Development of Endoplasmic Reticulum and Glyoxysomal Membrane Redox Activities during Castor Bean Germination

Redox activities, NADH:ferricyanide reductase, NAD(P)H:cytochrome reductases, and NADH:ascorbate free-radical reductase, are present in endoplasmic reticulum (ER) and glyoxysomal membranes from the endosperm of germinating castor bean (Ricinus comminus L. var Hale). The development of these functions was followed in glyoxysomes and ER isolated on sucrose gradients from castor bean endosperm daily from 0 through 6 days of germination. On a per seed basis, glyoxysomal and ER protein, glyoxysomal and ER membrane redox enzyme activities, and glyoxylate cycle activities peaked at day 4 as did the ER membrane content of cytochrome P-450. NADH:ferricyanide reductase was present in glyoxysomes and ER isolated from dry seed. This activity increased only about twofold in glyoxysomes and threefold in ER during germination relative to the amount of protein in the respective fractions. The other reductases, NADH:cytochrome reductase and NADH:ascorbate free-radical reductase, increased about 10-fold in the ER relative to protein up to 4 to 5 days, then declined. NADPH:cytochrome reductase reached maximum activity relative to protein at day 2 in both organelles. The increases in redox activities during germination indicate that the membranes of the ER and glyoxysome are being enriched with redox proteins during their development. The development of redox functions in glyoxysomes was found to be coordinated with development of the glyoxylate cycle.     

Ascorbate free-radical reduction by glyoxysomal membranes

Glyoxysomal membranes from germinating castor bean (Ricinus communis L. cv Hale) endosperm contain an NADH dehydrogenase. This enzyme can utilize extraorganellar ascorbate free-radical as a substrate and can oxidize NADH at a rate which can support intraglyoxysomal demand for NAD⁺. NADH:ascorbate free-radical reductase was found to be membrane-associated, and the activity remained in the membrane fraction after lysis of glyoxysomes by osmotic shock, followed by pelleting of the membranes. In whole glyoxysomes, NADH:ascorbate free-radical reductase, like NADH:ferricyanide reductase and unlike NADH:cytochrome c reductase, was insensitive to trypsin and was not inactivated by Triton X-100 detergent. These results suggest that ascorbate free-radical is reduced by the same component which reduces ferricyanide in the glyoxysomal membrane redox system. NADH:ascorbate free-radical reductase comigrated with NADH:ferricyanide and cytochrome c reductases when glyoxy-somal membranes were solubilized with detergent and subjected to rate-zonal centrifugation. The results suggest that ascorbate free-radical, when reduced to ascorbate by membrane redox system, could serve as a link between glyoxysomal metabolism and other cellular activities.     

Orientation of electron transport activities in the membrane of intact glyoxysomes isolated from castor bean endosperm

Intact glyoxysomes were isolated from castor bean endosperm on isometric Percoll gradients. The matrix enzyme, malate dehydrogenase, was 80% latent in the intact glyoxysomes. NADH:ferricyanide and NADH:cytochrome c reductase activities were measured in intact and deliberately broken organelles. The latencies of these redox activities were found to be about half the malate dehydrogenase latency. Incubation of intact organelles with trypsin eliminated NADH:cytochrome c reductase activity, but did not affect NADH:ferricyanide reductase activity. NADH oxidase and transhydrogenase activities were negligible in isolated glyoxysomes. Mersalyl and Cibacron blue 3GA were potent inhibitors of NADH:cytochrome c reductase. Quinacrine, Ca²⁺ and Mg²⁺ stimulated NADH:cytochrome c reductase activity in intact glyoxysomes. The data suggest that some electron donor sites are on the matrix side and some electron acceptor sites are on the cytosolic side of the membrane.     

Characterization of membrane-bound electron transport enzymes from castor bean glyoxysomes and endoplasmic reticulum

