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.

Interaction of alpha-lactalbumin with dimyristoyl phosphatidylcholine vesicles. II. A fluorescence polarization study.

The interaction of alpha-lactalbumin with dimyristoyl phosphatidylcholine vesicles was studied as a function of temperature, pH and the molar ratio of phospholipid to protein. The method consisted of measuring the fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene used as a probe embedded in the vesicles. After incubation of the protein with the phospholipid for 2 h at 23 degrees C, the polarization of the light emitted by this probe shifted to higher values; the shift was greater at acidic pH than at neutral pH. After incubation at 37 degrees C, no shift in polarization was found at pH 7, 6 and 5 while a strong increase occurred at pH 4. Lowering the temperature, after incubation at 37 degrees C, had little effect on the polarization at neutral pH. At pH 5, however, and in the transition range of the phospholipid, the polarization increased greatly. A kinetic study of the interaction carried out around the transition temperature of dimyristoyl phosphatidylcholine as a function of pH shows that the speed of complex formation between alpha-lactalbumin and the lipid increases from neutral to acidic pH. From the present results and in agreement with our earlier calorimetric and fluorescence data (Hanssens, I., Houthuys, C., Herreman, W. and van Cauwelaert, F.H. (1980) Biochim. Biophys, Acta 602, 539--557), it is concluded that at neutral pH the interaction mechanism is probably different from that at acidic pH. At neutral pH and at all temperatures, alpha-lactalbumin is mainly absorbed electrostatically to the outer surface of the vesicle with little or no influence on the transition temperature of the phospholipid. At this pH, only around the transition temperature is penetration possible. At pH 4, however, the protein is able to penetrate the vesicle at all temperatures and to interact hydrophobically with the phospholipid fatty acid chains. As a result of this interaction, the transition temperature is increased by about 4 degrees C. This different behaviour changes progressively upon acidification: at pH 5, penetration seems to be impossible at temperatures far above the transition temperature but occurs rapidly around the transition temperature.

Interaction of alpha-lactalbumin with dimyristoyl phosphatidylcholine vesicles. I. A microcalorimetric and fluorescence study.

alpha-Lactalbumin and dimyristoyl phosphatidylcholine were used as a prototype to study the influence of a protein conformational change, induced by the pH, on the interaction between that protein and a phospholipid. The enthalpy changes associated with the interaction of alpha-lactalbumin with dimyristoyl phosphatidylcholine vesicles were measured as a function of the molar ratio of phospholipid to protein, pH and temperature. Gel-filtration, electron-microscopic and fluorescence data for the same experimental conditions were also obtained. At pH 4 and 5, the enthalphy changes (delta H) are not only larger than at physiological pH, but also show a maximum at aobut 23 degrees C in the delta H vs. temperature graph. At pH 6 and 7, on the contrary, delta H increases with decreasing temperature without a maximum in the curve. Gel-chromatographic and electron-microscopic data show that at pH 6 and 7, the morphological characteristics of the vesicles are unchanged upon addition of alpha-lactalbumin, while at pH 4 and 5 at 23 degrees C an extra peak appears in the gel-filtration graphs between the pure vesicles and alpha-lactalbumin. The new fraction contains lipid-protein complexes. Electron micrographs show that bar-shaped entities are formed. A red shift at 23 degrees C and a blue shift at 37 degrees C, both to 336 nm, are observed for lambda max of the fluorescence emission spectra at pH 4 when alpha-lactalbumin is brought into contact with the phospholipid. At the same time, a strong increase in the fluorescence intensity is observed. The chromatographic and fluorescence data indicate that a lipid-protein complex with a molar ratio of approx. 80 is formed. At pH 7 and different temperatures, the emission maximum remains at the wavelength of pure alpha-lactalbumin, the change in the fluorescence intensity, however, indicates that interaction with the lipid occurs. The results can be explained on the basis of an electrostatic interaction at pH 6 and 7, and a hydrophobic interaction at pH 4 and 5.