The mechanisms of reduction of protein mixed disulfides (dethiolation) in cardiac tissue

Dethiolation of proteins (reduction of protein mixed disulfides) by NADPH-dependent and glutathione (GSH)-dependent enzymes, and by nonenzymatic reaction with GSH, was studied by electrofocusing methodology with glycogen phosphorylase b and creatine kinase as substrates. Phosphorylase b was not rapidly dethiolated by reduced glutathione alone, but a cardiac extract catalyzed rapid dethiolation by both an NADPH-dependent and a GSH-dependent process. In contrast, creatine kinase was actively dethiolated by GSH. This GSH-dependent dethiolation was not enhanced by a soluble extract of bovine heart. Creatine kinase was also not dethiolated by an NADPH-dependent process. Partial purification of the phosphorylase dethiolases showed that the NADPH-dependent dethiolase had both a high-molecular-weight and a low-molecular-weight component The properties of these components were similar to those of thioredoxin and thioredoxin reductase. These two components were sensitive to inhibition by phenylarsine oxide and inhibition was reversed by addition of a dithiol. In contrast, GSH-dependent dethiolation required a single component of low molecular weight. This process was less sensitive to phenylarsine oxide inhibition. These studies show that two cytosolic proteins, phosphorylase b and creatine kinase, were dethiolated by different mechanisms. Phosphorylase b was dethiolated by both NADPH-dependent and GSH-dependent enzymes found in a soluble extract of bovine heart. In contrast, creatine kinase was rapidly dethiolated nonenzymatically by GSH alone.     

Flavan-3-ol Biosynthesis : The Conversion of (+)-Dihydroquercetin and Flavan-3,4-cis-Diol (Leucocyanidin) to (+)-Catechin by Reductases Extracted from Cell Suspension Cultures of Douglas Fir

Extracts of cell suspension cultures from Douglas fir (Pseudotsuga menziesii) needles catalyzed the production of (+)-catechin (2,3-trans flavan-3-o1) from the 2,3-trans-flavan,3,4-cis-diol (leucocyanidin) in a NADPH-dependent reductase reaction at pH 7.4. Catechin was also produced, along with the 3,4-cis-diol, in a double step reduction from (+)-dihydroquercetin. It was necessary to eliminate any thiol such as 2-mercaptoethanol or dithiothreitol from the extract or assay mixture because these thiols apparently formed thioethers with the 3,4-diols.     

The function of gamma-glutamylcysteine and bis-gamma-glutamylcystine reductase in Halobacterium halobium

gamma-Glutamylcysteine and bis-gamma-glutamylcystine reductase appear to function in the halobacteria in a fashion analogous to GSH and glutathione reductase in other cells. Bis-gamma-glutamylcystine reductase (GCR), a NADPH-dependent dimer of Mr 122,000 recently purified to homogeneity from Halobacterium halobium (Sundquist, A.R., and Fahey, R.C. (1988) J. Bacteriol., 170, 3459-3467), was found to be highly specific for bis-gamma-glutamylcystine and to be present in cell extract at a level sufficient to maintain gamma-glutamylcysteine predominantly in its thiol form [( thiol]/[disulfide] approximately 50). Bis-gamma-glutamylcystine reductase is similar to glutathione reductase in many respects; GCR demonstrated a FAD:subunit stoichiometry of 1, inhibition by heavy metal ions, and a pH optimum near neutrality. However, GCR exhibited no activity with GSSG and was most active at salt levels exceeding 2 M. A turnover number of 1,700 mumol min-1 mumol-1 FAD and apparent Km values of 0.8 mM for bis-gamma-glutamylcystine and 0.29 mM for NADPH were determined for GCR. The effect of salt on the autoxidation rates of gamma-glutamylcysteine, GSH, and Cys was also studied. In the absence of added salt, Cys oxidized more rapidly than gamma-glutamylcysteine, which in turn oxidized more rapidly than GSH. The presence of 4.3 M chloride (K+ and Na+) significantly slowed the autoxidation of all three thiols. The rate of autoxidation of gamma-glutamylcysteine in 4.3 M chloride proved slower than that of GSH in the absence of added chloride. Thus, gamma-glutamylcysteine is at least as stable under halophilic conditions as GSH is under nonhalophilic conditions, explaining why halobacteria utilize gamma-glutamylcysteine rather than GSH.     

