Effect of divalent cations on NADH-dependent and NADPH-dependent cytochrome b5 reduction by hepatic microsomes

The effect of Ca2+ or Mg2+ on cytochrome b5 reduction by porcine liver microsomes was examined using trypsin-solubilized cytochrome b5 as a substrate. The reduction of exogenous cytochrome b5 by microsomes was low at 1.2 microM cytochrome b5 (3.9 or 2.7 nmol/min/mg protein, respectively, with NADH or NADPH). The addition of CaCl2 greatly enhanced either NADH-dependent or NADPH-dependent cytochrome b5 reduction. At 2 mM CaCl2, the reduction rate was increased to 23- or 18-fold of control, respectively with NADH or NADPH. The concentration for half-maximal effect (EC50) was 0.5 or 0.6 mM in the NADH or NADPH systems, respectively. MgCl2 also stimulated cytochrome b5 reduction with a EC50 value of 1.0 mM in the NADH system or 0.6 mM in the NADPH system. The comparison with the result with KCl indicated that the activation by CaCl2 or MgCl2 is caused mainly by their divalent cation moiety. The Km value for cytochrome b5 was decreased and the Vmax was increased by calcium with either the NADH- or the NADPH-dependent system. NADH-ferricyanide reductase activity was not affected by calcium, but NADPH-ferricyanide reductase activity was stimulated as well as NADPH-cytochrome c reductase activity. In the presence of Triton X-100, divalent cations were inhibitory in NADH-dependent cytochrome b5 reduction, and in contrast, stimulative in NADPH-dependent reaction. These findings suggest that the activation of cytochrome b5 reduction by divalent cations in the NADH system is mainly due to an increasing accessibility of the substrate, and in the NADPH system, in addition to this, a direct effect of divalent cations on NADPH-cytochrome P450 reductase is also involved.     

Engineering and characterization of a NADPH-utilizing cytochrome b5 reductase

Microsomal cytochrome b(5) reductase (EC 1.6.2.2) catalyzes the reduction of ferricytochrome b(5) using NADH as the physiological electron donor. Site-directed mutagenesis has been used to engineer the soluble rat cytochrome b(5) reductase diaphorase domain to utilize NADPH as the preferred electron donor. Single and double mutations at residues D239 and F251 were made in a recombinant expression system that corresponded to D239E, S and T, F251R, and Y, D239S/F251R, D239S/F251Y, and D239T/F251R, respectively. Steady-state turnover measurements indicated that D239S/F251Y was bispecific while D239T, D239S/F251R, and D239T/F251R were each NADPH-specific. Wild-type (WT) cytochrome b(5) reductase showed a 3700-fold preference for NADH whereas the mutant with the highest NADPH efficiency, D239T, showed an 11-fold preference for NADPH, a 39200-fold increase. Wild-type cytochrome b(5) reductase only formed a stable charge-transfer complex with NADH while D239T formed complexes with both NADH and NADPH. The rates of hydride ion transfer, determined by stopped-flow kinetics, were k(NADH-WT) = 130 s(-1), k(NADPH-WT) = 5 s(-1), k(NADH-D239T) = 180 s(-1), and k(NADPH-D239T) = 73 s(-1). K(s) determinations by differential spectroscopy demonstrated that D239T could bind nonreducing pyridine nucleotides with a phosphate or a hydroxyl substituent at the 2' position, whereas wild-type cytochrome b(5) reductase would only bind 2' hydroxylated molecules. Oxidation-reduction potentials (E degrees ', n = 2) for the flavin cofactor were WT = -268 mV, D239T = -272 mV, WT+NAD(+) = -190 mV, D239T+NAD(+) = -206 mV, WT+NADP(+) = -253 mV, and D239T+NADP(+) = -215 mV, which demonstrated the thermodynamic contribution of NADP(+) binding to D239T. The crystal structures of D239T and D239T in complex with NAD(+) indicated that the loss of the negative electrostatic surface that precluded 2' phosphate binding in the wild-type enzyme was primarily responsible for the observed improvement in the use of NADPH by the D239T mutant.     

