Coupling of photoactivatable glycolipid probes to apolipoproteins A-I and A-II in human high density lipoproteins 2 and 3.
High density lipoprotein (HDL) from human serum was subfractionated into HDL2 and HDL3 by rate-zonal density gradient ultracentrifugation. The orientation of apoproteins (apo) A-I and A-II in these subfractions was investigated by use of the photosensitive glycolipid probes, 2-(4-azido-2-nitrophenoxy)-palmitoyl[1-14C]glucosamine (compound A) and 12-(4-azido-2-nitrophenoxy)-stearoyl[1-14C]glucosamine (compound B). Both probes were added to the HDL-structures in a ratio of two or three probe molecules per particle and were photoactivated by irradiation at a wavelength above 340 nm. After delipidation the probe-apoprotein adducts were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Both the "shallow" probe (compound A) and the "depth" probe (compound B) were coupled for 10-14% (of the label added) to apoA-I and apoA-II from HDL3 and for about 6% to apoA-I and apoA-II from HDL2. By taking into account the relative amounts of apoA-I and apoA-II, it was estimated that the "shallow" probe labeled apoA-I 40% more effectively than apoA-II in both HDL2 and HDL3; the "depth" probe labeled apoA-I and apoA-II equally well in both subfractions. The data suggest that towards the surface HDL2 and HDL3 contain a relatively larger portion of apoA-I than apoA-II, whilst towards the core both subfractions are occupied by an equal portion of apoA-I and apoA-II. Application of these photolabels has failed to point out differences in the structural organization of HDL2 and HDL3.
D000345:Affinity Labels; D016632:Apolipoprotein A-I; D016633:Apolipoprotein A-II; D001054:Apolipoproteins A; D006017:Glycolipids; D006801:Humans; D008075:Lipoproteins, HDL / Q000302:isolation & purification; D046911:Macromolecular Substances; D008968:Molecular Conformation; D010777:Photochemistry
D000345:Affinity Labels; D016632:Apolipoprotein A-I; D016633:Apolipoprotein A-II; D001054:Apolipoproteins A; D006017:Glycolipids; D008075:Lipoproteins, HDL; D046911:Macromolecular Substances