NADPH-dependent reduction of 2,6-dichlorophenol-indophenol by the phagocytic vesicles of pig polymorphonuclear leucocytes.

NADPH-dependent 2,6-dichlorophenol-indophenol (DCIP) reductase activity in the homogenate of phagocytosing pig polymorphonuclear leucocytes was twice that of the resting cells and the activity in the phagocytic vesicles corresponded to the activity increment due to phagocytosis. The apparent Km value of the reductase activity in the vesicles for NADPH was 30 microM, which is similar to that of the NADPH-dependent superoxide (O2-) formation. Increasing the DCIP reductase activity by increasing the DCIP concentration caused a decrease in the O2- -forming activity, the NADPH oxidation rate being constant and independent of the dye concentration. p-Chloromercuribenzoate and cetyltrimethylammonium bromide at low concentrations inhibited the O2- -forming activity of the vesicles without inhibiting the DCIP reductase. Quinacrine inhibited both O2- formation and DCIP reduction. The DCIP reductase activity could be extracted with a mixture of deoxycholate and Tween-20, which extracts the O2- -forming activity. The reductase activity in the extract was enhanced 2-fold by the addition of FAD, and its apparent Km was 0.085 microM. These results indicate that the NADPH-dependent DCIP reductase activity of the phagocytic vesicles is catalysed by a flavin-containing component of the O2- -forming system.     

Structural analysis revealed a novel conformation of the NTRC reductase domain from Chlamydomonas reinhardtii

In plant chloroplasts, thiol regulation is driven by two systems. One relies on the activity of thioredoxins through their light dependent reduction by ferredoxin via a ferredoxin-thioredoxin reductase (FTR). In the other system, a NADPH-dependent redox regulation is driven by a NADPH-thioredoxin reductase C (NTRC). While the thioredoxin system has been deeply studied, a more thorough understanding of the function of this plant specific NTRC is desirable. NTRC is a single polypeptide harbouring a thioredoxin domain (Trx) at the C-terminus of a NADPH-dependent Thioredoxin reductase (TrxR). To provide functional and structural insights, we studied the crystal structure of the TrxR domain of the NTRC from Chlamydomonas reinhardtii (CrNTRC, Cre01.g054150.t1.2) and its Cys136Ser (C136S) mutant, which is characterized by the mutation of the resolving cysteine in the active site of the TrxR domain. Furthermore, we confirmed the role of NTRC as electron donor for 2-Cys peroxiredoxin (PRX) also in C. reinhardtii. The structural data of TrxR were employed to develop a scheme of action which addresses electron transfer between TrxR and Trx of NTRC and between NTRC and its substrates.     

A new and suitable reconstructed system for NADPH-dependent microsomal lipid peroxidation

In order to evaluate the O-2 participation in NADPH-dependent microsomal lipid peroxidation, we used reconstructed system which contained detergent-solubilized NADPH-dependent cytochrome P-450 reductase, cytochrome P-450, phospholipid liposomes, NADPH and Fe3+-ADP. Lipid peroxidation, monitored by the formation of thiobarbituric acid-reactive substance, was increased with increasing concentration of detergent-solubilized NADPH cytochrome P-450 reductase, cytochrome P-450 or Fe3+-ADP. Cytochrome P-450-dependent lipid peroxidation was parallel to O-2 generation monitored by chemiluminescence probe with 2-methyl-6-(p-methoxyphenol)-3,7-dihydroimidazo[1,2-a]pyrazin++ +-3-one. Lipid peroxidation was significantly inhibited by superoxide dismutase, but not by catalase or sodium benzoate. The reconstructed system herein described is considered to be very close to NADPH-dependent microsomal lipid peroxidation system.     

Selenite is a substrate for calf thymus thioredoxin reductase and thioredoxin and elicits a large non-stoichiometric oxidation of NADPH in the presence of oxygen.

