The interaction of the ion channel polypeptide gramicidin A with the L-a-lysophosphatidylcholine micelles in a membrane state association (approximative molar ratio 1 : 9) was investigated by Raman spectroscopy. Studies were carried out over the spectral ranges of 700-1700 cm-' and 2800-3100 cm-' at 10°C.The Raman spectrum of L-a-lysophosphatidylcholine micelles indicated a disordered structure of the lipid acyl chains by the high intensities of the gauche conformation vibrations. Changing from the micellar phase to the membrane state of association with gramicidin A, the intensities of all-trans stretching modes increased whereas the intensities of gauche conformation vibrations decreased, reflecting the emergence of ordered lipid chains.Hydrophobic interactions between the acyl chains and the polypeptide side chain residues were demonstrated. The absence of modifications in intensities of the very strong tryptophan vibrations in the complex spectrum indicated that, if the tryptophan-stacking interactions suggested by some authors exsist, they are very weak ones.Gramicidin antibiotics are excellent models of simple ion channels [I]. It has been shown that gramicidin A, a linear hydrophobic polypentadecapeptide, is able to form helical structures in the lipid bilayers with an internal channel of about 4 nm in diameter and an approximate length of 26 nm [2-71. The helical structure of gramicidin A has been described by Urry as a new type of helix, called initially the xL,D helix [2], a name which was later changed to j-helix because of the hydrogen-bonding relationshps to fl-pleated sheets [6]. Two single-stranded helical molecules associated amino end to amino end (head to head) by means of six intermolecular hydrogen bonds to form the complete channel.A variety of physical techniques (X-ray diffraction, differential scanning calorimetry, optical and electron microscopy, electron spin resonance spectroscopy) have been used in the study of the interaction of gramicidin A with phospholipid bilayers [S]. Recently, it has been demonstrated by NMR that gramicidin A promotes the formation of the hexagonal HI, phase in aqueous dispersion of phosphatidylethanolamine and phosphatidylcholine [9 -121. Further, conformational studies of gramicidin A incorporated into lysophosphatidylcholine micelles [13] showed that the channel assumes a stable conformation with a circular dichroisc pattern that is characteristic of left-handed helices. 23Na-and I3C-NMR studies have provided arguments that the stable heat-incorporated state is that of the functional transmembrane channel [14]. The incorporated polypeptide molecules are Abbreviation. ~-a-lyso PC, L-a-lysophosphatidylcholine.
The thermotropic state transition of the lipids in isolated platelet membranes has been studied by Ranian spectroscopy in the temperature range from -3 "C to + 45 "C using the (C -H) stretching vibrations, carotenoid (s) vibrations at 1530cmp' and 1160cm-', as well as the skeletal optical vibrations.1. The increase of temperature causes a decrease in intensity of the 2885 cm-* band relative to the 2855 cm-' one.The evaluation of the ratio 12885/Z2855 as a function of temperature indicates a double thermotropic state transition of platelet membrane lipids: the first one near 5°C and the second near 17.5"C.2. The ratio 11530/11160 shows that the intensity variations in the carotenoid(s) peaks follow the second lipid transition. Thus, it seems that the platelet membrane carotenoid(s) might be linked to the lipids which undergo transition near 17.5 "C.3. The spectral changes in the skeletal optical range suggest a considerable proportion of all trans chains in the membrane lipids at lower temperatures whereas gauche structures seem to be introduced at higher temperatures.There is evidence that the plasma membrane of blood platelets plays an important role in the platelet function [I].In an approach to the molecular organisation of platelet membranes we have undertaken Raman and infrared spectroscopic studies on intact platelet as well as on isolated platelet membranes [2-41. In the course of these studies, besides the presence of carotenoid(s) in platelet membranes [2], a thermotropic state transition of the platelet membrane lipid molecules has been signalized [4].Raman spectroscopy has provided information about thermotropic state transitions which occur in model membrane systems ([5] and references therein) and in lipids [6,7], as well as in plasma membranes isolated from various types of cells, such as erythrocytes [8], lymphocytes [9] or thymocytes [lo]. Therefore, in an attempt to know more about the characteristics and thermal response of platelet membrane components, we have investigated the variations of the Raman scattering as a function of temperature over the range -3 "C to + 45 "C.The present work analyses the main variations with temperature of Raman scattering which are due to (a) (C-H) stretching vibrations (2800-3000 cm-I ) ; (b) resonanceenhancedvibrations, v(-C=C-)andv(=C-C=)ofplatelet membrane-bound carotenoid(s) previously identified [2] and (c) skeletal optical v(C-C) vibrations (1000-1150cm-').Results are reported which show that the lipids in platelet membrane do undergo a double thermotropic state transition: the first one between 0 "C and 7 "C and the second one between 13°C and 20 "C. The spectral characteristics indicate that the carotenoid(s) present in platelet membranes, in addition to the carotenoid-protein complexes that are formed [2], might also be linked to the lipids which undergo transition between 13°C and 20 "C. MATERIALS AND METHODSPreparation of Platelet Membranes. Platelets were isolated by differential centrifugation from fresh human blood anticoagulated with citrate/d...
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