Preliminary X-ray data of NADH-cytochrome b5 reductase from human erythrocytes.
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Cytochrome Reductases↗
Biomedical subjects
T Yubisui
Publications and source records attributed to T Yubisui.
Molecular cloning of cDNAs of human liver and placenta NADH-cytochrome b5 reductase.
A cDNA coding for human liver NADH-cytochrome b5 reductase (cytochrome b5 reductase, EC 1.6.2.2) was cloned from a human liver cDNA library constructed in phage lambda gt11. The library was screened by using an affinity-purified rabbit antibody against NADH-cytochrome b5 reductase of human erythrocytes. A cDNA about 1.3 kilobase pairs long was isolated. By using the cDNA as a probe, another cDNA (pb5R141) of 1817 base pairs was isolated that hybridized with a synthetic oligonucleotide encoding Pro-Asp-Ile-Lys-Tyr-Pro, derived from the amino acid sequence at the amino-terminal region of the enzyme from human erythrocytes. Furthermore, by using the pb5R141 as a probe, cDNA clones having more 5' sequence were isolated from a human placenta cDNA library. The amino acid sequences deduced from the nucleotide sequences of these cDNA clones overlapped each other and consisted of a sequence that completely coincides with that of human erythrocytes and a sequence of 19 amino acid residues extended at the amino-terminal side. The latter sequence closely resembles that of the membrane-binding domain of steer liver microsomal enzyme.
Amino Acid Sequence↗
Structural comparison of bovine erythrocyte, brain, and liver NADH-cytochrome b5 reductase by HPLC mapping.
NADH-cytochrome b5 reductases purified from bovine erythrocytes and from bovine brain and liver microsomes solubilized with lysosomal protease were subjected to structural analysis by using HPLC mapping, amino acid analysis of the resulting peptides, and NH2-terminal sequence analysis of apoproteins. HPLC maps of the tryptic peptides derived from these enzymes were very similar to each other, and amino acid analysis of the HPLC-separated peptides indicated that the structures of these enzymes are identical except for the NH2-terminal region. The NH2-terminal sequence of the brain enzyme determined by automated Edman degradation was as follows: NH2-Phe-Gln-Arg-Ser-Thr-Pro-Ala-Ile-Thr-Leu-Glu-Asn-Pro-Asp- Ile-Lys-Tyr-Pro-Leu-Arg-Leu-Ile-Asp-Lys-Glu-Val-Ile- This sequence is identical to that of liver enzyme except that the liver enzyme started at the 3rd Arg or 4th Ser. The NH2-terminal amino acid residue of the soluble erythrocyte enzyme was not detected by automated Edman degradation. The sequence analysis of a tryptic peptide from the erythrocyte enzyme indicated that Leu is present before the NH2-terminal Phe of the brain enzyme. The recently reported sequence of the apparently identical protein (Ozols et al. (1985) J. Biol. Chem. 260, 11953-11961) differs in two amino acid assignments from our sequence.
Amino Acid Sequence↗
[Methemoglobin reducing systems].
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Animals↗
Characterization of a second form of NADPH-flavin reductase purified from human erythrocytes.
A second form of the NADPH-flavin reductase with an isoelectric point of 6.1 was purified to homogeneity from human erythrocytes. The enzyme showed NADPH-specific flavin reductase activity when FAD, FMN or riboflavin was used as an electron acceptor. Analyses of the amino acid compositions and immunological reactivities of the enzyme and the other flavin reductase with an isoelectric point of 8.1 revealed that the proteins of these two enzymes are indistinguishable to each other. Tightly bound NADP+, which was reducible by a NADPH-generating system, was specifically found in the second form of the enzyme.
Amino Acids↗
Complete amino acid sequence of NADH-cytochrome b5 reductase purified from human erythrocytes.