Membranes purified from castor bean endosperm glyoxysomes by washing with sodium carbonate exhibited integral NADH:ferricyanide and NADH:cytochrome c reductase activities. The enzyme activities could not be attributed to contamination by other endomembranes. Purified endoplasmic reticulum membranes also contained the redox activities; and marker enzyme analysis indicated minimum cross contamination between glyoxysomal and endoplasmic reticulum fractions. The glyoxysomal redox activities were optimally solubilized at detergent to protein ratios (weight to weight) of 10 (Triton X-100), 50 (3-[3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate), and 100 (octylglucoside). Detergent in excess of the solubilization optimum was stimulatory to NADH:ferricyanide reductase and inhibitory to NADH:cytochrome c reductase. Endoplasmic reticulum redox activity solubilization profiles were similar to those obtained for glyoxysomal enzymes using Triton X-100. Purification of the glyoxysomal and endoplasmic reticulum NADH:ferricyanide reductases was accomplished using dye-ligand affinity chromatography on Cibacron blue 3GA agarose. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis of NADH:ferricyanide reductase preparations purified by rate-zonal density gradient centrifugation, affinity chromatography, and nondenaturing electrophoresis of detergent-solubilized glyoxysomal and endoplasmic reticulum membranes consistently displayed 32- and 33-kDa silver-stained polypeptide bands, respectively.     

Spinach Nitrate Reductase : Effects of Ionic Strength and pH on the Full and Partial Enzyme Activities

Initial velocity studies of immunopurified spinach nitrate reductase have been performed under conditions of controlled ionic strength and pH and in the absence of chloride ions. Increased ionic strength stimulated NADH:ferricyanide reductase and reduced flavin:nitrate reductase activities and inhibited NADH:nitrate reductase, NADH:cytochrome c reductase and reduced methyl viologen:nitrate reductase activities. NADH:dichlorophenolindophenol reductase activity was unaffected by changes in ionic strength. All of the partial activities, expressed in terms of micromole 2 electron transferred per minute per nanomole heme, were faster than the overall full, NADH:nitrate reductase activity indicating that none of the partial activities included the rate limiting step in electron transfer from NADH to nitrate. The pH optimum for NADH:nitrate reductase activity was determined to be 7 while values for the various partial activities ranged from 6.5 to 7.5. Chlorate, bromate, and iodate were determined to be alternate electron acceptors for the reduced enzyme. These results indicate that unlike the enzyme from Chlorella vulgaris, intramolecular electron transfer between reduced heme and Mo is not rate limiting for spinach nitrate reductase.     

Electron transport in glyoxysomal membranes

Glyoxysomes were isolated from germinating castor bean endosperm by equilibrium density gradient centrifugation in a vertical rotor. To recover the membranes, glyoxysome ghosts were prepared by osmotic shock and then subjected to differential centrifugation. The glyoxysomal membranes and the endoplasmic reticulum (ER), isolated by the same methods, were assayed for electron transport components. Both organelles contained NADH ferricyanide reductase, NADH cytochrome c reductase, and cytochromes b₅ and P-420. The ER also contained cytochrome P-450. Pyridine hemochrome derivatives of the organelle membranes and hemin produced coincident difference spectra, indicating that only b-type cytochromes are present in glyoxysomal and ER membranes. The maximal activities of ferricyanide reductase and cytochrome c reductase in glyoxysomes, 2.19 and 0.33 μmol min^?1 mg membrane protein^?1, respectively, represent 30 and 18% of the activities in the ER. The cytochrome b₅ content of the glyoxysomal membrane is 0.108 nmol mg^?1, 31% of the level found in ER. The reductases from both organelles were resistant to solubilization by salt (0.2 m KCl) and were easily solubilized by detergent (1% Triton X-100). Flavin analysis of the organelles from germinating castor beam endosperm confirmed spectral evidence that the flavin content of glyoxysomes is quite high, 100 pmol mg protein^?1, more than twice that of mitochondria. Three-quarters of the glyoxysomal flavin was solubilized by KCl, but even after salt treatment the glyoxysomal membrane flavin content, 98 pmol mg membrane protein^?1, is three times greater than that of the ER.     

The oxidation-reduction characteristics of cytochrome b5 in hen liver microsomes.

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.     

Purification and Characterization of Microsomal Cytochrome b(5) and NADH Cytochrome b(5) Reductase from Pisum sativum

In this communication we document the reproducible protocols for the purification of milligram quantities of cytochrome b₅ and NADH-cytochrome b₅ reductase from the microsomal fraction of Pisum sativum. The cytochrome b₅ component of this NADH linked electron transport chain was found to have a molecular mass of 16,400 daltons and the reductase a molecular mass of 34,500 daltons. These components could be reconstituted into a functional NADH oxidase activity active in the reduction of exogenous cytochrome c or ferricyanide. In the latter assay the purified reductase exhibited a turnover number of 22,000 per minute. The amino-terminal amino acid sequence of the cytochrome b₅ component was determined by sequential Edmund degredation, thus providing crucial information for the efficient cloning of this central protein of plant microsomal electron transfer.     

