Skip to main content
NCBI home page
Biochemical Journal logoLink to Biochemical Journal
. 1996 Mar 1;314(Pt 2):587–593. doi: 10.1042/bj3140587

Purification and characterization of a plasma membrane ferricyanide-utilizing NADH dehydrogenase from Ehrlich tumour cells.

A del Castillo-Olivares 1, M A Medina 1, I Núñez de Castro 1, J Márquez 1
PMCID: PMC1217089  PMID: 8670074

Abstract

A ferricyanide-utilizing NADH dehydrogenase (NADH-ferricyanide oxidoreductase) from the plasma membrane of Ehrlich ascites tumour cells has been purified about 1500-fold to apparent homogeneity. The method comprises the isolation of an enriched plasma membrane fraction, solubilization with Triton X-100, ion-exchange chromatography, ammonium sulphate precipitation, Cibacron Blue chromatography and fast-protein liquid chromatography with a Superose-6 gel filtration column. The specific activity of the final pool was more than 61 units/mg protein. The pure enzyme examined by SDS/PAGE displayed only one type of subunit with an apparent molecular mass of 32.0 kDa. The molecular mass of the native protein (117.0 kDa) was estimated by gel filtration; these results suggest a protein composed of four subunits of identical molecular mass. The enzyme was stable in the pH interval between 6 and 9, with maximum activity at pH values from 7.5 to 8.5. The purified enzyme showed Michaelis-Menten kinetics for the substrates, with apparent K(m) values of 4.3 X 10(-5) M and 6.7 X 10(-5) M for NADH and ferricyanide respectively. The isolated protein was strongly inhibited by Zn2+ and the thio-specific reagents mersalyl and p-chloromercuribenzenesulphonic acid.

Full Text

The Full Text of this article is available as a PDF (439.9 KB).

