RESUMEN
The chemical synthesis of azido-substituted saturated, monoenoic and dienoic fatty acids of high specific tritium radioactivity, together with improved methods for their introduction into phosphatidylcholine, sphingomyelin and cholesteryl ester molecules are outlined. Phospholipid vesicles with these photosensitive probes were irradiated and the main crosslinking products isolated and characterized by high resolution mass spectroscopy.
Asunto(s)
Colesterol/análogos & derivados , Ácidos Grasos/síntesis química , Liposomas , Fosfatidilcolinas/síntesis química , Esfingomielinas/síntesis química , Azidas , Colesterol/síntesis química , Espectrometría de Masas , Métodos , Relación Estructura-ActividadRESUMEN
The molecular interactions and spatial arrangements of phospholipids and apoproteins of human high-density lipoprotein were studied by a chemical approach. Phosphatidylcholines and sphingomyelins substituted with fatty acyl residues of high specific radioactivity and labelled with the photosensitive azido group in specific positions were prepared by chemical synthesis. They were recombined with apolipoprotein A-I of human serum high density lipoprotein. The lipoprotein complexes containing either azido lecithins or azidosphingo-myelins were purified by agarose chromatography from excess lipids. The irradiation was performed under conditions which prohibit the interference with the apoprotein structure as proven by circular dichroism, fluorescence spectroscopy, immunodiffusion test and disc electrophoresis. Non-covalently bound lipid molecules were removed by Sephadex LH 20 chromatography. Mild alkaline treatment liberated radioactive fatty acids which were not directly linked to the polypeptide chain, but rather via neighbouring phospholipid molecules. The lipoprotein appeared as a single radioactive band in dodecylsulfate polyacrylamide gel electrophoresis as seen by radioscanning, which further proved the covalent linkage of the fatty acyl residues to the polypeptide chain. In the immunodiffusion test, there is no difference between covalently crosslinked phospholipid-apoLp A-I complex and the non-photolyticall treated complex. This is the first chemical proof of the spatial relationship of the hydrophobic side chains of the lipid and polypeptide chains in a lipoprotein complex.
Asunto(s)
Apolipoproteínas , Luz/efectos adversos , Fosfatidilcolinas , Esfingomielinas , Sitios de Unión , Dicroismo Circular , Reacciones Cruzadas , Humanos , Inmunodifusión , Lipoproteínas HDL , Peso MolecularRESUMEN
Chromatographyically and immunologically homogeneous apolipoprotein A-I (apoLp A-I) from human serum has been recombined in separate experiments with three species of sphingomyelin. The differed in the degree of saturation of their fatty acyl residues, stearoyl (18:0), oleoyl (18:1) and linoleoyl (18:2). The lipoprotein complexes formed were purified by CsCl density gradient centrifugation between 1.07 - 1.09 g/cm3 and by gel filtration. Stearoylsphingomyelin does not recombine with the apoprotein A-I below its phase transition temperature (tc = 41.5 degrees C). The lipoproteins eluted with the following apparent molecular weights: 18:0-sphingomyelin apoLp A-I, 8.0 X 10(5); 18:1-sphingomyelin apoLp A-I, 4.0 X 10(5); and 18:2-sphingomyelin apoLp A-I, 4.0 X 10(5). In electron microscopy the particles appear as discs of 160 - 170 A diameter and 50 - 60 A thickness. Their tendency to form stacked aggregates of discs decreases with the degree of their unsaturation. CD measurements underline the considerable increase in alpha-helicity of the secondary structure of apo A-I after recombination with the phospholipids. This increase in order is equal for the three sphingomyelin species (alpha-helicity of apoLP A-I = 0.46, after recombination 0.89). If the three sphingomyelin species are used in equal molar amounts in the recombination experiment, no preference for any one sphingomyelin species is observed. Recombination of apoLp A-I with sphingomyelin, labelled with the isotope 13C in the choline group, C-14 of stearic or linoleic, or C-11 of oleic acid, were performed for spin lattice relaxation time (T1) experiments. Compared with sphingomyelin liposomes, the polar head groups of these lipids in the lipoprotein particles possess a considerably higher mobility, whereas the changes in T-1-times of the C-atoms in the centre of the fatty acid chains of the lipids refer to their interactions with the polypeptide side chains. A model of the lipoprotein complexes formed is proposed on the basis of the experimental data.