NADH-cytochrome B5 Reductase Research Articles

Selective modification of rat hepatic microsomal fatty acid chain elongation and desaturation by fibrates: relationship with peroxisome proliferation

1. The time-course of the effect of clofibrate (CFB), bezafibrate (BFB) and gemfibrozil (GFB) on lipid plasma levels and palmitoyl-, palmitoleoyl- and gamma-linolenoyl-CoA elongase, delta-9, delta-6 and delta-5 desaturase activities, and microsomal electron transport chains, as well as the correlation with the peroxisomal proliferation phenomenon have been studied in male Sprague-Dawley rats. 2. As reported in our previous work, the three drugs behave as peroxisomal proliferators (the order of potency was BFB > CFB > or = GFB) and induced a clear reduction in both plasma cholesterol and triglyceride levels. 3. Palmitoyl-CoA elongation activity was increased by the three drugs (BFB = GFB > CFB), whereas palmitoleoyl-CoA elongation activity was only enhanced by GFB. Elongation activity was not modified by fibrates when gamma-linolenoyl-CoA was used as substrate. These results are in accordance with the existence of three different elongation systems for saturated, mono- and polyunsaturated fatty acids. 4. delta-9, delta-6 and delta-5 desaturase activities were increased by the three fibrates, with an order of potency BFB > CFB = GFB for delta-9 and delta-5, and GFB > BFB = CFB for delta-6. 5. Of the enzyme activities integrated in the microsomal electron transport chains, NADH cytochrome b5 reductase was not affected by fibrate treatment, NADPH cytochrome c reductase activity was enhanced (BFB = GFB > CFB), whereas NADH cytochrome c reductase activity was reduced by CFB and BFB. 6. The increase in Delta-9 and Delta-5 desaturase activities was highly dependent on the peroxisomal proliferation phenomena, whereas the increase in Delta-6 desaturase activity and the decrease in NADH cytochromec reductase was mainly independent. The modifications of palmitoyl-CoA elongase and NADPH cytochrome c reductase activities, as well as plasma lipid levels, were partially correlated with peroxisomal beta-oxidation, but the r2 values obtained point to the existence of additional independent mechanisms.7. As man is assumed to be a species refractory to peroxisomal proliferation, only those fibrate effectsnot absolutely related to this phenomenon are expected to appear after fibrate therapy.

Specific arrangement of three amino acid residues for flavin-binding barrel structures in NADH-cytochrome b5 reductase and the other flavin-dependent reductases

The structure of NADH-cytochrome b 5 reductase from pig liver microsomes has been refined to a crystallographic R factor of 0.223 at 2.4 Å resolution. A structural comparison between the flavin-binding β barrel domain of NADH-cytochrome b 5 reductase and those of the other flavin-dependent reductases, ferredoxin-NADP + reductase, phthalate dioxygenase reductase and nitrate reductase, indicated that the overall barrel foldings are similar to each other and that the specific arrangement of three amino acid residues (Arg, Tyr and Ser/Thr) is usually necessary for flavin-binding. These conserved residues overlap each other in their three-dimensional structures and stabilize the flavin-binding site in the four flavin-dependent reductases.

Crystal Structure of NADH-Cytochrome b5 Reductase from Pig Liver at 2.4 .ANG. Resolution

The three-dimensional structure of NADH-cytochrome b5 reductase from pig liver microsomes has been determined at 2.4 A resolution by X-ray crystallography. The molecular structure reveals two domains, the FAD binding domain and the NADH domain. A large cleft lies between these two domains and contains the binding site for the FAD prosthetic group. The backbone structure of the FAD binding domain has a great similarity to that of ferredoxin-NADP+ reductase [Karplus, P. A., Daniels, M. J., & Herriott, J. R. (1991) Science 251, 60-65], in spite of the relatively low sequence homology (about 15%) between the two enzymes. On the other hand, the structure of the NADH domain has several structural differences from that of the NADP+ domain of ferredoxin-NADP+ reductase. The size of the cleft between the two domains is larger in NADH-cytochrome b5 reductase than in ferredoxin-NADP+ reductase, which may be responsible for the observed difference in the nucleotide accessibility in the two enzymes.

