Laurence de La Fournière

Claude Bernard University Lyon 1, Villeurbanne, Rhône-Alpes, France

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Publications (5)22.81 Total impact

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    ABSTRACT: The secondary structures of peptides beta 25-35 (the active toxic fragment) and beta 35-25 (reverse sequence and non-toxic fragment), as well as of the amidated beta (25-35)-NH2 peptide were investigated in aqueous solution and in the solid state by means of Fourier-transformed infrared spectroscopy and circular dichroism spectroscopy. The conformations of the beta 25-35 and beta 35-25 in solid state were identical and contained mostly beta-sheet structures. In solid state the amidated beta (25-35)-NH2 peptide also contained mostly beta-sheet structures. Freshly prepared aqueous solutions of the beta 25-32 (0.5 - 3.8 mM) contained a mixture of beta-sheet and random coil structures. Within 30-60 min incubation at 37 degrees C in water or in phosphate-buffered saline solution (PBS), beta 25-35 was almost fully converted to a beta-sheet structure. Decreasing the temperature from 37 degrees C to 20 degrees C decreased the rate of conversion from random coil to beta-sheet structures, 1-2 h being required for complete conversion. In contrast beta 35-25 in water or in PBS buffer had mostly a random coil structure and remained so for 6 days. The amidated beta(25-35)-NH2 peptide in water (2.7 mM) was also mostly random coil. However, when this peptide (2-2.7 mM) was dissolved in PBS (pH 7.4) or in 140 mM NaCl, a gel was formed and its conformation was mostly beta-sheet. Decreasing the concentration of beta (25-35)-NH2 peptide in 140 mM NaCl aqueous solution from 2 mM to 1 mM or below favored the conversion from beta-sheet structures to random coil structures. The beta 25-35 was toxic to PC12 cells while beta 35-25 was not. The amidated peptide beta (25-35)-NH2 was at least 500-fold less toxic than beta 25-35. Structural differences between these beta peptides in aqueous solutions may explain the difference in their respective toxicities.
    Biochimica et Biophysica Acta 02/1996; 1315(1):40-6. DOI:10.1016/0925-4439(95)00102-6 · 4.66 Impact Factor
  • Laurence de La Fournière, Olivier Nosjean, René Buchet, Bernard Roux
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    ABSTRACT: The inactivation of alkaline phosphatase (AP) from bovine intestinal mucosa caused by lowering the p2H from 10.4 to 5.4 or by increasing the temperature from 25 degrees C to 70 degrees C were not followed by significant FTIR changes, indicating that the native conformation of AP was preserved under these conditions. Further decrease of p2H from 5.4 to 3.4 leaded to small infrared spectral changes of AP in the amide I' and amide II regions that were similar to the infrared spectral changes of AP induced by raising the temperature from 70 degrees C to 80 degrees C. The increase of temperature from 70 degrees C to 80 degrees C promoted the formation of intermolecular beta-sheets at the expense of some alpha-helix structures as evidenced by the appearance of the 1684 cm-1 and 1620 cm-1 component bands and the disappearance of the 1651-1657 cm-1 component band. This conformational change was followed by a sharp increase of the 2H/H exchange rate. CD spectra confirmed the FTIR results and were very sensitive to the variation of alpha-helix content while FTIR spectra were more receptive to the changes of beta-sheet structures.
    Biochimica et Biophysica Acta 05/1995; 1248(2):186-92. DOI:10.1016/0167-4838(95)00020-U · 4.66 Impact Factor
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    ABSTRACT: Several monoclonal antibodies (mAbs) were prepared against human pancreatic lipase (HPL). Two enzyme-linked immunosorbent assay (ELISA) procedures were set up for screening hybridomas producing specific antibodies. Four mAbs (81-23, 146-40, 315-25, and 320-24) of the IgG1 isotype were found to react with HPL in both simple sandwich and double sandwich ELISAs, while mAb 248-31, of the IgG2b isotype, reacted only with HPL in a double sandwich ELISA. The results of Western blot analysis carried out with native and SDS-denatured HPLs indicated that mAb 248-31 recognized only native HPL, while all the other mAbs recognized both forms of HPL. Since mAb 248-31 did not recognize SDS-denatured HPL, it was not possible to localize its epitope. To carry out epitope mapping along the primary sequence of HPL, four fragments (14, 26, 30, and 36 kDa) resulting from a limited chymotryptic cleavage of HPL were characterized by Western blotting as well as N-terminal amino acid sequence analysis. Of the above five anti-HPL mAbs, four (81-23, 248-31, 315-25, and 320-24) were found to inhibit the lipolytic activity of HPL (in both the presence and absence of bile salts and colipase), while mAb 146-40 had no inhibitory effects. The epitope recognized by mAb 146-40 was found to be located in the N-terminal domain (Lys1-Phe335). Combined immunoinactivation and epitope mapping studies showed that three inhibitory mAbs (81-23, 315-25, and 320-24) recognize overlapping epitopes from the hinge region between the N- and C-terminal domains of HPL, belonging to the 26-kDa fragment. In the presence of lipids, a significant decrease has been observed in the bending angle between the N- and C-terminal domains of the HPL tertiary structure (van Tilbeurgh, H., Egloff, M. P., Martinez, C., Rugani, N., Verger, R. and Cambillau, C. (1993) Nature 362, 814-820). From the present immunochemical data, we further propose that locking the hinge movement with mAbs may induce lipase immunoinactivation.
