ATPase activity of human ABCG1 is stimulated by cholesterol and sphingomyelin[S]
ATPase activity of human ABCG1 is stimulated by cholesterol and sphingomyelin[S]
复制标题
胆固醇和鞘磷脂刺激人ABCG1的ATP酶活性[S]
作者：
H. Hirayama;Yasuhisa Kimura;N. Kioka;M. Matsuo;K. Ueda
ATP结合盒蛋白G1（ABCG 1）对HDL的形成非常重要。然而，ABCG 1的生化特性尚未报道，ABCG 1如何参与HDL形成的机制仍不清楚。我们建立了一个程序来表达和纯化人ABCG 1使用悬浮适应的人细胞FreeStyle 293-F。ABCG 1在C端与绿色荧光蛋白和Flag肽融合，用正十二烷基-β-D-麦芽糖苷溶解，并通过单轮Flag-M2抗体亲和层析纯化。将纯化的ABCG 1重组于不同脂质组成的脂质体中，并分析ATP酶活性。ABCG 1重组在鸡蛋卵磷脂显示ATP酶活性（150 nmol/min/mg），这是抑制氟化铍。胆固醇和胆碱磷脂（尤其是鞘磷脂）可刺激重组于磷脂酰丝氨酸脂质体中的ABCG 1的ATP酶活性，且鞘磷脂的加入可增加ABCG 1对胆固醇的亲和力。这些结果表明，ABCG 1是一个积极的脂质转运蛋白，并具有不同的结合位点的胆固醇和鞘磷脂，这可能是协同耦合。
ATP-binding cassette protein G1 (ABCG1) is important for the formation of HDL. However, the biochemical properties of ABCG1 have not been reported, and the mechanism of how ABCG1 is involved in HDL formation remains unclear. We established a procedure to express and purify human ABCG1 using the suspension-adapted human cell FreeStyle293-F. ABCG1, fused at the C terminus with green fluorescent protein and Flag-peptide, was solubilized with n-dodecyl-β-D-maltoside and purified via a single round of Flag-M2 antibody affinity chromatography. The purified ABCG1 was reconstituted in liposome of various lipid compositions, and the ATPase activity was analyzed. ABCG1 reconstituted in egg lecithin showed ATPase activity (150 nmol/min/mg), which was inhibited by beryllium fluoride. The ATPase activity of ABCG1, reconstituted in phosphatidylserine liposome, was stimulated by cholesterol and choline phospholipids (especially sphingomyelin), and the affinity for cholesterol was increased by the addition of sphingomyelin. These results suggest that ABCG1 is an active lipid transporter and possesses different binding sites for cholesterol and sphingomyelin, which may be synergistically coupled.
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巴斯德毕赤酵母细胞中表达的 N-糖基化突变小鼠和人 P-糖蛋白的纯化和表征。
影响因子：
3.9
作者：
Urbatsch,IL;Wilke-Mounts,S;Gimi,K;Senior,AE
通讯作者：
Senior,AE
影响因子：
15.9
作者：
Yvan-Charvet, Laurent;Ranalletta, Mollie;Tall, Alan R.
通讯作者：
Tall, Alan R.
影响因子：
4.8
作者：
Herget, Meike;Kreissig, Nina;Abele, Rupert
通讯作者：
Abele, Rupert
影响因子：
2.9
作者：
Tsybovsky, Yaroslav;Wang, Benlian;Quazi, Faraz;Molday, Robert S.;Palczewski, Krzysztof
通讯作者：
Palczewski, Krzysztof
DOI：
10.1016/j.bbalip.2011.08.003
发表时间：
2012-03
期刊：
Biochimica et biophysica acta
影响因子：
--
作者：
Murphy AJ;Westerterp M;Yvan-Charvet L;Tall AR
通讯作者：
Tall AR