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Electron-microscopic and hydrodynamic characterization of recombinant apolipoprotein (a) and its association with LDL
M L Phillips1, A V Lembertas, V N Schumaker
1Department of Chemistry and Biochemistry, University of California, Los Angeles 90024.
Abstract:
A recombinant apo(a) containing 17 kringle 4 domains as well as the kringle 5 and protease domains of apo(a) was characterized by hydrodynamic studies and electron microscopy. Recombinant apo(a) is a monomer in solution with a molecular weight of 325,000 by sedimentation equilibrium and 320,000 by sedimentation and diffusion, and it is a highly asymmetric molecule with a frictional ratio of 2.2. In the electron microscope recombinant apo(a) is visualized as a flexible chain of domains approximately 800 A long. Sedimentation velocity studies also demonstrate that when it is mixed with LDL, recombinant apo(a) reversibly forms an Lp(a)-like complex with a 1:1 stoichiometry; moreover, complex formation is inhibited by 6-amino hexanoic acid. Hydrodynamic modeling and electron microscopy suggest that only a small portion of the r-apo(a) molecule interacts with the LDL and the rest of the chain extends into solution. Preliminary studies indicate that recombinant apo(a) also binds mouse LDL.
Insights
Researchers characterized recombinant apolipoprotein(a) [apo(a)], finding it forms Lp(a)-like complexes with LDL. This interaction is reversible and inhibited by 6-amino hexanoic acid, suggesting a specific binding site.
Area of Science:
- Biochemistry
- Molecular Biology
- Biophysics
Background:
- Apolipoprotein(a) [apo(a)] is a key component of Lp(a) particles, implicated in cardiovascular disease.
- Understanding apo(a) structure and function is crucial for developing targeted therapies.
Purpose of the Study:
- To characterize a recombinant apo(a) construct containing multiple Kringle 4 domains, Kringle 5, and protease domains.
- To investigate the interaction of recombinant apo(a) with low-density lipoprotein (LDL).
Main Methods:
- Hydrodynamic studies (sedimentation equilibrium, sedimentation velocity)
- Electron microscopy
- Biochemical assays
Main Results:
- Recombinant apo(a) exists as a monomer in solution with a molecular weight of ~325,000 and is highly asymmetric (frictional ratio 2.2).
- Electron microscopy revealed recombinant apo(a) as a flexible chain approximately 800 Å long.
- Recombinant apo(a) reversibly forms a 1:1 complex with LDL, inhibited by 6-amino hexanoic acid, indicating specific binding.
- Hydrodynamic modeling and microscopy suggest limited interaction between apo(a) and LDL, with most of the molecule extending into solution.
- Preliminary data show binding of recombinant apo(a) to mouse LDL.
Conclusions:
- The characterized recombinant apo(a) molecule possesses structural and functional properties similar to native apo(a).
- The study provides insights into the molecular interactions between apo(a) and LDL, relevant to Lp(a) assembly and function.
- The findings support a model where apo(a) interacts with LDL via a specific, limited region.