    Journal of Biological Chemistry 03/1995; 270(8):3932-7. · 4.60 Impact Factor
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    ABSTRACT: Several monoclonal antibodies (mAbs) were prepared against human pancreatic lipase (HPL). Two enzyme-linked immunosorbent assay (ELISA) procedures were set up for screening hybridomas producing specific antibodies. Four mAbs (81-23, 146-40, 315-25, and 320-24) of the IgG1 isotype were found to react with HPL in both simple sandwich and double sandwich ELISAs, while mAb 248-31, of the IgG2b isotype, reacted only with HPL in a double sandwich ELISA. The results of Western blot analysis carried out with native and SDS-denatured HPLs indicated that mAb 248-31 recognized only native HPL, while all the other mAbs recognized both forms of HPL. Since mAb 248-31 did not recognize SDS-denatured HPL, it was not possible to localize its epitope. To carry out epitope mapping along the primary sequence of HPL, four fragments (14, 26, 30, and 36 kDa) resulting from a limited chymotryptic cleavage of HPL were characterized by Western blotting as well as N-terminal amino acid sequence analysis. Of the above five anti-HPL mAbs, four (81-23, 248-31, 315-25, and 320-24) were found to inhibit the lipolytic activity of HPL (in both the presence and absence of bile salts and colipase), while mAb 146-40 had no inhibitory effects. The epitope recognized by mAb 146-40 was found to be located in the N-terminal domain (Lys1-Phe). Combined immunoinactivation and epitope mapping studies showed that three inhibitory mAbs (81-23, 315-25, and 320-24) recognize overlapping epitopes from the hinge region between the N- and C-terminal domains of HPL, belonging to the 26-kDa fragment. In the presence of lipids, a significant decrease has been observed in the bending angle between the N- and C-terminal domains of the HPL tertiary structure (van Tilbeurgh, H., Egloff, M. P., Martinez, C., Rugani, N., Verger, R. and Cambillau, C.(1993) Nature 362, 814-820). From the present immunochemical data, we further propose that locking the hinge movement with mAbs may induce lipase immunoinactivation.
    Journal of Biological Chemistry 02/1995; 270(8):3932-3937. · 4.60 Impact Factor
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    ABSTRACT: The kinetics of the adsorption of human gastric lipase (HGL) and human pancreatic lipase (HPL) were studied by recording the changes in the surface pressure with time in the absence and presence of an egg phosphatidylcholine (PC) monomolecular film spread at the air/water interface. In the presence of PC film, the tensioactivtty of HGL increased considerably compared with its behaviour at the air/water interface, whereas HPL exhibited a comparable degree of tensioactivity whether or not a phospholipid monolayer was present at the interface. This difference in surface behaviour is consistent with the higher penetration capacity attributed to HGL. Procolipase considerably increased both the initial adsorption rate and the final surface pressure reached by HPL compared with its adsorption without colipase.The kinetics of the hydrolysis of 1,2-didecanoyl-sn-glycerol (dicaprin) monolayers by HGL and HPL were measured using a “zero-order” trough. The large differences between the calculated characteristic adsorption times and the measured lag times indicate that the partition of the lipase molecules between the subsurface and the interface was probably limited by an energy barrier. The amplitude of this energy barrier can be partly attributed to the drastic conformational change in the enzyme, associated with the interfacial activation.The area per dicaprin molecule (56 Å2) corresponding to the maximal activity of HPL was compared with the dimension of the hydrophobic cleft surrounding the serine (Ser 152) of the catalytic triad of HPL, as recently demonstrated by H. Van Tilbeurgh and co-workers (Nature, 359 (1992) 159; 362 (1993) 814) in their studies on the “open” and “closed” forms of the respective three-dimensional crystalline structures. The catalytic triad was not accessible to a sphere 8.4 Å in diameter, mimicking the van der Waals envelope of the dicaprin molecule, due to the steric hindrance of the side chains of aromatic and cyclic residues F 215, F 77, Y 114 and H 263. It can be concluded that the substrate molecule must also undergo some conformational changes at the contact of the enzyme to be accommodated in the active site.
    Colloids and surfaces B: Biointerfaces 07/1994; DOI:10.1016/0927-7765(94)80069-3 · 4.29 Impact Factor

Publication Stats

68 Citations
22.81 Total Impact Points

Institutions

  • 1995
    • Claude Bernard University Lyon 1
      Villeurbanne, Rhône-Alpes, France
  • 1994
    • French National Centre for Scientific Research
      • Laboratoire de Architecture et Fonction des Macromolécules Biologiques
      Lutetia Parisorum, Île-de-France, France