Abstract
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.
Highlights
From the $Laboratory of Biochemistry, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892 and the VDefmrtmento.f~LifeScience, Korea Advanced Institute of Science and Technology, Daedok, 305-701 Korea
Comparison with PLC-81 and PLC-82 revealed that thededuced amino-terminal sequence of PLC-83 was incomplete by 10-20 amino acids
With the use of antibodies raised against synthetic peptides corresponding toPLC-83-specific amino acid sequences, we purified PLC-83 from a rat brain parspecies [6,7,8,9,10,11,12],two from Drosophilamelanogaster [13, 14], and one from Dictyostelium discoideum [15]) have been isolated
Summary
Vol 268, No., Issue of M m b 25, pp. 6654-8661, 1993 Printed in U.S.A. Cloning, Sequencing, Purification, andG,-dependent Activation of Phospholipase C-83”. Comparison of the predicted amino acid Protein isolation and molecular cloning studies have revealed sequence of this new PLC with other knownPLC iso- that PLC activities belong to a family of isozymes [4, 5]. Apparent molecular mass of 152 kDa on SDS-poly- All PLC isozymes contain -300 amino-terminal amino acrylamide gels, as compared with 160 and 140 kDa acids that precede the X domain. The distinct structural featuresof the different PLC types appear to be related to specific mechanisms of receptormediated enzyme activation. 580-bp clone contained a PLC-likesequence that was different from the G,a subunits represent the active components of known PLCs. the PT-insensitive G proteins that mediate receptor-stimulated hydrolysis of PtdIns 4,5-P2
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