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.
Connolly
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Cardiovascular Diseases Research Department, G. D. Searle Company, St. Louis, Missouri 63167, USA. dtoconn@ccmail.monsanto.com
Heuvelman
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D
Glenn
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D002731:4-Chloromercuribenzenesulfonate / Q000494:pharmacology; D000477:Alkylating Agents / Q000494:pharmacology; D000818:Animals; D001386:Azides / Q000494:pharmacology; D002352:Carrier Proteins / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000302:isolation & purification; D002460:Cell Line; D053480:Cholesterol Ester Transfer Proteins; D002788:Cholesterol Esters / Q000378:metabolism; D006224:Cricetinae; D003545:Cysteine; D004220:Disulfides / Q000494:pharmacology; D004229:Dithiothreitol / Q000494:pharmacology; D005033:Ethylmaleimide / Q000494:pharmacology; D006023:Glycoproteins; D006801:Humans; D007460:Iodoacetamide / Q000494:pharmacology; D007700:Kinetics; D011725:Pyridines / Q000494:pharmacology; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification; D013329:Structure-Activity Relationship; D013439:Sulfhydryl Reagents / Q000494:pharmacology*; D014162:Transfection
D000477:Alkylating Agents; D001386:Azides; C506642:CETP protein, human; D002352:Carrier Proteins; D053480:Cholesterol Ester Transfer Proteins; D002788:Cholesterol Esters; D004220:Disulfides; D006023:Glycoproteins; D011725:Pyridines; D011994:Recombinant Proteins; D013439:Sulfhydryl Reagents; C007542:4,4'-dipyridyl disulfide; C025369:4,4'-dithiobis(phenylazide); D002731:4-Chloromercuribenzenesulfonate; D003545:Cysteine; D005033:Ethylmaleimide; D004229:Dithiothreitol; D007460:Iodoacetamide