Annette Hayem

Inserm

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M Davril

Inserm

Anne Laine

Centres for Economic Development, Transport and the Environment

Dominique Vercaigne‐Marko

Polytech Lille

Marie Smyrlaki

Inserm

Catherine Figarella

Pathophysiology, Diagnosis and Treatment of Bone Diseases

Jacqueline Carrère

Hôpital Renée Sabran

ΈH

Έ. Hachulla

Université de Lille

Isabelle Van Seuningen

Inserm

J Mizon

Université de Lille

Bernard Bayard

Université de Montpellier

Cooperative Institutions

Inserm

Université de Lille

Centre National de la Recherche Scientifique

Centre Hospitalier Universitaire de Lille

Institut de Biologie de Lille

Laboratoire de Biochimie

Institut pour la Recherche sur le Cancer de Lille

Hôpital Renée Sabran

Hôpital Roger Salengro

Centre de Recherche Jean Pierre Aubert

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Column separation using Bio‐Gel P100 for the characterization of the products of human lung elastin degradation by leucocyte elastase and cathepsin G

Biomedical Chromatography (1986)

Marie Smyrlaki M Davril Annette Hayem

Abstract The solubilization of human lung elastin by leucocyte elastase and cathepsin G is described. Elastolysis kinetic studies clearly show that leucocyte elastase is more efficient in solubilizing elastin fibres than is cathepsin G. Cathepsin G can degrade elastin but at a much slower rate. Characterization of elastase and cathepsin G soluble elastin fragments, obtained after 24 h of digestion (enzyme‐substrate ratio, 1:100), was first performed by isoelectric focusing. Whole digests were focused as 6 bands in a pH range 4.2 to 4.7 and were found to have no significant differences in amino acid compositions. Biogel P‐100 gel filtration of the elastase digested fragments separated a major excluded fraction ( M r 's: 80 000 to 30 000) and a small retained one ( M r 's: 6000 to 4000). Conversely, cathepsin G digests were eluted as a minor excluded fraction and a more important retarded one ( M r 's: 6000 to 4000). Only the high molecular weight fractions of both enzymes digests contain crosslinked amino acids; this assigns a role for desmosines in the resistance of elastin to these proteases. These results are discussed in comparison with the data obtained by others.

Cathepsin G

Pancreatic elastase

Digestion

10.1002/bmc.1130010107

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Citations (3)

Chromatographic studies of human lung elastin digestion products obtained by leucocyte elastase: Comparison between newborn and adult soluble fragments

Biomedical Chromatography (1987)

Marie Smyrlaki M Davril Annette Hayem

Abstract Human insoluble elastin was prepared from newborn lungs and digested by leucocyte elastase. The soluble fragments were compared to those obtained from adult lung elastin in a previous work (Smyrlaki et al. , 1986). Gel filtration on a Bio‐Gel P‐100 column of newborn elastin allowed the separation of fraction F 1 N (M r 's 30 000–10 000) which was eluted later than the excluded fraction F 1 A (M r 's 80 000–30 000) previously isolated from adult elastin. The difference in the sizes of the large peptide fragments originating from both elastins was also shown on SDS‐PAGE. Reversed phase HPLC was performed on a C 18 column using a multi‐step gradient elution procedure. Different patterns were observed for the high (F 1 N) and the low (F 2 N) molecular size fragments of newborn elastin. The same peak distribution was obtained with adult elastin. Comparison of the amino acid compositions of the most retained peaks (3, 4 and 5), derived from fractions F 1 N and F 1 A, showed analogies for the contents of the major nonpolar amino acids and crosslinks. Thus, this procedure allowed the separation of typical fragments of elastin which might be released in vivo by leucocyte elastase during pulmonary diseases.

Digestion

Fraction (chemistry)

Amino Acid Analysis

10.1002/bmc.1130020204

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Citations (1)

Interaction between human serum α1-antichymotrypsin and human leukocyte cathepsin G complex formation and production of a modified inhibitor

Biochemical and Biophysical Research Communications (1982)

Anne Laine M Davril Annette Hayem

Cathepsin G

Molar ratio

Stoichiometry

Cathepsin C

Complex formation

10.1016/s0006-291x(82)80029-6

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Citations (26)

The microheterogeneity of serum α₁‐antichymotrypsin revealed by interaction with concanavalin A in crossed immunoaffinoelectrophoresis and in affinity chromatography

Electrophoresis (1989)

Anne Laine Έ. Hachulla Annette Hayem

Abstract In crossed immunoaffinoelectrophoresis with free concanavalin A in the first dimension, human serum α 1 ‐antichymotrypsin, purified or in whole serum, exhibited four peaks in presence of 0.02 M α‐methyl‐ D ‐glucoside added to the second‐dimensional gel. α 1 ‐Antichymotrypsin purified from the serum of a single healthy donor was separated by affinity chromatography into three fractions on a laboratory‐prepared concanavalin A‐Sepharose 4B column: a pass‐through fraction, a retarded fraction and a bound fraction, eluted from the column on addition of sugar to the buffer. These three fractions were analyzed by crossed immunoaffinoelectrophoresis and sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and isoelectric focusing before and after desialylation. The results of electrophoresis as well as chemical analyses indicate that these microheterogeneous forms carry glycans with decreasing degrees of branching from the concanavalin A‐pass‐through form to the concanavalin A‐bound form. This approach represents a first step towards the elucidation of the molecular basis of the microheterogeneity of α 1 ‐antichymotrypsin.

