Jhon D Y, Lee H H, Park D, Lee C W, Lee K H, Yoo O J, Rhee S G
Laboratory of Biochemistry, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892.
J Biol Chem. 1993 Mar 25;268(9):6654-61.
Six mammalian phospholipase C isozymes (PLC-beta 1, PLC-beta 2, PLC-gamma 1, PLC-gamma 2, PLC-delta 1, and PLC-delta 2) have been identified at both protein and DNA levels. Here, cDNAs corresponding to a previously unidentified PLC isozyme were isolated from a rat thyroid cell FRTL cDNA library. Comparison of the predicted amino acid sequence of this new PLC with other known PLC isozymes revealed a high degree of overall similarity with PLC-beta 1 and PLC-beta 2. Thus, the new PLC was named PLC-beta 3. Comparison with PLC-beta 1 and PLC-beta 2 also revealed that the deduced amino-terminal sequence of PLC-beta 3 was incomplete by 10-20 amino acids. With the use of antibodies raised against synthetic peptides corresponding to PLC-beta 3-specific amino acid sequences, we purified PLC-beta 3 from a rat brain particulate fraction. The purified enzyme exhibited an apparent molecular mass of 152 kDa on SDS-polyacrylamide gels, as compared with 150 and 140 kDa for PLC-beta 1 and PLC-beta 2, respectively. Studies of the activation of PLC-beta isozymes by three alpha subunits of Gq class G proteins, alpha q, alpha 11, and alpha 16 in the presence of guanosine 5-O-(3-thiotriphosphate) (GTP gamma S) revealed that the extent of activation decreased in the order of PLC-beta 1 > or = PLC-beta 3 >> PLC-beta 2 for all three alpha subunits, suggesting a certain degree of specificity in the interaction of Gq alpha subunits with different PLC-beta isozymes.
在蛋白质和DNA水平上已鉴定出六种哺乳动物磷脂酶C同工酶（PLC-β1、PLC-β2、PLC-γ1、PLC-γ2、PLC-δ1和PLC-δ2）。在此，从大鼠甲状腺细胞FRTL cDNA文库中分离出与一种先前未鉴定的PLC同工酶相对应的cDNA。将这种新PLC的预测氨基酸序列与其他已知PLC同工酶进行比较，发现它与PLC-β1和PLC-β2在整体上具有高度相似性。因此，这种新的PLC被命名为PLC-β3。与PLC-β1和PLC-β2的比较还表明，PLC-β3推导的氨基末端序列少了10 - 20个氨基酸。利用针对与PLC-β3特异性氨基酸序列相对应的合成肽产生的抗体，我们从大鼠脑微粒部分纯化了PLC-β3。在SDS-聚丙烯酰胺凝胶上，纯化的酶显示出明显的分子量为152 kDa，而PLC-β1和PLC-β2的分子量分别为150 kDa和140 kDa。在鸟苷5'-O-(3-硫代三磷酸)(GTPγS)存在的情况下，研究Gq类G蛋白的三个α亚基αq、α1α和α16对PLC-β同工酶的激活作用，结果表明，对于所有三个α亚基，激活程度按PLC-β1≥PLC-β3>>PLC-β2的顺序降低，这表明Gqα亚基与不同PLC-β同工酶相互作用存在一定程度的特异性。
