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. 1988 May 1;251(3):711–715. doi: 10.1042/bj2510711

The opposite effect of bivalent cations on cytochrome b5 reduction by NADH:cytochrome b5 reductase and NADPH:cytochrome c reductase.

M Tamura 1, T Yubisui 1, M Takeshita 1
PMCID: PMC1149062  PMID: 3137923

Abstract

The effects of bivalent cations on cytochrome b5 reduction by NADH:cytochrome b5 reductase and NADPH:cytochrome c reductase were studied with the proteinase-solubilized enzymes. Cytochrome b5 reduction by NADH:cytochrome b5 reductase was strongly inhibited by CaCl2 or MgCl2. When 1.2 microM-cytochrome b5 was used, the concentrations of CaCl2 and MgCl2 required for 50% inhibition (I50) were 8 and 18 mM respectively. The inhibition was competitive with respect to cytochrome b5. The extent of inhibition by CaCl2 or MgCl2 was much higher than that by KCl or other alkali halides. In contrast, cytochrome b5 reduction by NADPH:cytochrome c reductase was extremely activated by CaCl2 or MgCl2. In the presence of 5 mM-CaCl2, the activity was 24-fold higher than control when 4.4 microM-cytochrome b5 was used. The magnitude of activation by CaCl2 was 2-3-fold higher than that by MgCl2. The activation by these salts was much higher than that by KCl, indicating that bivalent cations play an important role in this activation. The mechanisms of inhibition and activation by bivalent cations of cytochrome b5 reduction by these two microsomal reductases are discussed.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bilimoria M. H., Kamin H. The effect of high salt concentrations upon cytochrome C, cytochrome B5, and iron-EDTA reductase activities of liver microsomal NADPH-cytochrome C reductase. Ann N Y Acad Sci. 1973;212:428–448. doi: 10.1111/j.1749-6632.1973.tb47612.x. [DOI] [PubMed] [Google Scholar]
  2. Black S. D., Coon M. J. Structural features of liver microsomal NADPH-cytochrome P-450 reductase. Hydrophobic domain, hydrophilic domain, and connecting region. J Biol Chem. 1982 May 25;257(10):5929–5938. [PubMed] [Google Scholar]
  3. Dailey H. A., Strittmatter P. Characterization of the interaction of amphipathic cytochrome b5 with stearyl coenzyme A desaturase and NADPH:cytochrome P-450 reductase. J Biol Chem. 1980 Jun 10;255(11):5184–5189. [PubMed] [Google Scholar]
  4. Dailey H. A., Strittmatter P. Modification and identification of cytochrome b5 carboxyl groups involved in protein-protein interaction with cytochrome b5 reductase. J Biol Chem. 1979 Jun 25;254(12):5388–5396. [PubMed] [Google Scholar]
  5. Enoch H. G., Strittmatter P. Cytochrome b5 reduction by NADPH-cytochrome P-450 reductase. J Biol Chem. 1979 Sep 25;254(18):8976–8981. [PubMed] [Google Scholar]
  6. Haniu M., Iyanagi T., Legesse K., Shively J. E. Structural analysis of NADPH-cytochrome P-450 reductase from porcine hepatic microsomes. Sequences of proteolytic fragments, cysteine-containing peptides, and a NADPH-protected cysteine peptide. J Biol Chem. 1984 Nov 25;259(22):13703–13711. [PubMed] [Google Scholar]
  7. Hildebrandt A., Estabrook R. W. Evidence for the participation of cytochrome b 5 in hepatic microsomal mixed-function oxidation reactions. Arch Biochem Biophys. 1971 Mar;143(1):66–79. doi: 10.1016/0003-9861(71)90186-x. [DOI] [PubMed] [Google Scholar]
  8. Holloway P. W., Wakil S. J. Requirement for reduced diphosphopyridine nucleotide-cytochrome b5 reductase in stearly coenzyme A desaturation. J Biol Chem. 1970 Apr 10;245(7):1862–1865. [PubMed] [Google Scholar]
  9. Keyes S. R., Cinti D. L. Biochemical properties of cytochrome b5-dependent microsomal fatty acid elongation and identification of products. J Biol Chem. 1980 Dec 10;255(23):11357–11364. [PubMed] [Google Scholar]
  10. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  11. Lu A. Y., Levin W., Selander H., Jerina D. M. Liver microsomal electron transport systems. III. The involvement of cytochrome b5 in the NADPH-supported cytochrome P-450-dependent hydroxylation of chlorobenzene. Biochem Biophys Res Commun. 1974 Dec 23;61(4):1348–1355. doi: 10.1016/s0006-291x(74)80432-8. [DOI] [PubMed] [Google Scholar]
