Multiple forms of carboxylesterases in the green bean (Phaseolus vulgaris L.) and pea (Pisum sativum L.) Public Deposited

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  • Esterase activity of an aqueous extract of the green bean was separated into fourteen bands, while aqueous extracted pea esterases revealed seven bands, by polyacrylamide-gel electrophoresis. The fourteen bands of bean esterase activity formed three groups; slow, intermediate and fast moving. α-Naphthyl acetate, propionate, and n-butyrate and AS naphthol acetate were hydrolyzed at various rates by the bean and pea esterases. No hydrolysis of β-naphthyl laurate was observed, indicating the absence of a lipase in the aqueous extracts of these vegetables. Since all the esterase bands active toward α-naphthyl acetate were inhibited by organophosphorus compounds (diisopropylphosphorofluoridate, diethyl p-nitrophenyl thiophosphate and diethyl p-nitrophenyl phosphate), these esterases were classified as carboxylesterases (carboxylic ester hydrolase, EC 3.1.1.1). To study the carboxylesterases of the green bean in greater detail, a protamine sulfate treated aqueous extract was separated into three fractions (S₁, S₂ and S₃) by chromatography on Sephadex G-100. Subsequent analysis of each fraction by polyacrylamide-gel electrophoresis demonstrated the presence of the slow moving group in fraction S₁, slow and intermediate moving groups in fraction S₂ and the fast moving group in fraction S₃. Hence, these studies suggest that the three groups of esterase activity in beans were dissimilar in molecular size and the relative molecular size was slow > intermediate > fast moving group. Chromatography of fraction S₁ on carboxymethyl (CM) cellulose with sodium chloride linear-gradient elution resulted in three fractions (CM₁, CM₂ and CM₃). Similarly, fraction S₂ yielded three fractions (DE₁, DE₂ and DE₃), while fraction S₃ produced two fractions (DE₄ and DE₅), by chromatography on microgranular diethylaminoethyl (DEAE) cellulose. Polyacrylamide-gel electrophoresis revealed the presence of the first three bands of the slow moving group in CM₁ and only the first two bands in CM₂. DE₁ possessed mainly the first two bands and DE₂ the last two bands of the five bands in the slow moving group. The five bands of the intermediate moving group of esterase activity was found only in fraction DE₃. The first two bands of the four bands of the fast moving group were separated into fraction DE₄, while the last two bands were in DE₅. Nine substrates and various concentrations of three inhibitors were used to characterize some of the fractions obtained from ion-exchange chromatography. Although most of the fractions hydrolyzed the substrates used in this study, each fraction differed to some extent in substrate specificity. Inhibitor studies indicated the presence of a sensitive and a resistant component of esterase activity in each fraction studied. These results suggest that the esterase fractions were composed of two enzymes. To account for the fourteen bands of esterase activity a hypothetical model of polymers consisting of two monomers was proposed. This hypothesized model suggests that the slow moving group contained six pentamers, the intermediate group five tetramers and the fast moving group four trimers. Most characteristics of the carboxylesterases of beans observed in these studies could be explained on the basis of the hypothetical model.
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