Evidence for N-terminal myristoylation of Tetrahymena arginine kinase using peptide mass fingerprinting analysis.
Evidence for N-terminal myristoylation of Tetrahymena arginine kinase using peptide mass fingerprinting analysis.
复制标题
使用肽质量指纹分析证明四膜虫精氨酸激酶 N 末端肉豆蔻酰化的证据。
DOI：
10.1007/s10930-016-9663-0
复制
发表时间：
2016
期刊：
影响因子：
--
通讯作者：
T.
中科院分区：
文献类型：
--
作者：
Motomura;S. and Suzuki;T.
在这项研究中，我们证实了N-末端豆蔻酰化的四膜虫pyriformisarginine激酶（AK 1）通过确定N-末端豆蔻酰化信号序列。使用昆虫无细胞蛋白质合成系统合成足够量的经修饰的酶，所述昆虫无细胞蛋白质合成系统含有脂肪酸进行转录后修饰所必需的所有元件。用胰蛋白酶消化后进行后续肽质量指纹图谱（PMF）分析。PMF数据覆盖39%（143个残基）的内部肽。目标N-豆蔻酰化肽的理论质量为832.4477，并且清楚地观察到实验质量（m/z-H+）为832.4747。两个质量之间的差异为0.0271，支持鉴定的准确性，并表明合成的T。pyriformisAK 1是豆蔻酰化的。T.用抗AK 1肽抗体和带有荧光发色团的二抗反应，用免疫荧光显微镜观察。与先前的蛋白质印迹分析一致，显微镜观察表明AK 1定位于纤毛中。本PMF和显微镜分析表明T. pyriformisAK 1可以通过N-末端肉豆蔻酰基定位和锚定到睫状膜上。
In this study, we confirmed N-terminal myristoylation ofTetrahymena pyriformisarginine kinase (AK1) by identifying a myristoylation signal sequence at the N-terminus. A sufficient amount of modified enzyme was synthesized using an insect cell-free protein synthesis system that contains all of the elements necessary for post-transcriptional modification by fatty acids. Subsequent peptide mass fingerprinting (PMF) analyses were performed after digestion with trypsin. The PMF data covered 39 % (143 residues) of internal peptides. The target N-myristoylated peptide had a theoretical mass of 832.4477 and was clearly observed with an experimental mass (m/z-H+) of 832.4747. The difference between the two masses was 0.0271, supporting the accuracy of identification and indicating that the synthesizedT. pyriformisAK1 is myristoylated. The fixed specimens ofT. pyriformiswere reacted with an anti-AK1 peptide antibody followed by a secondary antibody with a fluorescent chromophore and were observed using immunofluorescence microscope. In agreement with previous western blotting analyses, microscopic observations suggested that AK1 is localized in the cilia. The present PMF and microscopic analyses indicate thatT. pyriformisAK1 may be localized and anchored to ciliary membranes via N-terminal myristoyl groups.