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. 1978 Aug;62(2):197–203. doi: 10.1104/pp.62.2.197

NADH-Nitrate Reductase Inhibitor from Soybean Leaves 1

S Omata Jolly 1,2, N E Tolbert 1
PMCID: PMC1092089  PMID: 16660485

Abstract

A NADH-nitrate reductase inhibitor has been isolated from young soybean (Glycine max L. Merr. Var. Amsoy) leaves that had been in the dark for 54 hours. The presence of the inhibitor was first suggested by the absence of nitrate reductase activity in the homogenate until the inhibitor was removed by diethylaminoethyl (DEAE)-cellulose chromatography. The inhibitor inactivated the enzyme in homogenates of leaves harvested in the light. Nitrate reductases in single whole cells isolated through a sucrose gradient were equally active from leaves grown in light or darkness, but were inhibited by addition of the active inhibitor.

The NADH-nitrate reductase inhibitor was purified 2,500-fold to an electrophoretic homogeneous protein by a procedure involving DEAE- cellulose chromatography, Sephadex G-100 filtration, and ammonium sulfate precipitation followed by dialysis. The assay was based on nitrate reductase inhibition. A rapid partial isolation procedure was also developed to separate nitrate reductase from the inhibitor by DEAE-cellulose chromatography and elution with KNO3. The inhibitor was a heat-labile protein of about 31,000 molecular weight with two identical subunits. After electrophoresis on polyacrylamide gel two adjacent bands of protein were present; an active form and an inactive form that developed on standing. The active factor inhibited leaf NADH-nitrate reductase but not NADPH-nitrate reductase, the bacterial nitrate reductase or other enzymes tested. The site of inhibition was probably at the reduced flavin adenine dinucleotide-NR reaction, since it did not block the partial reaction of NADH-cytochrome c reductase. The inhibitor did not appear to be a protease. Some form of association of the active inhibitor with nitrate reductase was indicated by a change of inhibitor mobility through Sephadex G-75 in the presence of the enzyme. The inhibition of nitrate reductase was noncompetitive with nitrate but caused a decrease in Vmax.

The isolated inhibitor was inactivated in the light, but after 24 hours in the dark full inhibitory activity returned. Equal amounts of inhibitor were present in leaves harvested from light or darkness, except that the inhibitor was at first inactive when rapidly isolated from leaves in light. Photoinactivation of yellow impure inhibitor required no additional components, but inactivation of the purified colorless inhibitor required the addition of flavin.

Preliminary evidence and a procedure are given for partial isolation of a component by DEAE-cellulose chromatography that stimulated nitrate reductase. The data suggest that light-dark changes in nitrate reductase activity are regulated by specific protein inhibitors and stimulators.

