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Metabolism and Enzymology

Purification and Characterization of Pea Epicotyl -Amylase ¹

Department of Agronomy, University of Wisconsin, Madison, Wisconsin 53706-1597, U.S. Department of Agriculture, Agricultural Research Service, Cereal Crops Research Unit, Madison, Wisconsin 53706-1597

The most abundant -amylase (EC 3.2.1.2) in pea (Pisum sativum L.) was purified greater than 880-fold from epicotyls of etiolated germinating seedlings by anion exchange and gel filtration chromatography, glycogen precipitation, and preparative electrophoresis. The electrophoretic mobility and relative abundance of this -amylase are the same as that of an exoamylase previously reported to be primarily vacuolar. The enzyme was determined to be a -amylase by end product analysis and by its inability to hydrolyze -limit dextrin and to release dye from starch azure. Pea -amylase is an approximate 55 to 57 kilodalton monomer with a pl of 4.35, a pH optimum of 6.0 (soluble starch substrate), an Arrhenius energy of activation of 6.28 kilocalories per mole, and a K_m of 1.67 milligrams per milliliter (soluble starch). The enzyme is strongly inhibited by heavy metals, p-chloromer-curiphenylsulfonic acid and N-ethylmaleimide, but much less strongly by iodoacetamide and iodoacetic acid, indicating cysteinyl sulfhydryls are not directly involved in catalysis. Pea -amylase is competitively inhibited by its end product, maltose, with a K_i of 11.5 millimolar. The enzyme is partially inhibited by Schardinger maltodextrins, with -cyclohexaamylose being a stronger inhibitor than -cycloheptaamylose. Moderately branched glucans (e.g. amylopectin) were better substrates for pea -amylase than less branched or non-branched (amyloses) or highly branched (glycogens) glucans. The enzyme failed to hydrolyze native starch grains from pea and glucans smaller than maltotetraose. The mechanism of pea -amylase is the multichain type. Possible roles of pea -amylase in cellular glucan metabolism are discussed.

¹ This material was supported by the Cooperative State Research Service, U.S. Department of Agriculture, under agreement No. 87-CRCR-1-2324; the College of Agriculture and Life Sciences, University of Wisconsin-Madison; and the USDA Agricultural Research Service.

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