Characterization of α-Amylase from Shoots and Cotyledons of Pea (Pisum sativum L.) Seedlings

Abstract

The most abundant a-amylase (EC 3.2.1.1) in shoots and cotyledons from pea (Pisum sativum L.) seedlings was purified 6700-and 850-fold, respectively, utilizing affinity (amylose and cycloheptaamylose) and gel filtration chromatography and ultrafiltration. This a-amylase contributed at least 79 and 15% of the total amylolytic activity in seedling cotyledons and shoots, respectively. The enzyme was identified as an a-amylase by polarimetry, substrate specificity, and end product analyses. The purified aamylases from shoots and cotyledons appear identical. Both are 43.5 kilodalton monomers with pls of 4.5, broad pH activity optima from 5.5 to 6.5, and nearly identical substrate specificities. They produce identical one-dimensional peptide fingerprints following partial proteolysis in the presence of SDS. Calcium is required for activity and thermal stability of this amylase. The enzyme cannot attack maltodextrins with degrees of polymerization below that of maltotetraose, and hydrolysis of intact starch granules was detected only after prolonged incubation. It best utilizes soluble starch as substrate. Glucose and maltose are the major end products of the enzyme with amylose as substrate. This a-amylase appears to be secreted, in that it is at least partially localized in the apoplast of shoots. The native enzyme exhibits a high degree of resistance to degradation by proteinase K, trypsin/ chymostrypsin, thermolysin, and Staphylococcus aureus V8 protease. It does not appear to be a high-mannose-type glycoprotein.Common cell wall constituents (e.g. fi-glucan) are not substrates of the enzyme. A very low amount of this a-amylase appears to be associated with chloroplasts; however, it is unclear whether this activity is contamination or a-amylase which is integrally associated with the chloroplast.

Characterization and Subcellular Localization of Debranching Enzyme and Endoamylase from Leaves of Sugar Beet

“…However, higher concentrations were necessary for the enzyme thermostability. The pattern of Ca2' dependence of the enzyme activity and thermostability was similar to that for the endoamylase found in cereal grains and pea leaves (2,4).…”

“…4). This difference may result from the variation in the starch grain or the enzyme structures between two species.…”

Chloroplastic Regulation of Apoplastic α-Amylase Activity in Pea Seedlings

“…In crude enzyme preparations of some species, a-amylase is heat stable in the presence of Ca2 , whereas ,3-amylase is not (3,22). Heating crude extracts ofcontrol or photobleached leaves at 70°C inactivated fl-amylases but not the a2 a-amylase (Fig 7).…”

“…a-Amylase is also primarily extrachloroplastic in other plant species (22,28). To date, no apoplastic substrate for the pea a-amylase has been identified (3). NF (San 9789), a powerful inhibitor of carotenoid synthesis that causes photooxidation of chloroplasts in white light and inhibition of the expression of certain nuclear encoded genes (34,40), was found to enhance greatly the activity of the apoplastic a-amylase in pea, whereas other factors which limit plastid functions other than protein synthesis did not.…”

“…In pea, this enzyme is largely apoplastic (2,3) and, as is pea /3-amylase, is totally or almost totally extrachloroplastic (2,25,43). a-Amylase is also primarily extrachloroplastic in other plant species (22,28).…”