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1.
Multiple cytochromes b in Mycobacterium phlei 总被引:1,自引:0,他引:1
N S Cohen E Bogin T Higashi A F Brodie 《Biochemical and biophysical research communications》1973,54(2):800-807
Electron transport particles from contain at least 3 different active forms of cytochrome b, one reduced by NADH, with a λmax at 563 nm (bN563), and the other two reduced by either succinate or NADH, with λmax at 559 and 563 nm (bS559) and (bS563). Low temperature λmax for cytochrome b reduction with NADH or succinate are described. During steady state only bS563 was observed with succinate. In the presence of ATP, succinate reduced an increased amount of a b563. A branching of the NAD+-linked pathway and a convergence at the level of cytochrome c is suggested, with only one branch accessible to succinate. 相似文献
2.
Hen liver microsomes contained 0.20 nmol of cytochromeb5 per mg of protein. Upon addition of NADH about 95% cytochrome b5 was reduced very fast with a rate constant of 206 s?1When ferricyanide was added to the reaction system the cytochrome stayed in the oxidized form until the ferricyanide reduction was almost completed. The reduced cytochrome b5 in microsomes was oxidized very rapidly by ferricyanide. The rate constant of 4.5 × 108m?1 s?1, calculated on the basis of assumption that ferricyanide reacts directly with the cytochrome, was found to be more than 100 times higher than that of the reaction between ferricyanide and soluble cytochrome b5. To explain the results, therefore, the reverse electron flow from cytochrome b5 to the flavin coenzyme in microsomes was assumed.By three independent methods the specific activity of the microsomes was measured at about 20 nmol of NADH oxidized per s per mg of protein and it was concluded that the reduction of the flavin coenzyme of cytochrome b5 reductase by NADH is rate-limiting in the NADH-cytochrome b5 and NADH-ferricyanide reductase reactions of hen liver microsomes. In the NADH-ferricyanide reductase reaction the apparent Michaelis constant for NADH was 2.8 μm and that for ferricyanide was too low to be measured. In the NADH-cytochrome c reductase reaction the maximum velocity was 2.86 nmol of cytochrome c reduced per s per mg of protein and the apparent Michaelis constant for cytochrome c was 3.8 μm. 相似文献
3.
Vijay Tejwani Franz-Josef Schmitt Svea Wilkening Ingo Zebger Marius Horch Oliver Lenz Thomas Friedrich 《BBA》2017,1858(1):86-94
Ralstonia eutropha is a hydrogen-oxidizing (“Knallgas”) bacterium that can easily switch between heterotrophic and autotrophic metabolism to thrive in aerobic and anaerobic environments. Its versatile metabolism makes R. eutropha an attractive host for biotechnological applications, including H2-driven production of biodegradable polymers and hydrocarbons. H2 oxidation by R. eutropha takes place in the presence of O2 and is mediated by four hydrogenases, which represent ideal model systems for both biohydrogen production and H2 utilization. The so-called soluble hydrogenase (SH) couples reversibly H2 oxidation with the reduction of NAD+ to NADH and has already been applied successfully in vitro and in vivo for cofactor regeneration. Thus, the interaction of the SH with the cellular NADH/NAD+ pool is of major interest. In this work, we applied the fluorescent biosensor Peredox to measure the [NADH]:[NAD+] ratio in R. eutropha cells under different metabolic conditions. The results suggest that the sensor operates close to saturation level, indicating a rather high [NADH]:[NAD+] ratio in aerobically grown R. eutropha cells. Furthermore, we demonstrate that multicomponent analysis of spectrally-resolved fluorescence lifetime data of the Peredox sensor response to different [NADH]:[NAD+] ratios represents a novel and sensitive tool to determine the redox state of cells. 相似文献
4.