The thioredoxin system, comprising NADPH, thioredoxin reductase and thioredoxin reduces protein disulfides via redox-active dithiols. We have discovered that sodium selenite is a substrate for the thioredoxin system; 10 microM selenite plus 0.05 microM calf thymus thioredoxin reductase at pH 7.5 caused a non-stoichiometric oxidation of NADPH (100 microM after 30 min). In contrast, thioredoxin reductase from Escherichia coli showed no direct reaction with selenite, but addition of 3 microM E. coli thioredoxin also resulted in non-stoichiometric oxidation of NADPH, consistent with oxidation of the two active-site thiol groups in thioredoxin to a disulfide. Kinetically, the reaction was complex with a lag phase at low selenite concentrations. Under anaerobic conditions the reaction stopped after 1 mol selenite had oxidized 3 mol NADPH; the admission of air then resulted in continued consumption of NADPH consistent with autooxidation of selenium intermediate(s). Ferricytochrome c was effectively reduced by calf thymus thioredoxin reductase and selenite in the presence of oxygen. Selenite caused a strong dose-dependent inhibition of the formation of thiol groups from insulin disulfides with either the E. coli or calf-thymus thioredoxin system. Thus, under aerobic conditions selenite catalyzed, NADPH-dependent redox cycling with oxygen, a large oxygen-dependent consumption of NADPH and oxidation of reduced thioredoxin inhibiting its disulfide-reductase activity.     

Purification and properties of NADPH-dependent aldehyde reductase from human liver.

An aldehyde reductase (EC 1.1.1.2) from human liver has been purified to homogeneity. The enzyme is NADPH-dependent, prefers aromatic to aliphatic aldehydes as substrates, and is inhibited by barbiturates and hydantoins. The following physicochemical parameters were determined: molecular weight, 36,200; sedimentation coefficient, 2.9 S; Stokes radius, 2.65 nm; isoelectric point, pH 5.3; extinction coefficient at 280 nm, 54,300 M-1 cm-1. Results from polyacrylamide gel electrophoresis with and without sodium dodecyl sulfate, gel filtration, and ultracentrifugation suggest a monomeric structure. On molecule of NADPH binds to the enzyme causing a red shift of the coenzyme absorption maximum from 340 to 352 nm. The amino acid composition has been determined and a partial specific volume of 0.74 was computed from these data. An alpha-helicity of 7 and 18% was estimated from the ellipticities at 208 and 222 nm, respectively. Combination of the most reactive thiol group with p-mercuribenzoate does not cause loss of catalytic activity. Inactivation occurs when more than one thiol group is modified. The presence of NADPH or NADP+ prevents loss of activity by thiol modification. The comparison of structural features of aldehyde reductase with other monomeric and oligomeric dehydrogenases suggest similarities of aldehyde reductase with octopine dehydrogenase.     

In vitro restoration of nitrate reductase: investigation of Aspergillus nidulans and Neurospora crassa nitrate reductase mutants.

Extracts of Aspergillus nidulans wild type (bi-1) and the nitrate reductase mutant niaD-17 were active in the in vitro restoration of NADPH-dependent nitrate reductase when mixed with extracts of Neurospora crassa, nit-1. Among the A. nidulans cnx nitrate reductase mutants tested, only the molybdenum repair mutant, cnxE-14 grown in the presence of 10-minus 3 M Na2 MoO4 was active in the restoration assay. Aspergillus extracts contained an inhibitor(s) which was measured by the decrease in NADPH-dependent nitrate reductase formed when extracts of Rhodospirillum rubrum and N. crassa, nit-1 were incubated at room temperature. The inhibition by extracts of A. nidulans, bi-1, cnxE-14, cnxG-4 and cnxH-3 was a linear function of time and a logarithmic function of the protein concentration in the extract. The molybdenum content of N. crassa wild type and nit-1 mycelia were found to be similar, containing approx. 10 mu g molybdenum/mg dry mycelium. The NADPH-dependent cytochrome c reductase associated with nitrate reductase was purified from both strains. The NADPH-dependent cytochrome c reductase associated with nitrate reductase was purified from both strains. The enzyme purified from wild-type N. crassa contained more than 1 mol of molybdenum per mol of enzyme, whereas the enzyme purified from nit-1 contained negligible amounts of molybdenum.     

