Park D, Jhon D Y, Kriz R, Knopf J, Rhee S G
Laboratory of Biochemistry, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892.
J Biol Chem. 1992 Aug 15;267(23):16048-55.
cDNAs corresponding to a previously uncharacterized phospholipase C were isolated from an HL-60 cell cDNA library. The cDNAs encodes a putative polypeptide of 1181 amino acids with a calculated molecular mass of 133,700 daltons. Comparison of the amino acid sequence of the predicted protein with those of five mammalian phospholipase C isoforms (PLC-beta 1, PLC-gamma 1, PLC-gamma 2, PLC-delta 1, and PLC-delta 2) revealed that the new enzyme is most closely related to PLC-beta 1 with an overall amino acid sequence identity of 48%. Thus, the new phospholipase C was named PLC-beta 2. The least similarity between PLC-beta 1 and PLC-beta 2 is apparent in the carboxyl-terminal 450 amino acids. Both PLC-beta 1 and PLC-beta 2 were purified from extracts of HeLa cells that had been transfected with vaccinia virus containing the corresponding cDNAs. Like other mammalian PLC isoforms, including PLC-beta 1, the catalytic activity of PLC-beta 2 was entirely dependent on Ca2+, and PLC-beta 2 preferred phosphatidyl-inositol 4,5-bisphosphate to phosphatidylinositol as substrate. Recently, the alpha subunit of the pertussis toxin-insensitive G-protein alpha q has been shown to activate PLC-beta 1 but not PLC-gamma 1 and PLC-delta 1. When alpha q purified from bovine brain was reconstituted with PLC-beta 1 or PLC-beta 2, no stimulation of PLC-beta 2 was observed in the presence of either AlF4- or guanosine 5-O-(3-thiotriphosphate) (GTP gamma S), whereas PLC-beta 1 activity was enhanced markedly in the presence of AlF4- and less markedly but significantly in the presence of GTP gamma S. These results suggest that the receptor-dependent stimulation of PLC-beta 1 and that of PLC-beta 2 may require different G-protein alpha subunits. (see also accompanying article (Lee, C. H., Park, D., Wu, D., Rhee, S. G., and Simon, M. I. (1992) J. Biol. Chem. 267, 16044-16047).
从HL-60细胞cDNA文库中分离出与一种先前未被鉴定的磷脂酶C相对应的cDNA。这些cDNA编码一个推测的由1181个氨基酸组成的多肽，计算分子量为133,700道尔顿。将预测蛋白的氨基酸序列与五种哺乳动物磷脂酶C同工型（PLC-β1、PLC-γ1、PLC-γ2、PLC-δ1和PLC-δ2）的氨基酸序列进行比较，发现这种新酶与PLC-β1关系最为密切，总体氨基酸序列同一性为48%。因此，这种新的磷脂酶C被命名为PLC-β2。PLC-β1和PLC-β2之间最小的相似性在羧基末端的450个氨基酸中最为明显。PLC-β1和PLC-β2均从用含有相应cDNA的痘苗病毒转染的HeLa细胞提取物中纯化得到。与其他哺乳动物磷脂酶C同工型一样，包括PLC-β1，PLC-β2的催化活性完全依赖于Ca2+，并且PLC-β2以磷脂酰肌醇4,5-二磷酸作为底物比磷脂酰肌醇更具偏好性。最近，百日咳毒素不敏感的G蛋白αq亚基已被证明可激活PLC-β1，但不能激活PLC-γ1和PLC-δ1。当用从牛脑中纯化的αq与PLC-β1或PLC-β2重组时，在存在AlF4-或鸟苷5'-O-(3-硫代三磷酸)(GTPγS)的情况下，未观察到对PLC-β2的刺激，而在存在AlF4-时PLC-β1的活性显著增强，在存在GTPγS时活性增强程度较小但也显著。这些结果表明，PLC-β1和PLC-β2的受体依赖性刺激可能需要不同的G蛋白α亚基。(另见随附文章(Lee, C. H., Park, D., Wu, D., Rhee, S. G., and Simon, M. I. (1992) J. Biol. Chem. 267, 16044 - 16047)。