The complete amino acid sequence of soluble NADH-cytochrome b5 reductase purified from human erythrocytes was determined. The enzyme, which contained 8 methionine residues, was cleaved by cyanogen bromide. The resulting nine peptides were separated by gel filtration and purified further by high-performance liquid chromatography. The purified peptides were sequenced by automated Edman degradation. Three large CNBr peptides, residues 1-101, 109-151, and 169-231, were further fragmented with trypsin, Staphylococcus aureus V8 protease or a lysyl endopeptidase of Achromobacter lyticus. The peptides obtained from the tryptic digest of citraconylated FAD-depleted apoprotein completed the alignments of the other peptides. The enzyme was composed of 275 amino acid residues. The 4 functionally important cysteine residues were located in the COOH-terminal portion. The molecular weight of the protein was calculated to be 31,260 without FAD. A prediction of the secondary structure was made by the method of Chou and Fasman. The protein was hydrophilic as a whole (43% polarity), but some regions were rich in hydrophobic residues. From the sequence homology of this enzyme with the pyridine nucleotide-binding sites of other flavoproteins, three candidates for the FAD and NADH-binding domains were suggested.
Amino Acid Sequence↗
[Cell differentiation, maturation and enzyme abnormality--studies on NADH-cytochrome b5 reductase].
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Amino Acid Sequence↗
The inhibitory effect of halides and carboxylates on hepatic NADH: cytochrome b5 oxidoreductase.
The inhibitory effect of several halides and carboxylates on bovine liver microsomal NADH: cytochrome b5 oxidoreductase (EC 1.6.2.2) was examined. The magnitude of inhibition was altered by various anion species but not by cation species. The order of effectiveness was F- less than acetate less than Cl- less than Br- less than I- less than succinate congruent to citrate. The kinetics of these inhibitions was competitive with cytochrome b5, and non-competitive with NADH, indicating that these anions inhibit the interaction of the enzyme with cytochrome b5.
Animals↗
Palmitoyl-CoA elongation in brain microsomes: dependence on cytochrome b5 and NADH-cytochrome b5 reductase.
Experiments were performed to demonstrate the involvement of electron transport system in fatty acid elongation in rat brain microsomes. Mercuric chloride and p-chloromercuriphenylsulfonate, inhibitors on NADH-cytochrome b5 reductase, at 32 microM inhibited NADH-supported palmitoyl-CoA elongation to 30 and 60% of control activity, respectively, whereas NADPH-supported palmitoyl-CoA elongation was unaffected by these mercurials. An antibody to rat liver NADH-cytochrome b5 reductase inhibited brain microsomal NADH-cytochrome b5 reductase activity and NADH-dependent palmitoyl-CoA elongation. Treatment of brain microsomes with trypsin diminished the cytochrome b5 content; NADH- and NADPH-cytochrome c reductase activities were significantly decreased, but the decrease in NADH-cytochrome b5 reductase activity was relatively small. Whereas essentially no incorporation of malonyl-CoA into palmitoyl-CoA was observed with trypsin-treated microsomes, addition of detergent-solubilized cytochrome b5 resulted in a recovery of fatty acid elongation. These results indicate the presence of an electron transport system, NADH-NADH-cytochrome b5 reductase-cytochrome b5-fatty acid elongation, in brain microsomes.
4-Chloromercuribenzenesulfonate↗
Characteristics of b-type cytochromes in brain microsomes: comparison with liver microsomes.
Biochemical aspects of b-type cytochromes in swine cerebral microsomes were different from those of cytochrome b5 in liver microsomes, as well as the difference in absorption spectra. First, the kinetic constants, Km and Vmax, in rotenone-insensitive NADH-cytochrome c reductase activity were different from those of liver microsomes, and the activity of cerebral microsomes was higher than that of liver microsomes. Second, midpoint potentials (Em) of b-type cytochromes in cerebral microsomes were measured and compared with liver microsomal cytochrome b5. In cerebral microsomes two components of b-type cytochromes were resolved, and showed Em's of -30 and +50 mV, respectively, in the presence of 2 mM KCN. On the other hand, the Em of liver microsomal cytochrome b5 was -6 mV. The high-potential component of cerebral microsomal b-type cytochromes was identified as brain-b'5 [S. Yoshida, T. Yubisui, and M. Takeshita (1983) Biochem. Int. 7, 291-298] and the low-potential component as brain-b5. The significance of the difference between cerebral and liver microsomal b-type cytochromes was discussed.
Animals↗
Amino acid sequence of NADH-cytochrome b5 reductase of human erythrocytes.
