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 selectively removes Arg and/or Lys residues from the N terminus of several peptide substrates. Isolated and characterized from rat testes, this ubiquitous enzyme may participate in the final stages of precursor processing mechanisms. To test this hypothesis, we have investigated the secretion and subcellular localization of this enzyme in a rat cell line of pheochromocytoma (PC12 cells). By using a combination of biochemical and immunocytochemical methods, the following observations were made: (i) the level of aminopeptidase B detectable in the cell culture medium increased with time; (ii) 8-bromo-adenosine 3'-5'-cyclic monophosphate and the Ca2+ ionophore A23187 both stimulated enzyme liberation in the culture medium; (iii) brefeldin A, an inhibitor of vesicular transport from the endoplasmic reticulum to the Golgi apparatus, decreased enzyme secretion in a time-dependent manner; (iv) whereas nocodazole, a microtubule depolymerizing agent, inhibited enzyme secretion, cytochalasin D, a microfilament disruption agent, had no effect on released aminopeptidase B level; (v) immunofluorescence demonstrated the presence of aminopeptidase B in the Golgi apparatus; (vi) immunofluorescence, electron microscopy and tests of enzyme activity on intact cells showed an association of the peptidase with the external face of the plasma membrane. Together these data strongly argued in favour of the enzyme secretion by PC12 cells. It is concluded that aminopeptidase B may participate in processing events occurring either during its intracellular transport along the secretory pathway or at the plasma membrane level, or both.

DNA topoisomerase activities in concanavalin A-stimulated lymphocytes.

Topoisomerase activities have been measured in nuclear extracts of concanavalin A-stimulated lymphocytes. In parallel with the wave of DNA synthesis, type II topoisomerase activity was considerably increased. After 72 h treatment, this activity was stimulated approx. 20-fold over the activity in untreated cells. In contrast, type I topoisomerase was poorly stimulated after 24 h treatment, and 4-5-fold after 72 h. These findings, together with our previous results on regenerating rat liver, suggest a major role of topoisomerase II in DNA replication.