The Effect of GTP and Mg2’ on the GTPase Activity and the Fluorescent Properties of Go*

The structures of the guanosine 5'O-(3-thio)triphosphate (GTP gamma S)-containing guanine nucleotide-binding regulatory proteins (G proteins) are distinct from those of the GDP-containing forms. One indication of the conformational change caused by GTP gamma S is a Mg2+-sensitive increase in the intensity of the proteins' tryptophan fluorescence (Higashijima, T., Ferguson, K.M., Sternweis, P.C., Ross, E.M., Smigel, M.D., Gilman, A.G. (1987), J. Biol. Chem., 262, 762-766). GTP causes a similar change in the fluorescence of Go, a G protein from bovine brain. When Mg2+ is also present, the increase in fluorescence is transient, and the rate of decline in the intensity of the fluorescence is the same as the rate of GTP hydrolysis by the protein. The steady-state rate of hydrolysis of GTP by Go (0.3-0.4/min) is slower than the catalytic rate of the protein (2/min), because the rate-limiting step in the reaction is the release of GDP.

The G proteins' are a family of homologous, membraneassociated guanine nucleotide-binding regulatory proteins that act as transducers of receptor-mediated signals (1-3). Their properties are described briefly in the first of this series of papers. All of the G proteins hydrolyze GTP, albeit slowly, to GDP and Pi. Although the GTPase activity of the purified G proteins depends on the assay conditions and the particular protein, basal rates of hydrolysis are typically 0.01-1.5 min" (4,5). Receptor-mediated stimulation of GTPase activity has been demonstrated by reconstitution of purified G proteins with receptor-containing membranes (6)(7)(8)(9)(10)(11). Such reconstituted preparations yield hormone-stimulated activities that are similar to those noted in plasma membranes (12,13).
The GTPase activity of the G proteins is a crucial aspect of their regulatory mechanism. With regard to stimulation of adenylate cyclase, for example, it is believed that agonistbound receptors catalyze the exchange of tightly bound GDP on G, for GTP (14). GTP-bound G. stimulates adenylyl cy-~~ x This work was supported by United States Public Health Service Grants GM34497 and GM07062 and by American Cancer Society Grant CD225G. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
$ Present address: Dept. of Biophysics and Biochemistry, University of Tokyo, Japan.
Our previous experiments demonstrated that binding 0 1 GTPyS to either Gi or Go induced a change in the fluorescencc of these proteins. In the experiments reported here, we shoa that GTP causes a similar change in fluorescence, and thai this change can be used to measure the intrinsic rate of GTF hydrolysis by G,.
MATERIALS AND METHODS Protein Preparation-Go was purified as described (22). GDP-free G, was prepared by chromatography on Sephadex G-25 in ar (NH4)zS0,-containing buffer (23). The (NH4),S04 was removed bJ chromatography on Sephadex G-25 in buffer A (50 m M NaHepes, pE 8.0, 1 mM NaEDTA, 1 mM dithiothreitol, 0.1% Lubrol) containing 20% glycerol. GTP Hydrolysis-GTPase activity was measured as described (6, with some modifications. The G protein was incubated at 20 "C ir buffer A containing the indicated concentrations of [y-"PIGTP anc MgS04. For measurement of the time-dependent release of [32P]Pi the reaction was initiated by the addition of protein that had beer warmed to the temperature of the reaction. Aliquots (50 pl) were removed at the indicated intervals, added to 750 pl of 5% (w/v) Norii in 50 mM NaH2P04 (0 "C), and vortexed. The charcoal was removec by centrifugation (2000 rpm for 10 min in a Beckman JA 4.2 rotor) and the amount of radioactivity in a 400-pl aliquot of the supernatan was determined by liquid scintillation counting. The rate of protein. independent formation of ["PIP, was subtracted to determine the GTPase activity. [y3'P]GTP was prepared as described (24). All othel procedures have been described elsewhere (25).
Analysis of the Time-dependence of GTPase Activity-Estimate! for the dissociation rate of GDP ( k 2 ) from Go and the protein's r a~ of catalysis (kc,,,) were obtained by fitting the data to one of twc models: For GDP-free G protein: For GDP-containing G protein: In either case, the total amount of Go in the reaction was determinec by GTPyS binding as described (23). Thus, only k-z and kcat were permitted to vary during the nonlinear least squares parameter esti. mation procedure. Details of the derivation of these equations are provided in the Appendix.

