Chen Ku-Chung, Wang Li-Kuan, Chang Long-Sen
Institute of Biomedical Sciences, National Sun Yat-Sen University, Kaohsiung 804, Taiwan.
J Pept Sci. 2009 Feb;15(2):89-94. doi: 10.1002/psc.1097.
Size exclusion chromatographic analyses showed that Ca(2+)-free VILIP-1 contained both monomeric and dimeric forms, while no appreciable dimerization was noted with Ca(2+)-free VILIP-3. Swapping of EF-hands 3 and 4 of VILIP-1 with those of VILIP-3 caused the inability of the resulting chimeric protein to form dimeric protein. Nonreducing SDS-PAGE analyses revealed that most of the dimeric VILIP-1 was noncovalently bound together. Reduced glutathione (GSH)/oxidized glutathione (GSSG) treatment notably enhanced the formation of disulfide-linked VILIP-1 dimer, while Ca(2+) and Mg(2+) enhanced disulfide dimerization of VILIP-1 marginally in the presence of thiol compounds. Cys-187 at the C-terminus of VILIP-1 contributed greatly to form S-S-crosslinked dimer as revealed by mutagenesis studies. The ability of GSH/GSSG-treated VILIP-1 to activate guanylyl cyclase B was reduced by substituting Cys-187 with Ala. Together with disulfide dimer of VILIP-1 detected in rat brain extracts, our data may imply the functional contribution of disulfide dimer to the interaction of VILIP-1 with its physiological target(s).
尺寸排阻色谱分析表明，无钙的VILIP-1包含单体和二聚体形式，而无钙的VILIP-3未观察到明显的二聚化。将VILIP-1的EF手3和4与VILIP-3的进行交换，导致所得嵌合蛋白无法形成二聚体蛋白。非还原SDS-PAGE分析显示，大多数二聚体VILIP-1是非共价结合在一起的。还原型谷胱甘肽（GSH）/氧化型谷胱甘肽（GSSG）处理显著增强了二硫键连接的VILIP-1二聚体的形成，而在硫醇化合物存在的情况下，Ca(2+)和Mg(2+)对VILIP-1的二硫键二聚化有轻微增强作用。诱变研究表明，VILIP-1 C末端的Cys-187对形成S-S交联二聚体有很大贡献。用丙氨酸替代Cys-187会降低GSH/GSSG处理的VILIP-1激活鸟苷酸环化酶B的能力。连同在大鼠脑提取物中检测到的VILIP-1二硫键二聚体，我们的数据可能暗示二硫键二聚体对VILIP-1与其生理靶点相互作用的功能贡献。
