PubMed Health⌕ Search

Biomedical subjects

M Davril

Publications and source records attributed to M Davril.

At least 37 records · Page 2Linked to original sources
PubMed1987

Studies on the interactions of human pancreatic elastase 2 with human alpha 1-proteinase inhibitor and alpha 1-antichymotrypsin.

The interactions of human pancreatic elastase 2 with alpha 1-proteinase inhibitor and alpha 1-antichymotrypsin were compared by studies in vitro. The equimolar complexes obtained between the enzyme and either inhibitor were relatively stable at 25 degrees C since they could be visualized for up to 5 days by an electrophoretic method. However, in both cases, a slow dissociation occurred with release of active enzyme. As the kass. rate constants are of the same order of magnitude, with a slightly lower value for alpha 1-proteinase inhibitor when compared with alpha 1-antichymotrypsin [(5.6 +/- 1.2) X 10(5) and (8.9 +/- 1.3) X 10(5) M-1.s-1 respectively], partition of human pancreatic elastase 2 between both inhibitors in human plasma is mainly dependent on their respective concentrations. A comparative study by crossed immunoelectrophoresis of the interactions of this enzyme with the two inhibitors contained in normal human plasma and in a mimetic mixture of pure inhibitors was carried out. This allowed the visualization of complexes with either inhibitor. Formation of such a complex with alpha 1-antichymotrypsin had never been demonstrated previously. The patterns obtained are similar when working with normal plasma or with the synthetic mixture, suggesting that, in the conditions used, alpha 1-proteinase inhibitor and alpha 1-antichymotrypsin are the main inhibitors of human pancreatic elastase 2 in the plasma sample. However, it is also shown that part of the enzyme may be taken up by alpha 2-macroglobulin, which is responsible for the remaining enzyme activity on a synthetic substrate. The present work suggests that, according to the delay times of inhibition of human pancreatic elastase 2 calculated from the normal plasma concentrations of alpha 1-proteinase inhibitor and alpha 1-antichymotrypsin, a significant role can be assigned to both inhibitors. Moreover, the role of alpha 1-antichymotrypsin would be enhanced in alpha 1-proteinase-inhibitor deficiency.

Blood Proteins↗
PubMed1987

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

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 F1N (Mr's 30,000-10,000) which was eluted later than the excluded fraction F1A (Mr'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 C18 column using a multi-step gradient elution procedure. Different patterns were observed for the high (F1N) and the low (F2N) 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 F1N and F1A, 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.

Adult↗
PubMed1987

Photolysis and ozonolysis of (iso)desmosine-containing crosslinked peptides from porcine aorta elastin.

This report describes the use of photolysis and ozonolysis as a means of achieving complete cleavage of the pyridinium ring of (iso)desmosine in crosslinked elastin peptides. Although photolysis leads to the opening of the ring with concomitant formation of lysine, the peptide chains remain attached. Subsequent ozonolysis is able to completely achieve the cleavage of the rest of the ring skeleton, thus leading to the separation of the peptide chains. Formation of new amino acids, i.e. alpha-aminoadipic and glutamic acids, is emphasized. Localization of these amino acids within the released peptides should be of help in structural investigations on the crosslinking zones involving either isodesmosine or desmosine. However, other amino acids such as tyrosine and phenylalanine are sensitive to this procedure and side reactions occur which are responsible for peptide bond cleavage with the formation of breakdown products.

Amino Acids↗
PubMed1987

"In vivo" and "in vitro" inhibition of human pancreatic chymotrypsin A by serum inhibitors.

The interaction of human pancreatic chymotrypsin A with serum inhibitors was assessed by enzyme immunoassay, enzymatic activity and inhibitory capacity measurements and electrophoretic analyses. In normal serum, chymotrypsin A was detected in four forms: one form (Mr approximately equal to 25,000) which might be chymotrypsinogen A and three forms complexed to the main inhibitors present in serum, alpha 2-macroglobulin (alpha 2-M), alpha 1-proteinase inhibitor (alpha 1-PI) and alpha 1-antichymotrypsin (alpha 1-Achy). As chymotrypsin A remains to 90% active when bound to alpha 2-M, the chymotrypsin A/alpha 2-M complex was quantified by an enzymatic assay. The kinetic parameters of the interaction of chymotrypsin A with alpha 1-PI and alpha 1-Achy were determined. Using these data the partition of chymotrypsin A between the different inhibitors in serum was calculated. In acute pancreatitis, the chymotrypsin A plasma level follows the progression of the disease and in this case as well as in normal serum alpha 1-PI is the major antagonist of chymotrypsin A.