RESULTS
The Influence of GTP on the Fluorescence of G,,--Binding of GTPyS to Go, and G, changes the conformation of the proteins and causes an increase in the intensity of theil tryptophan fluorescence. A similar effect is caused by GTP, 757 GTPase When 10 pM GTP is added to a reaction containing 290 nM G,,,, there is a slow, exponential increase in the fluorescence intensity; steady state is achieved in 5 min ( Fig. 1). (The abrupt decrease in the intensity of the fluorescence emission immediately upon addition of GTP is due to the absorbance of the nucleotide.) The subsequent addition of 10 mM Mg2+ causes a further, rapid increase in the fluorescence intensity (>35% enhancement), which then declines exponentially (rate = 2.2 & 0.1 rnin") to a value that exceeds the baseline by 10%. The change in the fluorescence intensity caused by GTP in the absence of added Mg2+ is similar to that observed with GTP-yS in both time course and intensity (25), suggesting that the alteration is caused by the exchange of bound GDP for GTP. (Recall that Go, as purified contains 1 mol GDP/mol protein (Ref. 23).) Once steady state is achieved, addition of Mg2+ causes a rapid increase in the fluorescence intensity, also analogous to that observed when GTP-yS was used. Unlike the results obtained with GTP-yS, however, the Mg2"induced change in the presence of GTP is transient. These data suggest the following model: GTP is the form of the protein that has a 10% enhancement of the fluorescence intensity; Go,. GTP"$+ has a higher fluorescence intensity (35% enhancement), but, because of the hydrolysis of GTP, it has a short lifetime determined by kcat. Upon hydrolysis, the system returns to the GDP-bound form, which is the reference state for these experiments.
According to this model, the rate of approach to steady state is k+ + kc, when both Mg2+ and GTP are added to the G protein.
In the absence of Mg+, the rate is 12-2 . A consequence of the hydrolytic reaction is that the enhancement of steady-state fluorescence intensity in the presence of M e is f 2 k 2 / ( k -, + kcat), where f 2 is the increase in fluorescence characteristic of Go,,-GTP"?' . When 1 FM GTP is added to a reaction containing both Mg2' and G,,,, there is a rapid increase in the fluorescence intensity to a steady-state enhancement of 5% (Fig. 2). The rate of this increase (2-3 min-') is much faster than that observed when Mg2+ is omit-I ' . ted (Fig. 1). Given the magnitude of the increase in fluorescence (5%) and using estimates of f 2 =35% and kc, = 2 min", one would predict k-* to approximate 0.3 min" (see below). If this increase in fluorescence is caused by the steady-state concentration of Go, -GTP. M e , the addition of an excess of GDP should block the formation of Go,. GTP Mg", and the hydrolysis of GTP should cause a decrease in fluorescence. When 10 ~L M GDP is added to a reaction at steady state that contains Go,, 1 PM GTP, and 10 mM MgSO,, there is a rapid decline in the intensity of the fluorescence (Fig. 2). This relaxation process is complete in 1 min and is absent when 10 p~ GTP is substituted for GDP. The Rate of Hydrolysis of GTP-To test the hypothesis that hydrolysis causes the decline in fluorescence following the rapid increase upon addition of Mg2+ to GTP-containing Go, (Fig. l), Go, was incubated with 1 PM [-Y-~'P]GTP for 16 min to allow sufficient time for GTP to replace the GDP on G, . During this incubation there was little release of ["PIP, (Fig.  3). When 10 mM Mg2+ was added, there was a relatively rapid release of phosphate, followed by a slower rate of GTP hydrolysis. These data were analyzed as described under "Materials and Methods" to obtain estimates for kc, (1.8 Fig. 3. These data indicate that the release of GDP and not the hydrolysis of GTP is the ratelimiting step in the GTPase reaction at steady state.
The Effect of Bound GDP on the Hydrolysis of GTP-If the dissociation of GDP is rate-limiting, incubation of GDP-free Go with [T-~~PIGTP should cause a rapid release of phosphate. This hypothesis was tested by incubation of GDP-containing Go and GDP-free Go with 1 pM [y3'P]GTP (100 nM [-y-"P] GTP for GDP-free Go) and 10 mM MgS04 at 20 "C (Fig. 4). The release of ["PIPi catalyzed by GDP-containing Go lagged slightly before a steady-state rate of hydrolysis of 0.3 min" was achieved. The production of [32P]Pi in the reaction that contained GDP-free Go was initially faster and then slowed to the same steady-state rate of hydrolysis as found for GDPcontaining Go. Analysis of these data according to the equations described under "Materials and Methods" indicates that both forms of the protein catalyze hydrolysis at similar rates containing Go) and release GDP at the same rate (0.4 k 0.06 min" for GDP-free Go, 0.4 0.01 min" for GDP-containing GJ. The rate of dissociation of GDP from Go,, was measured directly using [a-"'PIGDP (see Table I, Ref. 26). This value averaged 0.3/min at 20 "C and was independent of the concentration of M e . It is in excellent agreement with those estimated above by consideration of the level of fluorescence enhancement achieved in the presence of M$+ and GTP (Fig.   2) and by examination of the rate of GTP hydrolysis (Figs. 3  and 4).

