Purification and characterization of a novel membrane-bound form of prolyl endopeptidase from bovine brain.
Prolyl endopeptidase has been predominantly described as a cytosolic activity capable of cleaving a number of important neuropeptides (including TRH, LHRH, Bradykinin, Angiotensin, Substance P, Neurotensin, Oxytocin and Vasopressin) on the carboxy side of proline. In this paper, we report, for the first time, on the complete purification and characterization of a membrane-bound form of prolyl endopeptidase. This novel activity has been isolated from the synaptosomal (plasma membranes) membranes of bovine brain. Following gel filtration, hydroxylapatite and hydrophobic interaction chromatographies, the prolyl endopeptidase activity was purified 1400-fold with a 23% recovery of activity. The enzyme was shown to have a relative molecular mass of 87 kDa and a Km of 60 microM for its specific fluorimetric substrate, Z-GlyProMCA. The purified enzyme demonstrated a relatively broad substrate specificity and a relatively high affinity for proline-containing neuropeptides. It was shown to be inhibited by certain thiol-protease inhibitors and by the metal chelator, 1,10-phenanthroline, thus possibly classifying it as a 'thimet' activity. The purified particular form of proyl endopeptidase displayed a similar substrate specificity to the previously reported cytosolic forms of the enzyme. However, there were differences between the two forms in term of their sensitivity to inhibitors, their affinities for the peptide substrates and their relative molecular masses. The different subcellular location (i.e. the synaptosomal membrane) of the particulate prolyl endopeptidase is also of potential physiological significance given that here it is more likely to come in contact with the vesicle-bound neuropeptides than is its cytosolic counterpart.
O'Leary
R M
RM
School of Biological Sciences, Dublin City University, Ireland.
Gallagher
S P
SP
O'Connor
B
B
D000818:Animals; D001921:Brain / Q000187:drug effects / Q000201:enzymology* / Q000648:ultrastructure; D002417:Cattle; D002462:Cell Membrane / Q000187:drug effects / Q000201:enzymology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D000085822:Prolyl Oligopeptidases; D012697:Serine Endopeptidases / Q000302:isolation & purification*; D013379:Substrate Specificity; D013574:Synaptosomes / Q000187:drug effects / Q000201:enzymology*
D004791:Enzyme Inhibitors; D012697:Serine Endopeptidases; D000085822:Prolyl Oligopeptidases