title: A prolyl endopeptidase from murine macrophages, its assay and specific inactivation.
abstract: The presence of a prolyl endopeptidase in the soluble fraction of murine peritoneal macrophages is reported. The prolyl endopeptidase is apparently highly specific for cleaving peptides after proline residues. A sensitive new fluorogenic assay substrate matching this specificity, benzyloxycarbonyl-Ala-Ala-Pro beta-methoxynaphthylamide, is described. The enzyme is rapidly inactivated by benzyloxycarbonyl-Ala-Ala-Pro diazomethyl ketone, one of a class of reagents specific for cysteine proteinases, and by diisopropyl fluorophosphate, an inhibitor of serine proteinases. Culture of macrophages with the addition of low levels of benzyloxycarbonyl-Ala-Ala-Pro diazomethyl ketone to the media allows the selective inhibition of the cytoplasmic enzyme as measured in lysates at the termination of culture. After exposure to inhibitor, macrophages resynthesize the enzyme over a period of days, a process which is inhibited by cycloheximide. Similar amounts of activity were found in both normal peritoneal macrophages and those elicited by prior injection of thioglycollate media. The enzyme from murine macrophages appears similar to that reported in bronchopulmonary lavage fluid and lung tissue and to those isolated from brain and pituitary tissues.
mesh_terms: D000818:Animals; D001202:Ascitic Fluid / Q000166:cytology; D055598:Chemical Phenomena; D002621:Chemistry; D003978:Diazomethane / Q000031:analogs & derivatives / Q000494:pharmacology; D010450:Endopeptidases / Q000302:isolation & purification* / Q000378:metabolism; D005260:Female; D007659:Ketones / Q000494:pharmacology; D008264:Macrophages / Q000201:enzymology*; D051379:Mice; D000085822:Prolyl Oligopeptidases; D012697:Serine Endopeptidases; D013347:Subcellular Fractions / Q000201:enzymology; D013379:Substrate Specificity
chemical_list: D007659:Ketones; D003978:Diazomethane; C038788:benzyloxycarbonylalanyl-alanyl-proline diazomethyl ketone; D010450:Endopeptidases; D012697:Serine Endopeptidases; D000085822:Prolyl Oligopeptidases