Purification and characterization of prolyl oligopeptidase from bovine lens.
Prolyl oligopeptidase (EC 3.4.21.26) has been purified 26,000-fold from bovine lens tissue by anion-exchange chromatography, gel filtration and isoelectric focussing, with an overall yield of 11%. The purified enzyme exhibited an isoelectric point of 4.8, a pH optimum of 7.5 and a molecular mass of 72 kDa under both native and denaturing conditions. The enzyme was inhibited by diisopropylfluorophosphate and N-ethylmaleimide, indicating the presence of an essential serine residue and an -SH group. The purified enzyme hydrolyzes the elastase substrate tboc-ala-ala-pNA an order of magnitude more rapidly than N-suc-gly-pro-MCA. It also hydrolyzes other elastase substrates including N-suc-ala-ala-ala-pNA, N-suc-ala-ala-pNA, N-suc-ala-ala-pro-ala-pNA and tboc-ala-ala-pro-ala-pNA, but at a slower rate. While the purified preparation of the lens enzyme rapidly hydrolyses bradykinin, ile-ser-bradykinin, insulin B chain, angiotensin and val-his-leu-thr-pro-val-glu-lys at the carboxyl side of proline, it does not hydrolyse either lens crysallins or bovine serum albumin. The amino acid sequence (GMFYNAYPQQDG) of a tryptic peptide from the enzyme is identical to the porcine brain prolyl oligopeptidase sequence 184-195. A similar activity has been identified in human lenses using both tboc-ala-ala-pNA and N-suc-gly-pro-MCA as substrates. The molecular weight, substrate specificity, inhibitor susceptibility and amino acid sequence data suggest that the bovine lens prolyl oligopeptidase is similar to prolyl endopeptidase isolated from other sources.
Sharma
K K
KK
Mason Institute of Ophthalmology, University of Missouri, Columbia 65212.
Ortwerth
B J
BJ
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D004591:Electrophoresis, Polyacrylamide Gel; D005033:Ethylmaleimide / Q000494:pharmacology; D007526:Isoelectric Point; D007531:Isoflurophate / Q000494:pharmacology; D007908:Lens, Crystalline / Q000201:enzymology*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D000085822:Prolyl Oligopeptidases; D012697:Serine Endopeptidases / Q000737:chemistry* / Q000302:isolation & purification* / Q000494:pharmacology; D013379:Substrate Specificity
D007531:Isoflurophate; D012697:Serine Endopeptidases; C000705287:PREPL protein, human; D000085822:Prolyl Oligopeptidases; D005033:Ethylmaleimide