May 2003
Volume 44, Issue 13
ARVO Annual Meeting Abstract  |   May 2003
Purification and Characterization of Phe-Ser Bond Cleaving Protease in Bovine Lens and its Identification as Endopeptidase 24.15
Author Affiliations & Notes
  • R. Chaerkady
    Ophthalmology, Univ Missouri, Columbia, MO, United States
  • K.K. Sharma
    Ophthalmology and Biochemistry, Univ Missouri, Columbia, MO, United States
  • Footnotes
    Commercial Relationships  R. Chaerkady, None; K.K. Sharma, None.
  • Footnotes
    Support  NIH Grants 11981, EY 09855, Award from RPB
Investigative Ophthalmology & Visual Science May 2003, Vol.44, 1238. doi:
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      R. Chaerkady, K.K. Sharma; Purification and Characterization of Phe-Ser Bond Cleaving Protease in Bovine Lens and its Identification as Endopeptidase 24.15 . Invest. Ophthalmol. Vis. Sci. 2003;44(13):1238.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract: : Purpose: To isolate and characterize an endopeptidase activity from bovine lens that cleaves Phe-Ser bonds in peptide substrates and determine its role in crystallin fragment accumulation in vivo. Methods: Young bovine lens homogenate was used to separate the new proteases. The protease activity was measured during purification steps using an internally quenched bradykinin (Mca-(Ala7,Lys(Dnp)9)-bradykinin) substrate. The protease was purified using several chromatographic steps. A tryptic digest of the purified protease was analyzed using MALDI-ToF. The specificity of the protease was determined using bradykinin and its analogues, as well as neurotensin and adipokinetic hormone G as substrates. The identity of the endopeptidase was also confirmed by using phosphinic peptide inhibitor specific for proteases EC Crystallin fragments isolated from aged bovine lenses was tested for their susceptibility to new endopeptidase action by measuring the generation of new amino groups Results: The endopeptidase activity, localized mainly in the outer cortex of the lens, was purified from young bovine lens extract. The peptide map finger printing showed that it has homology to the human thimet oligopeptidase. The purified protease cleaved bradykinin at phe5-ser6 and neurotensin at Arg8-Arg9. MALDI-ToF MS of the product of bradykinin and neurotensin hydrolysis confirmed the specificity of the protease. Basic or hydrophobic aminoacids at P1 and P1' position in the substrate were preferred over acidic residues. The phosphinic peptide inhibitor for EC (thimet oligopeptidase) and Biotinylated RPPGF chloromethylketone inhibitor completely inhibited the protease activity. The enzyme activity was also inhibited by higher concentration of DTT and ATP. The crystallin fragments obtained from aged bovine lenses were cleaved by the purified enzyme. Conclusions: This study shows the presence of thimet oligopeptidase in lens, which may be involved in the degradation of the peptides produced by the action of proteases in lens cortex. Since the level of peptides (crystallin fragments) increases in the nuclear region with age and cataract, it is likely that endopeptidase 24.15 activity plays an important role in this process.

Keywords: proteolysis • protein purification and characterization • cataract 

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