The Inhibitory Guanine Nucleotide-binding Protein (Ni) Purified from. Bovine Brain Is a High Affinity GTPase*. (Received for publication, September 18,1984).
THEJOURNAL OF
Vol. 260, No. 4, Issue of February 25, pp. 2057-2063,1985 Printed in U.S.A.
BIOLOGICAL CHEMISTRY
The Inhibitory Guanine Nucleotide-binding Protein (Ni)Purified from Bovine Brain Is a High Affinity GTPase* (Received for publication, September 18,1984)
Graeme Milligan and Werner A. Klee From the Laboratoryof Molecular Biology, National Instituteof Mental Health, Bethesda, Maryland20205
Using modifications of the methods of Bokochet d. (Bokoch, G. M., Katada, T., Northup, J. K., Ui, M., and Gilman, A. G. (1984) J. Biol. Chem. 259,3560-3567) and Codina et d. (Codina, J., Hildebrandt, J. D., Sekura, R. D., Birnbaumer, M., Bryan, J., Manclark, C. R., Iyengar, R., and Birnbaumer, L. (1984) J. Biol. Chem. 259,5871-5886), we have purified a pertussis toxin substrate with the expected characteristicsof the inhibitory guanine nucleotide-binding protein (Ni)essentially to homogeneity. The purified protein consists of 3 subunits of M,40,000,35,000, and 95%pure protein. Chromatography on octyl-Sepharoseprovided efficient separation of Ni from N. (the stimulatory guanine nucleotide-binding protein). Analytical ultracentrifugation indicates an M, of 82,000 and a sedimentation coefficient s20,u of 5.1. Theprotein is abletorestoreopiate-mediated inhibition of adenylate cyclase to membranes prepared from NG 108-15 cells which had been treated with pertussis toxin. Bovine brain Ni has the enzymatic properties of a low K,,, GTPase with a turnover number of 0.3 and affinities for nucleotides in the order GppNHp 2 GTP 2 GDP >> ATP, CTP, UTP, and GMP. Na+specifically stimulates the GTPase and low concentrations of Mg2+ ) inhibitory. SomeMg2+is apparently nec(
