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1.
The substrate stereospecificity of NADH-ferricyanide reductase activities in the inner mitochondrial membrane and peroxisomal membrane of potato (Solanum tuberosum L.) tubers, spinach (Spinacea oleracea L.) leaf plasma membrane, and red beetroot (Beta vulgaris L.) tonoplast were all specific for the [beta]-hydrogen of NADH, whereas the reductases in wheat root (Triticum aestivum L.) endoplasmic reticulum and potato tuber outer mitochondrial membrane were both [alpha]-hydrogen specific. In all isolated membrane fractions one or several polypeptides with an apparent size of 45 to 55 kD cross-reacted with antibodies raised against a microsomal NADH-ferricyanide reductase on western blots.  相似文献   

2.
Two monoclonal antibodies, 17(3)9 and 36(79)4, were preparedagainst nitrate reductase from Spinacia oleracea L. leaves.An enzyme-linked immunosorbent assay showed that 17(3)9, butnot 36(79)4, reacted more strongly to heat-denatured than nativeantigen. These antibodies inhibited NADH-nitrate reductase aswell as its various partial activities including reduced methylvilogen-nitrate reductase, reduced flavin mononucleotide-nitratereductase and NADH-cytochrome c reductase activities, but notNADH-ferricyanide reductase activity. Immunoblotting after electrophoreticseparation of nitrate reductase fragments obtained by Staphyrococcusaureus V8 protease digestion of native enzyme revealed thatthe two monoclonal antibodies bind to different epitopes locatedon the 28 kDa of the NADH-ferricyanide reductase domain. (Received October 2, 1987; Accepted June 9, 1988)  相似文献   

3.
The stereospecificity of NADH-ferricyanide reductase and NADH-cytochrome c reductase in the endoplasmic reticulum (ER) for the α-hydrogen on the nicotinamide ring is presented as a very sensitive and convenient assay to detect ER contamination in preparations of membranes lacking α-specific NADH-acceptor reductase, such as the plasma membrane and the tonoplast. The experimental details of the assay are given and the limitations explored (time-course, amount of protein, possible side reactions, speed, reproducibility, etc.). The NADH-ferricyanide reductase activity of plasma membranes from spinach and sugarbeet leaf was completely β-specific and always showed a latency (increase upon addition of Triton X-100), whereas the α-specificity in the ER was non-latent. This is consistent with the presence of mainly right-side-out vesicles in preparations of plasma membranes with the binding site for NADH and ferricyanide on the inner, cytoplasmic surface. In contrast, right-side-out ER vesicles have the binding site on the outer, cytoplasmic surface. The addition of as little as 1% of the α-specific ER (on an NADH-ferricyanide activity basis) to the spinach leaf plasma membrane could be detected with the stereospecificity assay. Wheat root plasma membrane showed some α-specificity (in addition to β-specificity) which was probably due to ER contamination since the activity was non-latent. The stereospecificity assay is also shown to be useful in monitoring the separation of tonoplast vesicles from ER vesicles by countercurrent distribution of a light microsomal fraction. It follows that the NADH-acceptor reductase activities in preparations of plasma membrane and tonoplast are due to distinct enzymes characteristic for those membranes.  相似文献   

4.
Potato microsomal membranes were solubilized by 0.5% sodium cholate solutions. Separation of lipids from proteins was realized by two successive gel filtrations on two different Sephadex columns. Lipid-free microsomal proteins maintained a high NADH-ferricyanide reductase activity but had a lowered (20%) NADH-cytochrome c reductase activity. The latter activity was strongly stimulated when lipid-free proteins were integrated, by sonication, into phosphatidylserine or phosphatidylinositol liposomes. Some stimulation was obtained also with phosphatidylcholine-lysophosphatidylcholine (7:3) mixtures. Other phospholipids were far less active or even inhibitory. Acidic phospholipids stimulate NADH-cytochrome c reductase activity by increasing noticeably the apparent affinities of enzymatic proteins for NADH or cytochrome c.  相似文献   