An antibody preparation elicited against purified, lysosomal-solubilized NADH-cytochrome b5 reductase from rat liver microsomes was shown to interact with methemoglobin reductase of human erythrocytes by inhibiting the rate of erythrocyte cytochrome b5 reduction by NADH. The ferricyanide reductase activity of the enzyme was not inhibited by the antibody, suggesting that the inhibition of methemoglobin reductase activity may be due to interference with the binding of cytochrorme b5 to the flavoprotein. Under conditions of limiting concentrations of flavoprotein, the antibody inhibited the rate of methemoglobin reduction in a reconstituted system consisting of homogeneous methemoglobin reductase and cytochrome b5 from human erythrocytes. This inhibition was due to the decreased level of reduced cytochrome b5 during the steady state of methemoglobin reduction while the rate of methemoglobin reduction per reduced cytochrome b5 stayed constant, suggesting that the enzyme was not concerned with an electron transport between the reduced cytochrome b5 and methemoglobin.An antibody to purified, trypsin-solubilized cytochrome b5 from rat liver microsomes was shown to inhibit erythrocyte cytochrome b5 reduction by methemoglobin reductase and NADH to a lesser extent than microsomal cytochrome b5 preparations from rat liver (trypsin solubilized or detergent solubilized) and pig liver (trypsin solubilized). The results presented establish that soluble methemoglobin reductase and cytochrome b5 of human erythrocytes are immunochemically similar to NADH-cytochrome b5 reductase and cytochrome b5 of liver microsomes, respectively. 相似文献
5.
Hirofumi Fukushima Shigenobu Umeki Takehito Watanabe Yoshinori Nozawa 《Biochemical and biophysical research communications》1982,105(2):502-508
With the use of detergents and successive column chromatographies, Tetrahymena b-type cytochrome was purified from microsomes to a specific content of 36.0 nmol per mg of protein. The purified form showed a single band on SDS-polyacrylamide gel with molecular weight of 22,000. The spectral properties of the reduced b-type cytochrome, the α-peak of which is situated at 560 nm and asymmetric with a shoulder at 556 nm, was different from that of rat liver microsomal cytochrome b5. However, it was reducible by NADH in the presence of NADH-cytochrome b5 reductase purified from rat liver microsomes.The results indicated that the microsomal b-type cytochrome should be designated as cytochrome b5 of a ciliated protozoan, Tetrahymena pyriformis. 相似文献
6.
Previous investigations have demonstrated that cells isolated from the outer zone (zona fasciculata + zona glomerulosa) of the guinea-pig adrenal cortex produce far more cortisol than those from the inner zone (zona reticularis). Studies were carried out to compare mitochondrial steroid metabolism in the two zones. Protein and cytochrome P-450 concentrations were similar in outer and inner zone mitochondria. However, the rate of 11 beta-hydroxylation was significantly greater in the outer zone despite the fact that substrates for 11 beta-hydroxylation (11-deoxycortisol, 11-deoxycorticosterone) produced larger type I spectral changes in inner zone mitochondria. The apparent affinities of 11-deoxycortisol and 11-deoxycorticosterone for mitochondrial cytochrome(s) P-450 were similar in the two zones. In both inner and outer zone mitochondria, 11 beta-hydroxylation was inhibited by metyrapone but unaffected by aminoglutethimide. Cholesterol sidechain cleavage activity, measured as the rate of conversion of endogenous cholesterol to pregnenolone, was far greater in outer than inner zone mitochondria. Addition of exogenous cholesterol or 25-hydroxycholesterol to the mitochondrial preparations did not affect pregnenolone production in either zone. Addition of pregnenolone to outer zone mitochondria produced a reverse type I spectral change (delta A 420-390 nm), suggesting displacement of endogenous cholesterol from cytochrome P-450. In inner zone mitochondria, pregnenolone induced a difference spectrum (delta A 425-410 nm) similar to the reduced vs oxidized cytochrome b5 spectrum. A b5-like cytochrome was found to be present in the mitochondrial preparations. Prior reduction of the cytochrome with NADH eliminated the pregnenolone-induced spectral change in inner zone mitochondria but had no effect in outer zone preparations. The results suggest that differences in mitochondrial steroid metabolism between the inner and outer adrenocortical zones account in part for the differences in cortisol production by cells in each zone. 相似文献
7.