Effect of KCl on the interactions between NADPH:cytochrome P-450 reductase and either cytochrome c, cytochrome b5 or cytochrome P-450 in octyl glucoside micelles.

Significant dissociation of FMN from NADPH:cytochrome P-450 reductase resulted in loss of the activity for reduction of cytochrome b5 as well as cytochrome c and cytochrome P-450. However, the ability to reduce these electron acceptors was greatly restored upon incubation of FMN-depleted enzyme with added FMN. The reductions of cytochrome c and detergent-solubilized cytochrome b5 by NADPH:cytochrome P-450 reductase were greatly increased in the presence of high concentrations of KCl, although the stimulatory effect of the salt on cytochrome P-450 reduction was less significant. No apparent effect of superoxide dismutase could be seen on the rate or extent of cytochrome reduction in solutions containing high-salt concentrations. Complex formation of the flavoprotein with cytochrome c, which is known to be involved in the mechanism of non-physiological electron transfer, caused a perturbation in the absorption spectrum in the Soret-band region of cytochrome c, and its magnitude was enhanced by addition of KCl. Similarly, an appreciable increase in ellipticity in the Soret band of cytochrome c was observed upon binding with the flavoprotein. However, only small changes were found in absorption and circular dichroism spectra for the complex of NADPH:cytochrome P-450 reductase with either cytochrome b5 or cytochrome P-450. It is suggested that the high-salt concentration allows closer contact between the heme and flavin prosthetic groups through hydrophobic-hydrophobic interactions rather than electrostatic-charge pairing between the flavoprotein and the cytochrome which causes a faster rate of electron transfer. Neither alterations in the chemical shift nor in the line width of the bound FMN and FAD phosphate resonances were observed upon complex formation of NADPH:cytochrome P-450 reductase with the cytochrome.     

Simultaneous purification and characterization of cytochrome b5 reductase and cytochrome b5 from sheep liver

Cytochrome b5 was purified from detergent solubilized sheep liver microsomes by using three successive DEAE-cellulose, and Sephadex G-100 column chromatographies. It was purified 54-fold and the yield was 23.5% with respect to microsomes. The apparent Mr of cytochrome b5 was estimated to be 16,200 +/- 500 by SDS-PAGE. Absolute absorption spectrum of the purified cytochrome b5 showed maximal absorption at 412 nm and dithionite-reduced cytochrome b5 gave peaks at 557, 526.5 and 423 nm. The ability of the purified sheep liver cytochrome b5 to transfer electrons from NADH-cytochrome b5 reductase to cytochrome c was investigated. The K(m) and Vmax values were calculated to be 0.088 microM cytochrome b5 and 315.8 microM cytochrome c reduced/min/mg enzyme, respectively. Also the reduction of cytochrome b5 by reductase was studied and K(m) and Vmax values were determined to be 5 microM cytochrome b5 and 5200 nmol cytochrome b5 reduced/min/mg enzyme, respectively. The K(m) and Vmax values for the cofactor NADH in the presence of saturating concentration of cytochrome b5 were found to be 0.0017 mM NADH and 6944 nmol cytochrome b5 reduced/min/mg enzyme, respectively. NADH-cytochrome b5 reductase was also partially purified from the same source, detergent solubilized sheep liver microsomes, by using two successive DEAE-cellulose, and 5'-ADP-agarose affinity column chromatographies. It was purified 144-fold and the yield was 7% with respect to microsomes. The apparent monomer Mr of reductase was estimated to be 34,000 by SDS-PAGE. When ferricyanide was used as an electron acceptor, reductase showed maximum activity between 6.8 and 7.5. The K(m) and Vmax values of the enzyme for ferricyanide were calculated as 0.024 mM ferricyanide and 673 mumol ferricyanide reduced/min/mg enzyme, respectively. The K(m) and Vmax values for the cofactor NADH in the presence of saturating amounts of ferricyanide were found to be 0.020 mM NADH and 699 mumol ferricyanide reduced/min/mg enzyme, respectively.     

