Partial purification and characterization of a diurnally fluctuating novel endoamylase from Arabidopsis thaliana leaves.

摘要

Zymograms obtained from samples of A. thaliana cv. Columbia grown under a 12 h light/12 h dark cycle showed that the onset of darkness induced a unique amylolytic enzyme in leaves, the activity of which was at its highest after about 90 min of darkness, and then rapidly disappeared from the soluble protein fraction. The peak of activity coincided with a decrease in starch content of the leaf. The dark-induced amylase was separated from other, non-inducible hydrolases, using polyethylene glycol precipitation, DE-52 ion exchange, Bio-Gel A-0.5 M gel filtration, and amylose affinity chromatography. The partially purified enzyme was characterized using the release of Remazol brilliant blue dye from starch azure, hydrolysis of beta-limit dextrin and periodated amylose, and the results obtained indicate that it is an alpha-amylase (endoamylase). Activity was lost by dialysis of the enzyme preparation against buffers containing EDTA or EGTA, and calcium protected from this loss of activity. This amylasediffers from other amylolytic enzymes in that it has a relatively high pH optimum for activity (pH 7.0). The possible role of this diurnally fluctuating endoamylase in the leaf starch metabolism is discussed.