Distribution of lipid-binding regions in human apolipoprotein B-100.
The distribution of lipid-binding regions of human apolipoprotein B-100 has been investigated by recombining proteolytic fragments of B-100 with lipids and characterizing the lipid-bound fragments by peptide mapping, amino acid sequencing, and immunoblotting. Fragments of B-100 were generated by digestion of low-density lipoproteins (LDL) in the presence of sodium decyl sulfate with either Staphylococcus aureus V8 protease, pancreatic elastase, or chymotrypsin. Particles with electron microscopic appearance of native lipoproteins formed spontaneously when detergent was removed by dialysis from enzyme digests containing fragments of B-100 and endogenous lipids, or from incubation mixtures of delipidated B-100 fragments mixed with microemulsions of exogenous lipids (cholesteryl oleate and egg phosphatidylcholine). Fractionation of the recombinant particles by isopycnic or density gradient ultracentrifugation yielded complexes similar to native LDL with respect to shape, diameter, electrophoretic mobility, and surface and core compositions. Circular dichroic spectra of these particles showed helicity similar to LDL but a somewhat decreased content of beta-structure. Most of the fragments of B-100 were capable of binding to lipids; 12 were identified by direct sequence analysis and 14 by reaction with antisera against specific sequences within B-100. Our results indicate that lipid-binding regions of B-100 are widely distributed within the protein molecule and that proteolytic fragments derived from B-100 can reassociate in vitro with lipids to form LDL-like particles.
Chen
G C
GC
Department of Anatomy, University of California, San Francisco 94143-0130.
Hardman
D A
DA
Hamilton
R L
RL
Mendel
C M
CM
Schilling
J W
JW
Zhu
S
S
Lau
K
K
Wong
J S
JS
Kane
J P
JP
D053299:Apolipoprotein B-100; D001055:Apolipoproteins B / Q000097:blood* / Q000235:genetics / Q000648:ultrastructure; D002478:Cells, Cultured; D002499:Centrifugation, Density Gradient; D002942:Circular Dichroism; D005347:Fibroblasts / Q000378:metabolism; D006801:Humans; D008854:Microscopy, Electron; D010446:Peptide Fragments / Q000302:isolation & purification / Q000378:metabolism; D011487:Protein Conformation; D011973:Receptors, LDL / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism / Q000648:ultrastructure; D014461:Ultracentrifugation
D053299:Apolipoprotein B-100; D001055:Apolipoproteins B; D010446:Peptide Fragments; D011973:Receptors, LDL; D011994:Recombinant Proteins