《BBA》2019,1860(10):148062
The hydrogen-oxidizing “Knallgas” bacterium Ralstonia eutropha can thrive in aerobic and anaerobic environments and readily switches between heterotrophic and autotrophic metabolism, making it an attractive host for biotechnological applications including the sustainable H2-driven production of hydrocarbons. The soluble hydrogenase (SH), one out of four different [NiFe]-hydrogenases in R. eutropha, mediates H2 oxidation even in the presence of O2, thus providing an ideal model system for biological hydrogen production and utilization. The SH reversibly couples H2 oxidation with the reduction of NAD+ to NADH, thereby enabling the sustainable regeneration of this biotechnologically important nicotinamide cofactor. Thus, understanding the interaction of the SH with the cellular NADH/NAD+ pool is of high interest. Here, we applied the fluorescent biosensor Frex to measure changes in cytoplasmic [NADH] in R. eutropha cells under different gas supply conditions. The results show that Frex is well-suited to distinguish SH-mediated changes in the cytoplasmic redox status from effects of general anaerobiosis of the respiratory chain. Upon H2 supply, the Frex reporter reveals a robust fluorescence response and allows for monitoring rapid changes in cellular [NADH]. Compared to the Peredox fluorescence reporter, Frex displays a diminished NADH affinity, which prevents the saturation of the sensor under typical bacterial [NADH] levels. Thus, Frex is a valuable reporter for on-line monitoring of the [NADH]/[NAD+] redox state in living cells of R. eutropha and other proteobacteria. Based on these results, strategies for a rational optimization of fluorescent NADH sensors are discussed. 相似文献
8.
The Respiratory Chain of Plant Mitochondria: VIII. Reduction Kinetics of the Respiratory Chain Carriers of Mung Bean Mitochondria with Reduced Nicotinamide Adenine Dinucleotide 总被引:7,自引:6,他引:1
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Storey BT 《Plant physiology》1970,46(4):625-630
Addition of 90 micromolar reduced nicotinamide adenine dinucleotide (NADH) in the presence of cyanide to a suspension of aerobic mung bean (Phaseolus aureus) mitochondria depleted with ADP and uncoupler gives a cycle of reduction of electron transport carriers followed by reoxidation, as NADH is oxidized to NAD+ through the cyanide-insensitive, alternate oxidase by excess oxygen in the reaction medium. Under these conditions, cytochrome b553 and the nonfluorescent, high potential flavoprotein Fpha of the plant respiratory chain become completely reduced with half-times of 2.5 to 2.8 seconds for both components. Reoxidation of flavoprotein Fpha on exhaustion of NADH is more rapid than that of cytochrome b553. There is a lag of 1.5 seconds after NADH addition before any reduction of ubiquinone can be observed, whereas there is no lag perceptible in the reduction of flavoprotein Fpha and cytochrome b553. The half-time for ubiquinone reduction is 4.5 seconds, and the extent of reduction is 90% or greater. About 30% of cytochrome b557 is reduced under these conditions with a half-time of 10 seconds; both cytochrome b562 and the fluorescent, high potential flavoprotein Fphf show little, if any, reduction. The two cytochromes c in these mitochondria, c547 and c549, are reduced in synchrony with a half-time of 0.8 second. These two components are already 60% reduced in the presence of cyanide but absence of substrate, and they become completely reduced on addition of NADH. These results indicated that reducing equivalents enter the respiratory chain from exogenous NADH at flavoprotein Fpha and are rapidly transported through cytochrome b553 to the cytochromes c; once the latter are completely reduced, reduction of ubiquinone begins. Ubiquinone appears to act as a storage pool for reducing equivalents entering the respiratory chain on the substrate side of coupling site 2. It is suggested that flavoprotein Fpha and cytochrome b553 together may act as the branching point in the plant respiratory chain from which forward electron transport can take place to oxygen through the cytochrome chain via cytochrome oxidase, or to oxygen through the alternate, cyanide-insensitive oxidase via the fluorescent, high potential flavoprotein Fphf. 相似文献
9.