Expression in Escherichia coli of Cytochrome c Reductase Activity from a Maize NADH:Nitrate Reductase Complementary DNA

A cDNA clone was isolated from a maize (Zea mays L. cv W64A×W183E) scutellum λgt11 library using maize leaf NADH:nitrate reductase Zmnr1 cDNA clone as a hybridization probe; it was designated Zmnr1S. Zmnr1S was shown to be an NADH:nitrate reductase clone by nucleotide sequencing and comparison of its deduced amino acid sequence to Zmnr1. Zmnr1S, which is 1.8 kilobases in length and contains the code for both the cytochrome b and flavin adenine dinucleotide domains of nitrate reductase, was cloned into the EcoRI site of the Escherichia coli expression vector pET5b and expressed. The cell lysate contained NADH:cytochrome c reductase activity, which is a characteristic partial activity of NADH:nitrate reductase dependent on the cytochrome b and flavin adenine dinucleotide domains. Recombinant cytochrome c reductase was purified by immunoaffinity chromatography on monoclonal antibody Zm2(69) Sepharose. The purified cytochrome c reductase, which had a major size of 43 kilodaltons, was inhibited by polyclonal antibodies for maize leaf NADH:nitrate reductase and bound these antibodies when blotted to nitrocellulose. Ultraviolet and visible spectra of oxidized and NADH-reduced recombinant cytochrome c reductase were nearly identical with those of maize leaf NADH:nitrate reductase. These two enzyme forms also had very similar kinetic properties with respect to NADH-dependent cytochrome c and ferricyanide reduction.     

Properties of the System for the Mixed Function Oxidation of Kaurene and Kaurene Derivatives in Microsomes of the Immature Seed of Marah macrocarpus: Electron Transfer Components

Cytochrome P-450 and cytochrome b₅ at levels of approximately 0.10 and 0.60 nanomole per milligram of microsomal protein were detected by spectral measurements in microsomes prepared from endosperm tissue of immature Marah macrocarpus seeds. TPNH-cytochrome c reductase, DPNH-cytochrome c reductase, andDPNH-cytochrome b₅ reductase activities were also present in these microsomes at levels of approximately 0.060, 0.22, and 0.52 unit per milligram of microsomal protein, respectively. (One unit of reductase is the amount of enzyme catalyzing the reduction of 1 micromole of electron acceptor per minute.) Treatments of microsomes with steapsin or trypsin were not effective in solubilizing any of these electron transport components in detectable form. However, treatment of a microsomal suspension in 25% glycerol with 1% sodium deoxycholate led to the release of about 60% of the protein and each of the above hemoproteins and electron transfer activities to the fraction which was not pelleted after centrifugation for 2 hours at 105,000g. Some ent-kaur-16-ene oxidase activity could be detected in the solubilized fraction after removal of the detergent. Cytochrome b₅ and DPNH-cytochrome b₅ reductase activity were largely separated from one another and from an overlapping mixture of TPNH-cytochrome c reductase and DPNH-cytochrome c reductase when the sodium deoxycholate-solubilized fraction was chromatographed on a DEAE-cellulose column. No cytochrome P-450 or cytochrome P-420 was detected in the column fractions and no ent-kaur-16-ene oxidase activity was detected when the column fractions were tested singly or in combination.     

The effect of o-diphenols upon the microsomal NADPH and NADH oxidase activities

Catechol and catecholamines have been assayed upon the microsomal NADPH and NADH oxidase activities. Epinephrine shows a catalytic effect on the NADPH oxidation characterized by a small lag. The two to threefold increase in rate can be suppressed by Superoxide dismutase if the enzyme is added before the reaction begins. The catalytic effect is ascribed to a quinone formed by two electron oxidation of epinephrine by the Superoxide ion. The quinone, which is not catalytically active in the NADH chain, appears to mediate electrons between the NADPH-cytochrome c reductase and oxygen. The four electron oxidation product adrenochrome is also active upon the NADPH chain but inactive upon the NADH chain.Epinephrine did not change the menadione-stimulated NADPH oxidase activity. Presumably, during this and the NADH oxidase activities, two electrons are simultaneously transferred to the oxygen molecule.Catechol and catecholamines doubled the rate of autoxidation of NADH in the presence of catalytic amounts of NADH-cytochrome b₅ reductase and cytochrome b₅, a result which suggests Superoxide ion formation in the autoxidation of the cytochrome.Epinephrine does not act upon the desaturation of endogenous substrate or upon endogenous lipid peroxidation.     

