Skip to main content
NCBI home page
Plant Physiology logoLink to Plant Physiology
. 1984 Oct;76(2):436–441. doi: 10.1104/pp.76.2.436

Separation Procedure and Partial Characterization of Two NAD(P)H Dehydrogenases from Cauliflower Mitochondria

Robert R Klein 1, John J Burke 1
PMCID: PMC1064306  PMID: 16663860

Abstract

A procedure was developed to separate and partially purify two NAD(P)H dehydrogenases from the inner membrane of cauliflower (Brassica oleracea L.) mitochondria. The procedure used Triton X-100 extraction followed by (NH4)2SO4 precipitation and gel filtration (Sepharose G-200 column) chromatography. The first dehydrogenase fraction (which eluted in the column void volume) was specific for NADH, was stimulated by KCl addition, and was inhibited by acidic pH, sulfhydryl reagents, and elevated temperature. This fraction contained two major polypeptides with molecular weights of about 57,600 and 32,600 daltons. The fraction exhibited electron paramagnetic resonance (EPR) signals associated with a reduced (ferredoxin-type) iron-sulfur center.

A second dehydrogenase fraction was eluted from the column after removal of the first dehydrogenase. This fraction oxidized NADH and NADPH, was stable at high temperatures, and had a broad pH optima that ranged from 6.0 to 7.8. Although it was relatively insensitive to additions of monovalent and divalent cations, its activity was sensitive to incubation with sulfhydryl reagents. The second dehydrogenase fraction contained five major polypeptides and lacked the iron-sulfur protein EPR signals shown by the first dehydrogenase fraction.

The dehydrogenase fractions represent three potential sites of entry to mitochondrial electron transport; two sites for NADH and a third site for NADPH.

Full text

PDF
436

Selected References

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

  1. Arron G. P., Edwards G. E. Oxidation of Reduced Nicotinamide Adenine Dinucleotide Phosphate by Potato Mitochondria: INHIBITION BY SULFHYDRYL REAGENTS. Plant Physiol. 1980 Apr;65(4):591–594. doi: 10.1104/pp.65.4.591. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bergsma J., Van Dongen M. B., Konings W. N. Purification and characterization of NADH dehydrogenase from Bacillus subtilis. Eur J Biochem. 1982 Nov;128(1):151–157. doi: 10.1111/j.1432-1033.1982.tb06945.x. [DOI] [PubMed] [Google Scholar]
  3. Burke J. J., Siedow J. N., Moreland D. E. Succinate dehydrogenase : a partial purification from mung bean hypocotyls and soybean cotyledons. Plant Physiol. 1982 Dec;70(6):1577–1581. doi: 10.1104/pp.70.6.1577. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Day D. A., Wiskich J. T. Isolation and properties of the outer membrane of plant mitochondria. Arch Biochem Biophys. 1975 Nov;171(1):117–123. doi: 10.1016/0003-9861(75)90014-4. [DOI] [PubMed] [Google Scholar]
  5. Douce R., Mannella C. A., Bonner W. D., Jr The external NADH dehydrogenases of intact plant mitochondria. Biochim Biophys Acta. 1973 Jan 18;292(1):105–116. doi: 10.1016/0005-2728(73)90255-7. [DOI] [PubMed] [Google Scholar]
  6. Hatefi Y. Preparation and properties of NADH: ubiquinone oxidoreductase (complexI), EC 1.6.5.3. Methods Enzymol. 1978;53:11–14. doi: 10.1016/s0076-6879(78)53006-1. [DOI] [PubMed] [Google Scholar]
  7. Huang P. C., Pharo R. L. A simple method for the purification of the mitochondrial NADH dehydrogenase. Biochim Biophys Acta. 1971 Aug 6;245(1):240–244. doi: 10.1016/0005-2728(71)90029-6. [DOI] [PubMed] [Google Scholar]
  8. Ikuma H., Bonner W. D. Properties of Higher Plant Mitochondria. I. Isolation and Some Characteristics of Tightly-coupled Mitochondria from Dark-grown Mung Bean Hypocotyls. Plant Physiol. 1967 Jan;42(1):67–75. doi: 10.1104/pp.42.1.67. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Imagawa T., Nakamura T. Properties and kinetics of salt activation of a membrane-bound NADH dehydrogenase from a marine bacterium Photobacterium phosphoreum. J Biochem. 1978 Sep;84(3):547–557. doi: 10.1093/oxfordjournals.jbchem.a132159. [DOI] [PubMed] [Google Scholar]
  10. Jackson C., Dench J. E., Hall D. O., Moore A. L. Separation of mitochondria from contaminating subcellular structures utilizing silica sol gradient centrifugation. Plant Physiol. 1979 Jul;64(1):150–153. doi: 10.1104/pp.64.1.150. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Koeppe D. E., Miller R. J. Oxidation of reduced nicotinamide adenine dinucleotide phosphate by isolated corn mitochondria. Plant Physiol. 1972 Mar;49(3):353–357. doi: 10.1104/pp.49.3.353. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. 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]
  13. Mains I., Power D. M., Thomas E. W., Buswell J. A. Purification of an NADH-(dichlorophenol-indophenol) oxidoreductase from Bacillus stearothermophilus. Biochem J. 1980 Nov 1;191(2):457–465. doi: 10.1042/bj1910457. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Møller I. M., Palmer J. M. Charge screening by cations affects the conformation of the mitochondrial inner membrane. A study of exogenous MAD(P)H oxidation in plant mitochondria. Biochem J. 1981 Jun 1;195(3):583–588. doi: 10.1042/bj1950583. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Møller I. M., Schwitzguébel J. P., Palmer J. M. Binding and screening by cations and the effect on exogenous NAD(P)H oxidation in Neurospora crassa mitochondria. Eur J Biochem. 1982 Mar;123(1):81–88. doi: 10.1111/j.1432-1033.1982.tb06501.x. [DOI] [PubMed] [Google Scholar]
  16. Ragan C. I. NADH-ubiquinone oxidoreductase. Biochim Biophys Acta. 1976 Nov 30;456(3-4):249–290. doi: 10.1016/0304-4173(76)90001-x. [DOI] [PubMed] [Google Scholar]
  17. Ruzicka F. J., Crane F. L. Quinone interaction with the respiratory chain-linked NADH dehydrogenase of beef heart mitochondria. II. Duroquinone reductase activity. Biochim Biophys Acta. 1971 Mar 2;226(2):221–233. doi: 10.1016/0005-2728(71)90089-2. [DOI] [PubMed] [Google Scholar]
  18. Tottmar S. O., Ragan C. I. The purification and properties of the respiratory-chain reduced nicotinamide--adenine dinucleotide dehydrogenase of Torulopsis utilis. Biochem J. 1971 Oct;124(5):853–865. doi: 10.1042/bj1240853. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Watrud L. S., Baldwin J. K., Miller R. J., Koeppe D. E. Induction of a Sensitive Response to Helminthosporium maydis Race T Toxin in Resistant Mitochondria of Corn (Zea mays L.) by Removal of the Outer Mitochondrial Membrane. Plant Physiol. 1975 Aug;56(2):216–221. doi: 10.1104/pp.56.2.216. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Plant Physiology are provided here courtesy of Oxford University Press

RESOURCES