Physical properties of recombinant apolipoprotein(a) and its association with LDL to form an LP(a)-like complex.
Recombinant apolipoprotein(a) has been studied by hydrodynamic techniques and electron microscopy. Recombinant apo(a) was primarily a monomer in solution with an s0(20,w) of 9.3 S, a D20,w of 2.29 ficks, and a molecular weight of 325,000 from sedimentation equilibrium and 318,000 from combining the sedimentation and diffusion coefficients. A small amount, approximately 10%, of the recombinant apo(a) was present as a high molecular weight aggregate. The Stokes radius of the monomer, determined either from the diffusion coefficient or by combining the sedimentation equilibrium data with the sedimentation velocity data, was 94 A. The frictional ratio was 2.2, suggesting a highly asymmetric or random coil structure. In the electron microscope, recombinant apolipoprotein(a) was visualized as a long, highly flexible chain of domains forming large, open coiled structures on the EM grid with contour lengths of about 800 A. Addition of 6-aminohexanoic acid at 50 mM, a concentration which should saturate the weak lysine binding sites, did not alter the sedimentation behavior. In vivo, apolipoprotein(a) is associated tightly with LDL to form a highly atherogenic lipoprotein, Lp(a). A single molecule of recombinant apo(a) also associated tightly with LDL to yield a 13.3-S Lp(a)-like complex. This complex dissociated upon the addition of 50 mM 6-aminohexanoic acid. A novel sucrose gradient centrifugation technique was employed to determine a dissociation constant for the reversible equilibrium between recombinant apo(a) and LDL; at physiological ionic strength the dissociation constant was 0.3 nM. Raising the salt concentration to 5 M NaBr caused the dissociation constant to increase to 500 nM.(ABSTRACT TRUNCATED AT 250 WORDS)
Phillips
M L
ML
Department of Chemistry and Biochemistry, University of California, Los Angeles 90024.
Lembertas
A V
AV
Schumaker
V N
VN
Lawn
R M
RM
Shire
S J
SJ
Zioncheck
T F
TF
D015119:Aminocaproic Acid / Q000494:pharmacology; D001053:Apolipoproteins / Q000737:chemistry* / Q000378:metabolism*; D053241:Apoprotein(a); D002460:Cell Line; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D004058:Diffusion; D006801:Humans; D017270:Lipoprotein(a) / Q000737:chemistry*; D008077:Lipoproteins, LDL / Q000378:metabolism*; D008854:Microscopy, Electron; D009994:Osmolar Concentration; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D014461:Ultracentrifugation
D001053:Apolipoproteins; D017270:Lipoprotein(a); D008077:Lipoproteins, LDL; D011994:Recombinant Proteins; D053241:Apoprotein(a); D015119:Aminocaproic Acid