Holzenburg A, Jones P C, Franklin T, Pali T, Heimburg T, Marsh D, Findlay J B, Finbow M E
Department of Biochemistry & Molecular Biology, University of Leeds, England.
Eur J Biochem. 1993 Apr 1;213(1):21-30. doi: 10.1111/j.1432-1033.1993.tb17730.x.
Electron microscopic analysis of gap-junction-like structures isolated from an anthropod (Nephrops norvegicus) and composed of a 16-kDa polypeptide, show the functional unit to be a star-shaped hexamer of protein arranged around a central channel which runs perpendicular to the plane of the membrane. Estimations of the molecular volume carried out on an averaged projection are consistent with a subunit mass of 16-18 kDa. Fourier transform infrared spectroscopy indicates a high alpha-helical content for the protein, supporting secondary-structure predictions of four transmembrane alpha helices/monomer. The averaged projection shows a close resemblance to a hexamer of the 16-kDa protein built on the basis of a four alpha-helical bundle [Finbow, M. E., Eliopoulos, E. E., Jackson, P. J., Keen, J. N., Meagher, L., Thompson, P., Jones, P. C. & Findlay, J. B. C. (1992) Protein Eng. 5, 7-15]. The reconstructed image is also similar to that obtained for gap-junction-like channels isolated from a related arthropod [Homarus americanus; Sikerwar, S. S., Downing, K. H. & Glaeser, R. M. (1991) J. Struct. Biol. 106, 255-263] whose protein content was unknown but which we demonstrate may be composed of a related 16-kDa protein. Previous studies have shown a high sequence identity of the Nephrops 16-kDa protein with the 16-kDa proteolipid subunit c of the vascular H(+)-ATPase, both of which in turn bear similarity to the 8-kDa proteolipid subunit of the F1F0-ATP synthase. Expression of cDNA coding for the Nephrops 16-kDa protein in Saccharomyces cerevisiae, in which the endogenous gene coding for the V-ATPase proteolipid has been inactivated, restores V-ATPase activity and cell growth.
对从一种节肢动物（挪威龙虾）分离出的、由一种16 kDa多肽组成的间隙连接样结构进行电子显微镜分析，结果显示其功能单元是围绕垂直于膜平面的中央通道排列的星形蛋白质六聚体。在平均投影上进行的分子体积估计与16 - 18 kDa的亚基质量一致。傅里叶变换红外光谱表明该蛋白质具有高α - 螺旋含量，支持四个跨膜α螺旋/单体的二级结构预测。平均投影显示与基于四个α - 螺旋束构建的16 kDa蛋白质的六聚体非常相似[芬博，M. E.，埃利奥普洛斯，E. E.，杰克逊，P. J.，基恩，J. N.，米agher，L.，汤普森，P.，琼斯，P. C. & 芬德利，J. B. C.（1992年）蛋白质工程5，7 - 15]。重建图像也与从相关节肢动物[美洲螯龙虾；西克瓦尔，S. S.，唐宁，K. H. & 格莱泽，R. M.（1991年）结构生物学杂志106，255 - 263]分离出的间隙连接样通道获得的图像相似，其蛋白质含量未知，但我们证明可能由相关的16 kDa蛋白质组成。先前的研究表明，挪威龙虾16 kDa蛋白质与血管H(+) - ATP酶的16 kDa蛋白脂质亚基c具有高度序列同一性，而这两者又都与F1F0 - ATP合酶的8 kDa蛋白脂质亚基相似。在酿酒酵母中表达编码挪威龙虾16 kDa蛋白质的cDNA，其中编码V - ATP酶蛋白脂质的内源基因已失活，可恢复V - ATP酶活性和细胞生长。
