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Bovine myelin proteolipid apoprotein (PLA), obtained in high yield and purity by a novel ultrafiltration procedure, has been used to study the perturbations produced by this protein on phosphatidylcholine bilayers, using infrared spectroscopy, nuclear magnetic resonance and fluorescence polarisation. PLA interacts with phospholipids in a similar manner to other intrinsic proteins. For bilayers in the fluid state, the fatty-acyl chain static order, as measured by deuterium NMR, is slightly increased in the presence of the protein, except at very high PLA concentrations. Phosphorus NMR reveals some perturbation of the phospholipid polar group by PLA, but to a smaller degree than occurs with other intrinsic proteins. An increase in static order above tc, (the onset temperature for gel-to-fluid transition) is also detected by infrared spectroscopy. Studies using steady-state polarisation of diphenylhexatriene fluorescence indicate that the microviscosity of the bilayer increases as a function of the protein mole fraction. From these data an estimation of the average number of lipids perturbed per protein monomer has been made, and a figure of 37 phospholipid molecules determined. The data are compatible with a picture of a hydrophobic polypeptide. perturbing the phospholipids close to it, but allowing rapid (> 104 s-1) exchange with all the lipid molecules in the system. [ABSTRACT FROM AUTHOR]

Solubilization of sonicated unilamellar vesicles by Triton X-100 is a complex process. Solubilization starts at low detergent concentrations, as compared to the case of large vesicles, and is accompanied by the simultaneous rapid formation of large multilamellar liposomes. Measurements of lipid and detergent distribution indicate that, at a 1:1 lipid:detergent mole ratio, about one-third of the lipid, with most of the detergent, is solubilized in the form of mixed micelles. The remaining two-thirds are in the form of multilamellar liposomes, virtually free of detergent. Higher detergent concentrations also bring about the solubilization of these liposomes. [ABSTRACT FROM AUTHOR]

The interaction of multilamellar phosphatidylcholine vesicles with the non-ionic detergent Triton X-100 has been studied under equilibrium conditions, specially in the sub-lytic range of surfactant concentrations. Equilibrium was achieved in less than 24 h. Estimations of detergent binding to bilayers, using [³H]Triton X-100, indicate that the amphiphile is incorporated even at very low concentrations (below its critical micellar concentration); a dramatic increase in the amount of bound Triton X-100 occurs at detergent concentrations just below those producing membrane solubilization. Solubilization occurs at phospholipid/detergent molar ratios near 0.65 irrespective of lipid concentration. The perturbation produced by the surfactant in the phospholipid bilayer has been studied by differential scanning calorimetry, NMR and Fourier-transform infrared spectroscopy. At low detergent concentration (lipid/detergent molar ratios above 3), a reduction in ²H-NMR quadrupolar splitting occurs, suggesting a decrease in the static order of the acyl chains; the same effect is detected by Fourier-transform infrared spectroscopy in the form of blue shifts of the methylene stretching vibration bands. Simultaneously, the enthalpy variation of the main phospholipid phase transition is decreased by about a third with respect to its value in the pure lipid/water system. For phospholipid/detergent molar ratios between 3 and 1, the decrease in lipid static order does not proceed any further; rather an increase in fluidity is observed, characterized by a marked decrease in the midpoint transition temperature of the gel-to-fluid phospholipid transition. At the same time an isotropic component is apparent in both 31P-NMR and ²H-NMR spectra, and a new low-temperature endotherm is detected in differential scanning calorimetric traces. When phospholipid and Triton X-100 are present at equimolar ratios some bilayer structure persists, as judged from calorimetric observations, but NMR reveals only one-component isotropic signals. At lipid/detergent molar ratios below unity, the NMR lines become narrower, the main (lamellar) calorimetric endotherm tends to vanish and solubilization occurs. [ABSTRACT FROM AUTHOR]

Discusses the purification of Escherichia coli pro-haemolysin, comparing its properties with that of the mature alpha-haemolysin. Aggregate formation in aqueous media; Direct measurement of protein binding to liposomal membranes; Intrinsic fluorescence emission of the protein upon layer binding.

Mixtures of di-(perdeuteropalmiotyl)-sn-glycero-3 choline ([²H62]Pam2GroPCho) with palmitoylcarnitine or palmitoyl-CoA in aqueous suspension have been examined by Fourier-transform infrared spectroscopy. The C-H (or C-D) region of the spectrum shows that an order-disorder transition exists in pure aqueous palmitoylcarnitine at 45°C; palmitoylcarnitine mixes with [²H62]Pam2GroPCho without perturbing the gel-fluid transition of the phospholipid event at [²H62]Pam2GroPCho/palmitoylcarnitine 1:2 molar ratios; and palmitoyl-CoA, however, at similar proportions, smears out the [²H62]Pam2GroPCho transition as detected from C-D stretching vibrations. Relevant data from the carbonyl region include; the high-frequency (non-hydrogen bound) carbonyl subpopulation, but not the low frequency one, detects the gel-to-fluid transition of the phospholipid; the carbonyl region detects the thermotropic transition over a wider temperature range than the methylene stretching region, i.e. detects changes starting well below and ending several degrees above the methylene transition temperature, and a significant interaction may occur between some coenzyme A group and the carbonyl groups of the phospholipid. The latter interaction may contribute to explain the conezyme A/carnitine exchange during mitochondrial fatty acid import. [ABSTRACT FROM AUTHOR]