AMINOPHOSPHOLIPID TRANSLOCATION IN ERYTHROCYTES - EVIDENCE FOR THE INVOLVEMENT OF A SPECIFIC TRANSPORTER AND AN ENDOFACIAL PROTEIN
AMINOPHOSPHOLIPID TRANSLOCATION IN ERYTHROCYTES - EVIDENCE FOR THE INVOLVEMENT OF A SPECIFIC TRANSPORTER AND AN ENDOFACIAL PROTEIN
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DOI：
10.1021/bi00453a005
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发表时间：
1990-01-09
期刊：
影响因子：
2.9
通讯作者：
SCHROIT, AJ
中科院分区：
文献类型：
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作者：
CONNOR, J;SCHROIT, AJ
在红细胞（RBC）中，外源性氨基磷脂荧光类似物从外叶向内叶的转运依赖于膜巯基的氧化状态。先前已经显示，吡啶基二硫代乙胺（PDA）对32-kDa膜蛋白上的巯基的氧化[Conner和Schroit（1988）Biochemistry 27，848-851]抑制NBD标记的磷脂酰丝氨酸（NBD-PS）的转运。在本研究中，其他巯基氧化剂进行了检查，以确定是否有其他网站参与运输过程。我们的研究结果表明，二酰胺抑制NBD-PS的运输通过一种机制，是独立的32 kDa的网站。这表现为二酰胺不能阻断用125 I标记的PDA标记32-kDa巯基，也不能防止PDA介导的NBD-PS转运抑制。二酰胺介导的抑制作用，而不是PDA介导的抑制作用，可以被半胱胺或内源性谷胱甘肽还原所逆转。（2-硝基苯甲酸），其消耗内源性谷胱甘肽并诱导内面蛋白的氧化[Reglinski等人（1988）J.Biol.Chem.263，12360-12366]，以类似于二酰胺的方式抑制NBD-PS转运。一旦建立，NBD-PS的不对称分布不能被任何一个位点的氧化所改变。这些数据表明，第二个网站的关键氨基磷脂的运输驻留在endofacial表面上，并表明，运输的氨基磷脂的红细胞双层膜依赖于一个协调和互补的过程之间的细胞骨架成分和32-kDa的膜多肽，都必须是可操作的运输进行。
The transport of exogenously supplied fluorescent analogues of aminophospholipids from the outer to inner leaflet in red blood cells (RBC) is dependent upon the oxidative status of membrane sulfhydryls. Oxidation of a sulfhydryl on a 32-kDa membrane protein by pyridyldithioethylamine (PDA) has been previously shown [Conner and Schroit (1988) Biochemistry 27, 848-851] to inhibit the transport of NBD-labeled phosphatidylserine (NBD-PS). In the present study, other sulfhydryl oxidants were examined to determine whether additional sites are involved in the transport process. Our results show that diamide inhibits the transport of NBD-PS via a mechanism that is independent of the 32-kDa site. This is shown by the inability of diamide to block labeling of the 32-kDa sulfhydryl with 125I-labeled PDA and to protect against PDA-mediated inhibition of NBD-PS transport. Diamide-mediated inhibition, but not PDA-mediated inhibition, could be reversed by reduction with cysteamine or endogenous glutathione, Similarly, treatment of RBC with 5,5''-dithiobis(2-nitrobenzoic acid), which depletes endogenous glutathione and induces oxidation of endofacial proteins [Reglinski et al. (1988) J. Biol. Chem. 263, 12360-12366], inhibited NBD-PS transport in a manner analogous to diamide. Once established, the asymmetric distribution of NBD-PS could not be altered by oxidation of either site. These data indicate that a second site critical to the transport of aminophospholipids resides on the endofacial surface and suggest that the transport of aminophospholipids across the bilayer membrane of RBC depends on a coordinated and complementary process between a cytoskeletal component and the 32-kDa membrane polypeptide, both must be operative for transport to proceed.