Relationship between Cottonseed Malate Synthase Aggregation Behavior and Suborganellar Location in Glyoxysomes and Endoplasmic Reticulum

Malate synthase (EC 4.1.3.2) (MS), an enzyme unique to the glyoxylate cycle, was studied in cotyledons of dark-grown cotton (Gossypium hirsutum, L.) seedlings. MS has generally been regarded as a peripheral membrane protein in glyoxysomes and believed by some to be synthesized on rough ER. Immunocyto-chemical localization of MS in both in situ and isolated cottonseed glyoxysomes, however, showed that MS was located throughout the matrix of glyoxysomes, not specifically associated with their membranes. Biochemical data also supported matrix localization. Isolated glyoxysomes were diluted in variously-buffered salt solutions (200 millimolar KCl or 100 millimolar K-phosphate) or detergents (0.1% Triton X-100, 10 millimolar deoxycholate, or 1.0% Triton X-114) and centrifuged to pellet membranes. Greater than 70% of the MS was recovered in supernatants after treatment with salt solutions, whereas generally less than 30% was released following detergent treatments. MS in pellets derived from glyoxysomes burst in low ionic strength buffer solutions was aggregated (observed on rate-zonal gradients). MS released following salt treatments was the 20S nonaggregated form indicating that salt solutions either disaggregated (or prevented aggregation of) glyoxysomal MS rather than releasing it from membranes. We confirmed reports by others that MS comigrated with ER (NADH: cytochrome c reductase) in sucrose (20-40% w/w) gradients buffered with 100 millimolar Tricine (pH 7.5) after 3 hours centrifugation. However, cottonseed MS did not comigrate with ER in gradients buffered with 10 millimolar Hepes (pH 7.0) or 20 millimolar K-phosphate (pH 7.2) after 3 hours centrifugation, or after 22 hours centrifugation in Tricine or Hepes. Collectively, our data with cotton seeds indicate that MS is not a peripheral membrane protein, and that the aggregation behavior of MS (in various buffers) very likely has led to misinterpretations of its putative associations with ER and glyoxysomal membranes.     

An electron-transport system associated with the outer membrane of liver mitochondria. A biochemical and morphological study

Preparations of rat-liver mitochondria catalyze the oxidation of exogenous NADH by added cytochrome c or ferricyanide by a reaction that is insensitive to the respiratory chain inhibitors, antimycin A, amytal, and rotenone, and is not coupled to phosphorylation. Experiments with tritiated NADH are described which demonstrate that this "external" pathway of NADH oxidation resembles stereochemically the NADH-cytochrome c reductase system of liver microsomes, and differs from the respiratory chain-linked NADH dehydrogenase. Enzyme distributation data are presented which substantiate the conclusion that microsomal contamination cannot account for the rotenone-insensitive NADH-cytochrome c reductase activity observed with the mitochondria. A procedure is developed, based on swelling and shrinking of the mitochondria followed by sonication and density gradient centrifugation, which permits the separation of two particulate subfractions, one containing the bulk of the respiratory chain components, and the other the bulk of the rotenone-insensitive NADH-cytochrome c reductase system. Morphological evidence supports the conclusion that the former subfraction consists of mitochondria devoid of outer membrane, and that the latter represents derivatives of the outer membrane. The data indicate that the electron-transport system associated with the mitochondrial outer membrane involves catalytic components similar to, or identical with, the microsomal NADH-cytochrome b₅ reductase and cytochrome b₅.     

Electron transport in purified glyoxysomal membranes from castor-bean endosperm

Glyoxysomes isolated from castor-bean (Ricinus communis L.) endosperm were treated with water, 0.2 M KCl, 1 M KCl, or 0.1 M Na₂CO₃. Glyoxysomal sacs, i.e. membranes which retained some visible matrix, resulted from the treatments with water and KCl. Glyoxysomal ghosts, i.e. intact membranes free of matrix, were only obtained following treatment with carbonate. The ghosts were free of activities of matrix enzymes, particularly palmitoyl-CoA oxidation, isocitrate dehydrogenase (EC 1.1.1.42) and isocitrate lyase (EC 4.1.3.1), and contained only negligible amounts of malate synthase (EC 4.1.3.2), malate dehydrogenase (EC 1.1.1.37), -hydroxyacyl-CoA dehydrogenase (EC 1.1.1.98) and catalase (EC 1.11.1.6). Distribution and appearance of membrane-associated particles in the protoplasmic and ectoplasmic faces of freeze-fracture replicas of the glyoxysomal membrane were the same in intact tissue, isolated glyoxysomes, and ghosts. Membranes purified by treatment with 0.2 M KCl or 0.1 M carbonate catalyzed the reduction of cytochrome-c when NADH or NADPH was provided as the electron donor. -Oxidation, localized in the matrix, could be linked to reduction of cytochrome-c or ferricyanide when purified membranes were combined with the matrix supernatant. Cytochrome-c could also be reduced by coupling enzyme activities in the matrix, NADP-isocitrate dehydrogenase or malate dehydrogenase, with those of the membrane. These results indicate that electrons from -oxidation, malate oxidation or isocitrate oxidation can be transferred directly to the redox components of the glyoxysomal membrane. We, therefore, conclude that any NADH and NADPH formed by enzymes in the matrix can be recycled continuously within the organelle.Abbreviations EF ectoplasmic face - ER endoplasmic reticulum - PF protoplasmic face     

