The uptake and degradation of low density lipoprotein (LDL) by human choriocarcinoma cells in culture was studied using [125I]iodo-LDL as the substrate. At 37 C, uptake of [125I]iodo-LDL was rapid, attaining a steady state within 4.5 h. After a lag time of about 1 h, degradation of [125I]iodo-LDL proceeded linearly for at least 30 h. The relationship of uptake and degradation to the concentration of [125I]iodo-LDL in the culture medium was consistent with the conclusion that both of these processes involved binding to a population of high affinity (apparent Kd, 4 × 10-9 M), low capacity cell surface receptors. High density lipoprotein caused little displacement of [125I]iodo- LDL from these receptor sites. At 4 C, the extent of uptake of [125I]iodo-LDL was reduced by 75%, whereas its degradation was completely inhibited. At 4 C, addition of heparin (10 mg/ml) to the culture medium bathing the cells caused dissociation of most of the bound [125I]iodo-LDL from the cells; but at 37 C, addition of heparin caused only a small dissociation of [125I]iodo-LDL from the cells. In the presence of chloroquine, the degradation of [125I]iodo-LDL was completely inhibited, but the extent of uptake of [125I]iodo-LDL was increased. Degradation of [125I]-iodo-LDL was inhibited by cyanide, azide, arsenate, carbonylcyanide trifluoromethoxyphenylhydrazone, ammonium chloride, and the protease inhibitors, L-l-tosylamide-2-phenylethylchloromethyl ketone and N-α-tosyl-L-lysine chloromethyl ketone. [125I]Iodo-high density lipoprotein was also taken up and degraded by human choriocarcinoma cells in a time- and concentration-dependent fashion, but the rate of degradation was considerably less than was the rate of degradation of [125I]iodo-LDL. These results are consistent with the following sequence of events in LDL metabolism. LDL first binds to specific high affinity receptors on the cell surface, and is then internalized by a temperature- and energy-dependent endocytotic process. Once internalized, the protein component of LDL is degraded by lysosomal proteases, and the cholesterol ester component of LDL is hydrolyzed. The resultant free cholesterol suppresses endogenous cholesterol synthesis and serves as a substrate for progesterone biosynthesis.
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