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).
Howell
S
S
Department of Biochemistry and Molecular Biology, University of Leeds, U.K.
Caswell
A M
AM
Kenny
A J
AJ
Turner
A J
AJ
D000595:Amino Acid Sequence; D001665:Binding Sites; D002116:Calcitonin / Q000378:metabolism* / Q000494:pharmacology; D002117:Calcitriol / Q000494:pharmacology; D002462:Cell Membrane / Q000201:enzymology*; D002478:Cells, Cultured; D010450:Endopeptidases / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay; D005455:Fluorescent Antibody Technique; D006020:Glycopeptides / Q000494:pharmacology; D006207:Half-Life; D006801:Humans; D006868:Hydrolysis; D008969:Molecular Sequence Data; D015260:Neprilysin / Q000037:antagonists & inhibitors / Q000378:metabolism*; D010006:Osteoblasts / Q000201:enzymology*; D010281:Parathyroid Hormone / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D006020:Glycopeptides; D010281:Parathyroid Hormone; D002116:Calcitonin; D010450:Endopeptidases; D015260:Neprilysin; D002117:Calcitriol; D013755:Tetradecanoylphorbol Acetate; C008890:phosphoramidon