The amino acid sequence of soluble NADH-cytochrome b5 reductase purified from normal human erythrocytes was determined as one approach to understand the hereditary disease of a deficiency of this enzyme. The protein is hydrophilic as a whole, but two regions, from Phe-36 to Ile-71 and from Met-231 to Phe-275, were found to be highly hydrophobic. The sequence of the latter region is particularly unique, and rich in proline (20%). The sequence of the amino-terminal region was very similar to the partial sequences of the corresponding regions of the enzymes from pig and steer liver microsomes.
Amino Acid Sequence↗
Circular dichroism spectra of NADH-cytochrome b5 reductase purified from rabbit erythrocytes.
Soluble oxidized NADH-cytochrome b5 reductase from rabbit erythrocytes showed characteristic negative CD bands at 285 and 460-490 nm and positive CD bands at 310 and 370-390 nm. By the anaerobic reduction of the enzyme with NADH, the sign of the CD spectrum was reversed. The CD spectrum of the NADPH-reduced enzyme was different from that of the NADH-reduced one and was closely similar to that of the dithionite-reduced one. Modifications of cysteine or tyrosine residues in the enzyme were shown to cause no release of flavin or to weaken the binding of flavin, as compared to the immediate release of flavin with HC1 or NaOH.
Animals↗
Microsomal electron-transport reductase activities and fatty acid elongation in rat brain. Developmental changes, regional distribution and comparison with liver activity.
Gestational and postnatal changes of microsomal NADH:cytochrome b5 reductase and NADPH:cytochrome c reductase activities were examined in rat brain. The specific activity of NADH:cytochrome b5 reductase was high at 18-19 days of gestational age, decreased to a minimum at 4 to 6 days after birth and increased thereafter. An essentially similar developmental pattern was observed for the specific activity of NADPH:cytochrome c reductase. In contrast, the specific activities of these reductases in liver microsomes were low, did not display a peak during gestation and increased steadily to a maximum at 40-50 days after birth. The rate of incorporation of [2-14C]malonyl-CoA into palmitoyl-CoA in brain microsomes was found to be high in the foetus, sharply decreased to a minimum at the time of birth and increased thereafter. The activity of fatty acid elongation in liver microsomes was much less than that in brain during gestation and increased rapidly after birth to values at 50-60 days 20-fold greater than the foetal activity. NADH and NADPH were equally effective for brain microsomal fatty acid elongation. Regional distribution of cytochrome reductase activities and the activity of fatty acid elongation showed the lowest specific activity in cerebellum. These results suggest that brain microsomal electron transport may be correlated with the developmental alteration in fatty acid elongation.
Animals↗
The heterogeneity of arylhydrocarbon hydroxylase in fetal liver microsomes of rats.
The characteristic of arylhydrocarbon hydroxylase system in fetal liver microsomes of rat was investigated. NADH-synergistic effect on NADPH-dependent arylhydrocarbon hydroxylase was observed in fetal liver microsomes of rat but not in maternal liver microsomes. NADH-synergistic effect decreased in parallel with the decrease of the ratio of cytochrome b5/cytochrome P-450 in liver microsomes. The cytochrome P-450 in arylhydrocarbon hydroxylase system in fetal liver microsomes of rat seemed to be different from that in offspring liver microsomes in respect of its dependency on cytochrome b5 system for its maximum activity.
4-Chloromercuribenzenesulfonate↗
Exponential decay of cytochrome b5 and cytochrome b5 reductase during senescence of erythrocytes: relation to the increased methemoglobin content.
Human erythrocytes were divided into age groups according to their density using phthalate esters as separating liquids. The concentration of cytochrome b5 and the activity of NADH-cytochrome b5 reductase decreased exponentially with the age of red cells. The apparent half-life of cytochrome b5 was estimated to be 44 days. The decline of cytochrome b5 seemed to be more rapid than the decline in the activities of glutamate-oxaloacetate transaminase and NADH-cytochrome b5 reductase whose apparent half-lives were 210 and 240 days, respectively. A biphasic decline of cytochrome b5 was observed on storage of erythrocytes at 4 degrees C. It was deduced from the kinetic results that the decrease of cytochrome b5 might be involved in the increase of the concentration of methemoglobin in senescent erythrocytes. Cytochrome b5 may be used as an indicator of mean red cell age.
Adult↗
Microsomal NADH-cytochrome b5 reductase of bovine brain: purification and properties.
