Purification of the apolipoprotein B-67-containing low density lipoprotein particle and its affinity for the low density lipoprotein receptor.
Naturally occurring mutant forms of apolipoprotein B (apoB)-100 may be able to provide valuable information on the structure-function relationships of apoB with the low density lipoprotein (LDL) receptor. ApoB-67, recently identified in a kindred displaying apoB levels 25% of normal (Welty et al. J. Clin. Invest. 1991. 87: 1748-1754), is predicted to contain 3040 amino acids and therefore, contains part of the epitope for antibody 4G3, which blocks binding of LDL to the LDL-receptor. To determine whether the amino terminal 67% of apoB-100 is important for binding to the LDL receptor, the apoB-67-containing lipoprotein particle was purified from plasma by gradient ultracentrifugation. The fractions containing apoB-67 were in the density range 1.049-1.070 g/ml. These fractions were pooled and adsorbed onto an affinity chromatography column containing the monoclonal antibody, MB-47. The epitope for MB-47 is two nonlinear domains between amino acids 3429 to 3453 and 3507 to 3523; therefore, apoB-100 will bind to the MB-47 column but apoB-67 will not. The resulting apoB-67-containing particles were completely devoid of apoB-100. In competitive binding studies, the apoB-67 lipoprotein particle did not compete with 125I-labeled apoB-100-containing LDL particles for binding, uptake, or degradation by normal human fibroblast monolayers. We conclude that the amino terminal 67% of apoB-100 in the naturally occurring lipoprotein particle does not appear to contain a functionally relevant epitope of the LDL-receptor binding domain.
Welty
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Jean Mayer USDA Human Nutrition Center on Aging, Tufts University, Boston, MA 02215, USA.
Seman
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Yen
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D001053:Apolipoproteins / Q000302:isolation & purification / Q000378:metabolism*; D001667:Binding, Competitive; D002478:Cells, Cultured; D002846:Chromatography, Affinity / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel; D005347:Fibroblasts / Q000378:metabolism; D006801:Humans; D011485:Protein Binding; D011973:Receptors, LDL / Q000378:metabolism*; D014461:Ultracentrifugation
D001053:Apolipoproteins; D011973:Receptors, LDL