McIntire W E, Dingus J, Schey K L, Hildebrandt J D
Department of Cell and Molecular Pharmacology and Experimental Therapeutics, Medical University of South Carolina, Charleston, South Carolina 29425, USA.
J Biol Chem. 1998 Dec 11;273(50):33135-41. doi: 10.1074/jbc.273.50.33135.
Go is the major G protein in bovine brain, with at least three isoforms, GoA, GoB, and GoC. Whereas alphaoA and alphaoB arise from a single Goalpha gene as alternatively spliced mRNAs, alphaoA and alphaoC are thought to differ by covalent modification. To test the hypothesis that alphaoA and alphaoC have different N-terminal lipid modifications, proteolytic fragments of alphao isoforms were immunoprecipitated with an N terminus-specific antibody and analyzed by matrix-assisted laser desorption ionization mass spectrometry. The major masses observed in immunoprecipitates were the same for all three alphao isoforms and corresponded to the predicted mass of a myristoylated N-terminal fragment. Structural differences between alphaoA and alphaoC were also compared before and after limited tryptic proteolysis using SDS-polyacrylamide gel electrophoresis containing 6 M urea. Based upon the alphao subunit fragments produced under activating and nonactivating conditions, differences between alphaoA and alphaoC were localized to a C-terminal fragment of the protein. This region, involved in receptor and effector interactions, implies divergent signaling roles for these two alphao proteins. Finally, the structural difference between alphaoA and alphaoC is associated with a difference of at most 2 daltons based upon measurements by electrospay ionization mass spectrometry.
Go是牛脑中主要的G蛋白，至少有三种亚型，即GoA、GoB和GoC。虽然αoA和αoB来自单一的Goα基因，是选择性剪接的mRNA，但αoA和αoC被认为在共价修饰方面存在差异。为了验证αoA和αoC具有不同的N端脂质修饰这一假设，用N端特异性抗体对αo亚型的蛋白水解片段进行免疫沉淀，并通过基质辅助激光解吸电离质谱分析。在所有三种αo亚型的免疫沉淀物中观察到的主要质量相同，且与豆蔻酰化N端片段的预测质量相对应。还使用含6 M尿素的SDS聚丙烯酰胺凝胶电泳，比较了有限胰蛋白酶消化前后αoA和αoC之间的结构差异。根据在激活和非激活条件下产生的αo亚基片段，αoA和αoC之间的差异定位于该蛋白的C端片段。这个参与受体和效应器相互作用的区域，意味着这两种αo蛋白具有不同的信号传导作用。最后，根据电喷雾电离质谱测量，αoA和αoC之间的结构差异最多为2道尔顿。
