Properties of phenylalanine ammonia-lyase from marrow cotyledons

Abstract

Salinity induced phenylalanine ammonia-lyase (PAL, EC. 4.3.1.1.) from cotyledons of Cucurbita pepo (marrow) up to 200 mM. The effect of stabilizing solutes, glycerol, proline, and betaine against salt in the assay medium on PAL activity was analyzed. Glycerol, betaine and proline enabled a great increase in substrate-dependent salt activation of the enzyme. PAL activity declined following polyethyleneglycol (PEG)-induced water stress. The enzyme was inhibited by β-chloroethyltrimethylammonium (CETA) and induced by GA 3. PAL was purified from marrow ( C. pepo) using (NH 4) 2SO 4 fractionation, ion exchange chromatography using DEAE–cellulose, Sephadex G-100 and Q-Sepharose. The enzyme was purified with 83-fold with specific activity of 26 U mg −1 protein. The enzyme was extremely unstable unless 30% glycerol was added. There was an abrupt discontinuity in Arrhenius plot and the values of activation energy were 2.6 and 12.2 kJ mol −1 for the upper and lower parts of the plot, respectively. PAL activity was strongly inhibited by ethylenediaminetetraacetate (EDTA) and o-phenanthroline. AMP, ADP and ATP inactivated PAL with ATP being the most potent inactivator. Treatment of the pure enzyme with phenylmethyl-sulphonyl fluoride (PMSF), N, N-dicyclohexylcarbodiimide (DCCD), and N-acetyl-imidazole (NAI) resulted in the inhibition of the enzyme and reveal that seryl, carboxyl and tyroysl residues are essential for the enzyme catalysis. The metal ion requirement of PAL was investigated and Mg 2+ particularly fulfilled this requirement for divalent cation. Ca 2+ decreased PAL activity below the control value.