Immunochemical study on the pathway of electron flow in reduced nicotinamide adenine dinucleotide-dependent microsomal lipid peroxidation.

NADH could support the lipid peroxidation of rat liver microsomes in the presence of ferric ions chelated by ADP(ADP-Fe). The reaction had a broad pH optimum (pH 5.8--7.4) and was more active in the acidic pH range. Antibodies to NADH-cytochrome b5 reductase [EC 1.6.2.2] and cytochrome b5 inhibited NADH-dependent lipid peroxidation in the presence of ADP-Fe, whereas the antibody against NADPH-cytochrome c reductase [EC 1.6.2.4] showed no inhibition. These oberservations suggest that the electron from NADH was supplied to the lipid peroxidation reaction via NADH-cytochrome b5 reductase and cytochrome b5. On the other hand, NADPH-supported lipid peroxidation was strongly inhibited by the antibody against NADPH-cytochrome c reductase, confirming the participation of this this flavoprotein in the NADPH-dependent reaction. In the presence of both ADP-Fe and ferric ions chelated by EDTA(EDTA-Fe), NADH-dependent lipid peroxidation was highly stimulated up to the level of the NADPH-dependent reaction. In this case, the antibody against cytochrome b5 could not inhibit the reaction, while the antibody against NADH-cytochrome b5 reductase did inhibit it, suggesting the direct transfer of electrons from NADH-cytochrome b5 reductase to EDTA-Fe complex.     

Assay of glutathione reductase in crude tissue homogenates using 5,5'-dithiobis(2-nitrobenzoic acid)

A method for assaying glutathione reductase (GSH; EC 1.6.4.2) in crude plant extracts is described. The method is based on the increase in absorbance at 412 nm when 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) is reduced by GSH. The effects of the following parameters on the assay were tested: various buffers, pH, buffer concentration, compounds commonly present in enzyme preparations, thiols, and the presence of another NADPH-dependent enzyme. The assay is more sensitive and less subject to interference than the widely used assay where NADPH oxidation is monitored. In particular, the specificity of DTNB allows assay of glutathione reductase in the presence of other NADPH-dependent enzymes and common protein extract contaminants.     

The thioredoxin system of Penicillium chrysogenum and its possible role in penicillin biosynthesis.

Penicillium chrysogenum is an important producer of penicillin antibiotics. A key step in their biosynthesis is the oxidative cyclization of delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine (ACV) to isopenicillin N by the enzyme isopenicillin N synthase (IPNS). bis-ACV, the oxidized disulfide form of ACV is, however, not a substrate for IPNS. We report here the characterization of a broad-range disulfide reductase from P. chrysogenum that efficiently reduces bis-ACV to the thiol monomer. When coupled in vitro with IPNS, it converts bis-ACV to isopenicillin N and may therefore play a role in penicillin biosynthesis. The disulfide reductase consists of two protein components, a 72-kDa NADPH-dependent reductase, containing two identical subunits, and a 12-kDa general disulfide reductant. The latter reduces disulfide bonds in low-molecular-weight compounds and in proteins. The genes coding for the reductase system were cloned and sequenced. Both possess introns. A comparative analysis of their predicted amino acid sequences showed that the 12-kDa protein shares 26 to 60% sequence identity with thioredoxins and that the 36-kDa protein subunit shares 44 to 49% sequence identity with the two known bacterial thioredoxin reductases. In addition, the P. chrysogenum NADPH-dependent reductase is able to accept thioredoxin as a substrate. These results establish that the P. chrysogenum broad-range disulfide reductase is a member of the thioredoxin family of oxidoreductases. This is the first example of the cloning of a eucaryotic thioredoxin reductase gene.     