Reduction of hexavalent chromium in a reconstituted system of cytochrome P-450 and cytochrome b5

The reduction of hexavalent chromium (Cr(VI] by the monooxygenase components was studied. Both a reconstituted system of cytochrome P-450 (P-450) and cytochrome b5 (b5) with NADPH was capable of reducing Na2CrO4 (30 microM) provided anaerobic atmosphere. The rates were 1.29 nmol Cr.min-1 nmol P-450(-1) and 0.73 nmol Cr.min-1 nmol b5(-1). Using NADH instead of NADPH gave very low reducing activities, confirming the enzymic nature of the P-450 dependent Cr(VI) reductase reaction. Oxygen, 22% (air) and 0.1% gave 89% and 69% inhibition of Cr(VI) reducing activity, respectively. Carbon monoxide (100%) caused an inhibition of about 37% and 44% for P-450 and b5, respectively. Externally added flavin mononucleotide (FMN) (3 microM) or Fe-ADP (10 microM) to the complete system stimulated the enzymatic reaction about 2-fold and 3-fold, respectively.     

Biodiversity of the P450 catalytic cycle: yeast cytochrome b5/NADH cytochrome b5 reductase complex efficiently drives the entire sterol 14-demethylation (CYP51) reaction

The widely accepted catalytic cycle of cytochromes P450 (CYP) involves the electron transfer from NADPH cytochrome P450 reductase (CPR), with a potential for second electron donation from the microsomal cytochrome b5/NADH cytochrome b5 reductase system. The latter system only supported CYP reactions inefficiently. Using purified proteins including Candida albicans CYP51 and yeast NADPH cytochrome P450 reductase, cytochrome b5 and NADH cytochrome b5 reductase, we show here that fungal CYP51 mediated sterol 14alpha-demethylation can be wholly and efficiently supported by the cytochrome b5/NADH cytochrome b5 reductase electron transport system. This alternative catalytic cycle, where both the first and second electrons were donated via the NADH cytochrome b5 electron transport system, can account for the continued ergosterol production seen in yeast strains containing a disruption of the gene encoding CPR.     

Transfer of cytochrome b 5 and NADPH cytochrome c reductase between membranes

NADPH-cytochrome c reductase also reduces cytochrome b 5. The reduction is very slow when the proteins are in solution or bound to different membranes. Only when both proteins share a common membrane, is cytochrome b 5 reduced rapidly by NADPH. The difference in reaction rates indicates recombination on a common membrane of cytochrome b 5 and NADPH reductase originally bound to different vesicles. The recombination of the two proteins occurs with a variety of biological membranes (previously enriched with either reductase or cytochrome b 5) as well as with liposomes. We explain this process as protein transfer rather than vesicle fusion for several reasons: 1. The vesicles do not alter shape or size during incubation. 2. The rate of this process corresponds to the rate of incorporation of the single proteins into liposomes carrying the 'complementary' protein. 3. The exchange of proteins between biological membranes and liposomes occupied by protein does not change the density of either membrane. Protein transfer between membranes appears to be limited to those proteins which had spontaneously recombined with a preformed membrane. In contrast, proteins incorporated into liposomes by means of a detergent were not transferred, nor were endogenous cytochrome b 5 and NADPH-cytochrome c reductase transferred from microsomes to Golgi membranes or lipid vesicles. We conclude that the endogenous proteins and proteins incorporated in the presence of a detergent are linked to the membrane in another manner than the same proteins which had been inserted into a preformed membrane.     

Gastric microsomal NADH-cytochrome b5 reductase: characterization and solubilization

NADH-cytochrome b5 reductase from hog gastric microsomes was studied with respect to substrate dependence, optimum pH, thermal denaturation as well as anti-cytochrome b5 antibodies and different ions. The reduction of potassium ferricyanide by the enzyme was specific for NADH. Using potassium ferricyanide or trypsin-solubilized liver cytochrome b5 (Tb5) as substrates, enzyme activity was inhibited by ADP and to a lesser extent by ATP. Tb5- (but not ferricyanide-) reductase was activated by ionic strength up to 0.05 ion equivalent per liter and inhibited at higher strengths whatever the ion used (Cl-, Na+, Ca2+, Mg2+). Enzyme solubilization occurred with Triton X100. The solubilization increased the Tb5- (but not the ferricyanide-) reductase activity up to a Triton:protein ratio of 15. We therefore suggest that gastric microsomes contain a Triton soluble membrane-bound NADH cytochrome b5 reductase which is in many respects similar to the liver and red cell enzymes.     