Fraction (chemistry)

Sodium dodecyl sulfate

10.1002/elps.1150100402

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Citations (8)

Interaction of human α₁‐antichymotrypsin with chymotrypsin

European Journal of Biochemistry (1984)

Anne Laine M Davril Annette Hayem

Human α 1 ‐antichymotrypsin reacts with bovine chymotrypsin to form an equimolar complex and this reaction is accompanied by the formation of a free, modified form of the inhibitor. Time‐course studies, performed on mixtures containing an excess of native inhibitor and kept at 0°C or at 25°C, show that the equimolar complex dissociates spontaneously; this dissociation results in the release of inactive modified α 1 ‐antichymotrypsin and of some active enzyme, which is able to recycle with active inhibitor in excess. When all the native inhibitor is used up, the released active enzyme degrades the remaining intact complex into intermediate forms. At the endpoint of the reaction only inactive modified inhibitor and some active chymotrypsin remain. Immunochemical data indicate that, in the complex, a steric hindrance of the antigenic determinants of the inhibitor prevents the formation of the precipitate with specific antiserum. Inactive modified inhibitor, which has dissociated from the complex, has retained antigenic determinants of the native α 1 ‐antichymotrypsin.

10.1111/j.1432-1033.1984.tb08072.x

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Citations (10)

Uniformity of Glycans within Molecular Variants of α_l‐Protease Inhibitor with Distinct Affinity for Concanavalin A

European Journal of Biochemistry (1982)

Bernard Bayard Jean‐Pierre Kerckaert Anne Laine Annette Hayem

Human a,‐protease inhibitor contains four asparagine‐linked carbohydrate chains per molecule. Three types of carbohydrate chains were released from the polypeptide backbone by hydrazinolysis:(a) biantennary (80%),(b) biantennary with an intercalated N‐acetylglucosamine residue (14%), and (c) triantennary (6%). Using concanavalin‐A‐affinity chromatography, native and S‐carboxymethylated α 1 ,‐protease inhibitor were fractionated into three distinctmolecular variants which were shown to contain only one type (a,b or c, respectively) of glycan per molecule. This and previous observations on other serum glycoproteins support the proposal of uniformity of glycan type within individual molecular variants of glycoproteins.

Residue (chemistry)

Protease inhibitor (pharmacology)

Oligosaccharide

10.1111/j.1432-1033.1982.tb06602.x

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Citations (27)

Human Urinary Proteinase Inhibitor: Inhibitory Properties and Interaction with Bovine Trypsin

Biological Chemistry Hoppe-Seyler (1985)

Malika Balduyck M Davril C Mizon Marie Smyrlaki Annette Hayem

The major urinary trypsin inhibitor (UTI) was found to inhibit bovine chymotrypsin and human leucocyte elastase strongly, cathepsin G weakly. No inhibition of porcine pancreatic elastase was observed. The stoichiometry of the inhibition of bovine trypsin by UTI was determined spectrophotometrically to be 1:2 (I/E molar ratio). After incubation of UTI with this enzyme in various molar ratios, two complexes (C1 and C2) could be visualized in alkaline polyacrylamide gel electrophoresis. C1 was isolated by affinity chromatography on Con-A Sepharose. In dodecyl sulfate polyacrylamide gel electrophoresis, C1 was dissociated to give an inhibitory band with the same electrophoretic mobility as native UTI. C2 released an active inhibitory fragment with Mr near 20000. A time-course study demonstrated that at a molar ratio I/E of 1.5:1, the C2 complex appears after two hours of incubation.

Cathepsin G

Pancreatic elastase

Sepharose

Sodium dodecyl sulfate

10.1515/bchm3.1985.366.1.9

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Citations (19)

HUMAN a₁-ANTICHYMOTRYPSIN : PURIFICATION, PROPERTIES AND REACTIONS WITH SOME SERINE PROTEASES

De Gruyter eBooks (1984)

Anne Laine Annette Hayem M Davril

10.1515/9783112328262-019

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Citations (0)

Strong ionic interactions between mucins and two basic proteins, mucus proteinase inhibitor and lysozyme, in human bronchial secretions

International Journal of Biochemistry (1992)

Isabelle Van Seuningen Nicole Houdret Annette Hayem M Davril

Differential centrifugation

10.1016/0020-711x(92)90262-y

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Citations (28)

Interaction of Human α₁-Proteinase Inhibitor with Human Leukocyte Cathepsin G

Biological Chemistry Hoppe-Seyler (1985)

Dominique Vercaigne‐Marko M Davril Anne Laine Annette Hayem

Interaction of human plasma alpha 1-proteinase inhibitor (alpha 1-PI) with human leukocyte cathepsin G was assessed by proteinase inhibitory capacity assays and electrophoretic analyses. Only a part of purified alpha 1-PI formed a 1 : 1 complex (Mr = 74 000) with leukocyte cathepsin G. The formation of this complex was accompanied by the appearance of a great amount of a free, proteolytically modified form of alpha 1-PI (Mr = 50 000) which had lost its inhibitory capacity. The complex was unstable with time and the only dead end product observed was a modified, inactive form of alpha 1-PI. During the breakdown of the complex, no release of active enzyme could be measured by spectrophotometric assays. In spite of the fact that the equimolarity of the complex was convincingly demonstrated, studies of the alpha 1-PI-cathepsin G reaction showed that total inhibition of this proteinase needed a large molar excess of alpha 1-PI. Nevertheless, alpha 1-PI acts as an efficient "suicide inhibitor" since no cathepsin G recovery was ever found.

Cathepsin G

Proteinase inhibitor

Alpha (finance)

10.1515/bchm3.1985.366.2.655

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Citations (13)