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1.
1. Heart and liver tissue samples were obtained from rats in various developmental stages from the 12-day-old embryo to the 120-day-old postnatal animal. 2. The body, heart and liver weights and percentage protein in the liver and heart of the prenatal and postnatal rat were determined. 3. The activities of NADH–, NADPH– and succinate–cytochrome c reductases and cytochrome oxidase were determined also. 4. The specific activities of all the enzymes increased in both heart and liver during late foetal development (16 days to term). The NADH– and succinate–cytochrome c-reductase activities in the heart increased threefold during the neonatal period (0 to 25 days post partum) and then remained constant to 120 days. All reductase activities increased in the liver three- to six-fold during the neonatal period. Cytochrome-oxidase activity in both tissues increased sixfold during this time but plateaued in the liver at 12 days rather than 25 days. 5. A sex difference was observed in NADH–cytochrome c-reductase activity in the liver. Up to 25 days post partum the activity was the same in both sexes, but from that time on the activity continued to increase in the female but remained unchanged in the male. 6. NADPH–cytochrome c-reductase activity increased only in the liver. 7. These results indicate that different electron-transport pathways predominate according to the tissue, developmental stage and sex of the animal.  相似文献   

2.
1. Aerobically grown yeast having a high activity of glyoxylate-cycle, citric acid-cycle and electron-transport enzymes was transferred to a medium containing 10% glucose. After a lag phase of 30min. the yeast grew exponentially with a mean generation time of 94min. 2. The enzymes malate dehydrogenase, isocitrate lyase, succinate–cytochrome c oxidoreductase and NADH–cytochrome c oxidoreductase lost 45%, 17%, 27% and 46% of their activity respectively during the lag phase. 3. When growth commenced pyruvate kinase, pyruvate decarboxylase, alcohol dehydrogenase, glutamate dehydrogenase (NADP+-linked) and NADPH–cytochrome c oxidoreductase increased in activity, whereas aconitase, isocitrate dehydrogenase (NAD+- and NADP+-linked), α-oxoglutarate dehydrogenase, fumarase, malate dehydrogenase, succinate–cytochrome c oxidoreductase, NADH–cytochrome c oxidoreductase, NADH oxidase, NADPH oxidase, cytochrome c oxidase, glutamate dehydrogenase (NAD+-linked), glutamate–oxaloacetate transaminase, isocitrate lyase and glucose 6-phosphate dehydrogenase decreased. 4. During the early stages of growth the loss of activity of aconitase, α-oxoglutarate dehydrogenase, fumarase and glucose 6-phosphate dehydrogenase could be accounted for by dilution by cell division. The lower rate of loss of activity of isocitrate dehydrogenase (NAD+- and NADP+-linked), glutamate dehydrogenase (NAD+-linked), glutamate–oxaloacetate transaminase, NADPH oxidase and cytochrome c oxidase implies their continued synthesis, whereas the higher rate of loss of activity of malate dehydrogenase, isocitrate lyase, succinate–cytochrome c oxidoreductase, NADH–cytochrome c oxidoreductase and NADH oxidase means that these enzymes were actively removed. 5. The mechanisms of selective removal of enzyme activity and the control of the residual metabolic pathways are discussed.  相似文献   

3.
The components involved in the respiratory system of the euryarcheon Halobacterium salinarum were investigated by spectroscopic and polarographic techniques. Previous results about the cytochrome composition could be verified. However, under low oxygen tension, the expression of a d-type cytochrome was detected. Membranes exerted an NADH– and succinate–cytochrome-c oxidoreductase as well as an NADH and succinate oxidase activity. These activities could be blocked by the following inhibitors: 7-jodocarboxylic acid, giving evidence for the presence of a type II NADH dehydrogenase, antimycin A, and myxothiazol, indicating the presence of a complex III analog, and the typical succinate dehydrogenase (SDH) and terminal oxidase inhibitors. Complex I inhibitors like rotenone and annonine were inactive, clearly excluding the presence of a coupled NADH dehydrogenase. In addition, no [Fe-S] resonances in the region of the NADH dehydrogenase (NDH) clusters could be observed after NADH addition. One of the terminal oxidases could be shown to act as a cytochrome-c oxidase with a K m value of 37 M and an activation energy of 23.7 kJ/mol. The relative molecular mass of the endogenous c-type cytochrome could be determined as 14.1 kD. The complex III analog could be enriched after detergent extraction with Triton X-100 and hydroxylapatite (HTP) chromatography. The partially purified complex contained a Rieske iron–sulfur cluster, b- and c-type cytochromes, and was catalytically active in the decylubiquinone–cytochrome-c oxidoreductase assay.  相似文献   

