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Metabolism and Enzymology

Purification of a -Amylase that Accumulates in Arabidopsis thaliana Mutants Defective in Starch Metabolism ¹

Department of Biochemistry, Michigan State University, East Lansing, Michigan 48824

Amylase activity is elevated 5- to 10-fold in leaves of several different Arabidopsis thaliana mutants defective in starch metabolism when they are grown under a 12-hour photoperiod. Activity is also increased when plants are grown under higher light intensity. It was previously determined that the elevated activity was an extrachloroplastic -(exo)amylase. Due to the location of this enzyme outside the chloroplast, its function is not known. The enzyme was purified to homogeneity from leaves of both a starchless mutant deficient in plastid phosphoglucomutase and from the wild type using polyethylene glycol fractionation and cyclohexaamylose affinity chromatography. The molecular mass of the -amylase from both sources was 55,000 daltons as determined by denaturing gel electrophoresis. Gel filtration studies indicated that the enzyme was a monomer. The specific activities of the purified protein from mutant and wild-type sources, their substrate specificities, and K_m for amylopectin were identical. Based on these results it was concluded that the mutant contained an increased level of -amylase protein. Enzyme neutralization studies using a polyclonal antiserum raised to purified -amylase showed that in each of two starchless mutants, one starch deficient mutant and one starch overproducing mutant, the elevated amylase activity was due to elevated -amylase protein.

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