Blood Proteins↗
PubMed1987

The effect of the glycosaminoglycan chain removal on some properties of the human urinary trypsin inhibitor.

The major urinary trypsin inhibitor UTI I is a proteoglycan. UTI c (Mr 26,000), produced by chrondroitin lyase digestion of UTI I, was isolated and characterized. About 90% of the glycosaminoglycan chain was removed by this treatment without proteolytic modification, as assessed by amino-acid composition and N-terminal sequence of UTI c. Its electrophoretic mobilities on alkaline and SDS-PAGE are identical with those of UTI II which occurs in urine during storage. To study the role of the glycosaminoglycan chain on the inhibitory properties of UTI I, UTI I and UTI c were compared using different proteinases as target enzymes. The inhibitory activity towards bovine trypsin and chymotrypsin as well as human granulocytic cathepsin G did not differ significantly. However, towards human granulocytic elastase, the equilibrium dissociation constant (Ki) is 5 times higher for UTI c than for UTI I. Weak inhibitory activities were measured on human plasmin, UTI c being more efficient than UTI I. The acid-stability of UTI I is not modified after chrondroitin lyase treatment. UTI I and UTI c are equally sensitive to trypsinolysis indicating that the covalently bound glycosaminoglycan chain does not play an important role for the stability of UTI I.

Cathepsin G↗
PubMed1986

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

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 (Mr's: 80,000 to 30,000) and a small retained one (Mr's: 6000 to 4000). Conversely, cathepsin G digests were eluted as a minor excluded fraction and a more important retarded one (Mr'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.

Amino Acids↗
PubMed1985

Human serum alpha 1-antichymotrypsin is an inhibitor of pancreatic elastases.

Incubation of human serum alpha 1-antichymotrypsin with human pancreatic elastase 2 or porcine pancreatic elastase results in the complete inhibition of each enzyme as determined by spectrophotometric assays. alpha 1-Antichymotrypsin reacts much more rapidly with the human than with the porcine enzyme. The inhibitor: enzyme molar ratio, required to obtain full inhibition of enzymatic activity, is equal to 1.25/1 when alpha 1-antichymotrypsin reacts with human pancreatic elastase 2 while it is markedly higher with porcine pancreatic elastase (5.5/1). Patterns obtained by SDS/polyacrylamide gel electrophoresis of the reaction products show the formation with both enzymes of an equimolar complex (Mr near 77 000) and the release of a fragment migrating as a peptide of Mr near 5000. Moreover a free proteolytically modified form of alpha 1-antichymotrypsin, electrophoretically identical with that obtained in the reaction with cathepsin G or bovine chymotrypsin, is produced in the reaction with each elastase but in a much greater amount when alpha 1-antichymotrypsin reacts with porcine elastase than with human elastase. As a consequence of our findings, the specificity of alpha 1-antichymotrypsin, so far limited to the inhibition of chymotrypsin-like enzymes from pancreas and leukocyte origin, has to be extended to the two pancreatic elastases investigated in this work. A contribution of alpha 1-antichymotrypsin to the regulatory balance between plasma inhibitors and human pancreatic elastase 2 in pancreatic diseases is suggested.

Animals↗
PubMed1985

Human urinary proteinase inhibitor: inhibitory properties and interaction with bovine trypsin.

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.

Animals↗
PubMed1985

Interaction of human alpha 1-proteinase inhibitor with human leukocyte cathepsin G.

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↗
PubMed1985

Photolysis and partial ozonolysis of kappa-elastin from ligamentum nuchae.

Photolysis and partial ozonolysis of guanidinated kappa-elastin led to the destruction of isodesmosine and desmosine up to 96.0 and 91.5% respectively. Simultaneously there was a 3.89-fold enrichment of the lysine content. Two lysine containing tripeptides and one dipeptide were isolated and characterized. Their amino acid sequences were Gly-Ala-Lys, Gly-Lys-Ala and Lys-Ala, respectively.

Amino Acid Sequence↗
PubMed1984

Interaction of human alpha 1-antichymotrypsin with chymotrypsin.

Human alpha 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 degree C or at 25 degrees C, show that the equimolar complex dissociates spontaneously; this dissociation results in the release of inactive modified alpha 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 alpha 1-antichymotrypsin.

