LOCALIZATION OF THE PALMITOYLATION SITE IN THE TRANSMEMBRANE PROTEIN P12E OF FRIEND MURINE LEUKEMIA-VIRUS
LOCALIZATION OF THE PALMITOYLATION SITE IN THE TRANSMEMBRANE PROTEIN P12E OF FRIEND MURINE LEUKEMIA-VIRUS
复制标题
DOI：
10.1111/j.1432-1033.1995.373zz.x
复制
发表时间：
1995-09-01
期刊：
影响因子：
--
通讯作者：
GEYER, R
中科院分区：
文献类型：
--
作者：
HENSEL, J;HINTZ, M;GEYER, R
在[9,10 - H - 3]棕榈酸存在的情况下，Friend鼠白血病病毒复合物在鼠细胞上增殖。从培养上清液中收集病毒颗粒，并用去污剂裂解。通过尺寸排阻色谱从裂解物中分离出病毒跨膜蛋白p12E，并通过窄径反相高效液相色谱（HPLC）纯化。通过基质辅助激光解吸/电离飞行时间质谱（MALDI - TOF - MS）对纯化产物进行分析，结果表明该蛋白被棕榈酰化，带有一个脂肪酸残基。在pH 7时，通过羟胺处理释放出放射性标记的脂肪酸，这表明酰化是通过硫酯键发生的。为了确定酰化位点，用胰蛋白酶消化p12E。所得的肽段要么直接进行MALDI - TOF - MS分析，要么在质谱分析之前通过微径反相HPLC进行分级分离。结果显示，Friend鼠白血病病毒的p12E在多肽假定跨膜锚定区的C末端一侧的半胱氨酸残基处被酰化。对纯化的酰化肽段进行脂肪酸分析表明，p12E几乎只携带棕榈酸。
Friend murine leukaemia virus complex was propagated on murine cells in the presence of [9,10-H-3]palmitic acid. Virus particles were harvested from the culture supernatant and lysed with detergents. The viral transmembrane protein, p12E, was isolated from the lysates by size-exclusion chromatography and purified by narrowbore reverse-phase HPLC. Analysis of the purified product by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) revealed that the protein is palmitoylated carrying one fatty acid residue. The radiolabelled fatty acid was released by hydroxylamine treatment at pH 7, indicating that acylation occurred via a thioester linkage. For allocation of the acylation site, p12E was digested with trypsin. The resulting peptides were either directly subjected to MALDI-TOF-MS or fractionated by microbore reverse-phase HPLC prior to mass spectrometry. The results revealed that p12E of Friend murine leukaemia virus is acylated at a cysteine residue situated at the C-terminal side of the putative transmembrane anchor of the polypeptide. Fatty acid analysis of the purified acylpeptide demonstrated that p12E carries almost exclusively palmitic acid.