Lipid-poor apolipoprotein A-I in Hep G2 cells: formation of lipid-rich particles by incubation with dimyristoylphosphatidylcholine.
Apolipoprotein A-I is a major secretory product of the human hepatoma cell line, Hep G2; approx. 70% of apolipoprotein A-I was separated from the medium as lipid-poor apolipoprotein A-I in the d greater than 1.21 g/ml fraction while 30% was associated with high-density lipoproteins (HDL) of d 1.063-1.21 g/ml. The lipid-poor apolipoprotein A-I contains 50% proapolipoprotein A-I which is similar to the isoform distribution in Hep G2 preformed HDL. We tested the ability of lipid-poor apolipoprotein A-I from Hep G2 to form complexes with dimyristoylphosphatidylcholine (DMPC) vesicles at DMPC/apolipoprotein A-I molar ratios of 100:1 and 300:1. Lipid-poor apolipoprotein A-I was recovered in complex form while at a 300:1 ratio, 68.8 +/- 6.3% was recovered. On electron microscopy, the former complexes were small discs 16.9 nm +/- 4.5 S.D. in diameter while the latter were larger discs 21.4 +/- 4.4 nm diameter. Non-denaturing gradient gel electrophoresis of complexes formed at a 100:1 ratio had a peak in the region corresponding to 9.64 +/- 0.08 nm; these particles possessed two apolipoprotein A-I molecules. At the higher ratio, 300:1, two distinct complexes were identifiable, one which banded in the 9.7 nm region and the other in the 16.9-18.7 nm region. The former particles contained two molecules of apolipoprotein A-I and the latter, three molecules. This study demonstrates that lipid-poor apolipoprotein A-I which is rich in more basic isoforms forms discrete lipoprotein complexes similar to those formed by mature apolipoprotein A-I. It is further suggested that, under the appropriate conditions, precursor or nascent HDL may be assembled extracellularly.
D016632:Apolipoprotein A-I; D001054:Apolipoproteins A / Q000378:metabolism*; D006528:Carcinoma, Hepatocellular; D002460:Cell Line; D002499:Centrifugation, Density Gradient; D004134:Dimyristoylphosphatidylcholine / Q000378:metabolism*; D006801:Humans; D007163:Immunosorbent Techniques; D007525:Isoelectric Focusing; D008075:Lipoproteins, HDL / Q000378:metabolism; D008099:Liver / Q000378:metabolism*; D008113:Liver Neoplasms; D046911:Macromolecular Substances; D008854:Microscopy, Electron; D011485:Protein Binding
D016632:Apolipoprotein A-I; D001054:Apolipoproteins A; D008075:Lipoproteins, HDL; D046911:Macromolecular Substances; D004134:Dimyristoylphosphatidylcholine