5.
Summary The stereospecificity of NADH-ferricyanide reductase activities in the inner mitochondrial membrane, peroxisomal membrane, plasma membrane and tonoplast are all specific for the -hydrogen of NADH whereas the reductases in the ER, the Golgi and the outer mitochondrial membrane are -specific. This shows unequivocally that the NADH-ferricyanide activity in the plasma membrane is not caused by ER contamination. In all the membranes one or several polypeptides with an apparent size of 45–50 kDa cross-react with antibodies raised against a microsomal NADH-ferricyanide reductase. An NADH-ferricyanide reductase was purified from spinach leaf plasma membranes. The enzyme was released from the membrane by CHAPS solubilization and purified 360-fold by ion-exchange chromatography followed by affinity chromatography and size exclusion chromatography on FPLC. A major band of 45 kDa was detected by SDS-PAGE and it cross-reacted with the anti-NADH-ferricyanide reductase antibodies. The native size of the enzyme is 160 kDa as determined by size-exclusion chromatography indicating that it is a tetramer. Isoelectric focusing revealed three isoenzymes between pH 5.3 and 5.6. The enzyme shows typical FAD fluorescence spectra with excitation peaks at 371 and 468 nm and an emission peak at 525 nm. It is specific for the -hydrogen of NADH and prefers NADH over NADPH as electron donor. It is highly specific for ferricyanide as electron acceptor and it is therefore unlikely to be the enzyme responsible for iron reduction on the outer surface of the plasma membrane.Abbreviations CHAPS 3-[(3-cholamidopropyl)dimethylammoniol]-1-propanesulfonate - DQ duroquinone - FPLC fast protein liquid chromatography; Ferricyanide hexacyanoferrate(III) - NEM N-ethylmaleimide - PCMB p-chloromercurobenzoate - SHAM salicylhydroxamic acid - SMP submitochondrial particles  相似文献   

6.
The role of NADH-cytochrome b5 reductase and cytochrome b5 as electron carriers in NADH-supported electron transport reactions in rat liver microsomes has been examined by measuring three enzyme activities: NADH-cytochrome P-450 reductase, NADH-peroxidase, and NADH-cytochrome c reductase. The first two reactions are known to involve the participation of an NADH-specific reductase and cytochrome P-450 whereas the third requires the reductase and cytochrome b5. Antibody prepared against NADH-cytochrome b5 reductase markedly inhibited the NADH-peroxidase and NADH-cytochrome c reductase activities suggesting the involvement of this NADH-specific reductase in these reactions. Liver microsomes prepared from phenobarbital-pretreated rats were digested with subtilisin to remove cytochrome b5 and the submicrosomal particles were collected by centrifugation. The specific content of cytochrome b5 in the digested particles was about 5% of that originally present in liver microsomes and all three enzyme activities showed similar decreases whereas NADH-ferricyanide reductase activity (an activity associated with the flavoenzyme NADH-cytochrome b5 reductase) remained virtually unchanged. Binding of an excess of detergent-purified cytochrome b5 to the submicrosomal particles at 37 °C for 20 min followed by centrifugation and enzymic measurements revealed a striking increase in the three enzyme activities. Further evidence for cytochrome b5 involvement in the NADH-peroxidase reaction was the marked inhibition by antibody prepared against the hemoprotein. These results suggest that in microsomal NADH-supported cytochrome P-450-dependent electron transport reactions, cytochrome b5 functions as an intermediate electron carrier between NADH-cytochrome b5 reductase and cytochrome P-450.  相似文献   

