A quantitative assay for the non-covalent association between apolipoprotein[a] and apolipoprotein B: an alternative measure of Lp[a] assembly.
Increasing evidence suggests that the assembly of lipoprotein[a] (Lp[a]) proceeds in two steps. In the first step, non-covalent interactions between apolipoprotein[a] (apo[a]) and apolipoprotein B (apoB) of low density lipoprotein (LDL) form a dissociable apo[a]:LDL complex. In the second step, a covalent disulfide linkage forms the stable Lp[a] particle. Several methods are currently used to study the assembly of Lp[a], however, these methods are laborious, time-consuming, and not suitable for a high throughput screening. We report here the development of a rapid and simple assay based on the binding of labeled LDL to a Lp[a]/apo[a] substrate which is immobilized on the surface of a microtiter plate. Quantification of bound LDL provides a measure of the extent of complex formation. Labeled LDL bound to both Lp[a] and apo[a] substrates with similar affinity. Plasma lipoproteins containing apoB as well as free apo[a] were capable of competing with LDL binding. The binding of LDL to Lp[a]/apo[a] was inhibited by L-proline and lysine analogs, which are known to inhibit the non-covalent association between apo[a] and apoB. Using this method we have found that nicotinic acid and captopril are able to inhibit the association of apo[a] with apoB. This method is compatible with automation and can be applied to a high throughput screening of inhibitors of Lp[a] formation.
Dardik
B N
BN
Novartis Institute for Biomedical Research, Novartis Pharmaceuticals Corporation, Summit, NJ 07901, USA.
Schwartzkopf
C D
CD
Stevens
D E
DE
Chatelain
R E
RE
D001053:Apolipoproteins / Q000097:blood*; D001055:Apolipoproteins B / Q000097:blood*; D053241:Apoprotein(a); D016650:Fluorescein-5-isothiocyanate; D006801:Humans; D007457:Iodine Radioisotopes; D017270:Lipoprotein(a) / Q000097:blood*; D008077:Lipoproteins, LDL / Q000037:antagonists & inhibitors; D008239:Lysine / Q000031:analogs & derivatives / Q000494:pharmacology; D011392:Proline / Q000031:analogs & derivatives / Q000494:pharmacology; D011485:Protein Binding; D011499:Protein Processing, Post-Translational
D001053:Apolipoproteins; D001055:Apolipoproteins B; D007457:Iodine Radioisotopes; D017270:Lipoprotein(a); D008077:Lipoproteins, LDL; D011392:Proline; D053241:Apoprotein(a); D016650:Fluorescein-5-isothiocyanate; D008239:Lysine