Purification of NADH-cytochromeb 5 reductase from rat liver plasma membranes

An NADH-cytochromeb5 reductase was purified from rat liver plasma membranes. Rat liver plasma membranes were prepared by aqueous two-phase partition. Peripheral proteins were removed by EDTA extraction and integral membrane proteins were solubilized with Triton X-100. The NADH-cytochromeb5 reductase was purified by hydroxyapatite, anion exchange, and gel filtration chromatographies. The purified preparation was homogeneous and estimated to have an apparent molecular weight of 32 kDa on SDS-polyacrylamide gel electrophoresis. Two tryptic peptides of the purified enzyme had sequence homologies with rat, human, and bovine NADH-cytochromeb5 reductases.

Localization of metmyoglobin-reducing enzyme (NADH-cytochrome b5 reductase) system components in bovine skeletal muscle

Localization of metmyoglobin-reducing enzyme system components (NADH-cytochrome b 5 reductase, cytochrome b 5 outer mitochondrial membrane cytochrome b) was demonstrated in bovine skeletal muscle by immunohistochemical techniques. Both NADH-cytochrome b 5 reductase and OM cytochrome b were located in red fibers. However, localization of cytochrome b 5 did not show a definite difference between fiber types. Using an immunoblotting technique. NADH-cytochrome b 5 reductase was found predominantly in the mitochondrial fraction, but it was also detected at lower levels in the microsomal fraction. OM cytochrome b was found predominantly in the mitochondrial fraction, but cytochrome b 5 was detected only in the microsomal fraction. The results from this study, along with previous work about the localization of myoglobin in muscle, suggest that NADH-cytochrome b 5 reductase reduces metmyoglobin by using OM cytochrome b at the mitochondrial surface and, in part, by using cytochrome b 5 at the sarcoplasmic reticulum.

DT-diaphorase as a determinant of sensitivity to adriamycin in non-small-cell lung-cancer cell lines.

We have reported the establishment of a mitomycin-C (MMC)-resistant non-small-cell lung-cancer cell line, PC-9/MC4. As determined by an MTT assay, this resistant cell line was found to be 4 times more sensitive to adriamycin (ADM) than was the parental PC-9. There were no significant differences in sensitivity to etoposide, mitoxantrone, daunomycin, epirubicin, pirarubicin, 9-aminoanthracycline or 3'-deamino-3'-morpholino-13-deoxo-10-hydroxy carminomycin. These data suggest that neither qualitative or quantitative changes in DNA topoisomerase II nor the enhanced repair of DNA can explain the differing sensitivity to ADM observed. No significant differences were found in the accumulation of ADM and glutathione (GSH) in these cell lines. Although total glutathione-S-transferase (GST) activity in PC-9/MC4 cells was lower than that observed in PC-9 cells and treatment with ethacrynic acid (EA) reduced sensitivity to ADM in both cell lines, relative resistance was unaffected. NADH-cytochrome b5 reductase (B5R) activity in PC-9/MC4 cells showed a 3-fold greater decrease than that in PC-9 cells, and DT-diaphorase (DTD) activity in PC-9/MC4 cells showed an approximately 200-fold greater decrease than that in PC-9 cells. Addition of dicumarol, an inhibitor of DTD, decreased the sensitivity of ADM of PC-9 but not of PC-9/MC4. DTD activity in the PC-9 cell line was inhibited by treatment with dicumarol while in PC-9/MC4 it remained unchanged. These data suggest that DT-diaphorase is a determinant of sensitivity to ADM in the 2 cell lines.

Stimulatory effects of lung cytochrome b5 on benzphetamine N-demethylation in a reconstituted system containing lung cytochrome P450 LgM2

1. 1. Cytochrome b5 was partially purified from sheep lung microsomes in the presence of detergents Emuigen 913 and cholate by three consecutive DEAE-cellulose and Sephadex G-100 gel filtration chromatographies. 2. 2. The specific content ofcytochrome b5 was 16.5 nmol/mg protein and purified cytochrome b5 fractions were free of cytochrome P450, NADPH-cytochrome P450 reductase and NADH-cytochrome b5 reductase activities. 3. 3. The influences of increasing concentrations of lung cytochrome b5 on benzphetamine N-demethylation reactions were examined in four different reconstitution systems containing lung cytochrome P 450 LgM2, lung cytochrome P450 reductase and lipid. In each system concentration of reductase was doubled with respect to former system. 4. 4. In all systems cytochrome b 5 stimulated benzphetamine Ndemethylase activity especially when cytochrome b5 was present at 0.5:1 molar ratio with respect to cytochrome / P450 LgM2. 5. 5. Besides, the greatest fold of increase in benzphetamine N-demethylation activity due to addition of cytochrome b5 was observed in System 1 with the lowest concentration of reductase.

