Molecular cloning, expression and regulatory activity of G alpha 11- and beta gamma-subunit-stimulated phospholipase C-beta from avian erythrocytes.
Molecular cloning, expression and regulatory activity of G alpha 11- and beta gamma-subunit-stimulated phospholipase C-beta from avian erythrocytes.
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禽类红细胞 G α 11 和 β γ 亚基刺激的磷脂酶 C-β 的分子克隆、表达和调节活性。
DOI：
10.1042/bj3160559
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发表时间：
1996
期刊：
影响因子：
--
通讯作者：
Harden,TK
中科院分区：
文献类型：
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作者：
Waldo,GL;Paterson,A;Boyer,JL;Nicholas,RA;Harden,TK
火鸡红细胞磷脂酶C （PLC）有助于描述g蛋白在受体调节的肌醇脂质信号传导中的作用。这种同工酶受Gq家族α-亚基和g蛋白βγ-亚基的独特调控。从火鸡红细胞cDNA文库中克隆了一个4819 bp的编码PLC的cDNA。该cDNA的开放阅读框编码一个1211个氨基酸的蛋白（计算分子质量139050 Da），包含从火鸡红细胞PLC中测序的16个肽的氨基酸序列。预测的火鸡PLC序列与先前克隆的PLC-β家族成员的序列具有相当大的相似性，与PLC-β2的同源性最高（71%），与PLC-β1（49%）、PLC-β3（46%）和PLC-β4（37%）的同源性较低。火鸡PLC-β和其他PLC-β同工酶之间最大的序列差异发生在c端结构域和X-和y -结构域之间的区域。火鸡同工酶和PLC-β2在g蛋白α-亚基的调控上有所不同，但在大约范围内只有44%的相似性。c端结构域的400个氨基酸残基与这些蛋白质的αq激活有关。重组火鸡PLC-β通过重组杆状病毒在Sf9昆虫细胞中表达得到纯化。重组酶在SDS/PAGE上的免疫反应性和迁移率与土鸡红细胞PLC-β相同。此外，重组酶的催化活性与天然火鸡红细胞PLC-β在胆酸盐和Ca2+存在下的催化活性，或用g α11或g蛋白β -亚基重组后的催化活性没有区别。火鸡PLC-β对g α11的激活比PLC-β2更敏感，对βγ-亚基的激活比PLC-β2和PLC-β1更敏感。
A turkey erythrocyte phospholipase C (PLC) has been instrumental in delineating the role of G-proteins in receptor-regulated inositol lipid signalling. This isoenzyme is uniquely regulated both by α-subunits of the Gq family and by G-protein βγ-subunits. A 4819 bp cDNA encoding this PLC has been cloned from a turkey erythrocyte cDNA library. The open reading frame of this cDNA encodes a 1211-amino-acid protein (calculated molecular mass 139050 Da) that contains amino acid sequences of 16 peptides sequenced from the turkey erythrocyte PLC. The predicted sequence of the turkey PLC shows considerable similarity with the sequences of previously cloned members of the PLC-β family, with the highest identity (71%) shared with PLC-β2 and lesser identities observed with PLC-β1 (49%), PLC-β3 (46%) and PLC-β4 (37%). The largest differences in sequence between the turkey PLC-β and other PLC-β isoenzymes occur in the C-terminal domain and in the region between the X- and Y-domains. The turkey isoenzyme and PLC-β2, which differ in their regulation by G-protein α-subunits, are only 44% similar across the approx. 400 amino acid residues of the C-terminal domain that has been implicated in αq activation of these proteins. Recombinant turkey PLC-β was purified to homogeneity following expression from a recombinant baculovirus in Sf9 insect cells. The immunoreactivity and mobility on SDS/PAGE of the recombinant enzyme were the same as observed with native turkey erythrocyte PLC-β. Moreover, the catalytic activities of the recombinant enzyme were indistinguishable from those of native turkey erythrocyte PLC-β in assays carried out in the presence of cholate and Ca2+, or in assays of activity after reconstitution with Gα11or G-protein βγ-subunits. The turkey PLC-β was more sensitive to activation by Gα11than was PLC-β2, and was more sensitive to activation by βγ-subunits than either PLC-β2 or PLC-β1.