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Titolo:
PURIFICATION AND CHARACTERIZATION OF PHOSPHOLIPASE C-BETA-1 MUTANTS EXPRESSED IN ESCHERICHIA-COLI
Autore:
MEIJ JTA; ROSS EM;
Indirizzi:
UNIV TEXAS,SW MED CTR,DEPT PHARMACOL,5323 HARRY HINES BLVD DALLAS TX 75235 UNIV TEXAS,SW MED CTR,DEPT PHARMACOL DALLAS TX 75235
Titolo Testata:
Biochemical and biophysical research communications
fascicolo: 3, volume: 225, anno: 1996,
pagine: 705 - 711
SICI:
0006-291X(1996)225:3<705:PACOPC>2.0.ZU;2-T
Fonte:
ISI
Lingua:
ENG
Soggetto:
GTPASE-ACTIVATING PROTEINS; HYDROLYSIS; FRAGMENTS; GQ/11;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Science Citation Index Expanded
Science Citation Index Expanded
Citazioni:
16
Recensione:
Indirizzi per estratti:
Citazione:
J.T.A. Meij e E.M. Ross, "PURIFICATION AND CHARACTERIZATION OF PHOSPHOLIPASE C-BETA-1 MUTANTS EXPRESSED IN ESCHERICHIA-COLI", Biochemical and biophysical research communications, 225(3), 1996, pp. 705-711

Abstract

In the M1-muscarinic receptor-G(q)-phospholipase C-beta 1 (PLC-beta 1) pathway, PLC-beta 1 is both the effector regulated by G(q) and acts as GTPase activating protein (GAP) for G(q). To rapidly evaluate in vitro PLC-beta 1 mutants constructed by oligonucleotide-directed mutagenesis, we established a quick expression and purification procedure. A pQE60/His(6)PLC-beta 1 construct was expressed in E. coli SG13009[pREP4]. Purification (similar to 160-fold) was obtained after high-sail extraction and chromatography over Ni+-agarose, Mono Q and Mono S columns. Several His(6)PLC-beta 1 mutants were equally responsive to alpha(q) . GTP gamma S, although the mutant His(6)PLC-beta 1-P57 (G758D) had only 2.5% the intrinsic PLC activity of the wild type. Also, His(6)PLC-beta 1 wild type and mutants acted as GAPs for G(q) in a reconstitution assay. Thus, the present procedure provides a method to quickly assess phospholipase activity, alpha q-responsiveness, and GAP activity of PLC-beta 1. (C) 1996 Academic Press, Inc.

ASDD Area Sistemi Dipartimentali e Documentali, Università di Bologna, Catalogo delle riviste ed altri periodici
Documento generato il 03/12/20 alle ore 15:48:09