Reduction of mitochondrial protein mitoNEET [2Fe–2S] clusters by human glutathione reductase

The human mitochondrial outer membrane protein mitoNEET is a newly discovered target of the type 2 diabetes drug pioglitazone. Structurally, mitoNEET is a homodimer with each monomer containing an N-terminal transmembrane α helix tethered to the mitochondrial outer membrane and a C-terminal cytosolic domain hosting a redox-active [2Fe–2S] cluster. Genetic studies have shown that mitoNEET has a central role in regulating energy metabolism in mitochondria. However, the specific function of mitoNEET remains largely elusive. Here we find that the mitoNEET [2Fe–2S] clusters can be efficiently reduced by Escherichia coli thioredoxin reductase and glutathione reductase in an NADPH-dependent reaction. Purified human glutathione reductase has the same activity as E. coli thioredoxin reductase and glutathione reductase to reduce the mitoNEET [2Fe–2S] clusters. However, rat thioredoxin reductase, a human thioredoxin reductase homolog that contains selenocysteine in the catalytic center, has very little or no activity to reduce the mitoNEET [2Fe–2S] clusters. N-ethylmaleimide, a potent thiol modifier, completely inhibits human glutathione reductase from reducing the mitoNEET [2Fe–2S] clusters, indicating that the redox-active disulfide in the catalytic center of human glutathione reductase may be directly involved in reducing the mitoNEET [2Fe–2S] clusters. Additional studies reveal that the reduced mitoNEET [2Fe–2S] clusters in mouse heart cell extracts can be reversibly oxidized by hydrogen peroxide without disruption of the clusters, suggesting that the mitoNEET [2Fe–2S] clusters may undergo redox transition to regulate energy metabolism in mitochondria in response to oxidative signals.     

Interaction of selenoprotein PA and the thioredoxin system, components of the NADPH-dependent reduction of glycine in Eubacterium acidaminophilum and Clostridium litorale [corrected]

Purification of protein PA of the glycine reductase complex from Eubacterium acidaminophilum and Clostridium litorale [corrected] was monitored by a new spectrophotometric assay. The procedure depended on a specific two- to threefold stimulation of a dihydrolipoamide dehydrogenase activity that is elicited by the interaction of a thioredoxin reductase-like flavoprotein and thioredoxin from both organisms. Protein PA isolated from E. acidaminophilum by 75Se labeling and monitoring of the dithioerythritol-dependent glycine reductase activity was identical in its biochemical, structural, and immunological properties to the protein isolated by using the stimulation assay. Proteins PA from both organisms were glycoproteins of Mr about 18,500 and exhibited very similar N-terminal amino acid sequences. Depletion of thioredoxin from crude extracts of E. acidaminophilum totally diminished the NADPH-dependent but not the dithioerythritol-dependent glycine reduction. The former activity could be fully restored by adding thioredoxin. Antibodies raised against the thioredoxin reductase-like flavoprotein or thioredoxin inhibited to a high extent NADPH-dependent but not dithioerythritol-dependent glycine reductase activity. These results indicate the involvement of the thioredoxin system in the electron flow from reduced pyridine nucleotides to glycine reductase.     

Reaction mechanism and regulation of mammalian thioredoxin/glutathione reductase

Thioredoxin/glutathione reductase (TGR) is a recently discovered member of the selenoprotein thioredoxin reductase family in mammals. In contrast to two other mammalian thioredoxin reductases, it contains an N-terminal glutaredoxin domain and exhibits a wide spectrum of enzyme activities. To elucidate the reaction mechanism and regulation of TGR, we prepared a recombinant mouse TGR in the selenoprotein form as well as various mutants and individual domains of this enzyme. Using these proteins, we showed that the glutaredoxin and thioredoxin reductase domains of TGR could independently catalyze reactions normally associated with each domain. The glutaredoxin domain is a monothiol glutaredoxin containing a CxxS motif at the active site, which could receive electrons from either the thioredoxin reductase domain of TGR or thioredoxin reductase 1. We also found that the C-terminal penultimate selenocysteine was required for transfer of reducing equivalents from the thiol/disulfide active site of TGR to the glutaredoxin domain. Thus, the physiologically relevant NADPH-dependent activities of TGR were dependent on this residue. In addition, we examined the effects of selenium levels in the diet and perturbations in selenocysteine tRNA function on TGR biosynthesis and found that expression of this protein was regulated by both selenium and tRNA status in liver, but was more resistant to this regulation in testes.     

