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. It converts small enkephalin-containing peptides into the corresponding enkephalins and inactivates biopeptides such as bradykinin and neurotensin in vitro. We investigated the presence of endooligopeptidase A in PC12 cells. This cell line was derived from a rat pheochromocytoma tumor 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. Endooligopeptidase A activity was measured in soluble cellular extracts using a specific 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 endooligopeptidase 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 PC12 endooligopeptidase A-like activity displayed identical elution profile as rabbit brain endooligopeptidase A in gel filtration and anion-exchange chromatography. In addition, an antiserum raised against rabbit brain endooligopeptidase A cross-reacted with a 71 kDa component from PC12 cell extracts in Western blotting and was also able to partially neutralize the PC12 endooligopeptidase A-like activity. Treatment of PC12 cells with basic fibroblast growth factor (bFGF), a neurotrophic factor for this cell line, did not modify the specific activity of this enzyme. However, cAMP analogs decreased the specific activity of the enzyme. These results indicate the presence of an endooligopeptidase A-like activity in PC12 cells which is modulated by cAMP but not by bFGF.
Ferro
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Departmento de Farmacologia, Universidade de São Paulo, Brazil.
Tambourgy
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Abreu
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Camargo
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Raw
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D015124:8-Bromo Cyclic Adenosine Monophosphate / Q000494:pharmacology*; D000310:Adrenal Gland Neoplasms; D000818:Animals; D015153:Blotting, Western; D001921:Brain / Q000201:enzymology; D003994:Bucladesine / Q000494:pharmacology*; D002454:Cell Differentiation; D002850:Chromatography, Gel; D000242:Cyclic AMP / Q000502:physiology*; D004228:Dithionitrobenzoic Acid / Q000494:pharmacology; D004229:Dithiothreitol / Q000494:pharmacology; D005029:Ethylenediamines; D016222:Fibroblast Growth Factor 2 / Q000494:pharmacology*; D007106:Immune Sera; D007700:Kinetics; D008666:Metalloendopeptidases / Q000302:isolation & purification / Q000378:metabolism*; D009842:Oligopeptides; D016716:PC12 Cells; D010618:Phenanthrolines / Q000494:pharmacology; D010673:Pheochromocytoma; D011817:Rabbits; D051381:Rats
D005029:Ethylenediamines; D007106:Immune Sera; D009842:Oligopeptides; D010618:Phenanthrolines; D016222:Fibroblast Growth Factor 2; C066641:QF-ERP7; D015124:8-Bromo Cyclic Adenosine Monophosphate; D003994:Bucladesine; D004228:Dithionitrobenzoic Acid; D000242:Cyclic AMP; D008666:Metalloendopeptidases; C022510:thimet oligopeptidase; D004229:Dithiothreitol; C025205:1,10-phenanthroline