Purification and Identification of a Plasma Membrane Associated Electron Transport Protein from Maize (Zea mays L.) Roots

Plasma membranes isolated from three-day-old maize (Zea mays L.) roots by aqueous two-phase partitioning were used as starting material for the purification of a novel electron transport enzyme. The detergent-solubilized enzyme was purified by dyeligand affinity chromatography on Cibacron blue 3G-A-agarose. Elution was achieved with a gradient of 0 to 30 micromolar NADH. The purified protein fraction exhibited a single 27 kilodalton silver nitrate-stained band on sodium dodecyl sulfate polyacrylamide gel electrophoretograms. Staining intensity correlated with the enzyme activity profile when analyzed in affinity chromatography column fractions. The enzyme was capable of accepting electrons from NADPH or NADH to reduce either ferricyanide, juglone, duroquinone, or cytochrome c, but did not transfer electrons to ascorbate free-radical or nitrate. The high degree of purity of plasma membranes used as starting material as well as the demonstrated insensitivity to mitochondrial electron transport inhibitors confirmed the plasma membrane origin of this enzyme. The purified reductase was stimulated upon prolonged incubation with flavin mononucleotide suggesting that the enzyme may be a flavoprotein. Established effectors of plasma membrane electron transport systems had little effect on the purified enzyme, with the exception of the sulfhydryl inhibitor p-chloromercuriphenyl-sulfonate, which was a strong inhibitor of ferricyanide reducing activity.     

Synthesis and Degradation of Nitrate Reductase during the Cell Cycle of Chlorella Sorokiniana

Studies on the diurnal variations of nitrate reductase (NR) activity during the life cycle of synchronized Chlorella sorokiniana cells grown with a 7:5 light-dark cycle showed that the NADH:NR activity, as well as the NR partial activities NADH:cytochrome c reductase and reduced methyl viologen:NR, closely paralleled the appearance and disappearance of NR protein as shown by sodium dodecyl sulfate gel electrophoresis and immunoblots. Results of pulse-labeling experiments with [³⁵S]methionine further confirmed that diurnal variations of the enzyme activities can be entirely accounted for by the concomitant synthesis and degradation of the NR protein.     

Oxidation of NADH by plant mitochondria: Kinetics and effects of calcium ions

The kinetics of NADH oxidation by the outer membrane electron transport system of intact beetroot (Beta vulgaris L.) mitochondria were investigated. Very different values for V_max and the K_m for NADH were obtained when either antimycin A-insensitive NADH-cytochrome c activity (V_max= 31 ± 2.5 nmol cytochrome c (mg protein)^?1 min^?1; K_m= 3.1 ± 0.8 μM) or antimycin A-insensitive NADH-ferricyanide activity (V_max= 1.7 ± 0.7 μmol ferricyanide (mg protein)^?1 min^?1; K_m= 83 ± 20 μM) were measured. As ferricyanide is believed to accept electrons closer to the NADH binding site than cytochrome c, it was concluded that 83 ± 20 μM NADH represented a more accurate estimate of the binding affinity of the outer membrane dehydrogenase for NADH. The low K_m determined with NADH-cytochrome c activity may be due to a limitation in electron flow through the components of the outer membrane electron transport chain. The K_m for NADH of the externally-facing inner membrane NADH dehydrogenase of pea leaf (Pisum sativum L. cv. Massey Gem) mitochondria was 26.7 ± 4.3 μM when oxygen was the electron acceptor. At an NADH concentration at which the inner membrane dehydrogenase should predominate, the Ca²⁺ chelator, ethyleneglycol-(β-aminoethylether)-N,N,-tetraacetic acid (EGTA), inhibited the oxidation of NADH through to oxygen and to the ubiquinone-10 analogues, duroquinone and ubiquinone-1, but had no effect on the antimycin A-insensitive ferricyanide reduction. It is concluded that the site of action of Ca²⁺ involves the interaction of the enzyme with ubiquinone and not with NADH.     

