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 Gα11 or G-protein βγ-subunits. The turkey PLC-β was more sensitive to activation by Gα11 than was PLC-β2, and was more sensitive to activation by βγ-subunits than either PLC-β2 or PLC-β1.
Molecular cloning, expression and regulatory activity of Gα11- and βγ-subunit-stimulated phospholipase C-β from avian erythrocytes
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Gary L. WALDO, Andrew PATERSON, José L. BOYER, Robert A. NICHOLAS, T. Kendall HARDEN; Molecular cloning, expression and regulatory activity of Gα11- and βγ-subunit-stimulated phospholipase C-β from avian erythrocytes. Biochem J 1 June 1996; 316 (2): 559–568. doi: https://doi.org/10.1042/bj3160559
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