Effect of Li + upon the Mg 2+ -Dependent Activation of Recombinant G i1 Nicole Minadeo,* Brian Layden,* Louis V. Amari,* Victoria Thomas,* Karrie Radloff,* Chandra Srinivasan,* Heidi E. Hamm,† and Duarte Mota de Freitas* ,1 *Department of Chemistry, Loyola University at Chicago, 6525 North Sheridan Road, Chicago, Illinois 60626; and †Department of Molecular Pharmacology and Biological Chemistry, Northwestern University Medical School, 320 East Superior Street, Chicago, Illinois 60611 Received October 2, 2000, and in revised form December 21, 2000; published online March 9, 2001 Although lithium salts have been used in the treat- ment and prophylaxis of manic-depressive or bipolar patients for 50 years, the mechanism of the pharmaco- logic action of Li  is unknown. Based on activity stud- ies of inhibitory and stimulatory guanine-binding (G) proteins in rat cortical membranes, it was proposed that Li  inhibition of G-proteins may account for its pharmacologic action. We used the purified  subunit of the recombinant inhibitory G-protein, rG i1 , and found that Li  at therapeutic levels significantly inhib- ited the formation of the GDP.AlF 4  .rG i1 complex. Be- cause our studies were conducted with a purified, metal-reconstituted G-protein rather than with cell membrane suspensions, our Li  inhibition results lend additional support to the G-protein hypothesis for Li  action. © 2001 Academic Press Key Words: G-proteins; rG i1 ; fluorescence spectros- copy; Li  /Mg 2 competition. Li + has both antimanic and antidepressive effects. Based on measurements obtained with rat cortical membrane suspensions, Avissar and coworkers (1) pro- posed that Li + modulates the enzymatic activity of G-proteins, which are key enzymes involved in signal transduction. Because G-proteins are Mg 2+ -activated, it was proposed that Li + might compete with Mg 2+ at the Mg 2+ -binding sites on G-proteins, rendering these proteins partially inactive (1– 4). It was shown (1) that, within the therapeutic dosage range, Li + inhibits the activities of the G-proteins, which inhibit and stimu- late adenylate cyclase, G i and G s ; Li + also inhibits the activity of G o , the G-protein that inhibits inositol mono- phosphatase. The addition of magnesium caused a re- versal of this inhibition (2). Thus, the G-protein hy- pothesis encompasses the inositol monophosphatase and the adenylate cyclase hypotheses, with G-proteins being the common site for the antimanic and antide- pressive effects of Li + . It has previously been shown that upon activation of G-proteins by GTP or GTP analogs, the intrinsic tryptophan fluorescence of the G-protein increases (5–7). In this study, the effect of Li + on the ability of Mg 2+ to activate a purified, metal- reconstituted G-protein, rG i1 , 2 was studied by fluores- cence spectroscopy. MATERIALS AND METHODS Chemicals. All chemicals were obtained from Aldrich or Sigma and were used without further purification. A colony of Escherichia coli genetically engineered to produce G i1 was obtained from Dr. Hamm’s laboratory. Preparation of rG i1 . Recombinant G i1 was isolated by the method of Lee et al. (8). The supernatant collected from this proce- dure was applied to a column (1.5  10 cm) containing the His- binding resin charged with Ni 2+ . The rG i1 was then eluted by the addition of 100 mM imidazole at pH 7.9. The presence of rG i1 in the fractions was confirmed by SDS–PAGE. The protein solution was then further purified by use of FPLC. rG i1 was then stored in a 50 mM Tris–Cl solution, pH 7.9, containing 40% glycerol and 10.0 M GDP at -20°. Preparation of rG i1 samples for fluorescent activity assay. Metal ion contaminants in the rG i1 samples were removed by dialyzing against 50.0 mM Tris–Cl, pH 7.9, 0.005% n-octyl--D-glucopyrano- side, 1.00 mM EDTA, 1.00 mM EGTA, 10.0 mM -mercaptoethanol, and 10.0 M GDP for 3 h (9). The protein samples were then dialyzed against 50.0 mM Tris–Cl solution, pH 7.9, containing 10.0 M GDP 1 To whom correspondence and reprint requests should be ad- dressed. Fax: (773) 508-3086. E-mail: dfreita@luc.edu. 2 Abbreviations used: FPLC, fast protein liquid chromatography; ICP-ES, inductively coupled plasma-emission spectrometer; rG i1 ,  subunit of recombinant inhibitory G-proteins; SDS–PAGE, sodium dodecylsufate–polyacrylamide gel electrophoresis. 0003-9861/01 $35.00 7 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved. Archives of Biochemistry and Biophysics Vol. 388, No. 1, April 1, pp. 7–12, 2001 doi:10.1006/abbi.2001.2282, available online at http://www.idealibrary.com on