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Title 

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

Authors 

Kyung Hyun ChoSo Jin AnWoo Song LeeY K PaikYoung Kook KimTae Sook Jeong

Publisher 

Elsevier

Issue Date 

2003

Citation 

Biochemical and Biophysical Research Communications, vol. 309, no. 4, pp. 864-872

Keywords 

acyl-CoA:cholesterol acyltransferaseatherosclerosishypercholesterolemiaisoenzymespecific inhibitorsviral expressioncholesterol acyltransferasecholesterol acyltransferase inhibitorDNA libraryenzyme regulation

Abstract 

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 50kDa- and a 46kDa-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 IC 50=0.64μM 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.

ISSN 

0006-291X

Link 

http://dx.doi.org/10.1016/j.bbrc.2003.08.077

Appears in Collections

1. Journal Articles > Journal Articles

Registered Date

2017-04-19


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