Selected References

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

  1. Barr R., Sandelius A. S., Crane F. L., Morré D. J. Redox reactions of tonoplast and plasma membranes isolated from soybean hypocotyls by free-flow electrophoresis. Biochim Biophys Acta. 1986 Dec 3;852(2-3):254–261. doi: 10.1016/0005-2728(86)90230-6. [DOI] [PubMed] [Google Scholar]
  2. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  3. CHANCE B., HESS B. Metabolic control mechanisms. I. Electron transfer in the mammalian cell. J Biol Chem. 1959 Sep;234:2404–2412. [PubMed] [Google Scholar]
  4. Crane F. L., Sun I. L., Barr R., Löw H. Electron and proton transport across the plasma membrane. J Bioenerg Biomembr. 1991 Oct;23(5):773–803. doi: 10.1007/BF00786001. [DOI] [PubMed] [Google Scholar]
  5. Crane F. L., Sun I. L., Clark M. G., Grebing C., Löw H. Transplasma-membrane redox systems in growth and development. Biochim Biophys Acta. 1985 Aug 1;811(3):233–264. doi: 10.1016/0304-4173(85)90013-8. [DOI] [PubMed] [Google Scholar]
  6. Del Castillo-Olivares A., Márquez J., Núez De Castro I., Medina M. A. Characterization of plasma membrane redox activity from Ehrlich cells. Cell Biochem Funct. 1994 Jun;12(2):149–152. doi: 10.1002/cbf.290120211. [DOI] [PubMed] [Google Scholar]
  7. Esmann M. ATPase and phosphatase activity of Na+,K+-ATPase: molar and specific activity, protein determination. Methods Enzymol. 1988;156:105–115. doi: 10.1016/0076-6879(88)56013-5. [DOI] [PubMed] [Google Scholar]
  8. Guerrini F., Valenti V., Pupillo P. Solubilization and Purification of NAD(P)H Dehydrogenase of Cucurbita Microsomes. Plant Physiol. 1987 Nov;85(3):828–834. doi: 10.1104/pp.85.3.828. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Hayashi M., Miyoshi T., Takashina S., Unemoto T. Purification of NADH-ferricyanide dehydrogenase and NADH-quinone reductase from Escherichia coli membranes and their roles in the respiratory chain. Biochim Biophys Acta. 1989 Oct 26;977(1):62–69. doi: 10.1016/s0005-2728(89)80009-x. [DOI] [PubMed] [Google Scholar]
  10. Henderson L. M., Chappell J. B., Jones O. T. Internal pH changes associated with the activity of NADPH oxidase of human neutrophils. Further evidence for the presence of an H+ conducting channel. Biochem J. 1988 Apr 15;251(2):563–567. doi: 10.1042/bj2510563. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Hjelmeland L. M., Chrambach A. Solubilization of functional membrane proteins. Methods Enzymol. 1984;104:305–318. doi: 10.1016/s0076-6879(84)04097-0. [DOI] [PubMed] [Google Scholar]
  12. Im W. B., Spector A. A. Sodium-dependent neutral amino acid transport in native and reconstituted membrane vesicles from Ehrlich cells. J Biol Chem. 1980 Jan 25;255(2):764–770. [PubMed] [Google Scholar]
  13. Kilberg M. S., Christensen H. N. Electron-transferring enzymes in the plasma membrane of the Ehrlich ascites tumor cell. Biochemistry. 1979 Apr 17;18(8):1525–1530. doi: 10.1021/bi00575a021. [DOI] [PubMed] [Google Scholar]
  14. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  15. Laporte F., Doussiere J., Mechin V., Vignais P. V. NADPH-cytochrome c reductase from rabbit peritoneal neutrophils. Purification, properties and function in the respiratory burst. Eur J Biochem. 1991 Feb 26;196(1):59–66. doi: 10.1111/j.1432-1033.1991.tb15785.x. [DOI] [PubMed] [Google Scholar]
  16. Le Maire M., Aggerbeck L. P., Monteilhet C., Andersen J. P., Møller J. V. The use of high-performance liquid chromatography for the determination of size and molecular weight of proteins: a caution and a list of membrane proteins suitable as standards. Anal Biochem. 1986 May 1;154(2):525–535. doi: 10.1016/0003-2697(86)90025-4. [DOI] [PubMed] [Google Scholar]
  17. Luque P., Márquez J., Núez de Castro I., Medina M. A. Sodium-dependent L-serine transport in plasma membrane vesicles isolated from Ehrlich cells by two-phase compartmentation. J Membr Biol. 1991 Sep;123(3):247–254. doi: 10.1007/BF01870407. [DOI] [PubMed] [Google Scholar]
  18. Luster D. G., Buckhout T. J. Purification and Identification of a Plasma Membrane Associated Electron Transport Protein from Maize (Zea mays L.) Roots. Plant Physiol. 1989 Nov;91(3):1014–1019. doi: 10.1104/pp.91.3.1014. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Markwell M. A., Haas S. M., Tolbert N. E., Bieber L. L. Protein determination in membrane and lipoprotein samples: manual and automated procedures. Methods Enzymol. 1981;72:296–303. doi: 10.1016/s0076-6879(81)72018-4. [DOI] [PubMed] [Google Scholar]
  20. Medina M. A., Núez de Castro I. Functional reconstitution of Ehrlich cell plasma membrane ferricyanide reductase. Biochem Biophys Res Commun. 1994 Dec 15;205(2):1109–1113. doi: 10.1006/bbrc.1994.2780. [DOI] [PubMed] [Google Scholar]
  21. Medina M. A., del Castillo-Olivares A., Márquez J., Núez de Castro I. Involvement of essential histidine residue(s) in the activity of Ehrlich cell plasma membrane NADH-ferricyanide oxidoreductase. Biochim Biophys Acta. 1994 Feb 23;1190(1):20–24. doi: 10.1016/0005-2736(94)90030-2. [DOI] [PubMed] [Google Scholar]
  22. Medina M. A., del Castillo-Olivares A., Schweigerer L. Plasma membrane redox activity correlates with N-myc expression in neuroblastoma cells. FEBS Lett. 1992 Oct 19;311(2):99–101. doi: 10.1016/0014-5793(92)81376-w. [DOI] [PubMed] [Google Scholar]
  23. Moreadith R. W., Fiskum G. Isolation of mitochondria from ascites tumor cells permeabilized with digitonin. Anal Biochem. 1984 Mar;137(2):360–367. doi: 10.1016/0003-2697(84)90098-8. [DOI] [PubMed] [Google Scholar]
  24. Márquez J., Sánchez-Jiménez F., Medina M. A., Quesada A. R., Núez de Castro I. Nitrogen metabolism in tumor bearing mice. Arch Biochem Biophys. 1989 Feb 1;268(2):667–675. doi: 10.1016/0003-9861(89)90335-4. [DOI] [PubMed] [Google Scholar]
  25. Nisimoto Y., Otsuka-Murakami H., Iwata S. NADPH-cytochrome c reductase from human neutrophil membranes: purification, characterization and localization. Biochem J. 1994 Feb 1;297(Pt 3):585–593. doi: 10.1042/bj2970585. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Park H. J., Reiser C. O., Kondruweit S., Erdmann H., Schmid R. D., Sprinzl M. Purification and characterization of a NADH oxidase from the thermophile Thermus thermophilus HB8. Eur J Biochem. 1992 May 1;205(3):881–885. doi: 10.1111/j.1432-1033.1992.tb16853.x. [DOI] [PubMed] [Google Scholar]
  27. Segura J. A., Aledo J. C., Gómez-Biedma S., Núez de Castro I., Márquez J. Tumor glutaminase purification. Protein Expr Purif. 1995 Jun;6(3):343–351. doi: 10.1006/prep.1995.1045. [DOI] [PubMed] [Google Scholar]
  28. Serrano A., Cordoba F., Gonzalez-Reyes J. A., Navas P., Villalba J. M. Purification and Characterization of Two Distinct NAD(P)H Dehydrogenases from Onion (Allium cepa L.) Root Plasma Membrane. Plant Physiol. 1994 Sep;106(1):87–96. doi: 10.1104/pp.106.1.87. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Villalba J. M., Canalejo A., Burón M. I., Córdoba F., Navas P. Thiol groups are involved in NADH-ascorbate free radical reductase activity of rat liver plasma membrane. Biochem Biophys Res Commun. 1993 Apr 30;192(2):707–713. doi: 10.1006/bbrc.1993.1472. [DOI] [PubMed] [Google Scholar]
  31. Villalba J. M., Navarro F., Córdoba F., Serrano A., Arroyo A., Crane F. L., Navas P. Coenzyme Q reductase from liver plasma membrane: purification and role in trans-plasma-membrane electron transport. Proc Natl Acad Sci U S A. 1995 May 23;92(11):4887–4891. doi: 10.1073/pnas.92.11.4887. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Walsh C. Chemical approaches to the study of enzymes catalyzing redox transformations. Annu Rev Biochem. 1978;47:881–931. doi: 10.1146/annurev.bi.47.070178.004313. [DOI] [PubMed] [Google Scholar]
  33. del Castillo-Olivares A., Márquez J., Núez de Castro I., Medina M. A. Two phases of ferricyanide reductase activity in Ehrlich cell plasma membranes. Z Naturforsch C. 1992 Nov-Dec;47(11-12):929–931. doi: 10.1515/znc-1992-11-1223. [DOI] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES