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Biomedical subjects

E Dufour

Publications and source records attributed to E Dufour.

48 records · Page 3Linked to original sources
PubMed1992

Binding of benzo(a)pyrene, ellipticine, and cis-parinaric acid to beta-lactoglobulin: influence of protein modifications.

The binding of benzo(a)pyrene, ellipticine, and cis-parinaric acid to native, esterified, and alkylated beta-lactoglobulin was followed by enhancement of the ligand fluorescence. Three studied ligands bind to native or modified beta-lactoglobulin in apparent molar ratios varying between 1/8 and 2/1, with apparent dissociation constants in the range of 10(-8) M for ligand/beta-lactoglobulin complexes. The studied, chemically modified beta-lactoglobulin derivatives display higher binding affinities for all studied ligands, cis-parinaric acid excluded. The reductive alkylation of epsilon-NH2 lysyl residues of beta-lactoglobulin increases the apparent molar ratios of benzo(a)pyrene and cis-parinaric acid, and decreases it for ellipticine. The esterified and native beta-lactoglobulin complexed to the investigated ligands display similar stoichiometries. Dynamic light scattering study of ligand-beta-lactoglobulin complexes in solution shows the formation of aggregates: the apparent hydrodynamic radius value of beta-lactoglobulin dimer (3.4 nm) reaches 49, 46, and 74 nm upon addition and binding of benzo(a)pyrene, ellipticine, and cis-parinaric acid, respectively.

Alkylation↗
PubMed1991

Binding of retinoids and beta-carotene to beta-lactoglobulin. Influence of protein modifications.

The binding of retinol, retinyl acetate, retinoic acid and beta-carotene to native, esterified and alkylated beta-lactoglobulin was followed by quenching of tryptophan fluorescence. Three studied retinoids bind to native or modified beta-lactoglobulin in 1:1 molar ratios, with apparent dissociation constants in the range of 10(-8) M. The maximum tryptophan fluorescence quenching of unmodified beta-lactoglobulin by beta-carotene is observed at the ligand/protein ratio of 1:2. Esterification and alkylation of beta-lactoglobulin shift the ratio of beta-carotene/protein to 1:1. In all the cases, except for retinoic acid binding to N-ethyllysyl-BLG, the performed chemical modifications of beta-lactoglobulin enhance protein binding affinity. Measured apparent dissociation constants of beta-carotene complexes with native and modified beta-lactoglobulin are an order of magnitude lower from binding constants of other studied retinoids.

Carotenoids↗
PubMed1991

Influence of pH on the structural changes of beta-lactoglobulin studied by tryptic hydrolysis.

The attempt to use trypsin in order to monitor pH (7.5-9.0) induced beta-lactoglobulin conformation changes has revealed differences in the cleavage of specific sites. The tryptic cleavage of two dibasic X-Lys-Lys-Y sites (Lys 69, 70 and 100, 101) shows slighter predominance of symmetrical cut at pH 7.5 and 8.0. Mostly asymmetrical cleavage yielding two C-terminal lysines can be observed at pH 8.5 and 9.0. Atypical cleavage of the Tyr-20-Ser-21 site, which at pH 9.0 is relatively negligible, increases substantially in pH 7.5-8.5. This implies that Tyr-20 probably is the tyrosine reported to be exposed on the surface of the protein during transformation of beta-lactoglobulin molecule occurring in the studied pH range (Tanford et al. (1959) J. Am. Chem. Soc. 81, 4032-4036).

Amino Acid Sequence↗
PubMed1990

Beta-lactoglobulin binds retinol and protoporphyrin IX at two different binding sites.

Measurement of tryptophan fluorescence quenching and the excitation energy transfer from tryptophanyl residues to the bound ligand indicates that beta-lactoglobulin binds tightly to hemin and protoporphyrin IX in a ligand-to-protein stoichiometric ratio. The apparent dissociation constants of hemin-beta-lactoglobulin and protoporphyrin IX-beta-lactoglobulin complexes are 2.5 x 10(-7) M and 4 x 10(-7) M, respectively. The addition of beta-lactoglobulin (final concentration = 10 microM, phosphate buffer 50 mM, pH 7.1) to the solution containing retinol and protoporphyrin IX triggers an energy transfer between beta-lactoglobulin tryptophan and protoporphyrin IX as well as between retinol and protoporphyrin IX. The efficiency of energy transfer depends on the distance between the donor (retinol) and the acceptor (protoporphyrin IX). Using the Förster theory, a retinolprotoporphyrin IX distance of 25 A was calculated. These results indicate that retinol and protoporphyrin IX are bound to the beta-lactoglobulin monomer at two different sites.

Animals↗
PubMed1990

Alcohol-induced changes of beta-lactoglobulin-retinol-binding stoichiometry.

It has been demonstrated using CD that ethanol induces important secondary structure changes of beta-lactoglobulin. CD spectra indicate that beta-lactoglobulin secondary structure, which is mainly composed of beta-strands, becomes mostly alpha-helical under the influence of the solvent polarity changes. The midpoint of beta-strand/alpha-helix transition in beta-lactoglobulin is observed at dielectric constant approximately 60 (35% ethanol; v/v). According to CD measurements, the ethanol-dependent secondary structure changes are reversible. The alkylation of lysines epsilon-NH2 in beta-lactoglobulin weakens the central beta-barrel structure, since the beta-strand/alpha-helix transition midpoint of alkylated beta-lactoglobulin is shifted to lower ethanol concentration (25% ethanol; v/v). beta-Lactoglobulin structural changes are triggering the dissociation of the beta-lactoglobulin-retinol complex as judged from complete quenching of its fluorescence in ethanol concentration greater than 30% (v/v). However, in 20% ethanol (v/v), beta-lactoglobulin still retains most of its native secondary structure as shown by CD and, in this condition, one beta-lactoglobulin molecule binds an additional second retinol molecule. This suggests that the highly populated species observed around 20% ethanol (v/v) might represent an intermediate state able to bind two molecules of retinol.

Alkylation↗
PubMed1989

Lysosomal proteinase-sensitive regions in fast and slow skeletal muscle myosins.

We investigated the limited proteolysis of fast and slow myosins purified from rabbit psoas major and semimembranosus proprius muscles, respectively, by the main lysosomal proteinases: cathepsins B, H, L, and D. In EDTA containing buffer, cathepsin D cleaved both myosins only at the rod-S1 junction leading to the formation of two S1 fragments of slightly higher Mr than the three forms obtained with chymotrypsin. On addition of MgCl2 instead of EDTA, myosin hydrolysis was markedly reduced. In contrast, irrespective of the presence of either MgCl2 or EDTA, cathepsin B hydrolysed both myosins into HMM and LMM. Cathepsin L digested myosins more extensively than cathepsins B and D and the main fragments generated were, in decreasing order of importance, rod, S1, S2, HMM, and LMM. In the incubation conditions tested, cathepsin H displayed nondetectable action on myosins. As fast and slow myosin digest patterns were compared, the main differences observed concerned the size of the proteolytic products and the rate of hydrolysis, which was about 4-fold higher for the fast than for the slow isoform. This appeared consistent whatever enzyme was considered.

Animals↗
PubMed1988

Delineation of chicken cathepsin L secondary structure; relationship between pH dependence activity and helix content.

The secondary structure of the recently sequenced chicken liver cathepsin L (EC 3.4.22.15) has been studied both by circular dichroism and a predictive method. The structural data provided by these approaches allow us to underline the extent of the structural similarities between cathepsin L and papain, one of the best known proteins in the cysteine proteinase family. The predictive method of Garnier et al. (J. Mol. Biol. 120 (1978) 97-120) is used to locate alpha-helix and beta-sheet segments in the cathepsin L sequence. An optimization of decision constants has been performed, using circular dichroism data, to improve good predictions. The combination of these approaches lead us to suggest that the location of ordered structures observed in papain is maintained in cathepsin L, but with an additional alpha-helix in the middle region (residues 85-108) of cathepsin L. Furthermore, we show that cathepsin L inactivation at neutral pH is correlated to the lost of alpha-helix content (40% at pH 5.8 and 17% at pH 7.0) in this protein. It appears that such an effect can be related to the change in the ionization state of histidine side-chains which are shown to be mainly located in the predicted alpha-helix regions.

Algorithms↗
PubMed1988

Proteolytic specificity of chicken cathepsin L on bovine beta-casein.

The proteolytic specificity of chicken cathepsin L was studied using bovine beta-casein as substrate. The peptide mixtures obtained after various times of hydrolysis were separated by RP-HPLC and ten peptides were identified. Chicken cathepsin L accepts proline residues in all positions except P'1. Looking at the amino acid residues on the amino side of the scissile bond we found three times the Tyr-Pro pair at P'1-P'2 positions and that the S'1 subsite can interact with modified amino acids such as phosphoserine.

Amino Acids↗
PubMed1988

Sequence homologies, hydrophobic profiles and secondary structures of cathepsins B, H and L: comparison with papain and actinidin.

The comparison of the amino acid sequences of 5 cysteine proteinases: papain, actinidin, rat cathepsins B and H and chicken cathepsin L, demonstrates a striking homology among their sequences. The N-terminal region (residues 1-70 in papain) and C-terminal region (residues 118-212 in papain) display the highest sequence homologies, whereas the lowest sequence homologies are observed in the middle region (residues 71-117 in papain); a segment where most insertions/deletions are observed. The highest sequence homology is observed between rat cathepsin H and chicken cathepsin L. As shown by X-ray studies, papain and actinidin have a clearly defined double domain structure. Each domain contains a core of non-polar side chains, which are retained in cathepsins B, H and L, except for the non-polar residue 203 of the core which is replaced by glutamic acid in cathepsin B. The percentage and the location of alpha-helix and beta-sheets of cathepsins B, H and L, assessed using the methods of Garnier et al. (1978, J. Mol. Biol. 120, 97-120) and Chou and Fasman (1974, Biochemistry 13, 222-245), show that the main ordered structures in papain and actinidin are probably retained in cathepsins B, H and L. The differences observed occur essentially in the middle region, a place where sequences display the lowest homologies and which is far removed from the active site.

Amino Acid Sequence↗
PubMed1988

[Action of muscular proteinases on fast and slow myosins. Relation with post-mortem proteolysis in muscles of variable contractility].

Sensitivity to proteolysis of fast and slow rabbit skeletal muscle myosins with cathepsins D, B, H and L and with calpains I and II was studied in various incubation conditions. Cathepsins D, B and L degraded both myosins and exhibited different specificities towards these proteins. Moreover, in each case slow myosin appeared to be less sensitive to proteolysis than fast myosin. This finding agrees well with the lower extent of protein hydrolysis observed during post-mortem ageing of meat in slow twitch pork muscles.

Animals↗
PubMed1987

Purification and amino acid sequence of chicken liver cathepsin L.

Cathepsin L was purified from chicken liver lysosomes by a two-step procedure. Cathepsin L exhibited a single band of Mr 27,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions, presented a high affinity for the substrate Z-Phe-Arg-NMec, was very unstable at neutral pH, and was inhibited by Z-Phe-Phe-CHN2. The complete amino acid sequence of cathepsin L has been determined and consists of 215 residues. The sequence was deduced from analysis of peptides generated by enzymatic digestions and by chemical cleavage at methionyl bonds. Comparison of the amino acid sequence of cathepsin L with those of rat liver cathepsins B and H and papain demonstrates a striking homology among their primary structures.

Amino Acid Sequence↗