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O.P. Srivastava, K. Srivastava; Association of Proteinase Activity with beta A3/A1–Crystallin . Invest. Ophthalmol. Vis. Sci. 2004;45(13):3964.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: The purpose of the study was to determine whether an Arg–bond hydrolyzing proteinase activity is associated with recombinant ßA3/A1–crystallin. Methods: The human recombinant ßA3/A1–crystallin (expressed in E.coli) was purified by a three–step procedure involving DEAE–Sephacel ion–exchange chromatography, butyl Sepharose hydrophobic interaction–chromatography and size–exclusion HPLC. The proteinase activity in the purified preparation was determined with either N–α benzyol DL–Arg p–nitroanilide or CBZ–L–Arg 7–amino 4–methyl coumarin as a substrate. The proteinase activity in the crystallin was activated following treatment with 2% sodium deoxycholate and size–exclusion HPLC. The properties of the crystallin proteinase was determined. Results: The purified ßA3/A1–crystallin showed a single protein band of ∼25 kDa on SDS–PAGE and also on 2D–gel electrophoresis. Following treatment of the crystallin preparation with sodium deoxycholate and size–exclusion chromatography, an Arg–bond hydrolyzing proteinase activity with the above two substrates was observed. On incubation of the crystallin preparation with sodium deoxycholate at 37°C in a time–course experiment, an activation of the proteinase activity with proteolysis of the crystallin followed by multimer formation among the proteolyzed fragments, was observed. The proteinase showed inhibition by serine–proteinase inhibitors, and proteolyzed lens αA– and αB–crystallins. Further analysis showed that the enzyme activity was associated with an N–terminally truncated dimeric form of the crystallin. Conclusions: A purified recombinant ßA3/A1–crystallin preparation, on deoxycholate treatment, exhibited an Arg–bong hydrolyzing proteinase activity.
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