Thyroliberin and luliberin degradation by enzymes in cultured cells of neural origin.

Two enzymes which can remove the N-terminal pyroglutamyl residue of Thyroliberin (Glp-His-ProNHz) have been identified in brain tissue of guinea-pig and other animals. Pyroglutamate aminopeptidase I is present in cytoplasm and requires DTT and EDTA for the assay of activity. [l] Pyroglutamate aminopeptidase II has been located in synaptosomal membranes and is inhibited by EDTA [2]. A number of enzymes have been identified in the soluble fraction of brain tissue which can introduce cleavage into the primary sequence of luliberin. Degrading activity has also been found in particulate fractions of both guinea-pig brain [4] and rat brain [5]. Both thyroliberin and luliberin have been suggested as neurotransmittors or neuromodulators in the CNS [6] and this study was undertaken to determine the cellular distribution of enzymes for the degradation of these peptides in cultured cells of neural origin. Neuroblastoma (New-2A) and glioma ( c 6 ) cells were cultured in Dubbecco's modified Eagles medium containing 10% foetal calf serum, 200 mM glutamine and gentamycin (100 pg/ml) and maintained at 37 OC in a humidified atmosphere of 9% C 0 2 The cells were seeded in tissue culture flasks at a density of lo4 cells/cm2 and allowed to reach confluency (3-4 days). The cells were harvested by scraping and washed once with Dulbecco's phosphate buffered saline. The final pellet (800 g x 10 min) was lyophilised and stored at -20 OC until assay. Prior to assay cells were resuspended in 1 ml of 150 mM potassium phosphate pH 7.5. Pyroglutamate aminopeptidase was measured using a modification of the method of Bauer and Kleinkauf [7] in which [Pro 3Hl Distribution of thyroliberin hydrolysing pyroglutmateaminopeptidases and of luliberin hydrolysing activity inneuroblastoma cells (neuro-2A) and in glioma (Cg) cells.