Localization of the two free thiol groups in the porcine pancreatic alpha-amylase I sequence |
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Authors: | L Pasero Y Mazzei B Abadie D Moinier M Fougereau G Marchis-Mouren |
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Affiliation: | 1. Institut de Chimie Biologique, Place Victor-Hugo, 13331 Marseille Cedex 3, France |
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Abstract: | Porcine pancreatic alpha-amylase I, a single 496 residue long polypeptide chain, contains 5 disulfide bridges and 2 free -SH groups. The conditions for specific blocking of native amylase either with radioactive N-ethyl maleimide or with labeled iodoacetic acid were determined. Under these conditions 2 moles of blocking reagent are incorporated per mole of amylase. [14C]-S-succinimido amylase was cleaved by CNBr and the resulting peptides were purified. Only one of them the CNBr 2 + 3 peptide (178 residues) was found labeled. Ts1 a 33-residue peptide containing the whole radioactivity was purified from the tryptic digest of this large fragment. After reduction and carboxymethylation Ts1A, (22 residues) was obtained which contains 2 moles of succinyl-Cys and one mole of CM-Cys per mole of peptide. Chymotryptic digestion of Ts1A yielded 2 equally labeled peptides: C1 (16 residues) and C2 (6 residues). Automated sequencing of both peptides and counting of the PTH-amino acids shows that the free cysteines are only 15 residues apart in the sequence. |
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Keywords: | PTH Phenylthiohydantoine CNBr Cyanogen bromide DTNB 5 5′-dithiobis-(2-nitrobenzoic acid) NEM N-ethyl maleimide |
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