Regulation and Immunolocalization of Acyl-Coenzyme A:Cholesterol Acyltransferase in Mammalian Cells as Studied with Specific Antibodies (*)
Regulation and Immunolocalization of Acyl-Coenzyme A:Cholesterol Acyltransferase in Mammalian Cells as Studied with Specific Antibodies (*)
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DOI：
10.1074/jbc.270.49.29532
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发表时间：
1995-12
期刊：
影响因子：
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通讯作者：
Chang Catherine C. Y.;Jun Chen;Matthew A. Thomas;D. Cheng;Del Priore Veronica A.;R. Newton;M. Pape;Ta-Yuan Chang
中科院分区：
文献类型：
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作者：
Chang Catherine C. Y.;Jun Chen;Matthew A. Thomas;D. Cheng;Del Priore Veronica A.;R. Newton;M. Pape;Ta-Yuan Chang
酰基辅酶A：胆固醇酰基转移酶（ACAT）催化各种组织中细胞内胆固醇酯的形成。我们最近报道了人巨噬细胞ACAT cDNA的克隆和表达。在本研究中，我们报告的生产特异性抗ACAT的多克隆抗体免疫兔的谷胱甘肽S-转移酶和ACAT蛋白的前131个氨基酸组成的重组融合蛋白。免疫印迹分析表明，抗体与来自各种人类细胞系的50 kDa蛋白带交叉反应。这些抗体免疫耗竭超过90%的洗涤剂溶解的ACAT活性从六种不同的人类细胞类型，表明50 kDa的蛋白质是这些细胞中的主要ACAT催化成分。在检查的多种人体组织中，抗体识别具有不同分子量的蛋白质条带。这些抗体还与中国仓鼠卵巢细胞中的ACAT蛋白发生交叉反应。免疫印迹分析表明，ACAT蛋白在人成纤维细胞，HepG 2细胞，或中国仓鼠卵巢细胞的含量不受影响的固醇在培养基中，表明在这些细胞中的ACAT活性的固醇依赖性调节的主要机制是不改变ACAT蛋白的含量。间接免疫荧光显微镜观察发现，ACAT蛋白在组织培养细胞中定位于内质网。这一发现，沿着与早期的研究，表明胆固醇浓度在内质网可能是主要的决定因素，为调节ACAT活性在完整的细胞。
Acyl-coenzyme A:cholesterol acyltransferase (ACAT) catalyzes the formation of intracellular cholesterol esters in various tissues. We recently reported the cloning and expression of human macrophage ACAT cDNA. In the current study, we report the production of specific polyclonal antibodies against ACAT by immunizing rabbits with the recombinant fusion protein composed of glutathione S-transferase and the first 131 amino acids of ACAT protein. Immunoblot analysis showed that the antibodies cross-reacted with a 50-kDa protein band from a variety of human cell lines. These antibodies immunodepleted more than 90% of detergent-solubilized ACAT activities from six different human cell types, demonstrating that the 50-kDa protein is the major ACAT catalytic component in these cells. In multiple human tissues examined, the antibodies recognized protein bands with various molecular weights. These antibodies also cross-reacted with the ACAT protein in Chinese hamster ovary cells. Immunoblot analysis showed that the ACAT protein contents in human fibroblast cells, HepG2 cells, or Chinese hamster ovary cells were not affected by sterol in the medium, demonstrating that the main mechanism for sterol-dependent regulation of ACAT activity in these cells is not change in ACAT protein content. As revealed by indirect immunofluorescent microscopy, the ACAT protein in tissue culture cells was located in the endoplasmic reticulum. This finding, along with earlier studies, suggests that cholesterol concentration in the endoplasmic reticulum may be the major determinant for regulating ACAT activity in the intact cells.