F. Van Cauwelaert

Shielding of phospholipid monolayers from phospholipase C hydrolysis by α-lactalbumin adsorption

Abstract α-Lactalbumin interacts more strongly with lecithin and cardiolipin monolayers at pH 3∼4 than at pH 7 to 10. At physiological pH this protein does not penetrate monolayers of DPPC and cardiolipin above pressures of 30 dynes/cm. Enzymatic hydrolysis of these monolayers by phospholipase C (Clostridium Welchii) is inhibited partially or totally when α-lactalbumin is injected in the subphase prior to the enzyme injection.

The thermotropic transition of large unilamellar (LUV) vesicles of dimyristoyl phosphatidylcholine by Raman spectroscopy

Abstract By observing Raman spectra in the 2800–3000 cm −1 region, we determined the gel to liquid transition characteristics of large unilamellar vesicles of dimyristoyl phosphatidylcholine. This transition occurs between 22°C and 28.5°C. The large unilamellar vesicles can be well distinguished from the small unilamellar vesicles, which gel to liquid transition is spread out from 10°C to 27°C. The multilamellar vesicles on the other hand melt in a highly cooperative phenomenon at 23.7°C. The intermediate character of the transition of large unilamellar vesicles is also illustrated by data of the lateral order parameter.

Tyrosine group behaviour in bovine α-lactalbumin as revealed by its Raman effect

Side group behaviour is often used for conformational studies of proteins. We have performed Raman spectroscopic measurements on the tyrosine groups of bovine α-lactalbumin. The 850/830 cm-1 doublet intensity ratio is a direct measure of the negative charge state of the phenolic oxygen and of the tyrosine environment. pH measurements confirm the existence of an acid conformer of BLA, that is comparable to, but clearly distinguishable from the apo-conformer. Following the Siamwiza theory, the Tyr groups in this partially unfolded state are situated in a more hydrophobic environment. Observation of Tyr groups behaviour in the denaturated states obtained by thermal or chemical treatment leads us to the same conclusion. However, the behavior of tryptophan groups is quite different. In an unfolded sate, the Trp residues are mostly exposed to the solvent.The stabilizing role of Ca2+ and Na+ ions in BLA is also investigated.

Study of a hydrophobic site on bovine α-lactalbumin by labeling with [125I]-TID

The hydrophobic, photoreactive probe 3-(trifluoromethyl)-3-(m-[125I]iodophenyl) diazirine ([125I]TID) labels apo-bovine alpha-lactalbumin but much less his Ca2+-form. The labeling of the apo-form is strong at protein concentrations of 0.5 mg ml-1 and increases with increasing concentration. Furthermore, increasing concentrations of NaCl, decrease the labeling of apo-alpha-lactalbumin with [125I]TID.

A circular dichroic study of Cu(II) binding to bovine α-lactalbumin

Abstract The visible and ultraviolet circular dichroic spectra resulting from the interaction of bovine α-lactalbumin with successive Cu(II) ions have been recorded under a variety of conditions. Analysis of the observed charge-transfer and d-d band transitions can be made in terms of two kinds of binding sites: at a histidyl group and at the N-terminal amino group, respectively. At basic pH the amide nitrogens of the peptide backbone progressively take part in the coordination. The occupation of the high affinity calcium binding site by Ca(II) and Mn(II) does not influence the Cu(II) binding process, suggesting that there is no direct interaction between this site and the Cu(II) binding sites.

Influence of acylcarnitines of different chain length on pure and mixed phospholipid vesicles and on sarcoplasmic reticulum vesicles

Palmitoyl-, myristoyl- and lauroylcarnitine destabilize small unilamellar vesicles of 1,2-dipalmitoyl-n-glycero-3-phosphorylcholine (DPPC) and 1,2-dimyristoyl-n-glycero-3-phosphorylcholine (DMPC) into multilamellar liposomes. Their effect on the bilayer is dependent on the acyl chain length of the acylcarnitine, the ratio of the lengths of the acyl chains of the phospholipid and the acylcarnitine and the molar ratio of the phospholipid to acylcarnitine but not the absolute concentration of the acylcarnitine in the solute. Sarcoplasmic reticulum vesicles are broken down by each of the acylcarnitines at concentrations below their critical micellar concentrations (CMC). These three acylcarnitines stimulate the Mg2+, Ca2+-ATPase activity in SR-vesicles to a certain maximum, after which a net inhibition is observed. The maximum degree of stimulation depends highly on acyl chain length: the shorter the chain length, the more effective. In the same concentration range where the Mg2+, Ca2+-ATPase activity is increased, the net Ca2+-uptake is markedly decreased.

Conformational aspects of the Cu2+ binding to α-lactalbumin. Characterization and stability of the Cu-bound state

By circular dichroism experiments the existence of a typical Cu2(+)-bound state is demonstrated for bovine- and for goat alpha-lactalbumin. As in the near-UV region an important ligand to metal charge-transfer band overlaps with the aromatic band of the protein, a subtraction method is developed in order to determine the net effect of Cu2+ ions on the protein conformation. The Cu2(+)-bound state, characterized by a vanishing tertiary structure and a substantial loss of secondary structure, clearly differs from the well-known Ca2(+)-, apo-, and acid conformers. At room temperature, the Cu2+ binding has already decreased the alpha-helix content of bovine alpha-lactalbumin to the extent that further unfolding by thermal or guanidine hydrochloride denaturation behaves in a non-cooperative way. Since for goat alpha-lactalbumin the Cu2+ binding to His-68 is much less important than for bovine alpha-lactalbumin, we observe a somewhat different conformational behaviour for goat alpha-lactalbumin. The results of this conformational circular dichroism study are confirmed by isothermal calorimetric data.

Calorimetric aspects of the heat activation of spores of Phycomyces blakesleeanus

Abstract When Phycomyces spores are heated in a differential scanning calorimeter (DSC) an exothermic process situated in the activation temperature range is detected. This process does not depend on oxygen and is absent in scans of activated spores. After activation the protein denaturation profile is also changed but this effect seems to depend on oxygen. Acetate activates spores in about 10 min, however longer treatment with this chemical is needed to affect the exothermic peak. The effects of some other chemicals that influence the activation mechanism, like D 2 O and FeCl 2 , are also reported.

Differential scanning calorimetric observations concerning the activation mechanism of spores of Phycomyces blakesleeanus.

Abstract Spores of Phycomyces were scanned in a Differential Scanning Calorimeter. The spectrum obtained was clearly influenced by previous activation of spores by heat or by acetate. When spores were allowed to return to dormancy the original spectrum of dormant spores was restored. The high temperature at which the difference in the spectrum between activated and dormant spores was found points to a protein denaturation. It is suggested therefore that the activation of spores is obtained through a conformational change of a protein.

Raman spectroscopic study of the environment of tryptophan residues in bovine α-lactalbumin

Abstractα-Lactalbumin is an important protein essential for lactose synthesis. The conformation, position and state of different amino acid side chains and the structural changes in environment are important parameters which affect the functional properties. In this paper, the use of Raman bands for the study of the environment of tryptophan residues is presented and discussed.