Investigations of human fetal adrenal gland microsomes indicated that a carbon monoxide binding pigment had an absorption maximum of 446 to 448 nm. This pigment, upon heat treatment at 37°C was degraded to the form of cytochrome p-420. NADPH reduced cytochrome p-450 slowly and completely. Typical concentrations of 0.75 and 0.16 nmoles/mg protein cytochrome P-450 and b5, respectively, were observed. Reduced ethylisocyanide spectra were similar to those of rat hepatic microsomes with absorption maxima at 430 as well as 454 nm. Typical type I spectral changes were observed with progesterone, 17-α-OH-progesterone, pregnenolone and androstenedione when these steroids were added to the sample cuvettes. Androstenedione exhibited an apparent spectral dissociation constant (KS) of 5×10−6M pregnenolone and progesterone exhibited higher affinities with apparent dissociation constants of 1.1×10−7M and 1.8×10−7M, respectively. The maximal absorbance change induced by androstenedione was lower (Emax = 0.027 per mg protien) than the changes in absorbance maxima induced by pregnenolone or progesterone (Emax = 0.060 and 0.047 per mg protein, respectively) when saturating concentrations of these steroids were added to the sample cuvettes. Ethylmorphine and aminopyrine (10−3M final concentrations) did not exhibit observable spectral changes; however, type II spectra could be elicited with aniline and nicotinamide and apparent dissociation constants of 3.5×10−2M and 2.5×10−2M, respectively, were obtained. 相似文献
10.
Metabolism of nitroxide spin labels in subcellular fraction of rat liver: I. Reduction by microsomes
Anna Iannone A. Tomasi V. Vannini Harold M. Swartz 《Biochimica et Biophysica Acta (BBA)/General Subjects》1990,1034(3):285-289
As part of an ongoing study of the role of subcellular fractions on the metabolism of nitroxides, we studied the metabolism of a set of seven nitroxides in microsomes obtained from rat liver. The nitroxides were chosen to provide information on the effects of the type of charge, lipophilicity and the ring on which the nitroxide group is locted Important variables that were studied included adding NADH, adding, induction of enzymed by intake of phenobarbital and the effects of oxygen. Reduction of nonparamagnetic derivatives and oxidation to paramagnetic derivatives were measured by electron-spin resonance spectroscopy. In general, the relative rates of reduction of nitroxides were similar to those observed with intact cells, but the effects of the various variables that were studied often differed from those observed in intact cells. The rates of reduction were very slow in the absence of added NADh or NADPH. The relative effect of these two nucleotides changed when animals were fed phenobarbital and paralleled the levels of NADPH cytochrome c reductase, cytochrome P-450, cytochrome b5 and NADH cytochrome c reductase; results with purified NADPH-cytochrome c reductase were consistent with these results. In microsomes from uninduced animals the rate of reduction was about 10-fold higher in the absence of oxygen. The products of reduction of nitroxides by microsomes were the corresponding hydroxylmines. We conclude that there are significant NADH- and NADPH-dependent paths for reduction of nitroxides by hepatic microsomes, probably involving cytochrome c reductases and not directly involving cytochrome P-450. From this, and from parallel studies now in progress in our laboratory, it seems likely that metabolism by microsomes is an important site of reduction of nitroxides. However, mitochondrial metabolism seems to play an even more important role in intact cells. 相似文献
11.
Hiroko Ohba Hiroshi Inano Bun-ichi Tamaoki 《Biochemical and biophysical research communications》1981,103(4):1273-1280
Cytochromes P-450 and 5 were observed in the microsomal fraction of interstitial tissue of rat testes. Microsomal cytochrome 5 was reduced by the NADH coupled with the activities of Δ5-3β-hydroxysteroid dehydrogenase with Δ5-Δ4 isomerase through conversion of pregnenolone to progesterone. Activities of NADPH-supported 17α-hydroxylase and C-17-C-20 lyase which converted progesterone to androstenedione were stimulated by either the presence of NADH or the oxidative reaction by the dehydrogenase upon Δ5-3β-hydroxysteroids. Androstenedione production enhanced by the reaction of the dehydrogenase was decreased by addition of the antibody against NADH-cytochrome 5 reductase which was purified from rat hepatic microsomes, suggesting the active participation of cytochrome 5 in the androgen synthesis. 相似文献
12.
Experimental evidence indicates that the major pathway of retinoic acid metabolism in hamster liver microsomes follows the sequence: retinoic acid → 4-hydroxy-retinoic acid → 4-keto-retinoic acid → more polar metabolites. Using all-trans-[10-3H]retinoic acid, it can be shown by reverse-phase high pressure liquid chromatographic analysis that the first and last steps of this sequence require NADPH, whereas the oxidation of 4-hydroxy to 4-keto-retinoic acid is NAD+ (or NADP+) dependent. Both NADPH-dependent steps, but not the NAD+-dependent dehydrogenase reaction, are strongly inhibited by carbon monoxide. The metabolism of retinoic acid but not of 4-hydroxy-retinoic acid is highly dependent on the vitamin A regimen of the animal. Retinoic acid is rapidly metabolized by liver microsomes either from vitamin A-normal hamsters or from vitamin A-deficient hamsters that have been pretreated with retinoic acid, but not by microsomes from vitamin A-deficient animals; in direct contrast, the rate of metabolism of 4-hydroxy-retinoic acid is equivalent in each of these microsomal preparations. Analysis of the kinetics of these reactions yields the following Michaelis constants with respect to the retinoid substrates: retinoic acid, 1 × 10?6m; 4-hydroxy-retinoic acid, 2 × 10?5m; and 4-keto-retinoic acid, 1 × 10?7m. The 4-hydroxy to 4-keto-retinoic acid oxidation has been shown to be experimentally irreversible, to have a KmNAD+of 2 × 10?5m, to be strongly inhibited by NADH, and to be unaffected by the presence of retinoic acid or its 4-keto-derivative in an equimolar ratio to the 4-hydroxy-substrate. 相似文献
13.
14.
The Structure of Calf Liver Cytochrome <Emphasis Type="Italic">b</Emphasis><Subscript>5</Subscript> at 2.8 Å Resolution 总被引:5,自引:0,他引:5
CYTOCHROME b5 is a haem-containing protein in the microsomes of liver tissue. It interacts specifically with a flavo-protein, cytochrome b5 reductase, which catalyses the transfer of electrons from NADH to the haem iron of the cytochrome1. The microsomal cytochrome b5 system has been implicated in fatty acid desaturation reactions2 and a similar system in erythrocytes may catalyse the reduction of methaemoglobin3. Calf liver cytochrome b5, solubilized by pancreatic lipase, has a molecular weight of 11,000 and consists of ninety-three amino-acids in the sequence shown in Fig. 1 (refs. 4 and 5). The haem group is non-covalently bound to the protein and can be removed reversibly by acid acetone treatment6. 相似文献
15.
Although microsomes prepared from rat kidney cortex contained significant concentrations of both NADH cytochrome b5 reductase and cytochrome b5, they did not catalyze cytochrome b5-dependent Δ9 oxidative lipid desaturation. However, incubation of kidney microsomes in the presence of control liver microsomes resulted in a two-fold increase in fatty acid desaturase activity over that seen with liver microsomes alone. Addition of kidney microsomes to liver microsomes prepared from animals maintained on a fat free diet resulted in an increased desaturase activity which was twice that seen with the control liver preparation. Kidney microsomes alone did not catalyze the cytochrome P-450-dependent N-demethylation of aminopyrine, and in contrast to the desaturate, no increase in demethylase activity was observed when kidney microsomes were added to liver microsomes. 相似文献
16.
Tsuyoshi Mouri Takeshi Shimizu Noriho Kamiya Masahiro Goto Hirofumi Ichinose 《Biotechnology progress》2009,25(5):1372-1378
A cytochrome P450BM3‐catalyzed reaction system linked by a two‐step cofactor regeneration was investigated in a cell‐free system. The two‐step cofactor regeneration of redox cofactors, NADH and NADPH, was constructed by NAD+‐dependent bacterial glycerol dehydrogenase (GLD) and bacterial soluble transhydrogenase (STH) both from Escherichia coli. In the present system, the reduced cofactor (NADH) was regenerated by GLD from the oxidized cofactor (NAD+) using glycerol as a sacrificial cosubstrate. The reducing equivalents were subsequently transferred to NADP+ by STH as a cycling catalyst. The resultant regenerated NADPH was used for the substrate oxidation catalyzed by cytochrome P450BM3. The initial rate of the P450BM3‐catalyzed reaction linked by the two‐step cofactor regeneration showed a slight increase (approximately twice) when increasing the GLD units 10‐fold under initial reaction conditions. In contrast, a 10‐fold increase in STH units resulted in about a 9‐fold increase in the initial reaction rate, implying that transhydrogenation catalyzed by STH was the rate‐determining step. In the system lacking the two‐step cofactor regeneration, 34% conversion of 50 μM of a model substrate (p‐nitrophenoxydecanoic acid) was attained using 50 μM NADPH. In contrast, with the two‐step cofactor regeneration, the same amount of substrate was completely converted using 5 μM of oxidized cofactors (NAD+ and NADP+) within 1 h. Furthermore, a 10‐fold dilution of the oxidized cofactors still led to approximately 20% conversion in 1 h. These results indicate the potential of the combination of GLD and STH for use in redox cofactor recycling with catalytic quantities of NAD+ and NADP+. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009 相似文献
17.
《Biochimica et Biophysica Acta (BBA)/Lipids and Lipid Metabolism》1981,663(1):194-202
According to Ohba et al. (Ohba M., Sato R., Yoshida Y., Nishino T. and Katsuki H. (1978) Biochem. Biophys. Res. Commun. 85, 21–27), yeast microsomes catalyze the removal of three methyl groups attached to the C-4 and C-14 positions of [1,7,15,22,26,30-14C]lanosterol (4,4,14α-trimethyl-5α-cholesta-8, 24-dien-3β-ol) in the presence of NADPH, NAD+ and molecular oxygen, concomitant with the liberation of 14CO2 derived from C-30 (one of the two methyl groups at the C-4 position). In this process the methyl group at the C-14 position is first removed in a cyanide-insensitive reaction and then the two methyl groups at the C-4 position are removed by a cyanide-sensitive enzyme system. In this study it was found that the 14CO2 formation from the 14C-labeled lanosterol was inhibited by antibodies to yeast cytochrome b5 and by palmitoyl-CoA, a substrate of the cytochrome b5-containing fatty acyl-CoA desaturase system of yeast microsomes. However, neither the antibodies nor palmitoyl-CoA inhibited the conversion of lanosterol to 4,4-dimethyl zymosterol (4,4-dimethyl-5α-cholesta-8,24-dien-3β-ol). It is concluded that cytochrome b5 and a cyanide-sensitive enzyme are involved in the 4-demethylation of 4,4-dimethylzymosterol, but not the 14α-demethylation of lanosterol, by yeast microsomes. It is suggested that a cyanide-sensitive enzyme acts as the terminal 4-demethylase and cytochrome b5 transfers reducing equivalents from NADPH to the terminal enzyme, as in the case of fatty acyl-CoA desaturation. The cyanide sensitivity of the 4-demethylation was, however, much greater than that of the desaturation. 相似文献
18.
