Chemical modification of acyl-CoA:cholesterol O-acyltransferase. 1. Identification of acyl-CoA:cholesterol O-acyltransferase subtypes by differential diethyl pyrocarbonate sensitivity.
Acyl-CoA:cholesterol O-acyltransferase (EC 2.3.1.26) (ACAT) catalyzes the intracellular synthesis of cholesteryl esters from cholesterol and fatty acyl-CoA at neutral pH. Despite the probable pathophysiologic role of ACAT in vascular cholesteryl ester accumulation during atherogenesis, its mechanism of action and its regulation remain to be elucidated because the enzyme polypeptide has never been identified or purified. Present chemical modification results identify two distinct tissue types of ACAT, based on marked differences in reactivity of an active-site histidine residue toward diethyl pyrocarbonate (DEP) and acetic anhydride. The apparent Ki of the DEP-sensitive ACAT subtype, typified by aortic ACAT, was 40 microM, but the apparent Ki of the DEP-resistant ACAT subtype, typified by liver ACAT, was 1500 microM, indicating a 38-fold difference in sensitivity to DEP. Apparent Ki's of aortic and liver ACAT for inhibition by acetic anhydride were also discordant (less than 500 microM and greater than 5 mM, respectively). On the basis of the reversibility of inhibition by hydroxylamine, a neutral pKa for maximal modification, and acetic anhydride protection against DEP inactivation, DEP and acetic anhydride appear to modify a common histidine residue. Oleoyl-CoA provided partial protection against inactivation by DEP and acetic anhydride, suggesting that the modified histidine is at or near the active site of ACAT. Systematic investigation of ACAT activity from 14 different organs confirmed the existence of 2 subtypes of ACAT on the basis of their different reactivities toward DEP and acetic anhydride.(ABSTRACT TRUNCATED AT 250 WORDS)
Kinnunen
P M
PM
Cardiology Division, Jewish Hospital, Washington University Medical Center, St. Louis, Missouri 63110.
DeMichele
A
A
Lange
L G
LG
D000085:Acetates / Q000494:pharmacology*; D000089:Acetic Anhydrides / Q000494:pharmacology*; D000818:Animals; D001011:Aorta / Q000201:enzymology / Q000648:ultrastructure; D001665:Binding Sites; D055598:Chemical Phenomena; D002621:Chemistry; D004047:Diethyl Pyrocarbonate / Q000494:pharmacology*; D005561:Formates / Q000494:pharmacology*; D006639:Histidine; D006863:Hydrogen-Ion Concentration; D019811:Hydroxylamine; D006898:Hydroxylamines / Q000494:pharmacology; D007700:Kinetics; D008297:Male; D008861:Microsomes / Q000201:enzymology; D008862:Microsomes, Liver / Q000201:enzymology; D011817:Rabbits; D002785:Sterol O-Acyltransferase / Q000037:antagonists & inhibitors* / Q000145:classification / Q000378:metabolism; D014018:Tissue Distribution
D000085:Acetates; D000089:Acetic Anhydrides; D005561:Formates; D006898:Hydroxylamines; C031800:acetic anhydride; D019811:Hydroxylamine; D006639:Histidine; D002785:Sterol O-Acyltransferase; D004047:Diethyl Pyrocarbonate