GAMMA-GLUTAMYL HYDROLASE FROM HUMAN SARCOMA HT-1080 CELLS - CHARACTERIZATION AND INHIBITION BY GLUTAMINE ANTAGONISTS

Elevated gamma-glutamyl hydrolase (GGH) activity as a contributing fac tor in mechanisms of acquired and intrinsic antifolate resistance has been reported for several cultured cell lines. Despite this, little is known about this enzyme, especially the human species. Using the huma n HT-1080 sarcoma line, we observed the secretion of GGH activity into media during culture (a phenomenon that could be markedly stimulated by exposure to NH4Cl) and an acidic pH optimum for in vitro catalytic activity of the enzyme. These properties are consistent with a lysosom al location for the enzyme. Unlike rodent GGH, preparations of HT-1080 enzyme (purified less than or equal to 2000-fold) displayed exopeptid ase activity in cleaving successive end-terminal gamma-glutamyl groups from poly-L-gamma-glutamyl derivatives of folate, methotrexate (MTX), and para-aminobenzoic acid substrates and a marked preference for lon g-chain polyglutamates (K-m values for glu(4) versus glu(1) derivative s were 17- and 15-fold lower for folate and MTX versions, respectively ). Using an in vitro assay screen, several glutamine antagonists [i.e. , 6-diazo-5-oxo-norleucine (DON), acivicin, and azaserine] were identi fied as human GGH inhibitors, with DON being the most potent and displ aying time-dependent inhibition. In cell culture experiments, simultan eous exposure of DON (10 mu M) and [H-3]MTX for 24 hr resulted in mode st elevations of the long-chain gamma-glutamyl derivatives of the anti folate for HT-1080 and another human sarcoma line. These compounds may serve as useful lead compounds in the development of specific GGH inh ibitors for use in examining the relationship between GGH activity and antifolate action and may potentially be used in clinical combination with antifolates that require polyglutamylation for effective cellula r retention.