7.
Plasma membrane-associated redox systems play important roles in regulation of cell growth, internal pH, signal transduction, apoptosis, and defense against pathogens. Stimulation of cell growth and stimulation of the redox system of plasma membranes are correlated. When cell growth is inhibited by antitumor agents such as doxorubicin, capsaicin, and antitumor sulfonylureas, redox activities of the plasma membrane also are inhibited. A doxorubicin-inhibited NADH-quinone reductase was characterized and purified from plasma membranes of rat liver. First, an NADH-cytochrome b(5) reductase, which was doxorubicin-insensitive, was removed from the plasma membranes by the lysosomal protease, cathepsin D. After removal of the NADH-cytochrome b(5) reductase, the plasma membranes retained a doxorubicin-inhibited NADH-quinone reductase activity. The enzyme, with an apparent molecular mass of 57 kDa, was purified 200-fold over the cathepsin D-treated plasma membranes. The purified enzyme had also an NADH-coenzyme Q(0) reductase (NADH: external acceptor (quinone) reductase; EC 1.6.5.) activity. Partial amino acid sequence of the enzyme showed that it was unique with no sequence homology to any known protein. Antibody against the enzyme (peptide sequence) was produced and affinity-purified. The purified antibody immunoprecipitated both the NADH-ferricyanide reductase activity and NADH-coenzyme Q(0) reductase activity of plasma membranes and cross-reacted with human chronic myelogenous leukemia K562 cells and doxorubicin-resistant human chronic myelogenous leukemia K562R cells. Localization by fluorescence microscopy showed that the reaction was with the external surface of the plasma membranes. The doxorubicin-inhibited NADH-quinone reductase may provide a target for the anthracycline antitumor agents and a candidate ferricyanide reductase for plasma membrane electron transport.  相似文献   

8.
A rabbit antiserum was prepared against rat liver microsomal cytochrome b5, and utilized in demonstrating the participation of this cytochrome in the microsomal stearyl-CoA desaturation reaction. The antiserum inhibited the NADH-cytochrome c reductase activity of rat liver microsorncs, but it did not inhibit either NADH-ferricyanide or NADPH-cytochrome c reductase activity of the microsomes. Thus, the inhibitory effect of the antiserum on the microsomal electron-transferring reactions seemed to be specific to those which require the participation of cytochrome b5.The NADH-dependent and NADPH-dependent desaturations of stearyl CoA by rat liver microsomes were strongly inhibited by the antiserum. The reduction of cytochrome b5 by NADH-cytochrome b5 reductase as well as the reoxidation of the reduced cytochrome b3 by the desaturase, the terminal cyanide-sensitive factor of the desaturation system, was also strongly inhibited by the antiserum. When about 90%, of cytochrome b5 was removed from rat liver microsomes by protease treatment, the desaturation activity of the microsomes became much more sensitive to inhibition by the antiserum. These results confirmed our previous conclusion that the reducing equivalent for the desaturation reaction is transferred from NAD(P)H to the cyanidesensitive factor mainly via cytochrome b5 in the microsomal membranes.  相似文献   

9.
《Plant science》1986,44(1):13-21
Catalytical hydrogenation of the unsaturated fatty acyl residues of microsomal lipids was realized for different times. Progress of the reaction was followed by calculating the progressive loss of double-bonds in 100 initial acyl residues (percentage of hydrogenation). The maximum loss observed was 45% after 60 min.The drop in polyunsaturated faty acid content was coupled with an increase in the amount of stearic acid and oleic acid.The order parameter of microsomal lipids, measured by ESR, increased parallely to the reduction of double bonds. Maximum hydrogenation of microsomal lipids strongly (200–250%) stimulated microsomal NADH-ferricyanide reductase activity. NADH-cytochrome c reductase, lysophosphatidylcholine-acyl-transferase and oleoyl-phosphatidylcholine desaturase were inhibited (40%, 100% and 100% respectively). These modifications of enzyme activities are discussed in conjunction with the changes observed in membrane fluidity, following hydrogenation of microsomal lipids  相似文献   

10.
Two nitrate reductase (NR) mutants were selected for low nitrate reductase (LNR) activity by in vivo NR microassays of M2 seedlings derived from nitrosomethylurea-mutagenized soybean (Glycine max [L.] Merr. cv Williams) seeds. The mutants (LNR-5 and LNR-6) appeared to have normal nitrate-inducible NR activity. Both mutants, however, showed decreased NR activity in vivo and in vitro compared with the wild-type. In vitro FMNH2-dependent nitrate reduction and Cyt c reductase activity of nitrate-grown plants, and nitrogenous gas evolution during in vivo NR assays of urea-grown plants, were also decreased in the mutants. The latter observation was due to insufficient generation of nitrite substrate, rather than some inherent difference in enzyme between mutant and wild-type plants. When grown on urea, crude extracts of LNR-5 and LNR-6 lines had similar NADPH:NR activities to that of the wild type, but both mutants had very little NADH:NR activity, relative to the wild type. Blue Sepharose columns loaded with NR extract of urea-grown mutants and sequentially eluted with NADPH and NADH yielded a NADPH:NR peak only, while the wild-type yielded both NADPH: and NADH:NR peaks. Activity profiles confirmed the lack of constitutive NADH:NR in the mutants throughout development. The results provide additional support to our claim that wild-type soybean contains three NR isozymes, namely, constitutive NADPH:NR (c1NR), constitutive NADH:NR (c2NR), and nitrate-inducible NR (iNR).  相似文献   

