Degradation Of Apo Research Articles

The conformation of apolipoprotein E isoforms in phospholipid complexes and their interaction with human Hep G2 cells

The human hepatoma cell line, Hep G2, has been used to compare the metabolism by isolated liver cells of purified isoforms of human apolipoprotein E (apo E). Complexes of [ 125I]apo E-3 3 , 2 2 , 3 2 and 4 3 with dimyristoyl phosphatidylcholine (DMPC) were prepared by a detergent-dialysis method: discoidal, bilayer complexes with a stoichiometry of 125 ± 15 mol DMPC/mol apo E resulted. The predominant phenotype apo E-3 3 , and the phenotype apo E-2 2 characteristic of patients with Type III hyperlipoproteinemia, interact similarly with DMPC and adopt the same conformation with 60–70% α-helix, as monitored by circular dichroism spectroscopy. The uptake and degradation at 37°C, and binding at 4°C by Hep G2 cells, of [ 125I]apo E-3/3/DMPC and [ 125I]apo E-2/2/DMPC complexes were compared. Apo E-3 3 was degraded more rapidly than apo E-2 2 suggesting that the diminished catabolism of the latter phenotype by intact livers is due to lack of recognition by the hepatocytes. The observed degradation of apo E was 3–4 times greater than that which could be attributed to fluid phase endocytosis and low-affinity adsorptive endocytosis. The degradation of [ 125I]apo A–I by Hep G2 cells can be accounted for by the above endocytotic mechanisms. The distinction between apo E-3 3 and apo E-2 2 isoforms is attributed to the presence of a cell-surface receptor on Hep G2 cells which binds apo E-3 3 with a higher affinity than apo E-2 2 .

Cholesterol esterification in macrophages. Stimulation by lipoproteins containing apo B isolated from human aortas.

A lipoprotein fraction possessing many of the characteristics of plasma low density lipoproteins (P-LDL) was isolated from homogenates of lesioned human aortas by affinity chromatography. In contrast to P-LDL, this fraction, termed A-LP, was found to be more electronegative than P-LDL and to stimulate cholesterol esterification in mouse peritoneal macrophages (MPM). This stimulation tended toward saturation at approximately 100 micrograms/ml lipoprotein cholesterol. Cholesterol esterification was partially inhibited by fucoidin, a competitive inhibitor of the scavenger receptor on MPM. These results suggest that A-LP is recognized by a high affinity binding site on MPM. Stimulation of cholesterol esterification in MPM by A-lP was inhibited by the lysosomotropic agent, chloroquine, indicating that degradation in lysosomes was a prerequisite for cholesterol esterification. Substantial degradation of apo B within the intact A-LP was demonstrated by SDS-PAGE and immunoblotting. This degradation could be responsible for the increased negative charge on A-LP, and the enhanced recognition of A-LP by a receptor on MPM. Stimulation of cholesterol esterification increased linearly over a 48-hour time period, suggesting that the receptor on MPM recognizing A-LP was not down-regulated. This unabated increase in cholesterol esterification resulted in massive accumulation of intracellular cholesteryl esters and a gradual increase in oil red O-positive droplets, giving the MPM the characteristic morphology of foam cells. If these mechanisms demonstrated in vitro also occur in the intact artery, they could explain how foam cells are formed within the fatty streak lesion.

Differential effects of ascorbate and EDTA on high-affinity binding of 3H-apomorphine and 3H-ADTN to calf caudate membranes

Recent reports describe effects of ascorbate on binding of dopaminergic agonists to membrane preparations of brain tissue. We now report that with calf caudate membranes, in the absence of EDTA, ascorbate (up to 10 mM) lowered the binding of [ 3H](−)apomorphine ( 3H-APO) by up to 34%, and the proportion “specific,” from 69% to 36%, and lowered binding of [ 3H](±)2-amino-6,7-dlhydroxy-1,2,3,4-tetrahydronaphthalene ( 3H-ADTN) by up to 38%, with little effect on the proportion of its specific binding. EDTA (5 mM), alone, also reduced binding of both ligands by 40–50% but had no effect on the proportion specific. With ascorbate plus EDTA, although total binding was lowered, the proportion of specific binding of 3H-APO rose to a maximum of 82% at 2.5 mM ascorbate, whereas that of 3H-ADTN was only slightly increased. Moreover, TLC revealed that the antioxidants were required during incubation to prevent temperature- and timedependent degradation of APO much more than ADTN. Thus, while each additive, alone, lowered binding of 3H-APO and of 3H-ADTN, ascorbate plus EDTA increased the proportion of specific binding, especially of 3H-APO, and protected both from degradation.