4.
A number of mutant forms of horse cytochrome c with single or double substitutions of lysine residues near the heme cavity involved in interaction of mitochondrial cytochrome c with ubiquinol:cytochrome c reductase (EC 1.10.2.2) (complex III) and cytochrome c oxidase (EC 1.9.3.1) (complex IV) were prepared.. The succinate:cytochrome c reductase and cytochrome c oxidase activities of mitoplasts of rat liver were measured in the presence of mutant forms of cytochrome c. The lysine residues in positions 8, 27, 72, 86, and 87 were shown to be the main contribution to the formation of a reactive complex with ubiquinol:cytochrome c reductase of the respiratory chain, whereas the lysine residues in positions 13, 79, 86, and 87 were predominantly responsible for the formation of a complex with cytochrome c oxidase.  相似文献   

5.
(1) The distributions of four oxidative enzymes were studied in crude brain fractions. (2) Freeze-thaw cycle treatment and frozen storage of homogenate fractions gave apparent enhancement of cytochrome oxidase and NADH cytochrome c reductase activities. (3) Deoxycholate released cytochrome oxidase and NADH cytochrome c reductase activities from low-speed precipitates. The NADH diaphorase was enhanced to a small degree while NADPH cytochrome c reductase was not affected by deoxycholate. (4) Distilled water coupled with a single homogenization released trapped soluble enzymes and mitochondria and gave nearly maximal cytochrome oxidase activity as judged by deoxycholate treatment. The total distilled water activity of NADH cytochrome c reductase was much less than that of deoxycholate-stimulated fractions. The activities of other enzymes were not markedly affected by distilled water although their distribution was changed.  相似文献   

6.
Abstract— The distributions of NADH2 dehydrogenase, NADH, cytochrome c reductase and cytochrome oxidase have been determined utilizing synaptosomal isolation techniques. Deoxycholate was used to determine compartmentation and/or ‘latency’ of these activities. NADPH, dehydrogenase proved to be a soluble and mitochondrial enzyme and the activity of this enzyme was not appreciably changed by deoxycholate treatment. NADHg cytochrome c reductase proved to be a mitochondrial enzyme with considerable activity in microsomal fractions. Deoxycholate treatment increased activity in the synaptosomal fraction 8.3-fold. A bimodal activation pattern was observed with synaptosomal and mitochondrial NADH, cyrochrome c reductase upon exposure to increasing concentrations of deoxycholate, with enhancement of activity at 0.25 % (w/v) and 0.50 % (w/v) deoxycholate. The enzyme was stable at concentrations of deoxycholate less than 0.25% (w/v) but was irreversibly inactivated at concentrations higher than 0.25% (w/v). The mechanism of this activation pattern appeared to be a combination of enzyme release and inactivation. Similar results were not observed in liver mitochondria. Cytochrome oxidase, a known mitochondrial marker, exhibited a 17-fold increase in synaptosomal activity with deoxycholate treatment. The synaptosomal cytochrome oxidase activity after deoxycholate treatment approached the activity in the free mitochondrial fraction. The percentage of mitochondrial protein in synaptosomal fractions was estimated to be about 30 per cent from a comparison of the respective total (deoxycholate-treated) activities. On the basis of these data we suggest that the synaptosomal fraction possesses a relatively sizable energy-producing potential which may be of significance in vivo.  相似文献   

