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Purification and biochemical characterization of ovine alpha-1-proteinase inhibitor: mechanistic adaptations and role of Phe350 and Met356.

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Relation http://ir.cftri.com/1820/
PEP-01-08
 
Title Purification and biochemical characterization of ovine alpha-1-proteinase inhibitor: mechanistic adaptations and role of Phe350 and Met356.
 
Creator Gupta, Vivek K
Appu Rao, A. G.
Gowda, Lalitha R
 
Subject 03 Proteins
 
Description The glycoprotein alpha-1-proteinase inhibitor (alpha-1-PI) is a member of the serpin super family that causes rapid and irreversible inhibition of redundant serine protease activity. A homogenous preparation of ovine alpha-1-PI, a 60 kDa protein was obtained by serially subjecting ovine serum to 40-70% (NH(4))(2)SO(4) precipitation, Blue Sepharose, size-exclusion, and concanavalin-A chromatography. Extensive insights into the trypsin, chymotrypsin, and elastase interaction with ovine alpha-1-PI, point towards the involvement of Phe(350) besides the largely conserved Met(356) in serine protease recognition and consequent inhibition. The N-terminal of C-terminal peptides cleaved on interaction with elastase, trypsin, and chymotrypsin prove the presence of diffused sub-sites in the vicinity of Met(356) and the strategically positioned Pro anchored peptide stretch. Further, human alpha-1-PI is more thermolabile compared to ovine alpha-1-PI, higher thermolability is mainly attributed to poorer glycosylation. The enzymatic deglycosylation of human and ovine alpha-1-PI results in diminished thermostability of the inhibitors, with sharp decrease in thermal transition temperatures but retaining their inhibitory potency. Homology modeling of the deduced amino acid sequence of ovine alpha-1-PI using the human alpha-1-PI template has been used to explain the observed inhibitor-protease interactions.
 
Date 2008
 
Type Article
PeerReviewed
 
Format application/pdf
 
Language en
 
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Identifier http://ir.cftri.com/1820/1/Protein_Expression_and_Purification%2C_Volume_57%2C_Issue_2%2C_February_2008%2C_Pages_290-302.pdf
Gupta, Vivek K and Appu Rao, A. G. and Gowda, Lalitha R (2008) Purification and biochemical characterization of ovine alpha-1-proteinase inhibitor: mechanistic adaptations and role of Phe350 and Met356. Protein Expression and Purification, 57 (2). pp. 290-302. ISSN 1046-5928