Transverse and lateral distribution of phospholipids and glycolipids in the membrane of the bacterium Micrococcus luteus.

The photodimerization of anthracene was used to investigate the transverse and lateral distribution of lipids in the membrane of the Gram-positive bacterium Micrococcus luteus. 9-(2-Anthryl)nonanoic acid (9-AN) is incorporated at a high rate into various membrane lipids of M. luteus. On irradiation of intact bacteria at 360 nm, anthracene-labeled lipids form stable photodimers which can be extracted and separated by thin-layer chromatography. We present here the results of a study on the distribution of two major lipids, phosphatidylglycerol (PG) and dimannosyldiacylglycerol (DMDG), within each leaflet of the membrane lipid bilayer. After metabolic incorporation of a tritiated derivative of 9-AN in M. luteus, the radioactivity associated with the photodimers issued from PG and DMDG was counted. In the bacterial membrane, the ratio of PG-DMDG heterodimer with respect to PG-PG and DMDG-DMDG homodimers is around half of what should be obtained for a homogeneous mixture of the two lipids. In order to find out whether this was due to an asymmetric distribution of the two lipids between the two membrane leaflets or a heterogeneous distribution of the two lipids within the same membrane leaflet, the transverse distribution of PG and DMDG was also investigated. This was carried out by following the kinetics of oxidation of the two lipids by periodic acid in the membrane of M. luteus protoplasts. PG predominated slightly in the outer layer (60%), while DMDG was found to be symmetrically distributed between the two leaflets. By itself, this lipid asymmetry cannot account for the lipid distribution determined from the photodimerization experiments. This indicates that PG and DMDG are not homogeneously distributed in the plane of the bacterial membrane.

Steady-state and time-resolved fluorescence anisotropy study of phospholipid molecular motion in the gel phase using 1-palmitoyl-2-[9-(2-anthryl)-nonanoyl] -sn-glycero-3-phosphocholine as probe.

1-Palmitoyl-2-[9-(2-anthryl)-nonanoyl]-sn-glycero-3-phosphocholine (Anthr-PC), a non-perturbing phospholipid probe [de Bony, J. and Tocane, J. F. (1983) Chem. Phys. Lipids 32, 105-121], has been designed in order to obtain insight into the membrane lipid organization at a 'microscopic' level, in terms of lateral distribution both in model and in natural membranes [de Bony, J. et al. (1984) Eur. J. Biochem. 143, 373-379; FEBS Lett. 174, 1-6]. In the present study, the molecular motions of this new fluorescent probe embedded in a lipid matrix have been investigated by fluorescence anisotropy techniques in steady-state and time-resolved modes. The results indicate that long axis rotation, monitored by the out-of-plane mode of rotation of the fluorophore, is fast even in the phospholipid gel state. It is moderately sensitive to the phase transition. The data suggest that this rotation is anisotropic. Cholesterol exhibits little effect on this rotation. The rotation of the long axis itself is sensitive to the transition. It is hindered as inferred from measurements at wavelength where both the in-plane and out-of-plane motions contribute to the depolarization of the emitted fluorescence light. Cholesterol restricts this motion. The behaviour of the free 9-(2-anthryl)-nonanoic acid is not significantly different from that of Anthr-PC. These results are discussed with respect to the influence of orientational constraints on the photodimerization process when this lipid probe is used to monitor phospholipid lateral distribution.

Photo-induced dimerization of anthracene phospholipids for the study of the lateral distribution of lipids in membranes.