Kinetic properties of purified sheep lung microsomal NADH-cytochrome b5 reductase.

1. Lung NADH-cytochrome b5 reductase was saturated with its artificial substrate, potassium ferricyanide at approximately 0.1 mM ferricyanide concentration, and the activity of the lung enzyme was inhibited by the higher concentrations of potassium ferricyanide. Ferricyanide at 0.5 and 1.0 mM inhibited the activity of the enzyme by about 20 and 61% respectively. The apparent Km value was calculated as 13.7 microM potassium ferricyanide and 4.3 microM NADH. 2. The Michaelis constants for cytochrome b5 and NADH were determined to be 1.67 and 7.7 microM from the Lineweaver-Burk plots. These results demonstrate that affinity of the lung reductase for its natural substrate is almost 10 times higher than that for potassium ferricyanide. 3. Addition of non-ionic detergent stimulated the rate of reductase-catalyzed reduction of lung cytochrome b5 up to 8.2-fold. 4. Kinetic studies performed with lung reductase by varying NADH and cytochrome b5 concentrations at different fixed concentrations at cytochrome b5 or NADH showed a series of parallel lines indicating a "ping-pong" type of kinetic mechanism for interaction of NADH and cytochrome b5 with lung cytochrome b5 reductase.     

Expression and characterization of a functional canine variant of cytochrome b5 reductase

Cytochrome b5 reductase (cb5r), a member of the flavoprotein transhydrogenase family of oxidoreductase enzymes, catalyzes the transfer of reducing equivalents from the physiological electron donor, NADH, to two molecules of cytochrome b5. We have determined the correct nucleotide sequence for the putative full-length, membrane-associated enzyme from Canis familiaris, and have generated a heterologous expression system for production of a histidine-tagged variant of the soluble, catalytic diaphorase domain, comprising residues I33 to F300. Using a simple two-step chromatographic procedure, the recombinant diaphorase domain has been purified to homogeneity and demonstrated to be a simple flavoprotein with a molecular mass of 31,364 (m/z) that retained both NADH:ferricyanide reductase and NADH:cytochrome b5 reductase activities. The recombinant protein contained a full complement of FAD and exhibited absorption and CD spectra comparable to those of a recombinant form of the rat cytochrome b5 reductase diaphorase domain generated using an identical expression system, suggesting similar protein folding. Oxidation-reduction potentiometric titrations yielded a standard midpoint potential (Eo') for the FAD/FADH2 couple of -273+/-5 mV which was identical to the value obtained for the corresponding rat domain. Thermal denaturation studies revealed that the canine domain exhibited stability comparable to that of the rat protein, confirming similar protein conformations. Initial-rate kinetic studies revealed the canine diaphorase domain retained a marked preference for NADH versus NADPH as reducing substrate and exhibited kcat's of 767 and 600 s(-1) for NADH:ferricyanide reductase and NADH:cytochrome b5 reductase activities, respectively, with Km's of 7, 8, and 12 microM for NADH, K3Fe(CN)6, and cytochrome b5, respectively. Spectral-binding constants (Ks) determined for a variety of NAD+ analogs indicated the highest and lowest affinities were observed for APAD+ (Ks=71 microM) and PCA+ (Ks=>31 mM), respectively, and indicated the binding contributions of the various portions of the pyridine nucleotide. These results provide the first correct sequence for the full-length, membrane-associated form of C. familiaris cb5r and provide a direct comparison of the enzymes from two phylogenetic sources using identical expression systems that indicate that both enzymes have comparable spectroscopic, kinetic, thermodynamic, and structural properties.     

Studies on methemoglobin reductase. Immunochemical similarity of soluble methemoglobin reductase and cytochrome b5 of human erythrocytes with NADH-cytochrome b5 reductase and cytochrome b5 of rat liver microsomes.

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.     

