The effect of phospholipids on apoB conformation and stability in reconstituted low density lipoproteins.

Abstract

This thesis is concerned with the mechanism that underlies the apparent atherogenic capacity of low density lipoproteins (LDL). This work provides a means for determining the effects of LDL phospholipids on the conformation of the protein moiety of LDL, apolipoprotein B (apoB). Reconstituted LDL (rLpB) particles were prepared from apoB and palmitoyloleoyl phosphatidylcholine (POPC). Intact apoB was isolated from native LDL retained $\sim$70% of the triglyceride (TG) from LDL. Aqueous soluble apoB-TG complexes exhibited a significantly reduced amphipathic $\alpha$-helical content (17%) and net negative charge ($-$2.9 mV) as compared to LDL-bound apoB (49% and $-$6 mV). Reconstitution of apoB-TG with phospholipid was accomplished through spontaneous complexation with POPC vesicles. apoB-TG was able to rapidly (10 min.) solubilize POPC at temperatures ranging from 4 to 24$\sp\circ$C. Electrophoresis on 4-15% gradient acrylamide gels showed the rLpB particles to be homogeneous and to exhibit a single discrete band. Inclusion of 300 molecules of POPC significantly increased the $\alpha$-helical content of apoB to 34% and the net negative charge to $-$4.9 mV. Phospholipidation of apoB also significantly decreased the effectiveness of GdnHCl in unfolding the apoprotein. Similar observations were seen with native LDL. The immunoreactivity of rLpB was assessed using various monoclonal antibodies. apoB-TG was immunoreactive with antibodies 1D1, 2D8, 3F5, 4G3 and 5E11. The inclusion of 300 molecules of POPC significantly increased the immunoreactivity of the conformation-specific antibodies 2D8 (p 0.001) and 4G3 (p 0.01) but had no major effect on the epitope accessibility or affinity of the other monoclonal antibodies studied. (Abstract shortened by UMI.)