Identification of the disulfide-linked homodimer of apolipoprotein E3 in plasma. Impact on receptor binding activity.
The nature of disulfide-linked structures of apolipoprotein (apo) E3 in the plasma of E3/3 subjects was examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis performed under nonreducing conditions followed by immunoblotting with apoE-specific antibodies. In addition to the expected presence of the heterodimer apoE3-A-II and monomeric apoE3, a band with an apparent Mr approximately 100,000 was also observed in plasma that had been treated with sulfhydryl-trapping reagents. This band and apoE3-A-II were both eliminated by disulfide reduction, which produced a corresponding increase in monomeric apoE3. Both bands were absent in plasma from a subject with the E4/4 phenotype. In spite of its apparent molecular weight on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the high molecular weight band was demonstrated to represent the disulfide-linked homodimer of apoE3. It was isolated from purified apoE3 preparations that had undergone oxygen-mediated dimerization and shown to elute from a Sephacryl S-300 column in a position with the expected molecular weight of a homodimer. The apoE3 dimer displayed a preference for high density lipoproteins, as determined by agarose chromatography of E3/3 plasma but was stripped from high density lipoproteins by ultracentrifugation. Quantitation of the relative ratios of homodimer, apoE3-A-II, and monomer in the plasma of 22 normolipidemic E3/3 subjects by immunoblotting revealed that the disulfide-linked structures accounted for the majority (approximately 55%) of plasma apoE. Both the homodimer and apoE3-A-II displayed a reduced ability to compete with low density lipoproteins for fibroblast low density lipoprotein receptors (20 and 30% of monomeric apoE3 binding activity, respectively). These results raise the possibility that the amount or availability of receptor-active apoE3 in E3/3 subjects may be rate limiting for metabolic events involving the low density lipoprotein receptor.
Weisgraber
K H
KH
Department of Pathology, University of California, San Francisco 94140-0608.
Shinto
L H
LH
D001057:Apolipoproteins E / Q000097:blood*; D001667:Binding, Competitive; D015153:Blotting, Western; D002850:Chromatography, Gel; D046508:Culture Techniques; D004220:Disulfides / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D010641:Phenotype; D011869:Radioligand Assay; D011973:Receptors, LDL / Q000187:drug effects*; D013438:Sulfhydryl Compounds / Q000737:chemistry; D014407:Tumor Cells, Cultured
D001057:Apolipoproteins E; D004220:Disulfides; D011973:Receptors, LDL; D013438:Sulfhydryl Compounds