Abstract:
PURPOSE:To isolate and characterize bovine lens endopeptidase activity that cleaves the Phe-Ser bond in peptide substrates. METHODS:The protease activity in young bovine lens homogenate was measured using the Mca-(Ala(7),Lys(Dnp)(9))-bradykinin substrate. Degradation of bradykinin and other unlabeled peptide substrates was monitored by reversed-phase HPLC on a C18 column. The protease was purified by means of several chromatography steps. An in-gel tryptic digest of the purified protease was analyzed by using matrix-assisted desorption ionization-time of flight mass spectrometry (MALDI-ToF-MS and nanospray quadrupole time of flight mass spectrometry (QqToF MS). The specificity of the protease was determined with bradykinin and its analogues. Crystallin fragments isolated from aged bovine lenses were tested for their susceptibility to degradation by a newly identified endopeptidase. RESULTS:Bradykinin hydrolyzing endopeptidase activity was localized mainly in the outer cortex of the lens. A characterization study showed that the purified protease was thiol and metal dependent. Peptide mass fingerprinting and tandem mass spectrometry (MS/MS) analysis of an in-gel tryptic digest matched the protein sequence of thimet oligopeptidase (TOP). The purified protease cleaved bradykinin specifically at Phe(5)-Ser(6) and neurotensin at Arg(8)-Arg(9). Basic or hydrophobic amino acids at P1 and P1' positions in the substrate were preferred over acidic residues. Enzyme activity was also inhibited by physiological levels of adenosine triphosphate (1 mM; ATP) and glutathione (3 mM; GSH). The crystallin fragments obtained from aged bovine lenses were cleaved by the purified enzyme. CONCLUSIONS:This study shows the presence of TOP in the bovine lens. Its unique substrate specificity and regulation of its activity by ATP and GSH suggest that TOP has an important role in peptide hydrolysis in the lens.
journal_name
Invest Ophthalmol Vis Scijournal_title
Investigative ophthalmology & visual scienceauthors
Chaerkady R,Sharma KKdoi
10.1167/iovs.03-0769keywords:
subject
Has Abstractpub_date
2004-04-01 00:00:00pages
1214-23issue
4eissn
0146-0404issn
1552-5783journal_volume
45pub_type
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