Reconstitution of the monooxygenase system in a solution and in an immobilized phospholipid layer

To clarify the molecular organization of NADH- and NADPH-dependent microsomal redox systems their isolated purified carriers were incorporated into immobilized azolectin layer with a higher viscosity than that of the liposomes. It was shown that the NADH-cytochrome c reductase activity characterizing the NADH-cytochrome b5 reductase and cytochrome b5 interaction sharply decreased in the immobilized system as compared to that in solution. However, the activity of hydroxylase reactions catalyzed by immobilized NADPH-cytochrome P-450 reductase and cytochrome P-450 was the same as in solution. This, the reconstitution in the immobilized phospholipid layer allowed to characterize NADH-cytochrome b5 reductase as a system operating on occasional collisions of its components. On the contrary, the diffusion of the NADPH-dependent redox chain carriers was not the rate-limiting step of the reaction.     

Purification and characterization of a novel enoyl coenzyme A reductase from Streptomyces collinus.

A novel NADPH-dependent enoyl reductase, catalyzing the conversion of 1-cyclohexenylcarbonyl coenzyme A (1-cyclohexenylcarbonyl-CoA) to cyclohexylcarbonyl-CoA, was purified to homogeneity from Streptomyces collinus. This enzyme, a dimer with subunits of identical M(r) (36,000), exhibits a Km of 1.5 +/- 0.3 microM for NADPH and 25 +/- 3 microM for 1-cyclohexenylcarbonyl-CoA. It has a pH optimum of 7.5, is most active at 30 degrees C, and is inhibited by both divalent cations and thiol reagents. Two internal peptide sequences were obtained. Ansatrienin A (an antibiotic produced by S. collinus) contains a cyclohexanecarboxylic acid moiety, and it is suggested that the 1-cyclohexenylcarbonyl-CoA reductase described herein catalyzes the final reductive step in the conversion of shikimic acid into this moiety.     

Study of the thiol/disulfide redox systems of the anaerobe Desulfovibrio vulgaris points out pyruvate:ferredoxin oxidoreductase as a new target for thioredoxin 1

Sulfate reducers have developed a multifaceted adaptative strategy to survive against oxidative stresses. Along with this oxidative stress response, we recently characterized an elegant reversible disulfide bond-dependent protective mechanism in the pyruvate:ferredoxin oxidoreductase (PFOR) of various Desulfovibrio species. Here, we searched for thiol redox systems involved in this mechanism. Using thiol fluorescent labeling, we show that glutathione is not the major thiol/disulfide balance-controlling compound in four different Desulfovibrio species and that no other plentiful low molecular weight thiol can be detected. Enzymatic analyses of two thioredoxins (Trxs) and three thioredoxin reductases allow us to propose the existence of two independent Trx systems in Desulfovibrio vulgaris Hildenborough (DvH). The TR1/Trx1 system corresponds to the typical bacterial Trx system. We measured a TR1 apparent K(m) value for Trx1 of 8.9 μM. Moreover, our results showed that activity of TR1 was NADPH-dependent. The second system named TR3/Trx3 corresponds to an unconventional Trx system as TR3 used preferentially NADH (K(m) for NADPH, 743 μM; K(m) for NADH, 5.6 μM), and Trx3 was unable to reduce insulin. The K(m) value of TR3 for Trx3 was 1.12 μM. In vitro experiments demonstrated that the TR1/Trx1 system was the only one able to reactivate the oxygen-protected form of Desulfovibrio africanus PFOR. Moreover, ex vivo pulldown assays using the mutant Trx1(C33S) as bait allowed us to capture PFOR from the DvH extract. Altogether, these data demonstrate that PFOR is a new target for Trx1, which is probably involved in the protective switch mechanism of the enzyme.     

