Brigitte Abadie

Complete amino acid sequence and location of the five disulfide bridges in porcine pancreatic α-amylase

Abstract Porcine pancreatic α-amylase (1,4-α- d -glucan glucanohydrolase EC 3.2.1.1), a single polypeptide chain, contains nine residues of methionine. Eight different fragments resulting from cleavage of this molecule by cyanogen bromide were characterized. The sequences of six of them have previously been reported. Two missing fragments, CN2 (82 residues) and CN3b1 (76 residues) were purified after breaking of the interpeptidic disulfide bridge and their complete sequence as well as that of the previously purified CN1 peptide (102 residues) are reported here. The location of the three disulfide bridges present in these peptides was determined. Ordering of the carboxymethylated cyanogen bromide fragments was carried out by pulse labeling the amylase chain in vivo. The complete sequence of the porcine pancreatic amylase chain (496 residues) and the location of its five disulfide bridges is presented. Comparison with human and mouse pancreatic and salivary α-amylases and with rat pancreatic amylase obtained from the corresponding cDNA nucleotidic sequences shows a high degree of homology between mammalian α-amylases.

Role of αvβ5 and αvβ6 integrin glycosylation in the adhesion of a colonic adenocarcinoma cell line (HT29‐D4)

We have previously characterized the expression of the αvβ5 and αvβ6 integrins as major receptors for the human colonic adenocarcinoma cell line (HT29‐D4), on vitronectin and fibronectin, respectively [Lehmann et al. (1994): Cancer Res 54:2102–2107]. In the present work we investigated the glycosylation role of these integrins in their adhesive functions. To this end, we used glycohydrolases to show that cell surface integrins were N‐glycosylated and sialylated, and that only the αv subunit carried some immature oligosaccharide side chains. To alter the glycosylation state of the cell surface αvβ5 and αvβ6 integrins, we used two oligosaccharide‐processing inhibitors: 1‐deoxymannojirimycin (dMNJ) and tunicamycin (TM). Following treatment of HT29‐D4 cells with dMNJ, cell surface αvβ5 and αvβ6 carried only high‐mannose‐type sugar chains, while TM‐treated cells expressed de‐N‐glycosylated integrins. Neither α/β heterodimers assembly nor cell surface expression were impaired in the presence of the drugs. Finally, we established that adhesion of dMNJ‐ or TM‐treated cells was altered on both vitronectin and fibronectin substrata, whereas the adhesion of these cells on laminin or collagen type I was virtually unchanged.

Localization of the two free thiol groups in the porcine pancreatic α-amylase I sequence

Abstract Porcine pancreatic α-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. Tsl a 33-residue peptide containing the whole radioactivity was purified from the tryptic digest of this large fragment. After reduction and carboxymethylation TslA, (22 residues) was obtained which contains 2 moles of succinyl-Cys and one mole of CM-Cys per mole of peptide. Chymotryptic digestion of TslA yielded 2 equally labeled peptides: Cl (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: C(SH) G S G A A A G T G T T C G S Y C(SH) N P G N R