Chemical proof of lipid-protein interactions by crosslinking photosensitive lipids to apoproteins. Intermolecular cross-linkage between high-density apolipoprotein A-I and lecithins and sphingomyelins.
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.
D001053:Apolipoproteins; D001665:Binding Sites; D002942:Circular Dichroism; D003429:Cross Reactions; D006801:Humans; D005779:Immunodiffusion; D008027:Light / Q000009:adverse effects*; D008075:Lipoproteins, HDL; D008970:Molecular Weight; D010713:Phosphatidylcholines; D013109:Sphingomyelins
D001053:Apolipoproteins; D008075:Lipoproteins, HDL; D010713:Phosphatidylcholines; D013109:Sphingomyelins