Reduced pyridine nucleotides and cytochrome b5 as electron donors for prostaglandin synthetase reconstituted in dimyristyl phosphatidylcholine vesicles

Prostaglandin synthetase has been reconstituted in dimyristyl phosphatidylcholine vesicles. These vesicles in either 80 or 2 microM flufenamate utilized NADH or NADPH as electron donor in the reductive peroxidative step of prostaglandin H2 formation. Vesicles containing bound cytochrome b5 reductase and cytochrome b5 in order to complete an NADH cytochrome b5 reductase system also utilized reduced cytochrome b5 as the electron donor for this peroxidative step. In systems containing cytochrome and reductase, the rate of NADH oxidation exceeded that of NADPH oxidation, indicating that reduced cytochrome b5 is an effective electron donor for prostaglandin H2 formation, enhancing both the initial rate and the extent of the reaction. The concentrations of reduced pyridine nucleotides and cytochrome b5 employed in these experiments to provide reductants for prostaglandin synthetase are within the concentration ranges that obtain under physiological conditions.     

Mutagenesis of Glycine 179 modulates both catalytic efficiency and reduced pyridine nucleotide specificity in cytochrome b5 reductase

Cytochrome b5 reductase (cb5r), a member of the ferredoxin:NADP+ reductase family of flavoprotein transhydrogenases, catalyzes the NADH-dependent reduction of cytochrome b5. Within this family, a conserved "GxGxxP" sequence motif has been implicated in binding reduced pyridine nucleotides. However, Glycine 179, a conserved residue in cb5r primary structures, precedes this six-residue "180GxGxxP185" motif that has been identified as binding the adenosine moiety of NADH. To investigate the role of G179 in NADH complex formation and NAD(P)H specificity, a series of rat cb5r variants were generated, corresponding to G179A, G179P, G179T, and G179V, recombinantly expressed in Escherichia coli and purified to homogeneity. Each mutant protein was found to incorporate FAD in a 1:1 cofactor/protein stoichiometry and exhibited absorption and CD spectra that were identical to those of wild-type cb5r, indicating both correct protein folding and similar flavin environments, while oxidation-reduction potentials for the FAD/FADH2 couple (n = 2) were also comparable to the wild-type protein (E(o)' = -272 mV). All four mutants showed decreased NADH:ferricyanide reductase activities, with kcat decreasing in the order WT > G179A > G179P > G179T > G179V, with the G179V variant retaining only 1.5% of the wild-type activity. The affinity for NADH also decreased in the order WT > G179A > G179P > G179T > G179V, with the Km(NADH) for G179V 180-fold greater than that of the wild type. Both Ks(H4NAD) and Ks(NAD+) values confirmed that the G179 mutants had both compromised NADH- and NAD+-binding affinities. Determination of the NADH/NADPH specificity constant for the various mutants indicated that G179 also participated in pyridine nucleotide selectivity, with the G179V variant preferring NADPH approximately 8000 times more than wild-type cb5r. These results demonstrated that, while G179 was not critical for either flavin incorporation or maintenance of the appropriate flavin environment in cb5r, G179 was required for both effective NADH/NADPH selectivity and to maintain the correct orientation and position of the conserved cysteine in the proline-rich "CGpppM" motif that is critical for optimum NADH binding and efficient hydride transfer.     

Binding of homogeneous cytochrome b5 to rat liver microsomes. Effect on N-demethylation reactions.

Incubation of rat homogeneous detergent-solubilized cytochrome b5 with rat liver microsomes resulted in specific binding of the hemoprotein which was rapidly reduced by NADH. The NADH cytochrome c reductase activity in these preparations increased in proportion to the amount of cytochrome bound. However, the extra-bound detergent-solubilized cytochrome b5 did inhibit NADPH-dependent N-demethylations, the NADH synergism and NADPH cytochrome P-450 reductase activity. Manganese protoporphyrin-apocytochrome complex when bound to microsomes in amounts equivalent to detergent-solubilised cytochrome b5 showed no effect on N-demethylation activity. Furthermore, the binding of cytochrome b5 preparations reconstituted from heme and apocytochrome b5 had no effect on either the NADPH-dependent N-demethylation of aminopyrene or ethylmorphine or the NADH synergism observed with rat liver microsomes. In addition, homogeneous cytochrome b5 eluted from three additional Sephadex G-100 columns showed no inhibitory effects when bound to liver microsomes. Spectral analyses of the acid-acetone extract of the hemoprotein showed an absorption peak at 278 nm suggesting that the homogeneous b5 contains contaminating amounts of tightly bound detergent which is responsible for the observed inhibition of mixed function oxidase activity and which is removed during extraction of the heme from the apocytochrome and during further gel filtration applications.     