Animals↗
PubMed1984

Arginine modification in elastase. Effect on catalytic activity and conformation of the calcium-binding site.

Chemical modification of 2 +/- 0.5 arginine residues of porcine pancreatic elastase by 1,2-cyclohexanedione leads to an 85 +/- 5% loss of activity with the specific substrate N-succinyltrialanine p-nitroanilide. Modification of additional arginines does not completely abolish the enzyme activity. The modification reaction is very fast (second order rate constant = 0.24 M-1 S-1) and involves only arginine residues. Acetyltetraalanine or trifluoroacetyltetraalanine decreases the rate of cyclohexanedione-induced inactivation of the enzyme but does not significantly change the number of modified arginine residues. Other dicarbonyl reagents, butanedione or phenylglyoxal, also react with elastase but at much lower rates. Cyclohexanedione-modified elastase is partially active against a series of synthetic substrates of varying chain length. The partial inhibition results from a 2- to 5-fold increase in Km while kappa cat is increased for most substrates. For N-succinyltrialanine p-nitroanilide both the acylation and deacylation rate constants are decreased. The Ki values of a series of acetylated and trifluoroacetylated inhibitors increase 2- to 5-fold. Modified elastase is still able to react with fibrous elastin and with plasma alpha 1-proteinase inhibitor but at significantly lower rates. Modification of one arginine residue alters the properties of the calcium-binding site of elastase as demonstrated by terbium luminescence experiments. The affinity of enzyme for terbium is decreased by a factor of 10 and the circularly polarized luminescence spectrum of the terbium-elastase complex is considerably flattened. Modification of further arginine residues does not increase the extent of these alterations. Circular dichroism shows that the overall conformation of elastase is not altered following arginine modification. We speculate that the two residues modified by cyclohexanedione are Arg 65, located at about 8 A from the metal ion-binding site, and Arg 217A, located at the S'3 subsite of elastase.

Animals↗
PubMed1981

Accessibility of epsilon-amino groups of lysine to guanidination in kappa-elastin from bovine ligamentum nuchae.

Kappa-Elastin contains 3.81 lysyl residues per 1000 residues of amino acids. Among these residues, free epsilon-amino groups represent about 16 p. 100, as revealed by guanidination, the buried lysyl residues (buried epsilon-amino groups) represent about 33 p. 100, as revealed by dansylation. After drastic reduction by borohydride, no aldimine bonds were detected and only 6 p. 100 of "deeply buried" amino groups of lysyl residues were detected by a second dansylation. The remaining lysines (about 46 p. 100) are engaged or inaccessible.

Animals↗
PubMed1981

[Results of leukocyte migration inhibition test against elastin in joint diseases (author's transl)].

UNLABELLED: The purpose of this work was to report the results of the test TML inhibition of leukocytes' migration, using elastin, by Soborg and Bendixen method, modified with elastin rates of 100 micrograms and 200 micrograms/ml, in joints disease. We verified that the test is generally negative in healthy controls (1 TML positive out of 26 cases); we studied 72 patients, and 38 out of them had a positive test. TML is frequently positive in Horton disease, spondylarthritis, rheumatoid polyarthritis without corticoids, collagen disease; in this cases there is a significant difference of migration between patients and controls group. We also noted a positive TML in infections arthritis and other inflammatory but not in joint's diseases with alteration of elastic tissue (scabs and lung's injury). IN CONCLUSION: TML positive test with elastin, is not a specific test of articular injury neither a proof of its immunologic nature but it contributes in diagnostic for joints diseases.

Adult↗
PubMed1981

Partial characterization of peptide fragment purified by isoelectrofocusing after organo alkaline hydrolysis of bovine ligamentum nuchae elastin.

Highly purified elastin from bovine ligamentum nuchae was submitted to partial alkaline hydrolysis (37 degree C, 72 H, 1 N KOH in 80 p. 100 aqueous ethanol). The non-coacervable fractions were submitted to isoelectrofocusing and five kappa-elastin fractions were obtained. The amino-acid compositions, the N-terminal amino-acids, the molecular weights and the thermolysin digests of each fraction were determined by various techniques. The average MW was about 14,500 (150 - 166 amino-acids). These results suggest that the distribution of cross-linking agents in fibrous elastin may not be uniform. The results also show that in certain cross-linked regions of similar molecular weight and size appearing to be composed of different polypeptides sequences containing different amounts of cross-links.

Animals↗