来自禽类红细胞的Gα11和βγ亚基刺激的磷脂酶C-β的分子克隆、表达及调节活性

Molecular cloning, expression and regulatory activity of G alpha 11- and beta gamma-subunit-stimulated phospholipase C-beta from avian erythrocytes.

作者信息

Waldo G L, Paterson A, Boyer J L, Nicholas R A, Harden T K

机构信息

Department of Pharmacology, University of North Carolina School of Medicine, Chapel Hill 27599-7365, USA.

出版信息

Biochem J. 1996 Jun 1;316 ( Pt 2)(Pt 2):559-68. doi: 10.1042/bj3160559.

DOI:10.1042/bj3160559

PMID:8687401

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC1217385/

Abstract

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 alpha-subunits of the Gq family and by G-protein beta gamma-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-beta family, with the highest identity (71%) shared with PLC-beta 2 and lesser identities observed with PLC-beta 1 (49%), PLC-beta 3 (46%) and PLC-beta 4 (37%). The largest differences in sequence between the turkey PLC-beta and other PLC-beta isoenzymes occur in the C-terminal domain and in the region between the X- and Y-domains. The turkey isoenzyme and PLC-beta 2, which differ in their regulation by G-protein alpha-subunits, are only 44% similar across the approx. 400 amino acid residues of the C-terminal domain that has been implicated in alpha q activation of these proteins. Recombinant turkey PLC-beta 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-beta. Moreover, the catalytic activities of the recombinant enzyme were indistinguishable from those of native turkey erythrocyte PLC-beta in assays carried out in the presence of cholate and Ca2+, or in assays of activity after reconstitution with G alpha 11 or G-protein beta gamma-subunits. The turkey PLC-beta was more sensitive to activation by G alpha 11 than was PLC-beta 2, and was more sensitive to activation by beta gamma-subunits than either PLC-beta 2 or PLC-beta 1.

摘要

火鸡红细胞磷脂酶C（PLC）在阐明G蛋白在受体调节的肌醇脂质信号传导中的作用方面发挥了重要作用。这种同工酶受到Gq家族的α亚基和G蛋白βγ亚基的独特调节。从火鸡红细胞cDNA文库中克隆出了一个编码该PLC的4819 bp cDNA。该cDNA的开放阅读框编码一个1211个氨基酸的蛋白质（计算分子量为139050 Da），其中包含从火鸡红细胞PLC中测序得到的16个肽段的氨基酸序列。预测的火鸡PLC序列与PLC-β家族先前克隆成员的序列有相当大的相似性，与PLC-β2的同一性最高（71%），与PLC-β1（49%）、PLC-β3（46%）和PLC-β4（37%）的同一性较低。火鸡PLC-β与其他PLC-β同工酶在序列上的最大差异出现在C末端结构域以及X结构域和Y结构域之间的区域。火鸡同工酶和PLC-β2在受G蛋白α亚基调节方面存在差异，在与这些蛋白质的αq激活有关的约400个氨基酸残基的C末端结构域中，它们的相似性仅为44%。重组火鸡PLC-β在杆状病毒重组体在Sf9昆虫细胞中表达后被纯化至同质。重组酶在SDS/PAGE上的免疫反应性和迁移率与天然火鸡红细胞PLC-β相同。此外，在胆酸盐和Ca2+存在的测定中，或在用Gα11或G蛋白βγ亚基重构后的活性测定中，重组酶的催化活性与天然火鸡红细胞PLC-β的催化活性没有区别。火鸡PLC-β比PLC-β2对Gα11的激活更敏感，并且比PLC-β2或PLC-β1对βγ亚基的激活更敏感。