DISCUSSION
Activation of G proteins by guanine nucleotides changes the proteins' conformation (8,(27)(28)(29)(30). Concomitant with this structural change is an increase in the intensity of fluorescence of tryptophan residues (25). Both GTPrS and GTP can cause this change, suggesting that a similar alteration in protein structure is caused by the different nucleotides. When GTP is added to GDP-containing Go,, there is a slow increase in the intensity of tryptophan fluorescence as GDP dissociates and GTP then binds. After GDP has been replaced with GTP, the addition of Mg2+ causes a rapid increase in fluorescence intensity, reflecting the formation of G,, . GTP -Mg2'. Unlike the complex formed in the presence of GTP+, the GTPcontaining form of the protein is transient, because GTP is hydrolyzed to GDP and Pi in the presence of M e . The hydrolysis is accompanied by a decrease in the fluorescence intensity, providing a direct measure of the catalytic reaction. The rate of the catalytic reaction (as distinguished from the rate of steady-state hydrolysis) found for G, and G: is similar to that noted for G, (4 rnin") and transducin (1 min") (5,7,18). The rate of GTP hydrolysis by Go, and Gi, measured at steady state is the same as the rate of dissociation of GDP. Another consequence of the relatively rapid kat is that the fraction of G protein with bound GTP and Mg2+ (presumably the active form of the protein) cannot exceed k-n/(k,,t + k J , even when very high concentrations of GTP are present. Thus, the hydrolytic reaction holds the protein in the GDPcontaining, presumably inactive form. Agonist-bound receptors increase the rate of dissociation of guanine nucleotide (7, 15,17), apparently by a catalytic mechanism that is analogous to that of the interaction of elongation factors Tu and Ts (31). As the rate of dissociation of GDP increases, the velocity of the GTPase reaction will increase to a limit of kcat and the fraction of the activated form of the G protein will approach 100%. Thus, the receptor alters the state of activation of the G protein merely by changing the rate of dissociation of GDP.
Even though kc,, is 5-to 20-fold greater (depending on the particular G protein)2 than the observed steady-state rate of GTP hydrolysis, it is still an exceedingly slow turnover rate. This too is an important part of the function of the G protein.
The turnover rate for adenylyl cyclase, estimated from the activity of the purified protein (32), is 1200 rnin". If GB,.

GDP Dissociation Lim
The derivative of Equation 6 evaluated at t = 0 equated with Equation 2 at t = 0 is clml + qmZ = Gt(lf ) E (8) where Gt is the total concentration of G protein, f is the fraction of the protein with bound GDP at t = 0, and Integration of Equation 6 between the limits of 0 and t gives When none of the G protein contains GDP at the start of the reaction ( f = 0) ~1 = -Gt ~2 E grkat/(kca + k-2)

In this case Equation 10 is
The rate of hydrolysis after steady state is attained is Gtkak-2 v . . =kcat + k-2 which is the same as for the previous case ( f = 1, Equations 12 and 13. Extrapolation of the steady-state solution to t = 0 shows that there is a burst of Pi release: