A study of prolyl endopeptidase in bovine serum and its relevance to the tissue enzyme.
Prolyl endopeptidase (PE) belongs to a group of enzymes that specifically recognise the imino acid proline. The characterisation of bovine serum PE was undertaken so that its relationship to its tissue counterparts could be considered. Using various chromatographic methods, PE was partially purified from bovine serum. This preparation was deemed to be enzymatically pure, based on its failure to hydrolyse a wide range of fluorimetric substrates. A native molecular mass of 69.7 kDa was estimated for the enzyme. PE was optimally active at pH 8.0-8.5, demonstrated a preference for phosphate buffer and remained stable over a pH range of 5.0-9.0. A narrowly focused optimal assay temperature of 37 degrees C was evident. Functional reagent studies indicated that this enzyme was a serine protease with a cysteine residue located near or at the active site. The enzyme was also sensitive to heavy metal inhibition. Substrate specificity investigations revealed that the bioactive peptides angiotensin II, bradykinin, luliberin and substance P were hydrolysed by the enzyme preparation, but lower specificities were evident towards these peptides in comparison with the enzyme's tissue counterparts. Specific inhibitor studies, using a range of compounds previously untested against a single PE source, indicated that alpha-ketobenzothiazole was the most effective PE inhibitor, with an IC50 value of 41 pM. In conclusion, the results presented in this paper indicate that bovine serum PE shares many of the characteristics associated with its tissue counterparts, with the exception of its specificity towards certain bioactive peptides.
Cunningham
D F
DF
School of Biological Sciences, Dublin City University, Ireland.
O'Connor
B
B
D000804:Angiotensin II / Q000378:metabolism; D000818:Animals; D001667:Binding, Competitive; D001920:Bradykinin / Q000378:metabolism; D002417:Cattle; D002729:Chloromercuribenzoates / Q000494:pharmacology; D003374:Coumarins / Q000737:chemistry / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D004795:Enzyme Stability / Q000187:drug effects / Q000502:physiology; D005470:Fluorometry; D007987:Gonadotropin-Releasing Hormone / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008670:Metals / Q000494:pharmacology*; D008970:Molecular Weight; D009479:Neuropeptides / Q000378:metabolism; D000085822:Prolyl Oligopeptidases; D012697:Serine Endopeptidases / Q000097:blood* / Q000737:chemistry / Q000187:drug effects*; D013373:Substance P / Q000378:metabolism; D013379:Substrate Specificity; D020245:p-Chloromercuribenzoic Acid
D002729:Chloromercuribenzoates; D003374:Coumarins; D004791:Enzyme Inhibitors; D008670:Metals; D009479:Neuropeptides; D000804:Angiotensin II; D013373:Substance P; D007987:Gonadotropin-Releasing Hormone; D020245:p-Chloromercuribenzoic Acid; C020988:N-carbobenzoxyglycyl-prolyl-4-methylcoumarinyl amide; D012697:Serine Endopeptidases; D000085822:Prolyl Oligopeptidases; D001920:Bradykinin