It is shown that photo-induced cross-linking reaction between anthracene-labelled phospholipids can be used for studying, at a molecular level, their lateral distribution in bilayer structures. A simple and versatile method is proposed. It is based on the property of anthracene to form covalently bound dimers upon irradiation in the near ultraviolet (360 nm) and on the possibility of separating the lipid photo-dimers from the lipid monomers by thin-layer chromatography. Identification of the photo-dimers is easily achieved since, upon illumination at a shorter wavelength (250-280 nm), they partially dissociate to the native monomer molecules. The feasibility of the method was tested by checking the effects of cations (sodium, calcium) on the homogeneity of 1/1 mixtures of anthracene-phosphatidylcholine, i.e. 1-acyl-2-[9-(2-anthryl)-nonanoyl]-sn-glycero-3-phosphocholin e (Anthr-PC) with anthracene-phosphatidic acid (Anthr-PA) and with anthracene-phosphatidylglycerol (Anthr-PG) in the form of liposomes. These lipids were anthracene-labelled by acylation of their glycerol backbone at the sn-2 position with the synthetic 9-(2-anthryl)-nonanoic acid. Data presented indicate a good miscibility of these lipids in the presence of sodium. For each lipid mixture, the lipid heterodimers were clearly identified and, quantitatively, they dominated the lipid homodimers, as expected for a regular distribution of the lipids in the 1/1 mixture. Addition of calcium ions to the lipid suspensions did not alter the miscibility properties of Anthr-PC and Anthr-PG. In contrast, calcium triggered a clear-cut phase separation in the Anthr-PC/Anthr-PA mixture as, in this case, only traces of the heterodimer form of the lipids remained observable on the chromatogram. The three anthracene-phospholipids, pure or mixed together, exhibit a clear-cut gel-to-liquid phase transition which was detectable by fluorescence intensity measurements. The analysis of the corresponding phase-transition temperatures confirms, at a 'macroscopic' level, the effects of sodium and calcium on the mixing properties of the anthracene phospholipids which were revealed at a 'microscopic' level by the dimerization procedure.

Evidence for a homogeneous lateral distribution of lipids in a bacterial membrane. A photo cross-linking approach using anthracene as a photoactivable group.

A new photo cross-linking method has been developed for the study of the lateral distribution of lipids in natural membranes, which uses anthracene as a photoactivable group. This method, which rests on the potentiality of anthracene to form covalently bound dimers upon irradiation around 340-380 nm has been applied to the membrane lipids (dimannosyl diacylglycerol, phosphatidylglycerol, phosphatidylinositol) of the bacterium Micrococcus luteus. These glyco- and phospholipids were anthracene labelled by metabolically incorporating the synthetic 9-(2-anthryl)nonanoic acid. The following sequential procedure was used: dimerization of the anthracene-labelled lipids in the membrane by irradiation of the intact cells at 360 nm; extraction of the lipids and thin-layer chromatography in the first dimension to separate the various lipid dimers from the monomers; partial dedimerization of the lipid dimers by illumination of the chromatogram at around 250-280 nm; chromatography in the second dimension to separate the native lipid monomers from the corresponding residual lipid dimers. On account of the occurrence of the 3 hetero dimers phosphatidylglycerol-dimannosyl diacylglycerol, phosphatidylinositol-dimannosyl diacylglycerol and phosphatidylglycerol-phosphatidylinositol after irradiating the cells, it is concluded that in this bacterial membrane, dimannosyl diacylglycerol, phosphatidylglycerol and phosphatidylinositol are homogeneously distributed.

Tryptophanyl-tRNA synthetase from beef pancreas. Spectroscopic analysis of the stoichiometry of formation of the enzyme-tryptophanyl-adenylate complex.

The dimeric enzyme tryptophanyl-tRNA synthetase from beef pancreas catalyses the stoichiometric formation of one mole of tryptophanyl-adenylate per subunit. This formation is associated with optical changes (absorbance, fluorescence, optical rotation) and is confirmed by analytical ultracentrifugation. An equal amplitude of the change is observed for each adenylation site at pH 8.0, 25 degrees C, regardless of the optical method used. The formation of two tryptophanyl adenylates per dimer corresponds to a molar absorbance change delta epsilon 291 = 12000 +/- 500 cm-1 M-1, to a fluorescence quenching of 24 per cent at 340 nm and to a variation in optical rotation of 6 per cent at 313 nm. The circular dichroic band of the adenosine moiety of ATP is strongly increased. The addition of sodium pyrophosphate to the tryptophanyl-adenylate-enzyme complex restores the absorbance and fluorescence amplitude observed prior to the addition of ATP to the enzyme. Magnesium ions are necessary to the reaction. A pertubation of the environment of both the protein and the substrates (tryptophan and ATP) have to be taken into account to explain the magnitude of the observed changes.

Lipid-protein interactions: NMR study of melittin and its binding to lysophosphatidylcholine.

Proton NMR of melittin differs according to the association state of the peptide in the monomer or tetramer. Melittin interacts with lysophosphatidyl-choline micelles, whatever the association state of melittin; well resolved superimposed spectra from both components for all the lipid to peptide molar ratios are observed. Within the complexes, local mobility and fast exchange occurs. On binding concomitant shifts on Trp19 indole lines and on the aliphatic CH2 protons of the lipids are detected. The lipid perturbation is maximum for methylene groups in a alpha and beta of the ester bond, this could allow positionning of Trp19 in the hydrophobic core of the lipids.