Purification of recombinant acyl-coenzyme A:cholesterol acyltransferase 1 (ACAT1) from H293 cells and binding studies between the enzyme and substrates using difference intrinsic fluorescence spectroscopy.
Acyl-coenzyme A:cholesterol acyltransferase 1 (ACAT1) is a membrane-bound enzyme utilizing long-chain fatty acyl-coenzyme A and cholesterol to form cholesteryl esters and coenzyme A. Previously, we had expressed tagged human ACAT1 (hACAT1) in CHO cells and purified it to homogeneity; however, only a sparse amount of purified protein could be obtained. Here we report that the hACAT1 expression level in H293 cells is 18-fold higher than that in CHO cells. We have developed a milder purification procedure to purify the enzyme to homogeneity. The abundance of the purified protein enabled us to conduct difference intrinsic fluorescence spectroscopy to study the binding between the enzyme and its substrates in CHAPS/phospholipid mixed micelles. The results show that oleoyl-CoA binds to ACAT1 with K(d) = 1.9 μM and elicits significant structural changes of the protein as manifested by the significantly positive changes in its fluorescence spectrum; stearoyl-CoA elicits a similar spectrum change but much lower in magnitude. Previously, kinetic studies had shown that cholesterol is an efficient substrate and an allosteric activator of ACAT1, while its diastereomer epicholesterol is neither a substrate nor an activator. Here we show that both cholesterol and epicholesterol induce positive changes in the ACAT1 fluorescence spectrum; however, the magnitude of spectrum changes induced by cholesterol is much larger than epicholesterol. These results show that stereospecificity, governed by the 3β-OH moiety in steroid ring A, plays an important role in the binding of cholesterol to ACAT1.
Chang
Catherine C Y
CC
Department of Biochemistry, Dartmouth Medical School, Hanover, New Hampshire 03755, United States. catherine.chang@dartmouth.edu
Miyazaki
Akira
A
Dong
Ruhong
R
Kheirollah
Alireza
A
Yu
Chunjiang
C
Geng
Yong
Y
Higgs
Henry N
HN
Chang
Ta-Yuan
TY
D000818:Animals; D016466:CHO Cells; D002478:Cells, Cultured; D002784:Cholesterol / Q000737:chemistry / Q000378:metabolism; D006224:Cricetinae; D003412:Cricetulus; D006801:Humans; D011994:Recombinant Proteins / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D002785:Sterol O-Acyltransferase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification*; D013379:Substrate Specificity
D011994:Recombinant Proteins; D002784:Cholesterol; D002785:Sterol O-Acyltransferase; C493492:sterol O-acyltransferase 1