Partial Purification and Characterization of the Major Endoamylase of Mature Pea Leaves

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Abstract
An endoamylase from leaves of pea (Piseum sativum) was purified to near homogeneity by affinity chromatography and ultrafiltration with a yield of about 20%. The purified protein had a specific activity of 686 to 1300 units per milligram protein. Molecular weights of 45 and 41 kilodaltons were determined by SDS-PAGE and molecular sieve chromatography, respectively. The purified protein exhibited an action pattern commensurate with that of an endoamylase and exhibited properties indicating it to be very similar to cereal grain .alpha.-amylases (calcium requirement, stability to heat, lability to low pH-values, insensitivity to sulfhydryl reagents). Leaf fractionation studies indicated that the enzyme was not primarily located in assimilatory mesophyll cells. Chloroplasts isolated from the leaves were found to contain endoamylases, but their activities represented only a small proportion of the totl amylolytic potential of the leaf and reflected for the most part properties quite different from those exhibited by the purified enzyme.