7.
The aerobic adaptation of anaerobically grownP. denitrificans carried out under conditions of limited growth is characterized by an exponential decrease of nitrite reductase activity and a sharp increase of cytochrome oxidase and a slow increase of NADH:cytochromec oxidase reductase and succinate dehydrogenase activities. The adaptation in a minimal adaptation medium under conditions of active or blocked protein synthesis showed that in addition to the degradation component of turnover during the aerobic adaptation other degradation enzyme(s), whose synthesis is induced by oxygen, are involved. This degradation system plays an essential role in the rapid disappearance of nitrite reductase and a pronounced decrease of the membranebound cytochromec oxidase activities during aerobic adaptation in the minimal adaptation medium.  相似文献   

8.
Summary Mutants of E. coli, completely devoid of nitrite reductase activity with glucose or formate as donor were studied. Biochemical analysis indicates that they are simultaneously affected in nitrate reductase, nitrite reductase, fumarate reductase and hydrogenase activities as well as in cytochrome c552 biosynthesis. The use of an antiserum specific for nitrate reductase shows that the nitrate reductase protein is probably missing. A single mutation is responsible for this phenotype: the gene affected, nir R, is located close to tyr R i.e. at 29 min on the chromosomal map.Abbreviations BV Benzyl-Viologen - NTG N-methyl-N-nitro-N-nitrosoguanidine - NR nitrate reductase - NIR nitrite reductase - FR fumarate reductase - HYD hydrogenase - CYT c552 cytochrome c552  相似文献   

9.
Cytochromes c 3 of different strains of sulfatereducing bacteria have been purified and tested for their capacity to reduce colloidal sulfur to hydrogen sulfide. The results are in good agreement with the activities reported for the whole cells. Cytochrome c 3 is the sulfur reductase of some strains of sulfate-reducing bacteria such as Desulfovibrio desulfuricans Norway 4 and sulfate-reducing bacterium strain 9974 from which the sulfur reductase activity can be purified with the cytochrome c 3. In contrast, Desulfovibrio vulgaris Hildenborough cytochrome c 3 is inhibited by the product of the reaction namely hydrogen sulfide. Chloramphenicol has no effect on the sulfur reductase activity of D. desulfuricans Norway 4 when resting cells grown on lactate-sulfate medium are put in the presence of colloidal sulfur. This shows that the sulfur reductase activity is constitutive and corresponds to the fact that colloidal sulfur grown cells do not contain more cytochrome c 3 (or another sulfur reductase) than lactate-sulfate-grown cells.  相似文献   

10.
Coronary artery disease (CAD) is the leading cause of mortality in diabetic patients. Mitochondrial dysfunction and increased production of reactive oxygen species (ROS) are associated with diabetes and CAD. Elevated levels of glycated LDL (glyLDL) were detected in patients with diabetes. Our previous studies demonstrated that glyLDL increased the generation of ROS and altered the activities of antioxidant enzymes in vascular endothelial cells (EC). This study examined the effects of glyLDL on oxygen consumption in mitochondria and the activities of key enzymes in the mitochondrial electron transport chain (ETC) in cultured porcine aortic EC. The results demonstrated that glyLDL treatment significantly impaired oxygen consumption in Complexes I, II/III, and IV of the mitochondrial ETC in EC compared to LDL or vehicle control detected using oxygraphy. Incubation with glyLDL significantly reduced the mitochondrial membrane potential, the NAD+/NADH ratio, and the activities of mitochondrial ETC enzymes (NADH-ubiquinone dehydrogenase, succinate cytochrome c reductase, ubiquinone cytochrome c reductase, and cytochrome c oxidase) in EC compared to LDL or control. The abundance of mitochondria-associated ROS and the release of ROS from EC were significantly increased after glyLDL treatment. The findings suggest that glyLDL attenuates the activities of key enzymes in the mitochondrial ETC, decreases mitochondrial oxygen consumption, reduces mitochondrial membrane potential, and increases ROS generation in EC, which potentially contribute to mitochondrial dysfunction in diabetic patients.  相似文献   

