Purification and characterization of recombinant G16 alpha from Sf9 cells: activation of purified phospholipase C isozymes by G-protein alpha subunits.
Purification and characterization of recombinant G16 alpha from Sf9 cells: activation of purified phospholipase C isozymes by G-protein alpha subunits.
复制标题
Sf9 细胞重组 G16 α 的纯化和表征：G 蛋白 α 亚基激活纯化的磷脂酶 C 同工酶。
DOI：
10.1073/pnas.90.19.9176
复制
发表时间：
1993
影响因子：
11.1
通讯作者：
Gilman,AG
中科院分区：
文献类型：
--
作者：
Kozasa,T;Hepler,JR;Smrcka,AV;Simon,MI;Rhee,SG;Sternweis,PC;Gilman,AG
利用重组杆状病毒在Sf9细胞中表达了编码异三聚体鸟嘌呤核苷酸结合蛋白α亚基G16 α的cDNA。在鸟苷5′-[γ -硫代]三磷酸存在下，Sf9细胞膜提取物中的G16 α激活了磷脂酶c - β 1 (plc - β 1)；Al3+、Mg2+和F-均不能激活该体系。Sf9细胞胞质部分的G16 α不刺激plc - β 1。g蛋白β - γ亚基复合物的同时表达增加了Sf9细胞膜中G16 α的含量。从表达G16 α、g蛋白β 2和γ 2亚基的细胞膜的胆酸提取物中纯化了鸟苷5'-[γ -硫代]三磷酸活化形式的G16 α。G16 α激活plc - β 1、plc - β 2和plc - β 3的方式与Gq α基本没有区别。G16 α介导的plc - β 1和plc - β 3的激活大大超过plc - β 2。G16 α没有激活PLC-gamma 1或PLC-delta 1。因此，Gq α家族的两个远亲成员Gq α和G16 α具有相同的激活已知plc - β亚型的能力。
A cDNA encoding G16 alpha, the alpha subunit of a heterotrimeric guanine nucleotide-binding protein, was expressed in Sf9 cells using recombinant baculovirus. G16 alpha in membrane extracts of Sf9 cells activated phospholipase C-beta 1 (PLC-beta 1) in the presence of guanosine 5'-[gamma-thio]triphosphate; the system could not be activated by Al3+, Mg2+, and F-. The G16 alpha in the cytosolic fraction of Sf9 cells did not stimulate PLC-beta 1. Concurrent expression of the G-protein beta gamma subunit complex increased the amount of G16 alpha in Sf9 cell membranes. The guanosine 5'-[gamma-thio]triphosphate-activated form of G16 alpha was purified from cholate extracts of membranes from cells expressing G16 alpha, and the G-protein beta 2 and gamma 2 subunits. G16 alpha activated PLC-beta 1, PLC-beta 2, and PLC-beta 3 in a manner essentially indistinguishable from that of Gq alpha. G16 alpha-mediated activation of PLC-beta 1 and PLC-beta 3 greatly exceeded that of PLC-beta 2. G16 alpha did not activate PLC-gamma 1 or PLC-delta 1. Thus, two distantly related members of the Gq alpha family, Gq alpha and G16 alpha, have the same ability to activate the known isoforms of PLC-beta.