title: Immunoblot analysis of low density lipoprotein receptors in fibroblasts from subjects with familial hypercholesterolemia.
abstract: This paper describes a sensitive method for study of the isoelectric point and molecular weight of immunoreactive low density lipoprotein (LDL) receptors of cultured human fibroblasts. The fibroblast receptors are solubilized with Triton X-100, partially purified by batch elution from DEAE-cellulose, and subjected to two-dimensional isoelectric focusing/sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The proteins are transferred electrophoretically to nitrocellulose paper which is then incubated with a mouse monoclonal antibody (IgG-C7) directed against the LDL receptor, followed by an 125I-labeled antibody against mouse IgG. The receptor-bound monoclonal antibody is localized by autoradiography. By this technique, the immunodetectable LDL receptors from normal human fibroblasts migrate as a single spot with an isoelectric point of 4.3 and a Mr of approximately 160,000. In one patient with homozygous familial hypercholesterolemia whose cells fail to bind 125I-labeled IgG-C7, no immunoreactive LDL receptor spot was detected after electrophoresis. We also studied LDL receptors from three homozygotes whose cells bind 125I-IgG-C7, i.e. cross-reacting material-positive mutants. Their immunodetectable receptors were indistinguishable from normal receptors in terms of isoelectric point and molecular weight. Similarly, the receptors from one patient with the internalization-defective form of familial hypercholesterolemia showed normal electrophoretic migration. The immunoblotting technique should prove useful in analyzing structural alterations, if they exist, in LDL receptors from other subjects with cross-reacting material-positive forms of familial hypercholesterolemia.
mesh_terms: D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D002478:Cells, Cultured; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D005347:Fibroblasts / Q000032:analysis; D006720:Homozygote; D006801:Humans; D006937:Hypercholesterolemia / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008077:Lipoproteins, LDL / Q000032:analysis*; D011956:Receptors, Cell Surface / Q000032:analysis* / Q000378:metabolism; D011973:Receptors, LDL
chemical_list: D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D008077:Lipoproteins, LDL; D011956:Receptors, Cell Surface; D011973:Receptors, LDL