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R. Tammali, D. Chandra, D.A. Greer, K.V. Ramana, S.K. Srivastava; Characterization Of Lens Fiber Cell Globulizing Aminopeptidase From Bovine Lens . Invest. Ophthalmol. Vis. Sci. 2006;47(13):2535.
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© ARVO (1962-2015); The Authors (2016-present)
We have recently identified a novel, bestatin sensitive, calcium independent fiber cell globulizing aminopeptidase (FCGAP) from rat lens and shown that inhibition of this protease prevents sugar–induced lens opacification. Now we have isolated and characterized FCGAP from bovine lens and investigated its role in lens fiber cell globulization.
DEAE Biogel–A, glutathione–affinity and hydroxylapetite–column chromatographic techniques were used to purify bovine lens FCGAP to homogeneity following the cleavage of BOC–Leu–Met–CMAC–SG. Mol.wt of the FCGAP was determined by SDS–PAGE and ESI/MS. The pH optima, PI and the effects of divalent cations and sulfhydryls on enzyme activity were determined. Substrate specificity was determined using various amino acid derivatives of p–nitroanilides.
Bovine lens FCGAP was purified to homogeneity with a specific activity of 3.1 U/mg protein. The FCGAP demonstrated a mol.wt of ∼26 kDa. ESI/MS demonstrated a single peak corresponding to M/Z value of 25,688. N–terminal analysis of FCGAP identified it to be same as GSTµ. The pH optima is 7.8 and the enzyme does not require divalent cations, but is inhibited by sulfhydryls. Protease inhibitors such as leupeptin, pepstain, antipain and E–64 did not inhibit FCGAP but aminopeptidase inhibitor bestatin is an excellent inhibitor. FCGAP cleaved p–nitroanilides of amino acids Met, Lys, Val, Pro and Phe. The Km values of FCGAP towards BOC–Leu–Met–CMAC–SG, Met–p–nitroanilide and Lys–p–nitroanilide were 121 ± 20 nM, 65±15 µM and 79± 9.7 µM, respectively. Inhibition of FCGAP with bestatin and uniblue–A prevented calcium –induced rat lens fiber cell globulization.
We have purified and characterized FCGAP from bovine lens and identified that this protease is distinct from calpain and leucine aminopeptidase but similar to GSTµ. Inhibition of FCGAP prevents fiber cell globulization and cataractogenesis indicating the therapeutic significance of FCGAP inhibitors.
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