Low-density lipoprotein binding assay using the calf adrenocortical low-density lipoprotein receptor.
The low-density lipoprotein (LDL) receptor was purified to a semipure solubilized form from calf adrenocortical tissue. This receptor was found to be a suitable substitute for the human LDL receptor for studying human LDL binding. The apparent dissociation constant of the receptor from calf adrenocortical cells, using human LDL as the ligand, was found to be 8.8 +/- 1.0 micrograms 125I-LDL/mL, similar to that reported for the human LDL receptor (4-10 micrograms LDL/mL). The calf adrenocortical LDL receptor demonstrated specificity toward human lipoprotein fractions that was identical with that of the human LDL receptor. A competitive binding assay was optimized using the semipurified solubilized calf adrenocortical receptor. This facilitated the study of nonenzymatically glycosylated human LDL by a binding assay that is much simpler and faster than previous studies, which used intact cultured cells. The present assay requires only a 1-h incubation of LDL with the receptor and a simple filtration procedure to remove unbound LDL. Using the present assay, it was shown that nonenzymatic glycosylation of LDL on the order of what is seen in diabetics, that is, modification of 2-5% of lysine residues, caused a decreased ability of the LDL to bind to the receptor.
Catomeris
P
P
Department of Chemistry and Biochemistry, University of Windsor, Ontario.
Thibert
R J
RJ
Draisey
T F
TF
D000302:Adrenal Cortex / Q000166:cytology / Q000378:metabolism*; D000818:Animals; D001667:Binding, Competitive; D001798:Blood Proteins / Q000378:metabolism; D015153:Blotting, Western; D002478:Cells, Cultured; D002848:Chromatography, DEAE-Cellulose; D003920:Diabetes Mellitus / Q000097:blood; D004591:Electrophoresis, Polyacrylamide Gel; D006031:Glycosylation; D006801:Humans; D007457:Iodine Radioisotopes; D008077:Lipoproteins, LDL / Q000097:blood* / Q000302:isolation & purification; D008239:Lysine / Q000378:metabolism; D011485:Protein Binding; D011973:Receptors, LDL / Q000378:metabolism*
D001798:Blood Proteins; D007457:Iodine Radioisotopes; D008077:Lipoproteins, LDL; D011973:Receptors, LDL; D008239:Lysine