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

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.