Skip to main content
PMC home page
Plant Physiology logoLink to Plant Physiology
. 1994 May;105(1):205–213. doi: 10.1104/pp.105.1.205

Purification and characterization of glutathione reductase isozymes specific for the state of cold hardiness of red spruce.

A Hausladen 1, R G Alscher 1
PMCID: PMC159347  PMID: 8029350

Abstract

Isozymes of glutathione reductase (GR) have been purified from red spruce (Picea rubens Sarg.) needles. Two isozymes could be separated by anion-exchange chromatography from both nonhardened or cold-hardened tissue. Based on chromatographic elution profiles, the isozymes were designated GR-1NH and GR-2NH in preparations from nonhardened needles, and GR-1H and GR-2H in preparations from hardened needles. N-terminal sequencing and immunological data with antisera obtained against GR-1H and GR-2H established that the isozymes from hardened needles are different gene products and show significant structural differences from each other. Chromatographic, electrophoretic, and immunological data revealed only minor differences between GR-2NH and GR-2H, and it is concluded that these isozymes are very similar or identical. Anion-exchange chromatography and native polyacrylamide gel electrophoresis also established that GR-1NH and GR-1H are different proteins. From these data we conclude that GR-1H is a distinct gene product, present only in hardened needles. Therefore, GR-1H can be considered to be a cold-hardiness-specific GR isozyme, and GR-1NH can be considered to be specific for nonhardened needles. It is proposed that GR-1H is a cold-acclimation protein.

Full Text

The Full Text of this article is available as a PDF (2.2 MB).

Selected References

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

  1. Anderson J. V., Chevone B. I., Hess J. L. Seasonal variation in the antioxidant system of eastern white pine needles : evidence for thermal dependence. Plant Physiol. 1992 Feb;98(2):501–508. doi: 10.1104/pp.98.2.501. [DOI] [PMC free article] [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. Connell J. P., Mullet J. E. Pea chloroplast glutathione reductase: purification and characterization. Plant Physiol. 1986 Oct;82(2):351–356. doi: 10.1104/pp.82.2.351. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Creissen G., Edwards E. A., Enard C., Wellburn A., Mullineaux P. Molecular characterization of glutathione reductase cDNAs from pea (Pisum sativum L.). Plant J. 1992 Jan;2(1):129–131. [PubMed] [Google Scholar]
  5. Ermler U., Schulz G. E. The three-dimensional structure of glutathione reductase from Escherichia coli at 3.0 A resolution. Proteins. 1991;9(3):174–179. doi: 10.1002/prot.340090303. [DOI] [PubMed] [Google Scholar]
  6. Hausladen A., Alscher R. G. Cold-hardiness-specific glutathione reductase isozymes in red spruce. Thermal dependence of kinetic parameters and possible regulatory mechanisms. Plant Physiol. 1994 May;105(1):215–223. doi: 10.1104/pp.105.1.215. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Kaplan J. C., Beutler E. Electrophoretic study of glutathione reductase in human erythrocytes and leucocytes. Nature. 1968 Jan 20;217(5125):256–258. doi: 10.1038/217256a0. [DOI] [PubMed] [Google Scholar]
  8. Karplus P. A., Schulz G. E. Refined structure of glutathione reductase at 1.54 A resolution. J Mol Biol. 1987 Jun 5;195(3):701–729. doi: 10.1016/0022-2836(87)90191-4. [DOI] [PubMed] [Google Scholar]
  9. 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]
  10. Matsudaira P. Limited N-terminal sequence analysis. Methods Enzymol. 1990;182:602–613. doi: 10.1016/0076-6879(90)82047-6. [DOI] [PubMed] [Google Scholar]
  11. Mehlhorn H., Cottam D. A., Lucas P. W., Wellburn A. R. Induction of ascorbate peroxidase and glutathione reductase activities by interactions of mixtures of air pollutants. Free Radic Res Commun. 1987;3(1-5):193–197. doi: 10.3109/10715768709069784. [DOI] [PubMed] [Google Scholar]
  12. Richmond R., Halliwell B. Formation of hydroxyl radicals from the paraquat radical cation, demonstrated by a highly specific gas chromatographic technique. the role of superoxide radical anion, hydrogen peroxide, and glutathione reductase. J Inorg Biochem. 1982 Oct;17(2):95–107. doi: 10.1016/s0162-0134(00)80078-1. [DOI] [PubMed] [Google Scholar]
  13. Schmidt A., Kunert K. J. Lipid peroxidation in higher plants : the role of glutathione reductase. Plant Physiol. 1986 Nov;82(3):700–702. doi: 10.1104/pp.82.3.700. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Smith I. K. Stimulation of glutathione synthesis in photorespiring plants by catalase inhibitors. Plant Physiol. 1985 Dec;79(4):1044–1047. doi: 10.1104/pp.79.4.1044. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES