The properties and function of γ-glutamyl hydrolase and poly-γ-glutamate

Abstract γ-Glutamyl hydrolase is a ubiquitous enzyme that has the capacity to cleave γ-glutamyl bonds of cellular folyl- and antifolylpoly-γ-glutamates. This study has revealed that the enzyme is secreted by primary cultures of rat hepatocytes and by H35 hepatoma cells. It was found that more than 99% of the total enzyme from H35 cells accumulated in the medium after 48 hr incubation with the serum-free medium. The cells were shown to remain intact during the secretion period since lactate dehydrogenase, dihydrofolate reductase and lysosomal hydrolases other than γ-glutamyl hydrolase were retained within the cell. When PteGlu 5 (folylGlu 4 ) is used as a substrate the initial product is PteGlu (folate), and there is no appearance of intermediate chain length pteroyl polyglutamates. Therefore, the secreted and cellular γ-glutamyl hydrolase from hepatoma cells appears to be an endopeptidase. Polyclonal antibodies to the poly-γ-glutamate substrates of the enzyme were prepared and characterized. The antibodies recognize the structural differences between α- and γ-glutamyl linkages but appear equally active with PteGlu 5 and its analogs such as 4-NH 2 -10-CH 3 PteGlu 5 and pABAGlu 5 . The affinity of the antibodies is related to the γ-glutamyl structure since l -glutamic acid, folate or p -aminobenzoic acid are inactive with the antibodies. Furthermore, poly-γ-glutamate has lower affinity for the antibodies than the poly-γ-glutamate derivatives of PteGlu, 4-NH 2 -10-CH 3 PteGlu or pABA.