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

Kyung Hyun Cho, Sojin An, Woo Song Lee, Young-Ki Paik, Young Kook Kim, Tae Sook Jeong

Research output: Contribution to journalArticle

41 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)864-872
Number of pages9
JournalBiochemical and Biophysical Research Communications
Volume309
Issue number4
DOIs
Publication statusPublished - 2003 Oct 3

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Substrate Specificity
Protein Isoforms
Substrates
Anilides
Diethyl Pyrocarbonate
Sterol O-Acyltransferase
Tissue
Cholic Acid
Dimers
Liver
Purification
Insects
Rats
Polyacrylamide Gel Electrophoresis
Screening
Fatty Acids
Binding Sites
Cholesterol
Derivatives
Sensitivity and Specificity

All Science Journal Classification (ASJC) codes

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

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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",
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.",
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Mass-production of human ACAT-1 and ACAT-2 to screen isoform-specific inhibitor : A different substrate specificity and inhibitory regulation. / Cho, Kyung Hyun; An, Sojin; Lee, Woo Song; Paik, Young-Ki; Kim, Young Kook; Jeong, Tae Sook.

In: Biochemical and Biophysical Research Communications, Vol. 309, No. 4, 03.10.2003, p. 864-872.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Mass-production of human ACAT-1 and ACAT-2 to screen isoform-specific inhibitor

T2 - A different substrate specificity and inhibitory regulation

AU - Cho, Kyung Hyun

AU - An, Sojin

AU - Lee, Woo Song

AU - Paik, Young-Ki

AU - Kim, Young Kook

AU - Jeong, Tae Sook

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AB - 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.

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