95] Folylpolyglutamate endopeptidase from chicken intestine: Isolation with the aid of affinity chromatography

Abstract

This chapter discusses the use of affinity chromatography for the isolation of folylpolyglutamate endopeptidase obtained from chicken intestine. Affinity chromatography provides functional specificity in fractionation of proteins and an increasing number of proteins are being purified by this method. In this study, this technique is used as an aid in the isolation of a specific endopeptidase from chicken intestine that catalyzes the initial step in the hydrolysis of pteroyl-γ-oligoglutamates. With PteGlu 7 as substrate, the sole products formed are PteGlu and γ-Glu. When PteGlu 5 is used as a substrate, the cleavage occurs in the same position with the release of PteGlu 2 and γ-Glu 3 . When PteGlu 3 , γ-Glu, and TNB-γ-Glu are used as substrates, under similar conditions, there is no hydrolysis. Thus, the enzyme is an endopeptidase that cleaves at the carboxyl position of pteroyldiglutamate. It has been shown that the hydrolysis of synthetic pteroylheptaglutamate, by a crude preparation of chicken intestine, occurs as a step by step consecutive hydrolytic reaction, involving a series of discrete enzymic activities. The first step in this mechanism is the cleavage between pteroyldiglutamate and the remaining γ-pentaglutamyl peptide. The endopeptidase purified performs that initial cleavage and other enzymes are responsible for subsequent hydrolysis to PteGlu and glutamate products.