Inactivation of cholesteryl ester transfer protein by cysteine modification.

The present studies examine the effects of various cysteine-modifying reagents on human recombinant cholesteryl ester transfer protein (CETP) activity. Dithiothreitol or other reducing agents had no effect on CETP transfer activity. Alkylating agents, including iodoacetamide and N-ethyl maleimide, also did not affect transfer activity. However, incubation of CETP with hydrophobic thiol-modifying reagents such as p-chloromercuriphenylsulfonic acid (IC50 = 0.02 microM), 4,4'-dithiodipyridine (IC50 = 0.5 microM), or 4,4'-dithiobis (phenyl azide) (IC50 = 0.5 microM) resulted in complete, time-dependent inactivation of both the cholesteryl ester and triglyceride transfer activities. Inactivation could be prevented by including dithiothreitol in the incubation. Long chain fatty acyl coenzyme A compounds were also found to be effective CETP inhibitors. The extent of inhibition was time-dependent, and proportional to the chain length of the fatty acyl portion of the molecule. These results suggest that CETP contains an essential free cysteine that resides in a hydrophobic environment within the protein.