AMINOPEPTIDASE-B - A PROCESSING ENZYME SECRETED AND ASSOCIATED WITH THE PLASMA-MEMBRANE OF RAT PHEOCHROMOCYTOMA (PC12) CELLS

Aminopeptidase B (Ap-B) is a Zn2+-dependent exopeptidase which selecti vely removes Arg and/or Lys residues from the N terminus of several pe ptide substrates, Isolated and characterized from rat testes, this ubi quitous enzyme may participate in the final stages of precursor proces sing mechanisms, To test this hypothesis, we have investigated the sec retion and subcellular localization of this enzyme in a rat cell line of pheochromocytoma (PC12 cells), By using a combination of biochemica l and immunocytochemical methods, the following observations were made : (i) the level of aminopeptidase B detectable in the cell culture med ium increased with time; (ii) 8-bromo-adenosine 3'-5'-cyclic monophosp hate and the Ca2+ ionophore A23187 both stimulated enzyme liberation i n the culture medium; (iii) brefeldin A, an inhibitor of vesicular tra nsport from the endoplasmic reticulum to the Golgi apparatus, decrease d enzyme secretion in a time-dependent manner; (iv) whereas nocodazole , a microtubule depolymerizing agent, inhibited enzyme secretion, cyto chalasin D, a microfilament disruption agent, had no effect on release d aminopeptidase B level; (v) immunofluorescence demonstrated the pres ence of aminopeptidase B in the Golgi apparatus; (vi) immunofluorescen ce, electron microscopy and tests of enzyme activity on intact cells s howed an association of the peptidase with the external face of the pl asma membrane. Together these data strongly argued in favour of the en zyme secretion by PC12 cells, It is concluded that aminopeptidase B ma y participate in processing events occurring either during its intrace llular transport along the secretory pathway or at the plasma membrane level, or both.