Mass-production of human ACAT-1 and ACAT-2 to screen isoform-specific inhibitor: a different substrate specificity and inhibitory regulation

被引:39
作者
Cho, KH
An, S
Lee, WS
Paik, YK
Kim, YK
Jeong, TS [1 ]
机构
[1] Korea Res Inst Biosci & Biotechnol, Lipid Metab Res Lab, Taejon 305333, South Korea
[2] Yonsei Univ, Dept Biochem, Seoul 131749, South Korea
关键词
Acyl-CoA : cholesterol acyltransferase; isoenzyme; atherosclerosis; hypercholesterolemia; viral expression; specific inhibitors;
D O I
10.1016/j.bbrc.2003.08.077
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Recently, acyl-CoA:cholesterol acyltransferase was found to be present as two isoforms, ACAT-1 and ACAT-2, in mammalian tissues with different metabolic functions and tissue-specific locations. In this study, the isoforms were mass-produced individually from insect cells to establish a more sensitive and reliable screening method for specific inhibitors against each isoform. The expressed hACAT-1 and hACAT-2 appeared as a 50 kDa- and a 46 kDa-band on SDS-PAGE, respectively, from Hi5 cells and they preferred to exist in oligomeric form, from dimer to tetramer, during the purification process. They also exhibited an approximate 3.4 to 3.7-fold increase in activities when compared to rat liver microsomal fractions at the same protein concentration. Known ACAT inhibitors, pyripyropene A, oleic acid anilide, and diethyl pyrocarbonate, were tested to evaluate the inhibitory specificity and sensitivity of the expressed enzymes. Interestingly, pyripyropene A inhibited only the hACAT-2 fraction with IC50 = 0.64 muM but not the hACAT-1 fraction; whereas the fatty acid anilide did not show a significant difference in inhibitory activity with either hACAT-1 or hACAT-2. Furthermore, cholesterol was more rapidly utilized by hACAT-1, but hACAT-2 esterified other cholic acid derivatives more efficiently. These results suggest that the specificity of each substrate and inhibitor was highly different, depending on each isoform from the viewpoint of the regulatory site and the substrate binding site location. (C) 2003 Elsevier Inc. All rights reserved.
引用
收藏
页码:864 / 872
页数:9
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