11.
Summary Outer mitochondrial membranes isolated by the swelling-shrinkage sonication procedure of Sottocasaet al. [19, 20] forms small sealed vesicles. If cytochromec is present during the procedure it is trapped inside these vesicles and can not be washed out nor is accessible to external enzymes, e.g., cytochrome oxidase (EC 1.9.3.1) or succinate-cytochromec reductase present as contamination by the inner membrane, but is fully accessible to rotenone-insensitive NADH-cytochromec reductase of the outer membrane. This indicates the impermeability of the outer mitochondrial membrane to cytochromec.A modification of the original procedure for the separation of the outer mitochondrial membrane is described.  相似文献   

12.
In this study, we purified and characterized the voltage-dependent anion channel (VDAC) from the Torpedo electric organ. Using immunogold labeling, VDAC was colocalized with the voltage-gated Ca2+ channel in the synaptic plasma membrane. By immunoblot analysis, five protein bands in synaptosomes isolated from the Torpedo electric organ cross reacted with two monoclonal anti-VDAC antibody. No more than about 7 to 10% mitochondrial contains could be detected in any synaptosomal membrane preparation tested. This was estimated by comparing the specific activity in mitochondria and synaptosomes of succinate–cytochrome-c oxidoreductase and antimycin-insensitive NADH–cytochrome-c oxidoreductase activities; mitochondrial inner and outer membrane marker enzymes, respectively. [14C]DCCD (dicyclohexylcarbodiimide), which specifically label mitochondrial VDAC, labeled four 30–35 kDa protein bands that were found to interact with the anti-VDAC antibody. The distribution of the Torpedo VDAC protein bands was different among membranes isolated from various tissues. VDAC was purified from synaptosomes and a separation between two of the proteins was obtained. The two purified proteins were characterized by their single channel activity and partial amino acid sequences. Upon reconstitution into a planar lipid bilayer, the purified VDACs showed voltage-dependent channel activity with properties similar to those of purified mitochondrial VDAC. Amino acid sequence of four peptides, derived from VDAC band II, exhibited high homology to sequences present in human VDAC1 (98%), VDAC2 (91.8%), and VDAC3 (90%), while another peptide, derived from VDAC band III, showed lower homology to either VDAC1 (88.4%) or VDAC2 (79%). Two more peptides show high homology to the sequence present in mouse brain VDAC3 (100 and 78%). In addition, we demonstrate the translocation of ATP into synaptosomes, which is inhibited by DCCD and by the anion transport inhibitor DIDS. The possible function of VDAC in the synaptic plasma membrane is discussed.  相似文献   

13.
Glyoxysomes isolated from castor bean (Ricinus communis L., var Hale) endosperm had NADH:ferricyanide reductase and NADH:cytochrome c reductase activities averaging 720 and 140 nanomole electrons/per minute per milligram glyoxysomal protein, respectively. These redox activities were greater than could be attributed to contamination of the glyoxysomal fractions in which 1.4% of the protein was mitochondrial and 5% endoplasmic reticulum. The NADH:ferricyanide reductase activity in the glyoxysomes was greater than the palmitoyl-coenzyme A (CoA) oxidation activity which generated NADH at a rate of 340 nanomole electrons per minute per milligram glyoxysomal protein. Palmitoyl-CoA oxidation could be coupled to ferricyanide or cytochrome c reduction. Complete oxidation of palmitoyl-CoA, yielding 14 nanomole electrons/per nanomole palmitoyl-CoA, was demonstrated with the acceptors, NAL, cytochrome c, and ferricyanide. Malate was also oxidized by glyoxysomes, if acetyl-CoA, ferricyanide, or cytochrome c was present. Glyoxysomal NADH:ferricyanide reductase activity has the capacity to support the combined rates of NADH generation by β-oxidation and the glyoxylate cycle.  相似文献   

