HIGHER-PLANT FOLYLPOLYGLUTAMATE SYNTHETASE .1. PURIFICATION, STABILITY AND REACTION REQUIREMENTS OF THE ENZYME FROM PEA-SEEDLINGS

Plant tissues contain highly conjugated forms of folate. Despite this, there have been no detailed reports on the isolation and properties of higher plant folylpolyglutamate synthetases. The present studies have examined the occurrence of this enzyme (FPGS) in extracts of pea (Pisum sativum L. cv. Homesteader) seedlings. Polyglutamate synthesis was measured in vitro, by incorporation of [H-3]-glutamate in the presence of [6R,S]-tetrahydrofolate monoglutamate and ATP. FPGS activity was detected in leaf and cotyledon tissues. In cotyledons, enzyme activity, on a per gram fresh weight and specific activity basis, remained relatively constant between day 1 and 4 of germination. Activity was also not appreciably changed when seeds were imbibed in 0.1 mM methotrexate, 3.5 mM cycloheximide or 3 mM chloramphenicol. The FPGS activity of 18-h-old cotyledons was purified 2700-fold with 40% recovery by a protocol involving streptomycin sulphate and ammonium sulphate fractionations followed by column chromatography on Sephacryl S-200, DEAE-cellulose and phenylagarose. The purified enzyme was unstable but additions of glycerol (50% v/v) reduced losses of activity at 4-degrees-C. In common with bacterial and mammalian FPGS, the synthetase of pea cotyledons had a molecular weight of 68,000, and polyglutamate synthesis was dependent on ATP and tetrahydrofolate.