  12. Nisimoto Y., Lambeth J. D. NADPH-cytochrome P-450 reductase-cytochrome b5 interactions: crosslinking of the phospholipid vesicle-associated proteins by a water-soluble carbodiimide. Arch Biochem Biophys. 1985 Sep;241(2):386–396. doi: 10.1016/0003-9861(85)90561-2. [DOI] [PubMed] [Google Scholar]
  13. Okayasu T., Ono T., Shinojima K. Involvement of cytochrome b5 in the oxidative desaturation of linoleic acid to gamma-linolenic acid in rat liver microsomes. Lipids. 1977 Mar;12(3):267–271. doi: 10.1007/BF02533345. [DOI] [PubMed] [Google Scholar]
  14. Omura T., Takesue S. A new method for simultaneous purification of cytochrome b5 and NADPH-cytochrome c reductase from rat liver microsomes. J Biochem. 1970 Feb;67(2):249–257. doi: 10.1093/oxfordjournals.jbchem.a129248. [DOI] [PubMed] [Google Scholar]
  15. PHILLIPS A. H., LANGDON R. G. Hepatic triphosphopyridine nucleotide-cytochrome c reductase: isolation, characterization, and kinetic studies. J Biol Chem. 1962 Aug;237:2652–2660. [PubMed] [Google Scholar]
  16. Prough R. A., Masters B. S. The mechanism of cytochrome b5 reduction by NADPH-cytochrome c reductase. Arch Biochem Biophys. 1974 Nov;165(1):263–267. doi: 10.1016/0003-9861(74)90163-5. [DOI] [PubMed] [Google Scholar]
  17. Reddy V. V., Kupfer D., Caspi E. Mechanism of C-5 double bond introduction in the biosynthesis of cholesterol by rat liver microsomes. J Biol Chem. 1977 May 10;252(9):2797–2801. [PubMed] [Google Scholar]
  18. STRITTMATTER P., VELICK S. F. The purification and properties of microsomal cytochrome reductase. J Biol Chem. 1957 Oct;228(2):785–799. [PubMed] [Google Scholar]
  19. Strittmatter P., Spatz L., Corcoran D., Rogers M. J., Setlow B., Redline R. Purification and properties of rat liver microsomal stearyl coenzyme A desaturase. Proc Natl Acad Sci U S A. 1974 Nov;71(11):4565–4569. doi: 10.1073/pnas.71.11.4565. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Sugiyama T., Miki N., Yamano T. The obligatory requirement of cytochrome b5 in the p-nitroanisole O-demethylation reaction catalyzed by cytochrome P-450 with a high affinity for cytochrome b5. Biochem Biophys Res Commun. 1979 Oct 12;90(3):715–720. doi: 10.1016/0006-291x(79)91886-2. [DOI] [PubMed] [Google Scholar]
  21. Takeshita M., Tamura M., Yoshida S., Yubisui T. Palmitoyl-CoA elongation in brain microsomes: dependence on cytochrome b5 and NADH-cytochrome b5 reductase. J Neurochem. 1985 Nov;45(5):1390–1395. doi: 10.1111/j.1471-4159.1985.tb07204.x. [DOI] [PubMed] [Google Scholar]
  22. Takesue S., Omura T. Purification and properties of NADH-cytochrome b5 reductase solubilized by lysosomes from rat liver microsomes. J Biochem. 1970 Feb;67(2):267–276. doi: 10.1093/oxfordjournals.jbchem.a129250. [DOI] [PubMed] [Google Scholar]
  23. Tamburini P. P., MacFarquhar S., Schenkman J. B. Evidence of binary complex formations between cytochrome P-450, cytochrome b5, and NADPH-cytochrome P-450 reductase of hepatic microsomes. Biochem Biophys Res Commun. 1986 Jan 29;134(2):519–526. doi: 10.1016/s0006-291x(86)80451-x. [DOI] [PubMed] [Google Scholar]
  24. Tamburini P. P., Schenkman J. B. Mechanism of interaction between cytochromes P-450 RLM5 and b5: evidence for an electrostatic mechanism involving cytochrome b5 heme propionate groups. Arch Biochem Biophys. 1986 Mar;245(2):512–522. doi: 10.1016/0003-9861(86)90244-4. [DOI] [PubMed] [Google Scholar]
  25. Tamburini P. P., White R. E., Schenkman J. B. Chemical characterization of protein-protein interactions between cytochrome P-450 and cytochrome b5. J Biol Chem. 1985 Apr 10;260(7):4007–4015. [PubMed] [Google Scholar]
  26. Tamura M., Yubisui T., Takeshita M., Kawabata S., Miyata T., Iwanaga S. Structural comparison of bovine erythrocyte, brain, and liver NADH-cytochrome b5 reductase by HPLC mapping. J Biochem. 1987 May;101(5):1147–1159. doi: 10.1093/oxfordjournals.jbchem.a121979. [DOI] [PubMed] [Google Scholar]
  27. Tamura M., Yubisui T., Takeshita M. Microsomal NADH-cytochrome b5 reductase of bovine brain: purification and properties. J Biochem. 1983 Nov;94(5):1547–1555. [PubMed] [Google Scholar]
  28. Tamura M., Yubisui T., Takeshita M. The inhibitory effect of halides and carboxylates on hepatic NADH: cytochrome b5 oxidoreductase. Biochem J. 1985 Aug 15;230(1):273–276. doi: 10.1042/bj2300273. [DOI] [PMC free article] [PubMed] [Google Scholar]

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