A simple binding assay for the determination of low-density lipoprotein receptors in cell homogenates.
A convenient binding assay has been developed for the determination of low-density lipoprotein (LDL) receptors in homogenates of cultured and freshly-isolated normal and malignant human cells. Cell homogenates were incubated with 125I-labeled LDL and the ligand bound to the homogenate particulates was separated from the unbound ligand by filtration. When the particulates of the homogenates were subsequently incubated with heparin, a fraction of the bound 125I-LDL was released. Previous studies on intact cells have shown that heparin exclusively releases LDL bound to its cell surface receptor. The heparin-sensitive binding of 125I-LDL to cell homogenate particulates represents LDL bound to its cell surface receptor as judged from the following criteria: (a) it was quantitatively similar to the heparin-sensitive binding of 125I-LDL to intact cells, (b) it showed a direct correlation to the receptor-mediated degradation of 125I-LDL by intact cells, (c) no heparin-sensitive binding could be detected in homogenates prepared from normal erythrocytes or from cultured fibroblasts from a patient with homozygous familial hypercholesterolemia (two types of cell lacking LDL receptors), (d) it was dependent on calcium and inhibited by EDTA, (e) it was susceptible to treatment with pronase, and (f) it was heat-labile. The assay developed should be of value in determining the number of LDL receptors in tissues, since it is far less time-consuming and requires less material than currently available methods.
D000328:Adult; D002118:Calcium / Q000494:pharmacology; D002460:Cell Line; D004492:Edetic Acid / Q000494:pharmacology; D005260:Female; D005347:Fibroblasts / Q000378:metabolism; D005910:Glioma / Q000378:metabolism; D006493:Heparin / Q000494:pharmacology; D006358:Hot Temperature; D006801:Humans; D006938:Hyperlipoproteinemia Type II / Q000378:metabolism; D008722:Methods; D011402:Pronase / Q000378:metabolism; D011973:Receptors, LDL / Q000378:metabolism*
D011973:Receptors, LDL; D006493:Heparin; D004492:Edetic Acid; D011402:Pronase; D002118:Calcium