HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Waldo, G. L. Articles by Harden, T. K. Search for Related Content PubMed PubMed Citation Articles by Waldo, G. L. Articles by Harden, T. K. Social Bookmarking                      What's this?

J. Biol. Chem., Vol. 266, Issue 22, 14217-14225, Aug, 1991

Purification of an AlF4- and G-protein beta gamma-subunit-regulated phospholipase C-activating protein

GL Waldo, JL Boyer, AJ Morris and TK Harden
Department of Pharmacology, University of North Carolina School of Medicine, Chapel Hill 27599.

A 150-kDa phospholipase C has previously been purified from turkey erythrocytes and has been shown by reconstitution with turkey erythrocyte membranes to be a receptor- and G-protein-regulated enzyme (Morris, A. J., Waldo, G. L., Downes, C.P., and Harden, T. K. (1990) J. Biol. Chem. 265, 13501-13507; Morris, A.J., Waldo, G.L., Downes, C.P., and Harden, T.K. (1990) J. Biol. Chem. 265, 13508-13514). Combination of this 150-kDa protein with phosphoinositide substrate-containing phospholipid vesicles prepared with a cholate extract from purified turkey erythrocyte plasma membranes resulted in conferrence of AlF4- sensitivity to the purified phospholipase C. Guanosine 5'-3-O- (thio)triphosphate also activated the reconstituted phospholipase C in a manner that was inhibited by guanosine 5'-2-O-(thio)-diphosphate. The magnitude of the AlF4- stimulation was increased with increasing amounts of plasma membrane extract, and was also dependent on the concentration of purified phospholipase C. Using reconstitution of AlF4- sensitivity as an assay, the putative G-protein conferring regulation to the 150-kDa phospholipase C was purified to near homogeneity by sequential chromatography over Q-Sepharose, Sephacryl S-300, octyl- Sepharose, hydroxylapatite, and Mono-Q. Reconstituting activity co- purified with an approximately 43-kDa protein identified by silver staining; lesser amounts of a 35-kDa protein was present in the final purified fractions, as was a minor 40-kDa protein. The 43-kDa protein strongly reacted with antiserum against a 12-amino acid sequence found at the carboxyl terminus of Gq and G11, the 35-kDa protein strongly reacted with G-protein beta-subunit antiserum, and the 40-kDa protein reacted with antiserum that recognizes Gi3. Immunoprecipitation of the 43-kDa protein resulted in loss of phospholipase C-stimulating activity of the purified fraction. The idea that this is a phospholipase C- regulating G-protein is further supported by the observation that co- reconstitution of G-protein beta gamma-subunit with the purified phospholipase C-activating fraction resulted in a beta gamma-subunit- dependent inhibition of AlF(4-)-stimulated phospholipase C activity in the reconstituted preparation.
CiteULike    Complore    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?

This article has been cited by other articles:

				M. D. Hains, M. R. Wing, S. Maddileti, D. P. Siderovski, and T. K. Harden G{alpha}12/13- and Rho-Dependent Activation of Phospholipase C-{epsilon} by Lysophosphatidic Acid and Thrombin Receptors Mol. Pharmacol., June 1, 2006; 69(6): 2068 - 2075. [Abstract] [Full Text] [PDF]

				L. Y. W. Bourguignon, E. Gilad, A. Brightman, F. Diedrich, and P. Singleton Hyaluronan-CD44 Interaction with Leukemia-associated RhoGEF and Epidermal Growth Factor Receptor Promotes Rho/Ras Co-activation, Phospholipase C{epsilon}-Ca2+ Signaling, and Cytoskeleton Modification in Head and Neck Squamous Cell Carcinoma Cells J. Biol. Chem., May 19, 2006; 281(20): 14026 - 14040. [Abstract] [Full Text] [PDF]

				J. P. Seifert, M. R. Wing, J. T. Snyder, S. Gershburg, J. Sondek, and T. K. Harden RhoA Activates Purified Phospholipase C-{epsilon} by a Guanine Nucleotide-dependent Mechanism J. Biol. Chem., November 12, 2004; 279(46): 47992 - 47997. [Abstract] [Full Text] [PDF]

				M. R. Wing, J. T. Snyder, J. Sondek, and T. K. Harden Direct Activation of Phospholipase C-{epsilon} by Rho J. Biol. Chem., October 17, 2003; 278(42): 41253 - 41258. [Abstract] [Full Text] [PDF]

				M. R. Wing, D. M. Bourdon, and T. K. Harden PLC-{varepsilon}: A Shared Effector Protein in Ras-, Rho-, and G{alpha}{beta}{gamma}-Mediated Signaling Mol. Interv., August 1, 2003; 3(5): 273 - 280. [Abstract] [Full Text] [PDF]

				J. T. Snyder, A. U. Singer, M. R. Wing, T. K. Harden, and J. Sondek The Pleckstrin Homology Domain of Phospholipase C-{beta}2 as an Effector Site for Rac J. Biol. Chem., May 30, 2003; 278(23): 21099 - 21104. [Abstract] [Full Text] [PDF]

				M. R. Wing, D. Houston, G. G. Kelley, C. J. Der, D. P. Siderovski, and T. K. Harden Activation of Phospholipase C-epsilon by Heterotrimeric G Protein beta gamma -Subunits J. Biol. Chem., December 14, 2001; 276(51): 48257 - 48261. [Abstract] [Full Text] [PDF]

				Y.-H. Kim, T.-J. Park, Y. H. Lee, K. J. Baek, P.-G. Suh, S. H. Ryu, and K.-T. Kim Phospholipase C-delta 1 Is Activated by Capacitative Calcium Entry That Follows Phospholipase C-beta Activation upon Bradykinin Stimulation J. Biol. Chem., September 10, 1999; 274(37): 26127 - 26134. [Abstract] [Full Text] [PDF]

				S. Miscia, A. Di Baldassarre, G. Sabatino, E. Bonvini, R. A. Rana, M. Vitale, V. Di Valerio, and F. A. Manzoli Inefficient Phospholipase C Activation and Reduced Lck Expression Characterize the Signaling Defect of Umbilical Cord T Lymphocytes J. Immunol., September 1, 1999; 163(5): 2416 - 2424. [Abstract] [Full Text] [PDF]

				R. M. Rajagopalan-Gupta, M. M. Rasenick, and M. Hunzicker-Dunn Luteinizing Hormone/Choriogonadotropin-Dependent, Cholera Toxin-Catalyzed Adenosine 5'-Diphosphate (ADP)-Ribosylation of the Long and Short Forms of Gs{alpha} and Pertussis Toxin-Catalyzed ADP-Ribosylation of Gi{alpha} Mol. Endocrinol., May 1, 1997; 11(5): 538 - 549. [Abstract] [Full Text]

				C. E. Austin, A. Faussner, H. E. Robinson, S. Chakravarty, D. J. Kyle, J. M. Bathon, and D. Proud Stable Expression of the Human Kinin B1 Receptor in Chinese Hamster Ovary Cells. CHARACTERIZATION OF LIGAND BINDING AND EFFECTOR PATHWAYS J. Biol. Chem., April 25, 1997; 272(17): 11420 - 11425. [Abstract] [Full Text] [PDF]

				J. S. Popova, J. C. Garrison, S. G. Rhee, and M. M. Rasenick Tubulin, Gq, and Phosphatidylinositol 4,5-Bisphosphate Interact to Regulate Phospholipase Cbeta 1 Signaling J. Biol. Chem., March 7, 1997; 272(10): 6760 - 6765. [Abstract] [Full Text] [PDF]

				P. A. Galvin-Parton, X. Chen, C. M. Moxham, and C. C. Malbon Induction of Galpha q-specific Antisense RNA in Vivo Causes Increased Body Mass and Hyperadiposity J. Biol. Chem., February 14, 1997; 272(7): 4335 - 4341. [Abstract] [Full Text] [PDF]

				D Wu, G. LaRosa, and M. Simon G protein-coupled signal transduction pathways for interleukin-8 Science, July 2, 1993; 261(5117): 101 - 103. [Abstract] [PDF]