11.
Peroxisomes and mitochondria were purified from potato tubers (Solanum tuberosum L. cv. Bintje) by differential centrifugation followed by separation on a continuous Percoll gradient containing 0.3 M sucrose in the lower half and 0.3 M mannitol in the upper half. The peroxisomes band at the bottom and the mitochondria in the middle of this type of gradient. Mitochondrial contamination of the peroxisomes was only 2% [as judged by cytochrome c oxidase (EC 1.3.9.1) activity]. Contamination by amyloplasts, plasma membrane and endoplasmic reticulum was also minimal. The peroxisomes were 80% intact as judged by malate dehydrogenase (MDH, NAD?-dependent; EC 1.1.1.37) latency. The specific activity of NADH-ferricyanide reductase and NADH-Cyt c reductase was 0.22 and 0.051 μmol (mg protein)?1 min?1 in freshly isolated peroxisomes, respectively. The active site of the reductase appeared to be on the inner surface of the membrane. The peroxisomes also contained a b-type cytochrome. Frozen peroxisomes were subfractionated by osmotic rupture followed by centrifugation to separate the soluble proteins from the peroxisomal membrane. About half the MDH and 30% of the NADH-ferricyanide reductase activity was associated with the membrane but only 6% of the catalase (EC 1.11.1.6) activity. A further wash removed 75% of the residual catalase with only a small loss of MDH or NADH-ferricyanide reductase activity. MDH appears to be closely associated with the peroxisomal membrane. When the purified peroxisomal membrane was analyzed by SDS-PAGE followed by silver staining, prominent bands at 22, 40, 41, 48, 53 and 74 kDa were observed. After immunoblotting the purified peroxisomal membrane, a band at 53 kDa showed strong cross-reactivity with antibodies raised against NADH-ferricyanide reductase. Since the NADH-ferricyanide reductase activity in the peroxisomal membrane could be shown to be specific for the β-hydrogen of NADH, the activity could not be due to contamination by endoplasmic reticulum where the reductase is α-specific. We conclude that the peroxisomal membrane contains a short redox chain, consisting of a NADH-ferricyanide reductase and a b-type cytochrome, similar to that of e.g. the plasma membrane. The role of this redox chain has yet to be elucidated.  相似文献   

12.
A rapid method of preparing plasma membranes from isolated fat cells is described. After homogenization of the cells, various fractions were isolated by differential centrifugation and linear gradients. Ficoll gradients were preferred because total preparation time was under 3 hr. The density of the plasma membranes was 1.14 in sucrose. The plasma membrane fraction was virtually uncontaminated by nuclei but contained 10% of the mitochondrial succinic dehydrogenase activity and 25–30% of the RNA and reduced nicotinamide adenine dinucleotide cytochrome c reductase activity of the microsomal fraction. Part of the RNA and NADH-cytochrome c reductase activity was believed to be native to the plasma membrane or to the attached endoplasmic reticulum membranes demonstrated by electron microscopy. The adenyl cyclase activity of the plasma membrane fraction was five times that of Rodbell's "ghost" preparation and retained sensitivity to epinephrine. The plasma membrane ATPase activity was five times that of the homogenate and microsomal fractions. Electron microscopic evidence suggested contamination of the plasma membrane fraction by other subcellular components to be less than the biochemical data indicated.  相似文献   