14.
Microbodies (peroxisomes and glyoxysomes), mitochondria, and microsomes from rat liver, dog kidney, spinach leaves sunflower cotyledons, and castor bean endosperm were isolated by sucrose density-gradient centrifugation. The microbody-limiting membrane and microsomes each contained NADH-cytochrome c reductase and had a similar phospholipid composition. NADH-cytochrome c reductase from plant and animal microbodies and microsomes was insensitive to antimycin A, which inhibited the activity in the mitochondrial fractions. The pH optima of cytochrome c reductase in plant microbodies and microsomes was 7.5–9.0, which was 2 pH units higher than the optima for the mitochondrial form of the enzyme. The activity in animal organelles exhibited a broad pH optimum between pH 6 and 9. Rat liver peroxisomes retained cytochrome c reductase activity, when diluted with water, KCl, or EDTA solutions and reisolated. Cytochrome c reductase activity of microbodies was lost upon disruption by digitonin or Triton X-100, but other peroxisomal enzymes of the matrix were not destroyed. The microbody fraction from each tissue also contained a small amount of NADH-cytochrome b5 reductase activity. Peroxisomes from spinach leaves were broken by osmotic shock and particles from rat liver by diluting in alkaline pyrophosphate. Upon recentrifugation liver peroxisomes yielded a core fraction containing urate oxidase at a sucrose gradient density of 1.23 g × cm−3, a membrane fraction at 1.17 g × cm−3 containing NADH-cytochrome c reductase, and soluble matrix enzymes at the top of the gradient.  相似文献   

15.
SYNOPSIS. Cell-free extracts of a streptomycin-bleached strain of Euglena gracilis var. bacillaris have been examined for enzyme systems primarily responsible for the oxidation of reduced pyridine nucelotides. NADH lipoyl dehydrogenase, NADH and NADPH oxidase, NADH and NADPH diaphorase, and NADH and NADPH cytochrome c reductase have been demonstrated. The NADPH-linked enzymes had lower activity rates and were less sensitive to N-ethyl maleimide and p-hydroxymercuribenzoate than their NADH-linked counterparts. NADH cytochrome c reductase was the most sensitive to antimycin A. Michaelis-Menten constants (Km) determined were as follows: NADH diaphorase, 350 μM; NADPH diaphorase, 200 μM; NADH cytochrome c reductase, 13 μM; NADPH cytochrome c reductase, 9 μM; NADH oxidase, 100 μM; NADPH oxidase 150 μM; NADH lipoyl dehydrogenase, 0.35 μM. Enzyme activities after storage at –5 C indicate that the diaphorases are less labile than the other tested enzymes, and the differential activities of the NADH and NADPH linked enzymes suggest that functionally they may have different roles.  相似文献   

16.
Exposure of aerobically-grown wild-type cells of Paracoccus denitrificans to a decreased aeration caused parallel increases in both PMS/ascorbate and succinate-linked activities of nitrite reductase. By contrast, the expression of the succinate-linked activity was considerably delayed in an insertion mutant specifically lacking the periplasmic 15 kDa cytochrome c-550. In this case the observed activity followed very closely the content of a 40 kDa cytochrome c. A subcellular fraction enriched in a haemoprotein of a similar apparent molecular weight showed the activity of cytochrome c peroxidase and was able to restore the antimycin-sensitive electron transport from membrane vesicles to nitrite reductase. It is concluded that P. denitrificans possesses an alternative nitrite-reducing pathway involving the 40 kDa cytochrome c instead of cytochrome c-550. This pathway branches from the respiratory chain after the cytochrome bc 1 segment.Abbreviations PAGE polyacrylamide gel electrophoresis - PMS phenazine methosulphate  相似文献   

17.
Evidence is presented about the dual location of NADPH-cytochrome c reductase in mitochondrial outer membranes as well as in microsomes, from pig heart.A high specific activity, was found in both fractions, even after their purification by washing, digitonin treatments, or passages on sucrose gradients. A large fraction of the total activity was associated with both mitochondria and microsomes.Mitochondrial outer membrane differs from microsomes by a low choline phosphotransferase activity and the absence of cytochrome P-450.The properties of mitochondrial and microsomal rotenone-insensitive NADH- and NADPH-cytochrome c reductases were studied. In microsomes, both activities have the same optimum pH (8.5) ; in contrast, in mitochondria they have a different one. The Km-NADPH were always much higher than those for NADH. In mitochondria the Km for NAD(P)H were dependent on cytochrome c concentration.The results show that the rotenone-insensitive NADH- and NADPH-cytochrome c reductases of mitochondria and microsomes have quite different behavior and do not appear to be supported by the same enzyme.  相似文献   

