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 C ETP transfer activity. Alkylating agents, including iodoacetamide and N-ethyl maleimide, also did not affect transfer activity. However, inc ubation of CETP with hydrophobic thiol-modifying reagents such as p-ch loromercuriphenylsulfonic acid (IC50 = 0.02 mu M), 4,4'-dithiodipyridi ne (IC50 = 0.5 mu M), or 4,4'-dithiobis (phenyl azide) (IC50 = 0.5 mu M) resulted in complete, time-dependent inactivation of both the chole steryl 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 proportio nal to the chain length of the fatty acyl portion of the molecule. The se results suggest that CETP contains an essential free cysteine that resides in a hydrophobic environment with in the protein. (C) 1996 Aca demic Press, Inc.