Bovine brain microsomal NADH-cytochrome b5 (cyt. b5) reductase [EC 1.6.2.2] was solubilized by digestion with lysosomes, and purified 8,500-fold with a 20% recovery by procedures including affinity chromatography on 5'-AMP-Sepharose 4B. The purified enzyme showed one band of a molecular weight of 31,000 on polyacrylamide gel electrophoresis with sodium dodecyl sulfate (SDS). Polyacrylamide gel electrophoresis of the purified enzyme without SDS revealed a major band with a faint minor band, both of which exhibited NADH-cyt. b5 reductase activity. The isoelectric points of these components were 6.0 (major) and 6.3 (minor). The apparent Km values of the purified enzyme for NADH and ferricyanide were 1.1 and 4.2 microM, respectively. The apparent Km value for cyt. b5 was 14.3 microM in 10 mM potassium phosphate buffer (pH 7.5). The apparent Vmax value was 1,190 mumol cyt. b5 reduced/min/mg of protein. The NADH-cyt. b5 reductase activity of the purified enzyme was inhibited by sulfhydryl inhibitors and flavin analogues. Inhibition by phosphate buffer or other inorganic salts of the enzyme activity of the purified enzyme was proved to be of the competitive type. These properties were similar to those of NADH-cyt. b5 reductase from bovine liver microsomes or rabbit erythrocytes, although the estimated enzyme content in brain was about one-twentieth of that in liver (per g wet tissue). An immunochemical study using an antibody to purified NADH-cyt. b5 reductase bovine liver microsomes indicated that NADH-cyt. b5 reductase from brain microsomes is immunologically identical to the liver microsomal enzyme.
Ammonium Sulfate↗
Presence of three B-type cytochromes in swine cerebral microsomes.
In swine cerebral microsomes purified with sucrose density gradient and glycerol-cholate gradient centrifugations, it was observed that a new b-type cytochrome which had alpha-peak at 560 nm and Soret peak at 428 nm at 23 degrees C was reduced preferentially by anaerobic NADPH in the presence of cyanide. The b5-type cytochromes were reduced completely by both NADH and NADPH anaerobically. Three b-type cytochromes were partially purified into two b-type, spectroscopically distinct from each other, and the new b-type (b560-5) cytochromes.
Animals↗
Purification and properties of soluble NADH-cytochrome b5 reductase of rabbit erythrocytes.
Soluble NADH-cytochrome b5 reductase was purified from rabbit erythrocytes to homogeneity by simple procedures developed in this study including fractionation with ammonium sulfate, gel filtration on a Sephadex G-75 column, and affinity chromatography on a 5'-AMP-Sepharose 4B column. The enzyme was purified about 12,000-fold from hemolysate in terms of NADH-cytochrome b5 reductase activity with a high yield of 40%. The purified enzyme has absorption maxima at 273, 390, and 462 nm, and shoulders at 370, 435, and 488 nm. The ratio of the absorbance at 273 nm to that at 462 nm of the purified enzyme was 5.6-5.8. The prosthetic group of the enzyme was found to be FAD, and the flavin content in the enzyme was 1 mol/mol of the enzyme. The molecular weight of the purified enzyme was estimated to be 33,000 and 32,000 by gel filtration on a Sephadex G-75 column, and by electrophoresis on polyacrylamide gel in the presence of sodium dodecyl sulfate, respectively. The NADH-cytochrome b5 reductase activity decreased strikingly as the buffer or salt concentration in the assay mixture was increased, and the optimal pH for the reduction of cytochrome b5 with NADH was determined to be 6.6 in Tris-maleate buffer of constant ionic strength. The maximum velocity of NADH-cytochrome b5 reductase activity of the purified enzyme was very high, 1,280 mumol/min/mg of protein in 10 mM phosphate buffer (pH 6.6). The Michaelis constants for NADH and cytochrome b5 were determined to be 2.5 and 4 microM, respectively. The reduction of cytochrome b5 with NADH by the enzyme was suggested to follow the ordered-type reaction mechanism based on the modes of product inhibition. From these results, and also from the estimated enzyme content in the erythrocytes (16-20 mg protein per liter of packed erythrocytes), the possible contribution of the enzyme to functions other than methemoglobin reduction in rabbit erythrocytes is discussed.
Animals↗