Purification and biochemical characterization of ovine alpha-1-proteinase inhibitor: mechanistic adaptations and role of Phe350 and Met356.
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.
Gupta
Vivek K
VK
Department of Protein Chemistry and Technology, Central Food Technological Research Institute, Mysore, Karnataka 570020, India.
Appu Rao
A G
AG
Gowda
Lalitha R
LR
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002850:Chromatography, Gel; D019075:Electrophoresis, Capillary; D006031:Glycosylation; D006801:Humans; D008715:Methionine / Q000378:metabolism*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010649:Phenylalanine / Q000378:metabolism*; D012697:Serine Endopeptidases / Q000378:metabolism; D034561:Sheep, Domestic; D040681:Structural Homology, Protein; D013329:Structure-Activity Relationship; D013816:Thermodynamics; D013997:Time Factors; D000515:alpha 1-Antitrypsin / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*
D000515:alpha 1-Antitrypsin; D010649:Phenylalanine; D008715:Methionine; D012697:Serine Endopeptidases