro by the microsomal fractions studied were not grossly affected by catabolite repression or loss of mitochondrial DNA.  相似文献   

14.
We studied phosphopeptidomannans (PPMs) of two Saccharomyces cerevisiae NCYC 625 strains (S. diastaticus): a wild type strain grown aerobically, anaerobically, and in the presence of antimycin and a [rho(0)] mutant grown aerobically and anaerobically. The aerobic wild-type cultures were highly flocculent, but all others were weakly flocculent. Ligands implicated in flocculation of mutants or antimycin-treated cells were not aggregated as much by concanavalin A as were those of the wild type. The [rho(0)] mutants and antimycin-treated cells differ from the wild type in PPM composition and invertase, acid phosphatase, and glucoamylase activities. PPMs extracted from different cells differ in the protein but not in the glycosidic moiety. The PPMs were less stable in mitochondrion-deficient cells than in wild-type cells grown aerobically, and this difference may be attributable to defective mitochondrial function during cell wall synthesis. The reduced flocculation of cells grown in the presence of antimycin, under anaerobiosis, or carrying a [rho(0)] mutation may be the consequence of alterations of PPM structures which are the ligands of lectins, both involved in this cell-cell recognition phenomenon. These respiratory chain alterations also affect peripheral, biologically active glycoproteins such as extracellular enzymes and peripheral PPMs.  相似文献   

15.
The enzymatic activities and the cytochrome components of the respiratory chain were investigated with membrane fractions from chemoheterotrophically growth 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 beta-types cytochromes b561, b560 and b558, and at least two c-type cytochromes c556 and c2 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 b560, 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 b561 with associated beta and gamma 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 c2 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   

16.
1. Subcellular fractionation of sphaeroplasts produced at different stages during the first 4h of respiratory adaptation of anaerobically grown glucose-de-repressed Saccharomyces carlsbergensis gave mitochondrial fractions that contained all the detectable c- and a-type cytochromes. 2. The rates of cytochrome formation were studied; individual cytochromes were produced at different rates so as to give respiratory chains having widely differing cytochrome ratios. A CO-reacting haemoprotein other than cytochrome a(3) also increased throughout 8h of respiratory adaptation. 3. Even after short periods of aeration, organisms contained mitochondria in which cytochrome-cytochrome interactions and the reaction of cytochrome a(3) with O(2) proceeded at rates almost as fast as in organelles from aerobically grown cells. 4. The technique of flow-flash photolysis enabled kinetic resolution of the reoxidation of cytochromes a(3) and a to be achieved and their individual contributions to extinction changes in the Soret region were assessed. The ratio cytochrome a(3)/cytochrome a increased over the early stages of adaptation.  相似文献   

17.
The addition of nitrate to cultures of Spirillum itersonii incubated under low aeration produced a diauxic growth pattern in which the second exponential phase was preceded by the appearance of nitrite in the medium. The organism also grew anaerobically in the presence of nitrate. Nitrate reductase activity could be demonstrated in cell-free extracts by use of reduced methyl viologen as the electron donor. The enzyme was located in the supernatant fraction after centrifugation of extracts for 2 hr at 40,000 x g, and it sedimented as a single peak when centrifuged in a sucrose gradient. Nitrate reductase activity was found in cells grown with low aeration without nitrate, but was increased about twofold by addition of nitrate. Enzyme activity was negligible in cells grown with high aeration. The proportion of soluble cytochrome c was increased two- to threefold in cells grown with nitrate. The specific activities of nitrate reductase and soluble cytochrome c rose when nitrate or nitrite was added to cell suspensions incubated with low aeration; nitrite was more effective than nitrate during the early stages of incubation. A nitrate reductase-negative mutant synthesized increased amounts of soluble cytochrome c in response to nitrate or to nitrite in the cell suspension system. It is concluded that enhanced synthesis of soluble cytochrome c does not require the presence of a functional nitrate reductase.  相似文献   

18.
Redox titration has been coupled to spectroscopic techniques, enzyme fractionation, and the use of mutants to examine the cytochrome composition of the membranes from cells grown aerobically and anaerobically with nitrate. A combination of techniques was found to be necessary to resolve the cytochromes. At least six b-type cytochromes were present. Besides cytochromes bfdh and bnr, components of the formate dehydrogenase-nitrate reductase pathway, cytochromes b556, b555, b562, and o, characteristic of aerobic respiratory pathways, were present. The midpoint oxidation-reduction potentials of the aerobic b-type cytochromes suggested that the sequence of electron transfer is: cytochrome b556 leads to b555 leads to b562 leads to O2.  相似文献   

19.
The kinetics of the ubiquinol-cytochrome c reductase reaction was examined using membrane fragments and purified bc(1) complexes derived from a wild-type (WT) and a newly constructed mutant (MUT) strains of Paracoccus denitrificans. The cytochrome c(1) of the WT samples possessed an additional stretch of acidic amino acids, which was lacking in the mutant. The reaction was followed with positively charged mitochondrial and negatively charged bacterial cytochromes c, and specific activities, apparent k(cat) values, and first-order rate constant values were compared. These values were distinctly lower for the MUT fractions using mitochondrial cytochrome c but differed only slightly with the bacterial species. The MUT preparations were less sensitive to changes of ionic strength of the reaction media and showed pure first-order kinetics with both samples of cytochrome c. The reaction of the WT enzyme was first order only with bacterial cytochrome c but proceeded with a non-linear profile with mitochondrial cytochrome c. The analysis of the reaction pattern revealed a rapid onset of the reaction with a successively declining rate. Experiments performed in the absence of an electron donor indicated that electrostatic attraction could directly participate in cytochrome c reduction.  相似文献   

20.
The phospholipid composition of various strains of the yeast, Saccharomyces cerevisiae, and several of their derived mitochondrial mutants grown under conditions designed to induce variations in the complement of mitochondrial membranes has been examined. Wild type and petite (cytoplasmic respiratory deficient) yeasts were fractionated into various subcellular fractions, which were monitored by electron microscopy and analyzed for cytochrome oxidase (in wild type) and phospholipid composition. 90% or more of the phospholipid, cardiolipin was found in the mitochondrial membranes of wild type and petite yeast. Cardiolipin content differed markedly under various growth conditions. Stationary yeast grown in glucose had better developed mitochondria and more cardiolipin than repressed log phase yeast. Aerobic yeast contained more cardiolipin than anaerobic yeast. Respiration-deficient cytoplasmic mitochondrial mutants, both suppressive and neutral, contained less cardiolipin than corresponding wild types. A chromosomal mutant lacking respiratory function had normal cardiolipin content. Log phase cells grown in galactose and lactate, which do not readily repress the development of mitochondrial membranes, contained as much cardiolipin as stationary phase cells grown in glucose. Cytoplasmic mitochondrial mutants respond to changes in the glucose concentration of the growth medium by variations in their cardiolipin content in the same way as wild type yeast does under similar growth conditions. It is concluded that cardiolipin content of yeast is correlated with, and is a good indicator of, the state of development of mitochondrial membrane.  相似文献   

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