Direct measurement of pO 2 distribution and bioreductive enzymes in human malignant brain tumors

Purpose : To measure the oxygen status of human malignant brain tumors in vivo and to determine the activities and expression of bioreductive enzymes in these same human brain tumor samples, as a means of assessing their suitability as targets for bioreductive drug therapy. Methods and Materials : A polarographic oxygen electrode was used to measure the intratumoral oxygen tension in twenty patients with malignant brain tumors during open brain surgery, performed under standard anaesthetic conditions. Six different tracks, each with a path length of 22 mm, were recorded per patient representing 192 readings. Following pO 2 measurements the tumors were resected and stored in liquid N 2 for subsequent bioreductive enzyme analysis. Eight human malignant brain tumors were assessed, by enzyme activity and western blot expression, for the presence of various bioreductive enzymes. These enzymes included DT-diaphorase, NADH cytochrome b5 reductase, and NADPH cytochrome P-450 reductase. Of these eight gliomas analyzed six samples were incubated with the bioreductive drug tirapazamine, in the presence of cofactor(s), to establish whether human brain tumors could metabolize this compound. Results : Both the high grade intrinsic and metastatic brain tumors showed significant regions of hypoxia. All the tumors subjected to enzyme profiling contained the bioreductive enzymes, DT-diaphorase, NADH cytochrome b5 reductase and NADPH cytochrome P-450 reductase. Also all six of the brain tumors investigated could metabolize tirapazamine to the two-electron reduction product. Conclusion : These findings would favor primary brain tumors as suitable targets for bioreductive therapy.

Stability and storage conditions of NADH-cytochrome b5 reductase cross-linked into gelatin by chromium (III) acetate

NADH-cytochrome b5 reductase was isolated and partially purified from rabbit liver microsomes. It was immobilized into gelatin by chemical cross-linking. Chromium (III) acetate was used as cross-linker. The effects of pH and temperature on the immobilized cytochrome b5 reductase were investigated. The reusability and storage stability of immobilized enzyme were also tested. Immobilized NADH-cytochrome b5 reductase activities were found to be stable for at least 72 d and 24 uses. The storage stability of NADH-cytochrome b5 reductase was improved with immobilization at 25°C.

Biosynthesis of N-glycolylneuraminic acid-containing glycoconjugates. Purification and characterization of the key enzyme of the cytidine monophospho-N-acetylneuraminic acid hydroxylation system.

We have proposed that cytidine monophospho-N-acetylneuraminic acid (CMP-NeuAc) hydroxylation is carried out by a multienzyme system involving CMP-NeuAc hydroxylase (the terminal enzyme of the system), cytochrome b5, and an NADH-dependent cytochrome b5-reducing factor (Kozutsumi, Y., Kawano, T., Yamakawa, T., and Suzuki, A. (1990) J. Biochem. 108, 704-706). The CMP-NeuAc hydroxylase was purified to homogeneity from the cytosolic fraction of mouse liver, using ion exchange columns, a Red-Sepharose column, and a soluble cytochrome b5-immobilized Sepharose column. The purified enzyme exhibited a single band (64 kDa) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and a single peak (58 kDa) on gel permeation chromatography, indicating that it is composed of a single polypeptide chain. The absorption spectrum did not indicate the presence of a heme prosthetic group in the enzyme. Atomic absorption spectrometry and an inhibition test using an iron chelator indicate that the enzyme contains non-heme iron as an electron acceptor. A reconstitution experiment with the purified CMP-NeuAc hydroxylase, soluble cytochrome b5, and recombinant NADH-cytochrome b5 reductase revealed that these three factors are essential for the reaction. The hydroxylase exhibited high affinity to CMP-NeuAc (Km = 5 microM) and was greatly stabilized by CMP-NeuAc. The molecular activity of the enzyme (approximately 500/min) is much lower than that reported for NADH-cytochrome b5 reductase, suggesting that the activity or amount of hydroxylase is rate-limiting in CMP-N-glycolylneuraminic acid (NeuGc) biosynthesis. These results, together with the previous observation that the level of CMP-NeuAc hydroxylase activity was associated with the expression of NeuGc in various tissues, support the notion that the enzyme is the key for regulation of the overall velocity of CMP-NeuAc hydroxylation and consequently for the expression of NeuGc-containing glycoconjugates.