Human tissue from uninvolved liver of cancer patients was fractionated using differential centrifugation and characterized for 11βHSD enzyme activity against corticosterone, dehydrocorticosterone, 7α- and 7β-hydroxy-dehydroepiandrosterone, and 7-oxo-dehydroepiandrosterone. An enzyme activity was observed in nuclear protein fractions that utilized either NADP+ or NAD+, but not NADPH and NADH, as pyridine nucleotide cofactor with Km values of 12 ± 2 and 390 ± 2 μM, compared to the Km for microsomal 11βHSD1 of 43 ± 8 and 264 ± 24 μM, respectively. The Km for corticosterone in the NADP+-dependent nuclear oxidation reaction was 102 ± 16 nM, compared to 4.3 ± 0.8 μM for 11βHSD1. The Kcat values for nuclear activity with NADP+ was 1687 nmol/min/mg/μmol, compared to 755 nmol/min/mg/μmol for microsomal 11βHSD1 activity. Inhibitors of 11βHSD1 decreased both nuclear and microsomal enzyme activities, suggesting that the nuclear activity may be due to an enzyme similar to 11βHSD Type 1 and 2. 相似文献
19.
Samuel Caito 《Biochemical and biophysical research communications》2010,392(3):264-998
Sirtuin1 (SIRT1) deacetylase and poly(ADP-ribose)-polymerase-1 (PARP-1) respond to environmental cues, and both require NAD+ cofactor for their enzymatic activities. However, the functional link between environmental/oxidative stress-mediated activation of PARP-1 and SIRT1 through NAD+ cofactor availability is not known. We investigated whether NAD+ depletion by PARP-1 activation plays a role in environmental stimuli/oxidant-induced reduction in SIRT1 activity. Both H2O2 and cigarette smoke (CS) decreased intracellular NAD+ levels in vitro in lung epithelial cells and in vivo in lungs of mice exposed to CS. Pharmacological PARP-1 inhibition prevented oxidant-induced NAD+ loss and attenuated loss of SIRT1 activity. Oxidants decreased SIRT1 activity in lung epithelial cells; however increasing cellular NAD+ cofactor levels by PARP-1 inhibition or NAD+ precursors was unable to restore SIRT1 activity. SIRT1 was found to be carbonylated by CS, which was not reversed by PARP-1 inhibition or selective SIRT1 activator. Overall, these data suggest that environmental/oxidant stress-induced SIRT1 down-regulation and PARP-1 activation are independent events despite both enzymes sharing the same cofactor. 相似文献
20.
The xanthine-oxidizing enzyme of rat liver has been purified as an NAD+-dependent dehydrogenase (type D) and as the O2-dependent oxidase (type O). The purified D and O variants are nearly homogenous as judged by polyacrylamide discontinuous gel electrophoresis and are indistinguishable on sodium dodecyl sulfate-urea gels. The absorption spectrum of the type D enzyme is indistinguishable from that of the type O enzyme and closely resembles the spectra of xanthine-oxidizing enzymes from other sources. The types D and O enzymes have essentially the same cofactor composition. Oxidation of xanthine by type D is stimulated by NAD+ with concomitant NADH formation. Type D is able to utilize NADH as well as xanthine as electron donor to various acceptors, in contrast to type O that is unable to oxidize NADH. Arsenite, cyanide and methanol completely abolish xanthine oxidation by the type D enzyme while affecting the activities with NADH to varying extents. In these respects rat liver xanthine dehydrogenase closely resembles chicken liver xanthine dehydrogenase. However, in contrast to the avian enzyme, the purified rat liver enzyme is unstable as a dehydrogenase and is gradually converted to an oxidase. This conversion is accompanied by an increase in the aerobic xanthine → cytochrome c activity. The native type D enzyme in rat liver extracts is precipitable with antibody prepared against purified type O. The Km for xanthine is not significantly different for the two forms. 相似文献