PHYSICAL AND KINETIC CHARACTERIZATION OF RECOMBINANT HUMAN CHOLESTERYL ESTER TRANSFER PROTEIN

Cholesteryl ester transfer protein (CETP) mediates the exchange of tri glycerides (TGs), cholesteryl esters (CEs) and phospholipids (PLs) bet ween lipoproteins in the plasma. In order to better understand the lip id transfer process, we have used recombinant human CETP expressed in cultured mammalian cells, purified to homogeneity by immunoaffinity ch romatography. Purified recombinant CETP had a weight-average relative molecular mass (M(w)) of 69 561, determined by sedimentation equilibri um, and a specific absorption coefficient of 0.83 litre . g(-1) . cm(- 1). The corresponding hydrodynamic diameter (D-h) of the protein, dete rmined by dynamic light scattering, was 14 nm, which is nearly twice t he expected value for a spheroidal protein of this molecular mass. The se data suggest that CETP has a non-spheroidal shape in solution. The secondary structure of CETP was estimated by CD to contain 32% alpha-h elix, 35% beta-sheet, 17% turn and 16% random coil. Like the natural p rotein from plasma, the recombinant protein consisted of several glyco forms that could be only partially deglycosylated using N-glycosidase F. Organic extraction of CETP followed by TLC showed that CE, unesteri fied cholesterol (UC), PL, TG and fatty acids (FA) were associated wit h the pure protein. Quantitative analyses verified that each mol of CE TP contained 1.0 mol of cholesterol, 0.5 mol of TG and 1.3 mol of PL. CETP mediated the transfer of CE, TG, PL, and UC between lipoproteins, or between protein-free liposomes. In dual-label transfer experiments , the transfer rates for CE or TG from HDL to LDL were found to be pro portional to the initial concentrations of the respective ligands in t he donor HDL particles. Kinetic analysis of CE transfer was consistent with a carrier mechanism, having a K-m of 700 nM for LDL particles an d of 2000 nM for HDL particles, and a k(eat) of 2 s(-1). The K-m value s were thus in the low range of the normal physiological concentration for each substrate. The carrier mechanism was verified independently for CE, TG, PL and UC in 'half-reaction' experiments.