13.
The objective of this study was to identify factors which limit leaf nitrate reductase (NR) activity as decline occurs during flowering and beginning seed development in soybean (Glycine max [L.] Merr. cv Clark). Level of NR enzyme activity, level of reductant, and availability of NO3 as substrate were evaluated for field-grown soybean from flowering through leaf senescence. Timing of reproductive development was altered within one genotype by (a) exposure of Clark to an artificially short photoperiod to hasten flowering and podfill, and (b) the use of an early flowering isoline. Nitrogen (N) was soil-applied to selected plots at 500 kilograms per hectare as an additional variable. Stem NO3 concentration and in vivo leaf NR activity were significantly correlated (R2 = 0.69 with nitrate in the assay medium and 0.74 without nitrate in the medium at P = 0.001) across six combinations of reproductive and soil N-treatment. The supply of NO3 from the root to the leaf tissue was the primary limitation to leaf NR activity during flowering and podfill. Levels of NR enzyme and reductant were not limiting to leaf NR activity during this period.  相似文献   

14.
ISOLATION AND PROPERTIES OF THE PLASMA MEMBRANE OF KB CELLS   总被引:3,自引:2,他引:1       下载免费PDF全文
Plasma membranes from KB cells were isolated by the method of latex bead ingestion and were compared with those obtained by the ZnCl2 method. Optimal conditions for bead uptake and the isolation procedure employing discontinuous sucrose gradient centrifugation are described. All steps of preparative procedure were monitored by electron microscopy and specific enzyme activities. The plasma membrane fraction obtained by both methods is characterized by the presence of the Na+ + K+-activated ATPase and 5'-nucleotidase, and contains NADPH-cytochrome c reductase and cytochrome b5. The latter two enzymes are also present in lower concentrations in the microsomal fraction. Unlike microsomes which are devoid of the Na+ + K+-activated ATPase and which contain only traces of 5'-nucleotidase activity, the plasma membrane fraction contains only trace amounts of the rotenone-insensitive NADH-cytochrome c reductase but no cytochrome P-450, both of which are mainly microsomal components. Morphologically the plasma membrane fraction isolated by the latex bead method is composed of vesicles of 0.1–0.3 µm in diameter. On the basis of the biochemical and morphological criteria presented, it is concluded that the plasma membrane fraction isolated by the above methods are of high degree of purity.  相似文献   

15.
In vivo nitrate reductase (NR) activity declined gradually either in absence or presence of Mg2+ In dark grown plants of spinach. The increased sensitivity of the extracted NR from the dark grown plants to Mg2+ and ATP is indicative of the post-translational modification as one of the mechanisms to control NR activity. The response of extracted NR was gradual and not instantaneous suggesting a complex interplay of NR regulation, as the dark acclimatized plants when exposed to light caused significant nitrate reduction within 15 min of light exposures even in the presence of Mg2+ and ATP.  相似文献   

16.
The importance of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase) in the regulation of sesquiterpenoid phytoalexin accumulation in potato (Solanum tuberosum L. cv Kennebec) was examined. Wounding of potato tubers produced a large temporary increase in HMG-CoA reductase activity of the microsomal and organelle fractions. Treatment of wounded tuber tissue with the sesquiterpenoid phytoalexin elicitor arachidonic acid further increased and prolonged the HMG-CoA reductase activity in the microsomal but not the organelle fraction. Incubation of elicitor-treated tuber tissue in white light reduced organelle and microsomal HMG-CoA reductase activity to 50% and 10%, respectively, of the activity of tissues held in darkness. Constant light also reduced overall phytoalexin accumulation 58% by greatly reducing levels of lubimin. Rishitin accumulation was not significantly altered by light. Application of nanomolar amounts of mevinolin, a highly specific inhibitor of HMG-CoA reductase, to elicitor-treated tuber tissue produced a large decline in lubimin accumulation and did not markedly alter rishitin accumulation. These results indicate that HMG-CoA reductase has a role in the complex regulation of sesquiterpenoid phytoalexin accumulation in potato.  相似文献   

