Isolation of Pteroyl-γ-oligoglutamyl Endopeptidase from Chicken Intestine with the Aid of Affinity Chromatography

Abstract

Abstract An enzyme which performs the first step in the catalytic release of folic acid from pteroyl-γ-oligoglutamates was isolated from chicken intestine by the use of affinity chromatography. The enzyme-interacting ligand system consisted of synthetic γ-oligoglutamyl peptides attached covalently to Sepharose 4B by cyanogen bromide activation. The retained enzyme was eluted from the affinity column with 1 m NaCl. DEAE-cellulose chromatography and gel filtration with Sephadex G-200 were required for a final purification of 1,200-fold with a 20 % recovery of the initial enzyme. The purified enzyme appeared as a single band on disc gel electrophoresis. The estimated molecular weight determined by gel filtration was 80,000, and the isoelectric point was 4.8. The isolated enzyme was characterized as specific endopeptidase which cleaves pteroyl-γ-oligoglutamates between pteroyl glutamyl-γ-glutamate (pteroyl diglutamate) and the remaining γ-glutamyl peptide. With pteroyl-γ-heptaglutamate as substrate, the Km(app) was determined as 6.3 x 10-5 m. The Vmax was 1.8 x 10-3 µmole per min.