Heterologous expression of an endogenous rat cytochrome b(5)/cytochrome b(5) reductase fusion protein: identification of histidines 62 and 85 as the heme axial ligands

The gene coding for expression of an endogenous soluble fusion protein comprising a b-type cytochrome-containing domain and a FAD-containing domain has been cloned from rat liver mRNA. The 1461-bp hemoflavoprotein gene corresponded to a protein of 493 residues with the heme- and FAD-containing domains comprising the amino and carboxy termini of the protein, respectively. Sequence analysis indicated the heme and flavin domains were directly analogous to the corresponding domains in microsomal cytochrome b(5) (cb5) and cytochrome b(5) reductase (cb5r), respectively. The full-length fusion protein was purified to homogeneity and demonstrated to contain both heme and FAD prosthetic groups by spectroscopic analyses and MALDI-TOF mass spectrometry. The cb5/cb5r fusion protein was able to utilize both NADPH and NADH as reductants and exhibited both NADPH:ferricyanide (k(cat) = 21.7 s(-1), K(NADPH)(m) = 1 microM. K(FeCN6)(m) = 8 microM) and NADPH:cytochrome c (k(cat) = 8.3 s(-1), K(NADPH)(m) = 1 microM. K(cyt c)(m) = 7 microM) reductase activities with a preference for NADPH as the reduced pyridine nucleotide substrate. NADPH-reduction was stereospecific for transfer of the 4R-proton and involved a hydride transfer mechanism with a kinetic isotope effect of 3.1 for NADPH/NADPD. Site-directed mutagenesis was used to examine the role of two conserved histidine residues, H62 and H85, in the heme domain segment. Substitution of either residue by alanine or methionine resulted in the production of simple flavoproteins that were effectively devoid of both heme and NAD(P)H:cytochrome c reductase activity while retaining NAD(P)H:ferricyanide activity, confirming that the former activity required a functional heme domain. These results have demonstrated that the rat cb5/cb5r fusion protein is homologous to the human variant and has identified the heme and FAD as the sites of interaction with cytochrome c and ferricyanide, respectively. Mutagenesis has confirmed the identity of both axial heme ligands which are equivalent to the corresponding residues in microsomal cytochrome b(5).     

Purification and characterization of two forms of soluble NADH cytochrome b5 reductases from human erythrocytes.

1. Two forms of soluble NADH cytochrome b5 reductase were purified from human erythrocytes. Two distinct fractions both having the NADH cytochrome b5 reductase activity eluted from the second DEAE-cellulose column were further purified by ultrafiltration and 5'-ADP-agarose affinity chromatography. 2. The final preparations were purified 9070- and 4808-fold, respectively, over hemolysate. Both reductases exhibited identical electrophoretic patterns when subjected to SDS-PAGE and apparent monomer Mr of each reductase was determined to be 32,000 +/- 1300. 3. Vmax values of reductase II for the various electron acceptors, namely, 2,6-dichlorophenolindophenol, ferricyanide and cytochrome c through cytochrome b5 were found to be 1.9, 1.8 and 2 times higher than those of reductase I. 4. Some differences were noted for reductase I and reductase II fractions. Their elution profiles from a second DEAE-cellulose column were quite different and that suggested that reductase II is more acidic than reductase I. Reductase II was found to be more sensitive to heat treatment than reductase I.     

