Casey, Patrick J. and Fong, Henry K. W. and Simon, Melvin I. and Gilman, Alfred G. (1990) Gz, a guanine nucleotide-binding protein with unique biochemical properties. Journal of Biological Chemistry, 265 (4). pp. 2382-2390. ISSN 0021-9258 http://resolver.caltech.edu/CaltechAUTHORS:CASjbc90
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Cloning of a complementary DNA (cDNA) for Gz alpha, a newly appreciated member of the family of guanine nucleotide-binding regulatory proteins (G proteins), has allowed preparation of specific antisera to identify the protein in tissues and to assay it during purification from bovine brain. Additionally, expression of the cDNA in Escherichia coli has resulted in the production and purification of the recombinant protein. Purification of Gz from bovine brain is tedious, and only small quantities of protein have been obtained. The protein copurifies with the beta gamma subunit complex common to other G proteins; another 26- kDa GTP-binding protein is also present in these preparations. The purified protein could not serve as a substrate for NAD-dependent ADP- ribosylation catalyzed by either pertussis toxin or cholera toxin. Purification of recombinant Gz alpha (rGz alpha) from E. coli is simple, and quantities of homogeneous protein sufficient for biochemical analysis are obtained. Purified rGz alpha has several properties that distinguish it from other G protein alpha subunit polypeptides. These include a very slow rate of guanine nucleotide exchange (k = 0.02 min^-1), which is reduced greater than 20-fold in the presence of mM concentrations of Mg2+. In addition, the rate of the intrinsic GTPase activity of Gz alpha is extremely slow. The hydrolysis rate (kcat) for rGz alpha at 30 degrees C is 0.05 min^-1, or 200-fold slower than that determined for other G protein alpha subunits. rGz alpha can interact with bovine brain beta gamma but does not serve as a substrate for ADP-ribosylation catalyzed by either pertussis toxin or cholera toxin. These studies suggest that Gz may play a role in signal transduction pathways that are mechanistically distinct from those controlled by the other members of the G protein family.
|Additional Information:||Copyright © 1990 by the American Society for Biochemistry and Molecular Biology. (Received for publication, August 9, 1989) We thank Drs. Michael Graziano, Michael Freissmuth, Maurine Linder, and Sussane Mumby for continued discussions and criticism of this work. We also thank Dr. Freissmuth for assistance with the ADP-ribosylations and Lisa Ortlepp for skillful technical assistance. This work was supported in part by United States Public Health Service Grants GM34497 (to A.G.G.), GM34236 (to M.I.S.), National Research Service Award GM11982 (to P.J.C.), and by American Cancer Society Grant BC555I. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. [H.K.W.F. was] [s]upported by the Jules and Doris Stein Research Fund to Prevent Blindness. [A.G.G. was] [s]upported by the Raymond and Ellen Willie Chair of Molecular Neuropharmacology, the Perot Family Foundation, and The Lucille P. Markey Charitable Trust.|
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