17.
B. A. Notton  R. J. Fido  G. Galfre 《Planta》1985,165(1):114-119
A set of monoclonal antibodies has been raised against NADH-nitrate reductase (NR; EC 1.6.6.1) from spinach (Spinacea oleracea L.) leaves. Antibodies were screened by enzyme-linked immunosorbent assay and by their ability to inhibit various activities of the enzyme. The six monoclonals selected (AFRC MAC 74 to 79) are all gamma globulins; four (MAC 74 to 77) inhibit all terminal donating activities (NADH-NR; flavin mononucleotide, reduced form (FMNH2)-NR; and methyl viologen, reduced form (MV)-NR) and two (MAC 78 and 79) inhibit the acceptor activities (NADH-NR, and NADH-cytochrome c reductase). MAC 74 to 77 inhibit the NADH-NR activity of crude extracts of a variety of species (mono- and dicotyledoneae) while MAC 78 and 79 are effective against spinach and marrow, but not oil-seed rape, cucumber, oats, wheat and barley.Abbreviations Cyt c Rase cytochrome c reductase - ELISA enzyme-linked immunosorbent assay - FAD(H2) flavin adenine dinucleotide (reduced form) - FMN(H2) flavin mononucleotide (reduced form) - McAb monoclonal antibody - MV methyl viologen reduced form - NR nitrate reductase  相似文献   

18.
Electron transport from untreated to mersalyzed microsomal vesicles at the level of NADH-cytochrome b5 reductase or cytochrome b5 has been demonstrated in the absence of added water-soluble electron carriers. A similar effect was shown in the systems “intact mitochondria — mersalyzed microsomes” and “mersalyzed mitochondria— untreated microsomes”. No measurable electron transport between intact and mersalyzed particles of inner mitochondrial membrane was found. The obtained data suggest that the capability to carry out intermembrane electron transfer is specific for NADH-cytochrome b5 reductase and/or cytochrome b5, localized in microsomal and outer mitochondrial membranes.  相似文献   

19.
R J Fido 《Phytochemistry》1991,30(11):3519-3523
Fragments of spinach nitrate reductase (NR) were prepared by limited proteolysis of immunopurified enzyme using both Staphylococcus aureus V8 protease and trypsin. Incubation of NR with V8 protease yielded two enzymically active fragments which could be size separated by FPLC on a Superose 12 column or subjected to further proteolysis while bound to a blue Sepharose affinity column. An NADH-ferricyanide (NADH-FR) active fragment bound to, and was eluted from, a blue Sepharose column by micromolar concentrations of NADH. A fragment with methyl viologen-NR activity was either eluted from the same column using 1 M KNO3 or on further treatment in situ on the blue Sepharose column with trypsin. Incubation of holo-NR with trypsin resulted in the loss of all terminal nitrate reducing activities but no loss in either NADH-FR activity or NADH-cytochrome c reductase activity. Two protease-sensitive regions of NR are shown which connect essentially between the flavin (FAD) and haem domains, and between the haem and molybdenum domains of NR. Amino acid analysis of the FAD- and FAD/haem-containing domains yielded two partial sequences which are compared with sequences deduced from complementary DNA (cDNA) of NR from Arabidopsis, tobacco and spinach. The deduced sequences from Arabidopsis and tobacco are found to be ca 80% and the spinach 100% homologous to the sequence obtained for spinach NR fragments.  相似文献   

20.
The activity of liver microsomal and Guerin??s carcinoma NADH-cytochrome b 5 reductase, the content and the rate of cytochrome b 5 oxidation-reduction have been investigated in tumor-bearing rats exposed to preliminary irradiation. Preliminary irradiation of rats (before transplantation of Guerin??s carcinoma) resulted in the decrease of NADH-cytochrome b 5 reductase activity and the content of cytochrome b 5 in the Guerin??s carcinoma microsomal fraction in the latent and logarithmic phases of oncogenesis compared with the non-irradiated tumor-bearing rats. The effect of irradiation preceding transplantation of the tumor to rats results in the increase of enzymatic activities of liver microsomal NADH-cytochrome b 5 reductase in the latent and logarithmic phases of tumor growth as compared with non-irradiated tumor-bearing rats. At the same time the content of cytochrome b 5 decreased, while the rate of its oxidation-reduction rate simultaneously increased in the liver microsomal fraction of tumor-bearing rats.  相似文献   

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