Effects of in vivo benzo(a)pyrene treatment on liver microsomal mixed-function oxidase activities of gilthead seabream ( Sparus aurata)

Benzo(a)pyrene [B(a)P] treatment of gilthead seabream, 25 mg/kg, i.p. for 5 consecutive days, did not cause any significant changes in ethylmorphine N-demethylase and aniline 4-hydroxylase activities of liver microsomes. The same treatment did not alter the liver microsomal cytochrome b5 content, NADH-cytochrome b5 reductase and NADPH-cytochrome P450 reductase activities. However, benzo(a)pyrene treatment caused a 2–3-fold increase in 7-ethoxyresorufin O-deethylase (7-EROD) activity of gilthead seabream liver microsomes. Although, upon treatment, total cytochrome P450 content of liver microsomes increased about 1.7-fold in 1990 fall, no such increase was observed in spring 1991. However, a new cytochrome P450 with an apparent M r of 58,000 was observed on SDS-PAGE of liver microsomes obtained from benzo(a)pyrene treated gilthead seabream. Besides, in vitro addition of 0.2 × 10 −6 M benzo(a)pyrene to the incubation mixture inhibited 7-ethoxyresorufin O-deethylase activity by 93%. Gilthead seabream liver microsomal 7-ethoxyresorufin O-deethylase activity was characterized with respect to substrate concentration, amount of enzyme, type of buffer used, incubation period and temperature.

Heterogeneity of the rat NADH-cytochrome-b5-reductase transcripts resulting from multiple alternative first exons.

In order to understand the mechanisms responsible for the generation of different isoforms (membrane-bound and soluble) of NADH-cytochrome b5 reductase, and the different clinical forms of recessive congenital methemoglobinemia due to the deficiency of this enzyme in humans (type I, without mental retardation; type II, with mental retardation), we have looked for mRNA heterogeneity in various rat tissues. We have found four types of mRNAs, each with a different first exon (1L, 1R, 1X and 1Y), all of which were precisely spliced to join the common second exon. Our results are consistent with a 5'-->3' 'scanning' mechanism for splice-site selection. The previously characterized 1L and 1R transcripts arise from the alternative use of either a ubiquitous promoter (Pr-L) or an erythroid-specific promoter (Pr-R). In addition, the X and Y RNA species are novel transcripts which are expressed ubiquitously and at a relatively low level. The first alternative exons 1X and 1Y are noncoding, such that the AUG codon present in the common second exon is functional, as it is in the R mRNA. Thus, the X and Y mRNAs are expected to be translated in vivo into a ubiquitous soluble enzyme. Consequently, the rat NADH-cytochrome-b5-reductase gene is expressed through the use of at least four different promoters, which are probably subjected to different forms of regulation. This model of gene expression in rat could be important in understanding the basis for the different types of the NADH-cytochrome-b5-reductase enzyme and their deficiency in man.

An evaluation of the measurement of the activities of complexes I-IV in the respiratory chain of human skeletal muscle mitochondria.

The measurement of individual respiratory chain complexes is an important component of the investigation of diseases due to mitochondrial dysfunction. We have evaluated assays which measure complexes I to IV in human skeletal muscle mitochondria and in addition optimized these assays to provide sensitive and reliable diagnostic techniques, particularly in situations where a partial interruption at a single complex needs to identified. Using several established methods of membrane disruption we have found that optimal activities of complexes I and II are obtained by freeze-thawing the mitochondria in hypotonic potassium phosphate buffer, whereas complex III and IV activities are markedly increased by the addition of the detergent n-dodecyl-beta-D-maltoside. Complex I activity is measured in the presence of 2.5 mg.ml-1 bovine serum albumin, which increases rotenone sensitivity, and we have shown that NADH-cytochrome b5 reductase makes an important contribution to the rotenone-insensitive NADH-ubiquinone oxidoreductase activity. Complex II activity is measured after preincubation of the mitochondrial fraction with succinate to fully activate the complex. Complex I and III activities are dependent upon the length of the isoprenoid chain of the ubiquinone and ubiquinol, respectively. These assays have been used to establish a control range.