Oxidative enzyme activities in rat brain subcellular fractions. The effect of deoxycholate, freeze-thaw, and water

(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.     

Mode of action of natural inactivator proteins from corn and rice on a purified assimilatory nitrate reductase

The molecular basis for the action of two natural inactivator proteins, isolated from rice and corn, on a purified assimilatory nitrate reductase has been examined by several physical techniques. Incubation of purified Chlorella nitrate reductase with either rice inactivator protein or corn inactivator protein results in a loss of NADH:nitrate reductase and the associated partial activity, NADH:cytochrome c reductase, but no loss in nitrate-reducing activity with reduced methyl viologen as the electron donor. The molecular weight of the reduced methyl viologen:nitrate reductase species, determined by sedimentation equilibrium in the Beckman airfuge after complete inactivation with rice inactivator protein or with corn inactivator protein, was 595,000 and 283,000, respectively, compared to a molecular weight of 376,000 for the untreated control determined under the same conditions. Two protein peaks were observed after molecular-sieve chromatography on Sephacryl S-300 of nitrate reductase inactivated by corn inactivator protein. The Stokes radii of these fragments were 68 and 24 Å, compared to a value of 81 Å for untreated nitrate reductase. The large fragment contained molybdenum and heme but no flavin, and had nitrate-reducing activity with reduced methyl viologen as electron donor. The small fragment contained FAD but had no NADH:cytochrome c reductase or nitrate-reducing activities. Molecular weights determined by sodium dodecyl sulfate-gel electrophoresis were 67,000 and 28,000 for the large and small fragments, respectively, compared to a subunit molecular weight of 99,000 determined for the untreated control. No change in subunit molecular weight of nitrate reductase after inactivation by rice inactivator protein was observed. These results indicate that rice inactivator protein acts by binding to nitrate reductase. The stoichiometry of binding is 1–2 molecules of rice inactivator protein to one tetrameric molecule of nitrate reductase. Corn inactivator protein, in contrast, acts by cleavage of a M_r 30,000 fragment from nitrate reductase which is associated with FAD. The remaining fragment is a tetramer of M_r 70,000 subunits which retains nitrate-reducing activity and contains molybdenum and heme but has no NADH:dehydrogenase activity. The action of rice inactivator protein was partially prevented by NADH and completely prevented by a combination of NADH and cyanide, while the action of corn inactivator protein was not significantly affected by these effectors.     

Resolution and reconstitution of succinate-cytochrome c reductase. Preparations and properties of high purity succinate dehydrogenase and ubiquinol—cytochrome c reductase

An improved method was developed to sequentially fractionate succinate-cytochrome c reductase into three reconstitutive active enzyme systems with good yield: pure succinate dehydrogenase, ubiquinone-binding protein fraction and a highly purified ubiquinol-cytochrome c reductase (cytochrome b-c₁ III complex).An extensively dialyzed succinate-cytochrome c reductase was first separated into a succinate dehydrogenase fraction and the cytochrome b-c₁ complex by alkali treatment. The resulting succinate dehydrogenase fraction was further purified to homogeneity by the treatment of butanol, calcium phosphate gel adsorption and ammonium sulfate fractionation under anaerobic condition in the presence of succinate and dithiothreitol. The cytochrome b-c₁ complex was separated into cytochrome b-c₁ III complex and ubiquinone-binding protein fractions by careful ammonium acetate fractionation in the presence of deoxycholate.The purified succinate dehydrogenase contained only two polypeptides with molecular weights of 70 000 and 27 000 as revealed by the sodium dodecyl sulfate polyacrylamide gel electrophoretic pattern. The enzyme has the reconstitutive activity and a low K_m ferricyanide reductase activity of 85 μmol succinate oxidized per min per mg protein at 38°C.Chemical composition analysis of cytochrome b-c₁ III complex showed that the preparation was completely free of contamination of succinate dehydrogenase and ubiquinone-binding protein and was 30% more pure than the available preparation.When these three components were mixed in a proper ratio, a thenoyl-trifluoroacetone- and antimycin A-sensitive succinate-cytochrome c reductase was reconstituted.