Murine retrovirus Pr65gag forms a 130K dimer in the absence of disulfide reducing agents.
Murine retrovirus Pr65gag forms a 130K dimer in the absence of disulfide reducing agents.
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鼠逆转录病毒 Pr65gag 在没有二硫键还原剂的情况下形成 130K 二聚体。
DOI：
10.1016/0042-6822(84)90164-8
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发表时间：
1984
期刊：
影响因子：
3.7
通讯作者：
R. Luftig
中科院分区：
文献类型：
--
作者：
Y. Yoshinaka;I. Katoh;R. Luftig
从慢性感染的HTG-2仓鼠细胞的组织培养液中获得的Gazdar-鼠肉瘤病毒（Gz-MSV）颗粒在形态上是不成熟的，并且含有未切割的Pr 65 gagas主要蛋白（>95%考马斯蓝染色）（A. Pinter和E. deHarven，1979，Virology，99，103-110; Y. Yoshinaka和R. B。Luftig，1982，Virology，118，380-388）。用1%十二烷基硫酸钠（SDS）破碎Gz-MSV颗粒，在无β-巯基乙醇（β-MSH）等还原剂存在下，用SDS-聚丙烯酰胺凝胶电泳（PAGE）分析，在130 K的Mr下，Pr 65 gag考马斯亮蓝染色的条带几乎有一半是一条带。用抗MuLV p30、p15、p12和p10的单特异性抗血清进行的电泳印迹研究表明，130 K条带与所有四种抗原交叉反应，表明它是Pr 65 gag的二聚体。二维（2D）SDS-PAGE，其中第一维在非还原条件下进行，第二维用β-MSH进行，支持了130 K条带是Pr 65 gag的二聚体复合物的论点。在SDS凝胶中还观察到少量的260 K和更高聚合形式的Pr 65 gag，表明聚合形式也可能存在。用[~（32）P]PPi体内标记HTG-2细胞获得的~（32）P标记的Gz-MSV颗粒在SDS-PAGE上显示，在130 K位置仅检测到10%的~（32）P标记。相比之下，在130 K下在2D凝胶上发现30%的考马斯亮蓝染色的Pr 65 gag材料。这表明未磷酸化的Pr 65 gag比莫洛尼鼠白血病病毒（M-MuLV）的磷酸化Pr 65 gagPr 65 gag更可能参与二聚体形成，磷酸化Pr 65 gag在SDS-PAGE上以次要（5%的染色）蛋白条带存在，也显示130 K二聚体。此外，在Gz-MSV的β-MSH缺陷SDS制备物中，经胰蛋白酶处理后，观察到一条32 K带，该带被p15抗血清染色，但不被p10、p12和p30抗血清染色。如果加入β-MSH，则该条带不再存在。因此，在未成熟的MuLV颗粒中Pr 65 gag二聚化似乎至少涉及多蛋白的p15区域。由于p15是一种非常疏水的蛋白质，当病毒装配过程中病毒体前体蛋白被带到细胞膜上时，可能会形成Pr 65 gagdimers。
Gazdar-murine sarcoma virus (Gz-MSV) particles, obtained from tissue culture fluids of chronically infected HTG-2 hamster cells are immature in morphology and contain uncleaved Pr65gagas the predominant protein (>95% Coomassie blue stain) (A. Pinter and E. deHarven, 1979,Virology,99, 103–110; Y. Yoshinaka and R. B. Luftig, 1982,Virology,118, 380–388). When Gz-MSV particles are disrupted in 1% sodium dodecyl sulfate (SDS) and then analyzed by SDS-polyacrylamide gel electrophoresis (PAGE) in theabsenceof reducing agents, such as β-mercaptoethanol (β-MSH) almost half of the Pr65gagCoomassie blue-stained band is detected as a band at aMrof 130K. Electrophoretic blotting studies with monospecific antisera against MuLV p30, p15, p12, and p10 showed that the 130K band cross-reacted with all four antigens suggesting that it was a dimer of Pr65gagTwo-dimensional (2D) SDS-PAGE where the first dimension was run under nonreducing conditions and the second with β-MSH, supported the contention that the 130K band was a dimeric complex of Pr65gagOne also saw minor amounts of a 260K and higher polymeric forms of Pr65gagon the SDS gels, suggesting that polymeric forms may exist as well. When32P-labeled Gz-MSV particles obtained byin vivolabeling of infected HTG-2 cells with [32P]PPi were electrophoresed on SDS-PAGE, only 10% of the32P label was detected at the 130K position. In contrast, 30% of the Coomassie blue-stained Pr65gagmaterial was found at 130K on the 2D gels. This suggests that unphosphorylated Pr65gagis more likely to participate in dimer formation than phosphorylated Pr65gagPr65gagof Moloney murine leukemia virus (M-MuLV), which is present as a minor (5% of stain) protein band on SDS-PAGE also showed 130K dimers. Further, in β-MSH-deficient SDS preparations of Gz-MSV, electrophoresed after trypsin treatment, a 32K band that stained with p15, but not p10, p12, nor p30, antisera was observed. If β-MSH was added, this band was no longer present. Thus Pr65gagdimerization in immature MuLV particles appears to at least involve the p15 region of the polyprotein. Since p15 is an extremely hydrophobic protein, formation of Pr65gagdimers may occur when virion precursor proteins are brought to the cell membrane during virus assembly.
通过一种新型肽图谱技术定位小鼠逆转录病毒 gag 前体中的脂质-蛋白质和蛋白质-蛋白质相互作用。
DOI：
--
发表时间：
1983
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
Pepinsky,RB
通讯作者：
Pepinsky,RB