CMP‐N‐acetylneuraminic acid hydroxylase from mouse liver and pig submandibular glands

In this report, the nature of the protein components involved in the functioning of cytidine-5'-monophosphate-N-acetylneuraminic acid (CMP-Neu5 Ac) hydroxylase in high-speed supernatants of mouse liver has been investigated. Fractionation and reconstitution experiments showed that this enzyme system consists of NADH-cytochrome b5 reductase, cytochrome b5 and a 56-kDa terminal electron acceptor having the CMP-Neu5 Ac hydroxylase activity. This enzyme system is extracted in a soluble protein fraction; however, the amphipathic, usually membrane-associated, forms of cytochrome b5 and the reductase were found to predominate and are presumably the forms which support the turnover of the hydroxylase in vivo. Although the majority of cellular cytochrome b5 and cytochrome b5 reductase is membrane-bound, the addition of intact microsomes elicited no significant increase in the hydroxylase activity of supernatants. Detergent-solubilised microsomes, however, potently activated the hydroxylase, probably due to the greater accessibility of the cytochrome b5. Accordingly, in reconstitution experiments, pure hydrophilic cytochrome b5 interacts more effectively with the hydroxylase than isolated amphipathic cytochrome b5. Studies on the CMP-Neu5 Ac hydroxylase system in fractionated porcine submandibular glands and bovine liver suggest that the composition of this enzyme system is conserved in all mammals possessing sialoglycoconjugates containing N-glycolylneuraminic acid.

An in-frame deletion of codon 298 of the NADH-cytochrome b5 reductase gene results in hereditary methemoglobinemia type II (generalized type). A functional implication for the role of the COOH-terminal region of the enzyme.

The nucleotide sequence was determined for the gene of NADH-cytochrome b5 reductase of a patient of type II hereditary methemoglobinemia found in Yokohama, Japan. An in-frame deletion of 3 base pairs corresponding to codon 298 (TTC) was identified in the patient. The patient was homozygous for the mutation as shown by hybridization experiments using allele-specific oligonucleotides. The mutation causes deletion of Phe-298, which is the third to the COOH-terminal residue, indicating that in this mutant enzyme the sequence of this region has changed from -Cys-Phe-Val-Phe-COOH to -Cys-Val-Phe-COOH. The mutant enzyme, whose Phe-298 was deleted (F298 delta), was prepared by means of a bacterial expression system and site-directed mutagenesis. The kcat/Km value (NADH) of the enzyme was 5.7 s-1 M-1, which corresponds to 0.4% of that of the wild type. Moreover, the enzyme was much less thermostable than the wild type. To examine further the role of the COOH-terminal portion of the enzyme, various mutant enzymes were also prepared and characterized. The enzymatic properties of F298L, F300L, and F298L/F300L were essentially the same as that of the wild type. The kinetic properties of F298A, and F300A were not greatly affected, but the stability of the enzymes was somewhat impaired. Since Val-299 is naturally Ala in steer enzyme, no specific residues in the carboxyl-terminal region (298-300) are essential to the enzyme function. The instability of the F298/F300A double mutant indicates that the hydrophobicity of the carboxyl-terminal region of the enzyme might be important to maintain the conformation of the enzyme. high impairment of the activity of the F298 delta, F298stop, and F300stop mutants might be caused by the loss of the residue(s) in the carboxyl-terminal portion. These results indicate that the hydrophobicity, but not the specific amino acid residues, of the carboxyl-terminal portion of the enzyme is important for the stability of the enzyme.

Effect of Vitamin B6Deficiency on Linoleic Acid Desaturation in the Arachidonic Acid Biosynthesis of Rat Liver Microsomes

The effect of vitamin B6 deficiency on the desaturation of linoleic acid (18:2n-6) to γ-linolenic acid (18:3n-6) in rat liver microsomes was studied. Rats fed with a vitamin B6-deficient 70% casein diet for 5 weeks displayed a striking decrease mainly in the activity of terminal Δ6-desaturase in the 18:2n-6 desaturation system, although the activity of the electron-transport enzyme, NADH-cytochrome b5 reductase, was also significantly decreased. The 18:2n-6 desaturation index, the arachidonic acid (20:4n-6)/ 18:2n-6 ratio in the liver microsomal total lipids and in their phosphatidylcholine (PC) fraction, decreased together with the microsomal Δ6-desaturase activity, these changes being quite faithfully reflected in the plasma lipid. Among the microsomal lipid components, the PC content significantly decreased. In addition, a highly positive correlation was found between the PC content and Δ6-desaturase activity in liver microsomes. In an in vitro experiment, the Δ6-desaturase activity decreased linearly with increasing rate of PC hydrolysis by phospholipase C. These results suggest that the altered PC content in microsomes induced by vitamin B6 deficiency in vivo may affect the 18:2n-6 desaturation and thus decrease the biosynthesis of 20:4n-6

Cytochrome b5 involvement in cytochrome P450 monooxygenase activities in house fly microsomes.

