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B De Foresta

Publications and source records attributed to B De Foresta.

4 recordsLinked to original sources
PubMed1996

Brominated detergents as tools to study protein-detergent interactions.

In order to study protein-detergent short-range interactions, we analyzed the quenching by brominated detergents of reticulum sarcoplasmic (SR) Ca(2+)-ATPase intrinsic fluorescence. For this purpose, 7,8-dibromododecyl beta-maltoside and 2-O-(10,11-dibromoundecanoyl)sucrose, brominated analogs of two non-ionic detergents, the frequently used dodecylmaltoside and the newly synthesized 2-O-lauroylsucrose respectively, were prepared. Rayleigh scattering measurements showed that the brominated detergents efficiently and rapidly solubilized SR vesicles like their non-brominated analogs although at slightly higher concentrations. Similarly, each analog had a slightly higher critical micellar concentration than its parent detergent. The partition coefficient K (expressed as the ratio of the molar fraction of detergent in the SR lipid phase to that in the aqueous phase, at pH 7.5 and 20 degrees C) was similar for brominated and non-brominated dodecyl maltoside (3.5-4 x 10(5)) and slightly lower for dibromoundecanoylsucrose (approximately 10(5)) than for lauroylsucrose (approximately 2 x 10(5)). At detergent concentrations too low to solubilize the membrane, the brominated detergents rapidly inserted (within seconds) into SR vesicles. In this concentration range, Ca(2+)-ATPase fluorescence quenching steadily increased with detergent concentration. When the membrane was saturated with detergent, the residual fluorescence was about half of its initial value, indicating significant protein-detergent, contacts, possibly due to a slightly higher affinity of Ca(2+)-ATPase for these detergents than for phospholipids. For higher detergent concentrations, solubilizing the membrane, the fluorescence continued to decrease with detergent concentration, with no evidence for a dramatic change in the average hydrophobic environment of the protein during the transition from bilayers to a soluble state. For still higher detergent concentrations, above that necessary for membrane solubilization, the fluorescence was further quenched to a residual relative value of about 20%, corresponding to further delipidation of the protein surface, in agreement with previous results [de Foresta, B., le Maire, M., Orlowski, S., Champeil, P., Lund, S., Møller, J.V., Michelangeli, F. & Lee, A.G. (1989) Biochemistry 28, 2558-2567]. Fluorescence quenching for solubilized Ca(2+)-ATPase was quickly reversed upon addition of excess non-brominated detergent. The effects of the four detergents on the Ca(2+)-ATPase hydrolysis of p-nitrophenyl phosphate were similar and correlated with the protein-detergent contacts evidenced above. In conclusion, both these brominated detergents appear to be promising tools to study protein-detergent interactions at the hydrophobic surface of a membrane protein, either in a membrane or in solubilized complexes.

Animals↗
PubMed1994

Cancellation of the cooperativity of Ca2+ binding to sarcoplasmic reticulum Ca(2+)-ATPase by the non-ionic detergent dodecylmaltoside.

The perturbation of the kinetics of the sarcoplasmic reticulum (SR) membranous Ca(2+)-ATPase cycle by the non-ionic detergent dodecylmaltoside (DM) has been shown to exhibit specific features which were not observed with the related detergents octa(ethylene glycol) monododecylether and Triton X-100 [de Foresta, B., Henao, F. & Champeil, P. (1992) Eur. J. Biochem. 209, 1023-1034]. This previous study has been completed here by a detailed analysis of the perturbation by DM of the interaction of Ca2+ with membranous ATPase, both in its unphosphorylated and phosphorylated form. Equilibrium binding measurements, performed at pH 7.5 and 20 degrees C, showed that only one 45Ca2+ was bound with high affinity to the ATPase in the presence of maximally perturbing concentrations of DM, as compared to two 45Ca2+ in the absence of detergent. This binding was also assessed by a small decrease in the tryptophan fluorescence intensity. Binding of a second Ca2+ occurred only with a much lower affinity. In the presence of DM, the pCa dependence of the phosphorylation by [gamma-32P]ATP of the ATPase shifted towards 50-fold higher Ca2+ concentrations than in its absence. Furthermore, DM completely inhibited the cooperativity of this dependence. This shift strongly suggests that the phosphorylation of DM-perturbed ATPase requires the binding of this second, low-affinity Ca2+. In order to assess this, samples of ATPase were intramolecularly cross-linked with glutaraldehyde. This treatment stabilized the phosphorylated intermediated with occluded Ca2+ [Ross, D. C., Davidson, G.A. & McIntosh, D. B. (1991) J. Biol. Chem. 266, 4613-4621]. Both in the absence and presence of DM, the cross-linked enzyme occluded close to two Ca2+/phosphorylated molecule. Finally, the pCa dependences of the ATPase hydrolytic activity, measured with two different high-energy substrates, ATP or p-nitrophenylphosphate (PNpP), were also found to shift towards higher Ca2+ concentrations in the presence of DM, which was again consistent with a normal coupling ratio, i.e. two bound Ca2+/substrate hydrolyzed. As compared to other detergents, the maltoside head group of DM might favor a stronger interaction with membranous ATPase, resulting in its high perturbing effect on Ca2+ binding. The loss of cooperativity of Ca2+ binding evidenced here makes DM a useful tool in the analysis of the sequence of events occurring during Ca2+ binding.

Animals↗
PubMed1979

Study of fluorescent tryptophyl residues and extrinsic probes for the characterization of molecular domains of Folch-Pi apoprotein.

The highly hydrophobic myelin Folch-Pi apoprotein can be solubilized in organic as well as in aqueous media. In order to understand the molecular organization changes consecutive to changes in the solvent medium, the environment of intrinsic probes and extrinsic labels has been studied by fluorescence and accessibility to some reagents. In acqueous solution, only two tryptophan residues per protein molecule of 23,500 molecular weight have been shown to fluoresce, and their fluorescence characterisitics indicate an hydrophobic and/or constrained environment. Two ANS binding sites have also been observed having a high quenching effect on the intrinsic chromophore fluorescence. A large accessibility has been evidenced for the protein sulfhydryl groups in chloroform-methanol 2:1 (v/v), both by kinetic study of the protein reaction with a specific reagent, N-(1-anilino-naphtyl-4) maleimide, and by the fluorescence characteristics of this probe once linked to the protein. The free sulfhydryl groups were still reactive in acqueous solution, but extrinsic fluorescence of the labelled apoprotein transferred from chloroform-methanol 2:1 (v/v) into water gave evidence of constraints on the probe or on its environment. Such constraints may contribute to the solubilization in acqueous solution of this highly hydrophobic protein.

Apoproteins↗