Abstract
Using modifications of the methods of Bokoch et al. (Bokoch, G.M., Katada, T., Northup, J. K., Ui, M., and Gilman, A. G. (1984) J. Biol. Chem. 259, 3560-3567) and Codina et al. (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 characteristics of the inhibitory guanine nucleotide-binding protein (Ni) essentially to homogeneity. The purified protein consists of 3 subunits of Mr 40,000, 35,000, and less than 10,000. The Mr 40,000 band is found, upon close examination, to consist of a poorly resolved doublet. Starting with the membranes from 1,320 g of bovine forebrain we purified the protein some 100-fold with approximately 20% yield to obtain 13 mg of a greater than 95% pure protein. Chromatography on octyl-Sepharose provided efficient separation of Ni from Ns (the stimulatory guanine nucleotide-binding protein). Analytical ultracentrifugation indicates an Mr of 82,000 and a sedimentation coefficient S20,w of 5.1. The protein is able to restore opiate-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 Km GTPase with a turnover number of 0.3 and affinities for nucleotides in the order GppNHp greater than or equal to GTP greater than or equal to GDP much greater than ATP, CTP, UTP, and GMP. Na+ specifically stimulates the GTPase and low concentrations of Mg2+ (less than 50 microM) are inhibitory. Some Mg2+ is apparently necessary because EDTA, but not EGTA, abolishes the GTPase activity.
Highlights
M, 40,000 band is found, upon close examination, to consist of a poorly resolved doubletS. tarting with the membranes from 1,320 g of bovine forebrain we purified the protein some 100-fold with -20% yield to Reconstitution-As described in the Miniprint Supplement, we purified bovine brain Ni by enrichment of the pertussis toxin substrate
The adenylate cyclase of membranes preon octyl-Sepharoseprovided efficient separation of Ni pared from pertussis toxin-treated NG 108-15 cells is relafrom N..Analyticalultracentrifugationindicates an other inhibitory hormones, because of ADP-ribosylation of Ni
The protein purified from bovine brain membranes in this work is Ni because it is asubstrate for ADP-ribosylation, Control EDTA EGTA
Summary
Vol 260, No 4, Issue of February 25, ppP. r2i0n5te7d-2i0n63U,.1S9.8A5. The Inhibitory Guanine Nucleotide-binding Protein (Ni)Purified from Bovine Brain Is a High Affinity GTPase*. Vol 260, No 4, Issue of February 25, ppP. The Inhibitory Guanine Nucleotide-binding Protein (Ni)Purified from Bovine Brain Is a High Affinity GTPase*. Graeme Milligan and WerneAr. Klee From the Laboratoryof Molecular Biology, National Instituteof Mental Health, Bethesda, Maryland20205. Using modifications of the methods of Bokochet d. In thisreport we describe the purification of Nifrom bovine K., Ui, M., and brain, a rich source. 259,3560-3567) properties of this protein and itsenzymatic activity as a high and Codina et d.(Codina, J., Hildebrandt, J. D., Birnbaumer, M., Bryan, J., Manclark, C.
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