Selective induction of cytochrome b5 and NADH cytochrome b5 reductase by propylthiouracil

Both the cytochrome b₅ level and NADH cytochrome b₅ reductase activity in rat liver microsomes were increased 2-fold by repeated i.p. administration of 1.5 mmol/kg propylthiouracil (PTU) for 2 weeks, but neither the cytochrome P-450 level nor NADPH cytochrome P-450 reductase activity were affected by the treatment. Liver microsomes from PTU-treated rats showed a significant decrease in aminopyrine N-demethylation, but not in benzphetamine N-demethylation, aniline hydroxylation or 7-ethoxycoumarin O-deethylation. A single administration of the compound had no effect on any components of the system. $\text{In vitro}$, drug hydroxylation activities were not affected by PTU up to 1.0 mM. From the above evidence, repeated administration of PTU selectively induced cytochrome b₅ and NADH cytochrome b₅ reductase in rat liver microsomes.     

Covalent cross-linking of the active sites of vesicle-bound cytochrome b5 and NADH-cytochrome b5 reductase

A water-soluble carbodiimide has been used to promote the formation of amide bonds between carboxyl residues on cytochrome b5 and lysyl residues on cytochrome b5 reductase. The visible and UV absorption spectrum of the purified cross-linked complex was identical with the sum of the spectra of the individual enzymes, and the average apparent molecular weight of the complex, determined by sodium dodecyl sulfate-gel electrophoresis, was within 12% of the sum of the apparent molecular weights of the two monomeric enzymes, indicating that the cross-linked derivative was a dimer containing one molecule each of cytochrome b5 and cytochrome b5 reductase. When reconstituted into phospholipid vesicles, the amphipathic derivative showed substantially reduced Vmax values with the soluble electron acceptors potassium ferricyanide, cytochrome b5 heme peptide and cytochrome c, and with the membrane-bound acceptors amphipathic cytochrome b5 and stearyl-CoA desaturase. The soluble catalytic fragment of the derivative, produced by limited digestion with subtilisin Carlsberg, showed similar decreases in Vmax values with the above soluble acceptors. In contrast, intradimer electron transfer in the soluble fragment, measured by stopped flow spectrophotometry at 2 degrees C was very efficient. Ninety per cent of the cytochrome b5 in the derivative was reduced with a first order rate constant of 51 s-1 upon the addition of NADH; the transfer of electrons from NADH to the reductase FAD prosthetic group, which is known to be the rate-limiting step in the reductase reaction mechanism, proceeded with an apparent rate constant of 57 s-1 under these conditions. These kinetic data show that the enzymes in the complex are cross-linked together at the surfaces involved in protein-protein contacts during electron transfer in an orientation similar to that assumed during electron transfer between the free proteins.     

Effects of domain-specific erythrocyte membrane modulators on acetylcholinesterase and NADH:cytochrome b5 reductase activities

Previously, we showed using electron paramagnetic resonance that the physical state of one side of erythrocyte membranes could be modulated by agents which interact with the opposite side (reviewed in Butterfield, 1989, Biological and Synthetic Membranes, A. R. Liss, Inc., New York). The present study was undertaken to determine whether membrane-bound enzymes would exhibit a similar transmembrane modulation effect. The effects of known, domain-specific modulators of the physical state of erythrocyte membranes on the activity of two membrane-bound enzymes were investigated. Acetylcholinesterase, an enzyme having its active site situated on the extracellular side of the membrane, seemed to be unaffected by most of the modulators employed in this study, with the exception of reversible inhibition by benzyl alcohol. Conversely, the activity of NADH:cytochrome b5 reductase, an enzyme whose active site is located on the cytoplasmic side of the erythrocyte membrane, was increased by those agents that interact primarily with skeletal proteins to increase skeletal protein-protein interactions; however, those agents which interact primarily with the skeleton to decrease protein-protein interactions decreased the activity of NADH:cytochrome b5 reductase. This enzyme's activity was also significantly altered by lectins which bind specifically to the external face of glycophorin A on the opposite side of the membrane, but it's activity was unaffected by concanavalin A, a lectin which binds to the external face of band 3. The results of these biochemical studies suggested that NADH:cytochrome b5 reductase can interact with and its activity can be modulated by skeletal or transmembrane proteins. In addition, these results support the hypothesis that in transmembrane signaling processes, biophysical and biochemical changes are correlated.