GAMMA-GLUTAMYL HYDROLASE FROM PEA COTYLEDONS

Folylpolyglutamates are substrates of gamma-glutamyl hydrolases (GGH) (EC 3.4.22.12). A GGH protein of Pisum sativum, cv. Homesteader was extracted from six-day-old cotyledons and purified over 10000-fold. The purification protocol involved streptomycin sulphate and (NH4)2SO4 fractionations followed by chromatography on DEAE-Sephacel, Sephacryl S-200, Matrex Green A and heparin agarose. Activity, based on the hydrolysis of [C-14]-glutamyl-labelled pteroyltriglutamic acid (PteGlu3), was maximal at pH 6 and associated with a M(r) 55000 protein. Differential centrifugation of mannitol-buffered tissue extracts showed that GGH activity was mainly cytosolic with only minor amounts detected in the vacuolar and mitochondrial fractions. The major properties of this hydrolase were similar to those reported for the intracellular GGH proteins of mammals. Pea GGH had endopeptidase action as indicated by the conversion of PteGlU3 to PteGlu and diglutamyl peptide. PteGlu5, the major polyglutamate chain length of this tissue, was converted to PteGlu and smaller amounts of PteGlu2. Hydrolysis of PteGlu3 by the purified enzyme was strongly inhibited by PteGlu5, zinc ions, phenylmethylsulphonylfluoride and p-hydroxymercuribenzoate but not by 2-mercaptomethylglutaric acid (0.04-4 mM). Although N-carbobenzoxyphenylalanyl-alanine and gamma-glutamyl tripeptide were not hydrolysed by the enzyme, the conversion of p-aminobenzoyltriglutamate (p-ABAGlu3) to p-ABAGlu occurred at ca 13% of the rate observed for PteGlu3.