Membrane peptidases on human osteoblast-like cells in culture: hydrolysis of calcitonin and hormonal regulation of endopeptidase-24.11.

Five membrane peptidase activities have been identified on cultured human osteoblast-like cells. These consisted of the four exopeptidases aminopeptidase-A, aminopeptidase-N, aminopeptidase-W and carboxypeptidase-M, and the endopeptidase, endopeptidase-24.11. The presence of endopeptidase-24.11 was confirmed immunochemically by immunofluorescent staining and by enzyme-linked immunosorbent assay. The inclusion of phosphoramidon partially inhibited the hydrolysis of human calcitonin by a membrane fraction prepared from osteoblast-like cell membranes, thus implicating endopeptidase-24.11 in its inactivation. Another metallopeptidase also contributed substantially to calcitonin hydrolysis. Purified porcine endopeptidase-24.11 (1 microgram) was shown to hydrolyse calcitonin with a half-life of 23 min, which compared to a half-life of 0.5 min for substance P under similar conditions. Sequence data revealed that the initial site of hydrolysis of calcitonin was between residues Lys18 and Phe19. The expression of endopeptidase-24.11 by cultured osteoblast-like cells was shown to be modified by various agents: expression was decreased by phorbol 12-myristate-13-acetate (160 nM for 48 h) and increased in the presence of calcitonin (1.5 nM for 48 h) and 1,25-dihydroxyvitamin D3 (0.01-1 microM for 72 h).