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Title: A Kunitz-type glycosylated elastase inhibitor with one disulfide bridge
Authors: Sumikawa, J. T.
Nakahata, A. M.
Fritz, H.
Mentele, R.
Sampaio, M. U.
Oliva, MLV
Universidade Federal de São Paulo (UNIFESP)
Max Planck Inst Biochem
Keywords: Bauhinia rufa
elastase/antagonists and inhibitors
Kunitz inhibitor
serine endopeptidases
Issue Date: 1-Apr-2006
Publisher: Georg Thieme Verlag Kg
Citation: Planta Medica. Stuttgart: Georg Thieme Verlag Kg, v. 72, n. 5, p. 393-397, 2006.
Abstract: A glycosylated Bauhinia rufa elastase inhibitor (gBrEI) was purified and characterized using acetone precipitation, affinity chromatography on concanavalin A-Sepharose, ion-exchange chromatography on a HiTrap Q column, size exclusion chromatography on a Superdex 200 column and reverse-phase chromatography on a C-18 column. gBrEI inhibited pancreatic porcine elastase with an equilibrium dissociation constant (K-i) of 6.18 x 10(-8) M, but it did not inhibit human neutrophil elastase, bovine trypsin, human plasma kallikrein or porcine pancreatic kallikrein. On SDS-electrophoresis, gBrEI appeared as a single 20-kDa band, also after reduction. Schiff reagent staining indicated a carbohydrate portion in the protein, which was confirmed by mass spectrometry. the glycosylated site was Asn(38), and a carbohydrate portion of 1.17 kDa was identified. gBrEI was found to contain 144 amino acid residues, and a FASTA database analysis showed that it belongs to the plant Kunitz-type inhibitor family. Val(66) was identified as reactive site P1 residue by comparison of conserved positions in the sequences. Since gBrEI harbors a single disulfide bridge, it may be considered a new type of Kunitz inhibitor, intermediate between the classical kunitz inhibitors, which contain two disulfide bridges, and those from B. bauhinioides, which do not have such bridges.
ISSN: 0032-0943
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Appears in Collections:Em verificação - Geral

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