The disulfide linkage and the free sulfhydryl accessibility of acyl-coenzyme A : Cholesterol acyltransferase 1 as studied by using mPEG5000-maleimide
文献类型：期刊论文
| 作者 | Guo, ZY; Chang, CCY; Lu, XH; Chen, J; Li, BL; Chang, TY |
| 刊名 | BIOCHEMISTRY
 |
| 出版日期 | 2005 |
| 卷号 | 44期号:17页码:6537-6546 |
| 通讯作者 | Chang, TY (reprint author), Dartmouth Coll Sch Med, Dept Biochem, Hanover, NH 03755 USA.,Ta.Yuan.Chang@dartmouth.edu |
| 英文摘要 | Acyl-coenzyme A:cholesterol acyltransferase (ACAT) is a membrane protein located in the endoplasmic reticulum (ER). It plays important roles in cellular cholesterol homeostasis. Human ACAT1 (hACAT1) contains nine cysteines (C). To quantify and map its disulfide linkage, we performed thiol-specific modifications by mPEG(5000)-maleimide (PEG-mal) and iodoacetamide (IA) under denatured condition, using extracts that contain wild-type or various single C to A mutant hACATIs. With the wild-type enzyme, seven Cs could be modified before dithiothreitol (DTT) treatment; nine Cs could be modified after DTT treatment. With the C528A or the C546A enzyme, all eight Cs could be modified before or after DTT treatment. With all other remaining single C to A mutant enzymes, six Cs could be modified before DTT treatment, and eight Cs could be modified after DTT treatment. We next performed Lys-C protease digestion on hACATI with a hernagglutinin (HA) tag at the C-terminus. The digests were treated with or without DTT and analyzed by SDS-PAGE and Western blotting. The two predicted C-terminal fragments (K496-K531 and N532-F550-HA tag) were trapped as a single peptide band, but only when the digests were treated without DTT. Thus, C528 and C546 near the enzyme's C-terminus form a disulfide. PEG-mal is impermeable to ER membranes. We used PEG-mal to map the localizations of the seven free sulfhydryls and the disulfide bond of hACATI present in microsomal vesicles. The results show that C92 is located on the cytoplasmic side of the ER membrane and the disulfide is located in the ER lumen, while all other free Cs are located within the hydrophobic region(s) of the enzyme. |
| 学科主题 | Biochemistry & Molecular Biology |
| 类目[WOS] | Biochemistry & Molecular Biology |
| 关键词[WOS] | HAMSTER OVARY CELLS ; ENDOPLASMIC-RETICULUM ; HUMAN LIVER ; CHEMICAL MODIFICATION ; O-ACYLTRANSFERASE ; IN-VITRO ; COA ; MEMBRANE ; ACAT-1 ; IDENTIFICATION |
| 收录类别 | SCI |
| 语种 | 英语 |
| WOS记录号 | WOS:000228731700016 |
| 版本 | 出版稿 |
| 源URL | [http://202.127.25.143/handle/331003/1979]  |
| 专题 | 上海生化细胞研究所_上海生科院生化细胞研究所 |
| 推荐引用方式 GB/T 7714 | Guo, ZY,Chang, CCY,Lu, XH,et al. The disulfide linkage and the free sulfhydryl accessibility of acyl-coenzyme A : Cholesterol acyltransferase 1 as studied by using mPEG5000-maleimide[J]. BIOCHEMISTRY,2005,44(17):6537-6546. |
| APA | Guo, ZY,Chang, CCY,Lu, XH,Chen, J,Li, BL,&Chang, TY.(2005).The disulfide linkage and the free sulfhydryl accessibility of acyl-coenzyme A : Cholesterol acyltransferase 1 as studied by using mPEG5000-maleimide.BIOCHEMISTRY,44(17),6537-6546. |
| MLA | Guo, ZY,et al."The disulfide linkage and the free sulfhydryl accessibility of acyl-coenzyme A : Cholesterol acyltransferase 1 as studied by using mPEG5000-maleimide".BIOCHEMISTRY 44.17(2005):6537-6546. |
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