Purification and characterization of glutathione reductase isozymes specific for the state of cold hardiness of red spruce (Picea rubens sarg.)

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Virginia Tech

Isozymes of glutathione reductase (GR) have been purified from red spruce. A major isozyme is present throughout the year, while one isozyme is present only during summer in non-hardened needles. A third isozyme is present only during winter in hardened needles. The isozymes present in non-hardened and hardened needles have been designated GR-INH and GR-IH, respectively. The major GR isoform has been designated GR-2NH or GR-H, depending on whether it was purified from nonhardened or hardened needles.

GR-2NH and GR-2H have been purified to homogeneity, as judged by SDS polyacrylamide gel electrophoresis. GR-lNH and GR-IH showed several contaminating proteins in the final preparations. GR-2NH and GR-2H could each be further separated into five charge isomers by isoelectric focussing, and the relative abundance of these charge isomers differs between preparations from non-hardened and hardened needles. GR-1NH and GR-1H differ from GR-2NH and GR-2H with respect to their kinetic, immunological, and physical characteristics. GR-1NH appears different from GR-1H based on chromatographic and electrophoretic behaviour. However, no differences in the temperature dependence of kinetic parameters between either isozyme have been found. The amino-terminal sequences of GR-1H and GR-2H show a high degree of homology with GR's from other organisms. Oligonucleotides derived from the amino-terminal sequences of GR-1H and GR-2H, or from conserved regions within other GR's have been derived and used to amplify cDNA by the polymerase chain reaction. A 500 base pair cDNA, produced with oligonucleotides expected to be specific for GR-1H has been used to transform E.coli.

The differences between the isozymes are discussed with respect to temperature adaptation of enzyme function in a species that experiences extreme temperature differences during its life cycle.