Purification of low density lipoprotein receptor from liver and its quantification by anti-receptor monoclonal antibodies.
The low density lipoprotein (LDL) receptor has been purified to homogeneity from rabbit liver by a combination of DEAE-Sephacel chromatography, LDL-Sepharose 4B chromatography and preparative SDS/polyacrylamide-gel electrophoresis. The receptor protein had a pI of 4.45 and an Mr of 120 x 10(3)-125 x 10(3) in SDS gels under non-reducing conditions. Incubation of the LDL receptor with neuraminidase decreased its Mr to 105 x 10(3)-110 x 10(3) and increased its pI from 4.45 to 5.25. The purified receptor exhibited all the properties of the membrane-bound receptor including Ca2+-dependent binding of rabbit and human LDL but not of methylated LDL or high density lipoprotein. The amount of LDL receptor present in rabbit liver was measured by a quantitative blotting procedure employing a newly developed rat anti-receptor monoclonal antibody. The affinity and specificity of this monoclonal antibody allowed the quantification of the LDL receptor in detergent extracts of liver homogenate, thus eliminating the loss of receptor associated with the preparation of membrane fractions prior to receptor assay. Livers from adult female New Zealand White rabbits contained 149 +/- 13 ng of LDL receptor/mg of liver protein. Administration of pharmacological doses of 17 alpha-ethinyloestradiol raised the concentration of LDL receptor in liver to 312 +/- 25 ng/mg of liver protein.
D000818:Animals; D000911:Antibodies, Monoclonal; D016632:Apolipoprotein A-I; D001054:Apolipoproteins A / Q000097:blood; D001055:Apolipoproteins B / Q000097:blood; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D004997:Ethinyl Estradiol / Q000494:pharmacology; D005260:Female; D007122:Immunoelectrophoresis; D008099:Liver / Q000032:analysis* / Q000187:drug effects / Q000378:metabolism; D008297:Male; D009929:Organ Size / Q000187:drug effects; D011817:Rabbits; D011973:Receptors, LDL / Q000187:drug effects / Q000276:immunology / Q000302:isolation & purification*
D000911:Antibodies, Monoclonal; D016632:Apolipoprotein A-I; D001054:Apolipoproteins A; D001055:Apolipoproteins B; D011973:Receptors, LDL; D004997:Ethinyl Estradiol