Purification by affinity chromatography of 2,4-dienoyl-CoA reductases from bovine liver and Escherichia coli

1. Dye-ligand chromatography using immobilized Cibacron blue F3GA (blue Sepharose CL-6B) and Procion red HE3B (Matrex gel red A) as matrices and general ligand chromatography employing immobilized 2',5'-ADP (2',5'-ADP-Sepharose 4B) and immobilized 3',5'-ADP (3',5'-ADP-Agarose) were employed for purification of NADPH-dependent 2-enoyl-CoA reductase and 2,4-dienoyl-CoA reductase from bovine liver (formerly called 4-enoyl-CoA reductase [Kunau, W. H. and Dommes, P. (1978) Eur. J. Biochem. 91, 533-544], as well as 2,4-dienoyl-CoA reductase from Escherichia coli. 2. The NADPH-dependent 2-enoyl-CoA reductase from bovine liver mitochondria was separated from 2,4-dienoyl-CoA reductase by dye-ligand chromatography (Matrex gel red A/KCl gradient) as well as by general ligand affinity chromatography (2',5'-ADP-Sepharose 4B/NADP gradient). The enzyme was obtained in a highly purified form. 3. The NADPH-dependent 2,4-dienoyl-CoA reductase from bovine liver mitochondria was purified to homogeneity using blue Sepharose CL-6B, Matrex gel red A, and 2',5'-ADP-Sepharose 4B chromatography. 4. The bacterial 2,4-dienoyl-CoA reductase was completely purified by ion-exchange chromatography on DEAE-cellulose followed by a single affinity chromatography step employing 2',5'-ADP-Sepharose 4B and biospecific elution from the column with a substrate, trans,trans-2,4-decadienoyl-CoA. 5. The application of dye-ligand and general ligand affinity chromatography for purification of NADPH-dependent 2,4-dienoyl-CoA reductases taking part in the beta-oxidation of unsaturated fatty acids is discussed. It is concluded that making use of coenzyme specificity for binding and substrate specificity for elution is essential for obtaining homogeneous enzyme preparations.     

Comparative study of monomeric reconstituted and membrane microsomal monooxygenase systems of the rabbit liver. II. Kinetic parameters of reductase and monooxygenase reactions.

The kinetic parameters of NADPH-dependent cytochrome P450 LM2 (2B4) reduction and substrate oxidation in the monomeric reconstituted system, consisting of purified NADPH-cytochrome P450 reductase and cytochrome P450 LM2 monomers, and in phenobarbital-induced rabbit liver microsomes were compared. In the absence of benzphetamine, NADPH-dependent reduction of cytochrome P450 LM2 was monophasic in the monomeric reconstituted system and biphasic in the microsomes. The presence of the substrate in the monomeric reconstituted system caused the appearance of the fast phase. In this system substrate-free cytochrome P450 LM2 was entirely low-spin, and the addition of benzphetamine shifted the spin equilibrium to a high state very weakly. No correlation between high-spin content and the proportion of the fast phase of NADPH-dependent LM2 reduction was found in the system. Vmax values for the oxidation of type I substrates (benzphetamine, dimethylaniline, aminopyrine) in the monomeric reconstituted system were higher or the same as in the microsomes, whereas Km values for the substrates and NADPH were lower in the microsomes. Maximal activity of the monomeric reconstituted system was observed at a 1:1 NADPH-cytochrome P450 reductase/cytochrome P450 LM2 ratio. Measurements of benzphetamine oxidation as a function of NADPH-cytochrome P450 reductase/cytochrome P450 LM2 ratio at a constant total protein concentration allowed the Kd of the NADPH-cytochrome P450 reductase/cytochrome P450 LM2 complex to be estimated as 6.4 +/- 0.5 microM. Complex formation between the NADPH-cytochrome P450 reductase and cytochrome P450 LM2 monomers was not detected by recording the difference binding spectra of the reductase monomers with LM2 monomers or by treatment the mixture of the monomers of the proteins with the crosslinking reagent, water-soluble carbodiimide.