18.
1. Mitochondria isolated from rat liver were disrupted with 0.3 per cent deoxycholate and a number of subfractions were isolated from this preparation by differential centrifugation. 2. The protein N, RNA and phospholipide content, as well as the succinoxidase, cytochrome c oxidase, adenylate kinase, and DPNH-cytochrome c reductase of these fractions were determined. 3. Two of these subfractions, found to consist of mitochondrial membranes (2), contained ~ 12 per cent of the protein N and ~ 35 per cent of the phospholipide of the whole mitochondria and accounted for ~ 70 per cent of the succinoxidase and cytochrome c oxidase activity of the original mitochondrial preparation. There was no discernible adenylate kinase, DPNH-cytochrome c reductase, or phosphorylating activities in these fractions, nor could they oxidize other substrates of the Krebs's cycle. 4. The most active fraction (60 minutes at 105,000 g pellet) had a higher phospholipide/protein value than the whole mitochondria and showed a seven-to elevenfold concentration of succinoxidase and cytochrome c oxidase activities. 5. Evidence has been given to indicate that the various components of the succinoxidase complex are present in this membrane fraction in the same relative proportions as in the whole mitochondria. 6. The implications of these findings are discussed.  相似文献   

19.
Derepression of mitochondria and their enzymes in yeast: regulatory aspects   总被引:27,自引:0,他引:27  
We have performed a detailed analysis of the properties of glucose-repressed cells of a commercial strain of Saccharomyces cerevisiae. They contain measurable amounts of the respiratory enzymes NADH oxidase, cytochrome c oxidase, succinate dehydrogenase, succinate:cytochrome c reductase and NADH:cytochrome c reductase (antimycin A-sensitive) as well as the dehydrogenases for l-malate, l-glutamate, and l8-isocitrate. Cytochromes b, c1, and aa3 are present in amounts that may be in excess of those required for cytochrome-linked enzyme activities. Enzymes and cytochromes are localized in large, presumably mitochondrial organelles among which no compositional or functional heterogeneity could be detected.We have also analyzed the kinetics of synthesis of respiratory enzymes and cytochromes during the release from catabolite(glucose) repression. All activities assayed except for cytochrome c oxidase begin their derepression before the external glucose concentration falls below 0.4%; derepression of cytochrome oxidase occurs only after the glucose concentration falls below 0.1%. The earlier events comprise the “fermentative” phase of derepression while the later events comprise the “oxidative” phase. The two phases can be distinguished operationally by their sensitivity to antimycin A. Only the oxidative phase is blocked by the inhibitor. Respiratory enzymes and cytochromes appear to fall into two classes distinguishable by their increase during derepression. An apparently constitutive one consists of cytochrome c oxidase, ATPase, and cytochromes aa3, b, and c1; these entities increase in amount per cell but not in amount per unit of mitochondrial mass and are of the order of 5-fold or less. The second class consists of those activities that increase by more than 6-fold and may be considered derepressible in the strict sense. Thus, proliferation and differentiation of mitochondria both contribute to the cellular changes associated with derepression.The fermentative phase of derepression does not require mitochondrial function, mitochondrial protein, or RNA synthesis, or the gradual accumulation of regulatory elements for either its initiation or persistence. This phase of derepression also occurs in cytoplasmic petites. In contrast, the oxidative phase of derepression requires mitochondrial function. Mitochondrial gene expression is required for the biogenesis of fully functional mitochondria but, except for cytochrome c, it plays little or no role in regulating the expression of nuclear genes the products of which are localized in mitochondria.  相似文献   

20.
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   

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