The involvement of cytochrome b5 in different cytochrome P450 monooxygenase and palmitoyl CoA desaturase activities in microsomes from insecticide-resistant (LPR) house flies was determined using a specific polyclonal antiserum developed against house fly cytochrome b5. Anti-b5 antiserum inhibited the reduction of cytochrome b5 by NADH-cytochrome b5 reductase. The antiserum also inhibited palmitoyl CoA desaturase, methoxycoumarin-O-demethylase (MCOD), ethoxycoumarin-O-deethylase (ECOD), and benzo[a]pyrene hydroxylase (aromatic hydrocarbon hydroxylase, AHH) activities. However, methoxyresorufin-O-demethylase (MROD) and ethoxyresorufin-O-deethylase (EROD) activities were not affected by this antiserum. These results demonstrate that cytochrome b5 is involved in fatty acyl CoA desaturase activities and in certain cytochrome P450 monooxygenase activities (i.e., MCOD, ECOD, and AHH) in LPR house fly microsomes. Other cytochrome P450 monooxygenase activities (i.e., MROD and EROD) may not require cytochrome b5. The results suggest that cytochrome b5 involvement with cytochrome P450 monooxygenase activities is dependent upon the cytochrome P450 isoform involved.

Interaction of Microsomal Cytochrome P-450 s and N-Phenylcarbamates that Induce Flowering in Asparagus Seedlings

n-Propyl N-(3,4-dichlorophenyl)carbamate, which induces flowering while it inhibits a step or steps in the phenylpropanoid metabolism in Asparagus officinalis L. seedlings, was found to retard the conversion of t-cinnamic acid to p-coumaric acid by high-pressure liquid chromatography of the metabolites in the shikimic acid pathway. The concentrations of the metabolites preceding t-cinnamic acid on the pathway in treated and untreated seedlings were the same, but those of p-coumaric acid and later metabolites were significantly lower in treated plants. The carbamate inhibited phenylpropanoid metabolism when used to treat stem segments that included a shoot apex primordium, where flowers are induced, and when added to a 100,000 × g microsomal fraction prepared from such segments. NADPH-cytochrome P-450 and NADH-cytochrome b5 reductases in the 100,000 × g fraction were not inhibited by the carbamate. The results showed that this compound has its site of action on the endoplasmic reticulum and that it inhibits cytochrome P-450s, including t-cinnamic acid 4-hydroxylase. We examined the flower-inducing activity of known cytochrome P-450 inhibitors, and found that piperonyl butoxide also causes flowering.

Purification and Partial Characterization of Microsomal NADH-Cytochrome b5 Reductase from Higher Plant Catharanthus roseus

A simple three step procedure was used to purify microsomal NADH-cytochrome b5 (ferricyanide) reductase to homogeneity from the higher plant C. roseus. The microsomal bound reductase was solubilized using zwitterionic detergent-CHAPS. The solubilized reductase was subjected to affinity chromatography on octylamino Sepharose 4B, blue 2-Sepharose CL-6B and NAD+-Agarose. The homogeneous enzyme has an apparent molecular weight of 33,000 as estimated by SDS-PAGE. The purified enzyme catalyzes the reduction of purified cytochrome b5 from C. roseus in the presence of NADH. The reductase also readily transfers electrons from NADH to ferricyanide (Km 56 μM), 2,6-dichlorophenolindophenol (Km 65 μM) and cytochrome c via cytochrome b5 but not to menadione.

Interaction of non-myristoylated NADH-cytochrome b5 reductase with cytochrome b5-dimyristoylphosphatidylcholine vesicles.

An expression vector for NADH-cytochrome b5 reductase containing a thrombin cleavage site directly before the N-terminal glycine residue of the flavoprotein was used to isolate the non-myristoylated enzyme by thrombin cleavage of the initial fusion protein of a short segment of the multiple cloning site of the plasmid vector and the reductase. This flavoprotein preparation, containing only the 28-residue N-terminal peptide segment of the membrane-binding domain of the mammalian enzyme, binds to phospholipid vesicles and interacts with membrane-bound cytochrome b5. The effect of N-myristoylation of the enzyme therefore appears to be limited to facilitating and stabilizing interactions with phospholipid vesicles. However, the relatively short intervening peptide sequence that separates the crucial peptide membrane-binding domain from lysine 41, which has been implicated in the active-site interaction with cytochrome b5 (Strittmatter, P., Kittler, J. M., Coghill, J. E., and Ozols, J. (1992) J. Biol. Chem. 267, 2519-2523), provides some limitation of the distance from the membrane surface for the interactions required for rapid electron transfer from the flavin of the reductase to the heme of cytochrome b5.