Isolation of aspartate kinase from Coix lacryma-jobi
--Juverlandi Lugli and Ricardo A. Azevedo

Coix lacryma-jobi, together with maize, Tripsacum and sorghum, belongs to the grass tribe Andropogoneae. This cereal is native to Southeast Asia and has been used as a food source for humans and livestock, in the production of alcoholic beverages, and as a medicinal plant. Seeds of Coix lacryma-jobi contain around 29% protein, the major constituent of which is a prolamin called coixin. Like other cereal prolamins, the coixin polypeptides contain very low levels of lysine and tryptophan (Ottoboni et al., J. Agric. Food Chem. 38:631, 1990).

This cereal is now under biochemical investigation in order to study the biosynthesis of lysine, threonine, methionine and isoleucine (the aspartate family of amino acids).

The enzyme aspartate kinase, which has been isolated and purified in many higher plants, was extracted from Coix endosperms at different stages of development with 50 mM Tris buffer (pH 7.4) containing 200 mM KCl, 2 mM lysine, 2 mM threonine, 1 mM DTT, 0.1 mM EDTA and 15% (v/v) glycerol. Proteins from crude extracts were precipitated with ammonium sulphate (35-60%) and desalted on a Sephadex G50 column equilibrated with 25 mM Tris buffer (pH 7.4) containing 50 mM KCl, 1 mM DTT, 0.1 mM lysine, 0.1 mM threonine and 10% (v/v) glycerol. The desalted sample was applied to a Fast Flow Q Sepharose column equilibrated in the same buffer and eluted "step-wise" with 100 mM, 200 mM, 300 mM, 400 mM and 500 mM KCl in the same buffer. Aspartate kinase activity was determined by the optimized hydroxamate assay (Azevedo et al., Phytochemistry 31:3725, 1992) and protein by Bradford.

Aspartate kinase activity was extracted from 5 g of endosperm from each developmental stage. Stages 1 and 2 presented the highest levels of activity, and in both stages the amino acids threonine and lysine, at a concentration of 5 mM each, partially inhibited the activity of aspartate kinase, showing an additive effect when the amino acids were added together. Stage 2 was selected for further experiments based on the amount of endosperm that can be obtained in comparison to stage 1.

The anion exchange chromatography step showed that aspartate kinase could be eluted with 300 mM KCl, and the peak was also inhibited by threonine and lysine. These results indicated the presence of at least two forms of the enzyme in Coix; one sensitive to threonine inhibition, which corresponds to around 50% of the total activity (the major component) and the other sensitive to lysine (around 30% of the total activity). This result is different from those obtained in other plants, since in the majority the isoenzyme sensitive to lysine represented the major component. In other plants, aspartate kinase activity could be eluted with around 200 mM KCl, which was not enough to elute aspartate kinase from Coix. The peak containing aspartate kinase activity eluted from the Fast Flow Q Sepharose column was concentrated with 70% ammonium sulphate and is being tested in a Sephacryl S200 gel filtration column for molecular weight determination. 


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