CHARACTERIZATION OF AN ENDOOLIGOPEPTIDASE A-LIKE PROTEIN IN PC12 CELLS - ACTIVITY MODULATION BY CAMP BUT NOT BY BASIC FIBROBLAST GROWTH-FACTOR

Endooligopeptidase A is a putative neuropeptide-metabolizing enzyme. I t converts small enkephalin-containing peptides into the corresponding enkephalins and inactivates biopeptides such as bradykinin and neurot ensin in vitro. We investigated the presence of endooligopeptidase A i n PC12 cells. This cell line was derived from a rat pheochromocytoma t umor and resembles fetal chromaffin cell. Depending on the supplements added to the cell culture, this cell line can be differentiated into mature chromaffin cell or sympathetic neuron-like cell. Endooligopepti dase A activity was measured in soluble cellular extracts using a spec ific fluorogenic substrate QF-ERP7. The PC12 endooligopeptidase A-like activity shared similar but not identical biochemical properties with rabbit brain endooligopeptidase A. Similarly to rabbit brain endoolig opeptidase A, the PC12 endooligopeptidase A-like activity was enhanced by DTT, totally inhibited by DTNB and 1-10 Phenanthroline, partially inhibited by cFP-AAF-pAb, and not affected by PMSF. Furthermore, the P C12 endooligopeptidase A-like activity displayed identical elution pro file as rabbit brain endooligopeptidase A in gel filtration and anion- exchange chromatography. In addition, an antiserum raised against rabb it brain endooligopeptidase A cross-reacted with a 71 kDa component fr om PC12 cell extracts in Western blotting and was also able to partial ly neutralize the PC12 endooligopeptidase A-like activity. Treatment o f PC12 cells with basic fibroblast growth factor (bFGF), a neurotrophi c factor for this cell line, did not modify the specific activity of t his enzyme. However, cAMP analogs decreased the specific activity of t he enzyme. These results indicate the presence of an endooligopeptidas e A-like activity in PC12 cells which is modulated by cAMP but not by bFGF. (C) 1995 Wiley-Liss, Inc.