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Selected References

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

  1. Beevers L., Schrader L. E., Flesher D., Hageman R. H. The Role of Light and Nitrate in the Induction of Nitrate Reductase in Radish Cotyledons and Maize Seedlings. Plant Physiol. 1965 Jul;40(4):691–698. doi: 10.1104/pp.40.4.691. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. DAVIS B. J. DISC ELECTROPHORESIS. II. METHOD AND APPLICATION TO HUMAN SERUM PROTEINS. Ann N Y Acad Sci. 1964 Dec 28;121:404–427. doi: 10.1111/j.1749-6632.1964.tb14213.x. [DOI] [PubMed] [Google Scholar]
  3. Dalling M. J., Tolbert N. E., Hageman R. H. Intracellular location of nitrate reductase and nitrite reductase. I. Spinach and tobacco leaves. Biochim Biophys Acta. 1972 Dec 14;283(3):505–512. doi: 10.1016/0005-2728(72)90266-6. [DOI] [PubMed] [Google Scholar]
  4. Garrett R. H., Nason A. Involvement of a B-type cytochrome in the assimilatory nitrate reductase of Neurospora crassa. Proc Natl Acad Sci U S A. 1967 Oct;58(4):1603–1610. doi: 10.1073/pnas.58.4.1603. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. HARTLEY B. S. Proteolytic enzymes. Annu Rev Biochem. 1960;29:45–72. doi: 10.1146/annurev.bi.29.070160.000401. [DOI] [PubMed] [Google Scholar]
  6. Ingle J. Nucleic acid and protein synthesis associated with the induction of nitrate reductase activity in radish cotyledons. Biochem J. 1968 Aug;108(5):715–724. doi: 10.1042/bj1080715. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Jolly S. O., Campbell W., Tolbert N. E. NADPH- and NADH-nitrate reductases from soybean leaves. Arch Biochem Biophys. 1976 Jun;174(2):431–439. doi: 10.1016/0003-9861(76)90371-4. [DOI] [PubMed] [Google Scholar]
  8. KILSHEIMER G. S., AXELROD B. Inhibition of prostatic acid phosphatase by alpha-hydroxycarboxylic acids. J Biol Chem. 1957 Aug;227(2):879–890. [PubMed] [Google Scholar]
  9. Kadam S. S., Gandhi A. P., Sawhney S. K., Naik M. S. Inhibitor of nitrate reductase in the roots of rice seedlings and its effect on the enzyme activity in the presence of NADH. Biochim Biophys Acta. 1974 May 20;350(1):162–170. doi: 10.1016/0005-2744(74)90214-9. [DOI] [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. Massey V., Brumby P. E., Komai H. Studies on milk xanthine oxidase. Some spectral and kinetic properties. J Biol Chem. 1969 Apr 10;244(7):1682–1691. [PubMed] [Google Scholar]
  12. Massey V., Palmer G. On the existence of spectrally distinct classes of flavoprotein semiquinones. A new method for the quantitative production of flavoprotein semiquinones. Biochemistry. 1966 Oct;5(10):3181–3189. doi: 10.1021/bi00874a016. [DOI] [PubMed] [Google Scholar]
  13. Schrader L. E., Ritenour G. L., Eilrich G. L., Hageman R. H. Some characteristics of nitrate reductase from higher plants. Plant Physiol. 1968 Jun;43(6):930–940. doi: 10.1104/pp.43.6.930. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Siccardi A. G., Lazdunski A., Shapiro B. M. Interrelationship between membrane protein composition and deoxyribonucleic acid synthesis in Escherichia coli. Biochemistry. 1972 Apr 25;11(9):1573–1582. doi: 10.1021/bi00759a005. [DOI] [PubMed] [Google Scholar]
  15. Travis R. L., Jordan W. R., Huffaker R. C. Evidence for an Inactivating System of Nitrate Reductase in Hordeum vulgare L. during Darkness That Requires Protein Synthesis. Plant Physiol. 1969 Aug;44(8):1150–1156. doi: 10.1104/pp.44.8.1150. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Wallace W. A nitrate reductase inactivating enzyme from the maize root. Plant Physiol. 1973 Sep;52(3):197–201. doi: 10.1104/pp.52.3.197. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Wallace W. Effects of a nitrate reductase inactivating enzyme and NAD(P)H on the nitrate reductase from higher plants and Neurospora. Biochim Biophys Acta. 1975 Feb 19;377(2):239–250. doi: 10.1016/0005-2744(75)90306-x. [DOI] [PubMed] [Google Scholar]
  18. Wallace W. Purification and properties of a nitrate reductase-inactivating enzyme. Biochim Biophys Acta. 1974 Mar 21;341(1):265–276. doi: 10.1016/0005-2744(74)90087-4. [DOI] [PubMed] [Google Scholar]
  19. Wallace W. The distribution and characteristics of nitrate reductase and glutamate dehydrogenase in the maize seedling. Plant Physiol. 1973 Sep;52(3):191–196. doi: 10.1104/pp.52.3.191. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Weber K., Osborn M. The reliability of molecular weight determinations by dodecyl sulfate-polyacrylamide gel electrophoresis. J Biol Chem. 1969 Aug 25;244(16):4406–4412. [PubMed] [Google Scholar]

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