Zardeneta G, Horowitz P M
Department of Biochemistry, University of Texas Health Science Center, San Antonio 78284-7760.
Eur J Biochem. 1992 Dec 15;210(3):831-7. doi: 10.1111/j.1432-1033.1992.tb17486.x.
The interaction was studied between the mitochondrial enzyme thiosulfate sulfurtransferase and liposomes, in the form of large unilamellar vesicles (LUV), prepared from either cardiolipin (CL), PtdCho or PtdSer. At equivalent concentrations of lipid, more partially folded thiosulfate sulfurtransferase bound to CL/LUV than to PtdSer/LUV, and only traces were bound to PtdCho/LUV. Native thiosulfate sulfurtransferase did not bind to any of these LUV. We show that CL/LUV-sequestered thiosulfate sulfurtransferase is inactive but may be reactivated (approximately 56%) with the aid of detergents, thiosulfate, beta-mercaptoethanol and phosphate buffer. Reactivations in the presence of PtdSer/LUV or PtdCho/LUV was only 9% or 1%, respectively. Analysis of the complex by protease digestion and fluorescence spectroscopy indicated that thiosulfate sulfurtransferase was held by CL/LUV and PtdSer/LUV as a folding intermediate. Data presented here suggest that detergents may not interact directly with the protein, but, rather, their primary role in reactivation is to disrupt the LUV, allowing flexibility to the anchored thiosulfate sulfurtransferase molecule, thereby promoting folding. These studies complement other reports which imply a possible role for CL in protein translocation across the mitochondria, since we find that CL binds to thiosulfate sulfurtransferase and sequesters it in a translocation-competent prefolded conformation, which may readily lead to a correctly folded enzyme.
研究了线粒体酶硫代硫酸盐硫转移酶与脂质体（以大单室囊泡（LUV）形式存在）之间的相互作用，这些脂质体由心磷脂（CL）、磷脂酰胆碱（PtdCho）或磷脂酰丝氨酸（PtdSer）制备而成。在脂质浓度相等的情况下，与PtdSer/LUV相比，更多部分折叠的硫代硫酸盐硫转移酶与CL/LUV结合，而与PtdCho/LUV的结合量仅为痕量。天然硫代硫酸盐硫转移酶不与任何这些LUV结合。我们发现，被CL/LUV隔离的硫代硫酸盐硫转移酶没有活性，但在去污剂、硫代硫酸盐、β-巯基乙醇和磷酸盐缓冲液的帮助下可能会重新激活（约56%）。在PtdSer/LUV或PtdCho/LUV存在的情况下重新激活的比例分别仅为9%或1%。通过蛋白酶消化和荧光光谱对复合物进行分析表明，硫代硫酸盐硫转移酶以折叠中间体的形式被CL/LUV和PtdSer/LUV固定。此处呈现的数据表明，去污剂可能不会直接与蛋白质相互作用，相反，它们在重新激活中的主要作用是破坏LUV，使锚定的硫代硫酸盐硫转移酶分子具有灵活性，从而促进折叠。这些研究补充了其他报告，这些报告暗示CL在蛋白质跨线粒体转运中可能发挥作用，因为我们发现CL与硫代硫酸盐硫转移酶结合，并将其隔离在具有转运能力的预折叠构象中，这可能很容易导致形成正确折叠的酶。
