| 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. |
