Lysine-ketoglutarate reductase in normal and opaque-2 endosperm --Marcia R. Brochetto-Braga, Adilson Leite and Paulo Arruda An inbred line, L1038, homozygous normal, and the corresponding mutant L1038, homozygous opaque-2, were used to investigate the activity of lysine-ketoglutarate reductase. The enzyme catalyses the reaction: Lysine + a-ketoglutarate + NADPH to saccharopine + NADP+.

The enzyme showed a typical developmental pattern of activity for both genotypes. The activity increased with the onset of seed development, reached a peak around 20 days after pollination and then decreased towards seed maturity. The enzyme activity of the mutant endosperm was 2 to 3 times lower than the normal endosperm during seed development.

Enzyme extracts from twenty endosperms of each genotype were used for ion-exchange chromatography on DEAE-cellulose columns (Fig. 1). The patterns of enzyme elution were very similar for normal and opaque-2 endosperms, but the enzyme amount of normal endosperm was two times higher than the opaque-2 endosperm. Since we used the same amount of protein, corresponding to the same amount of endosperm for both genotypes, we concluded that the opaque-2 gene reduces the synthesis of the enzyme when in homozygous recessive form.

Figure 1. Elution patterns of enzyme activity and protein from DEAE-cellulose columns: (o___o) lysine-ketoglutarate reductase activity; (___) protein measured by absorbance at 280 nm; (___) potassium chloride concentration gradient.

Polyclonal antibodies raised against the purified enzyme from normal endosperm were used to study the immunological properties of normal and mutant enzymes. Western blot analysis of purified extracts from both genotypes revealed a single common band of 134kDa. Based on these results we concluded that the lysine-ketoglutarate reductase gene is under the transcriptional or translational control of the opaque-2 gene.


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