A GTPase-activating Protein for the G Protein Galpha z
IDENTIFICATION, PURIFICATION, AND MECHANISM OF ACTION*

(Received for publication, September 20, 1996, and in revised form, November 21, 1996)

Jun Wang , Yaping Tu , Jimmy Woodson , Xiaoling Song and Elliott M. Ross Dagger

From the Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, Texas 75235-9041

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
Acknowledgments
REFERENCES


ABSTRACT

A GTPase-activating protein (GAP) specific for Galpha z was identified in brain, spleen, retina, platelet, C6 glioma cells, and several other tissues and cells. Gz GAP from bovine brain is a membrane protein that is refractory to solubilization with most detergents but was solubilized with warm Triton X-100 and purified up to 50,000-fold. Activity is associated with at least two separate proteins of Mr ~22,000 and 28,000, both of which have similar specific activities. In an assay that measures the rate of hydrolysis of GTP pre-bound to detergent-soluble Galpha z, the GAP accelerates hydrolysis over 200-fold, from 0.014 to 3 min -1 at 15 °C, or to >= 20 min-1 at 30 °C. It does not alter rates of nucleotide association or dissociation. When co-reconstituted into phospholipid vesicles with trimeric Gz and m2 muscarinic receptor, Gz GAP accelerates agonist-stimulated steady-state GTP hydrolysis as predicted by its effect on the hydrolytic reaction. In the single turnover assay, the Km of the GAP for Galpha z-GTP is 2 nM. Its activity is inhibited by Galpha z-guanosine 5'-O-thiotriphosphate (Galpha z-GTPgamma S) or by Galpha z-GDP/AlF4 with Ki ~1.5 nM for both species; Galpha z-GDP does not inhibit. G protein beta gamma subunits inhibit Gz GAP activity, apparently by forming a GTP-Galpha zbeta gamma complex that is a poor GAP substrate. Gz GAP displays little GAP activity toward Galpha i1 or Galpha o, but its activity with Galpha z is competitively inhibited by both Galpha i1 and Galpha o at nanomolar concentrations when they are bound to GTPgamma S but not to GDP. Neither phospholipase C-beta 1 (a Gq GAP) nor several adenylyl cyclase isoforms display Gz GAP activity.


INTRODUCTION

G proteins mediate numerous cellular processes by traversing a cycle of GTP binding and hydrolysis. Bound GTP activates a G protein such that it can stimulate a downstream effector protein. Activation is terminated when the bound GTP is hydrolyzed to GDP, which does not activate. Each step of the cycle is controlled, such that both steady-state GTPase activity and the concentrations of the active and inactive forms are highly regulated.

Activation of heterotrimeric G proteins is promoted by seven-span cell-surface receptors that facilitate GDP release and GTP binding. Small monomeric G proteins (Ras, Rac, Rho, Arf, etc.) are activated by cytosolic proteins that similarly facilitate GTP binding.

In many cases, hydrolysis of bound GTP, the deactivation step, is accelerated by GTPase-activating proteins, or GAPs1 (1-4). GAPs appear to fulfill at least one of four definable roles. Some GAPs for monomeric signaling G proteins, such as Ras GAP, appear to attenuate G protein signal amplitude in response to inputs from inhibitory signaling pathways (1, 2, for review). GAPs for the monomeric G proteins involved in cytoplasmic vesicle trafficking are thought to act by terminating a G protein-dependent assembly or transit step. Some effector proteins that are regulated by heterotrimeric G proteins also act as GAPs for their G protein regulators. The GAP activities of these effectors, such as phospholipase C-beta (5, 6) and the cyclic GMP phosphodiesterase gamma  subunit (7, 8), may allow effector-specific modulation of response times or may enhance the selectivity of receptor-G protein signaling (3). A fourth class of GAPs, also for the trimeric G proteins, includes members of the recently identified RGS protein family (4, 9-13). Little is known of the physiology of RGS proteins, but they can contribute to desensitization toward a prolonged signal (Sst2p in yeast; Refs. 10, 14, 15) or act as long term attenuators of signal amplitude (Egl-10 protein in Caenorhabditis elegans; Ref. 9).

G protein GAP activity can potentially be used to identify and purify regulators of G protein function or to point to novel inputs to G protein signaling pathways. For GAPs that are also effectors, their identification can indicate what downstream signals the G protein mediates.

We began a search for new GAPs for heterotrimeric G proteins by looking for a GAP for Gz, a pertussis toxin-insensitive member of the Gi family that is abundant in brain, adrenal medulla, and platelets (16-19). There were three reasons for this choice. Although Gz can mediate inhibition of adenylyl cyclase (20-22) and respond to receptors that regulate other Gi family members (21, 23), the signaling pathway(s) mediated by Gz remains unknown, and a Gz GAP might be an effector. Second, Gz hydrolyzes bound GTP very slowly (16). Its activation lifetime is about 7 min, which seems incompatible with normal signaling functions unless a GAP accelerates deactivation. Last, the slow hydrolytic rate simplifies design of an assay for a GAP. We describe here the detection of Gz GAP activity in brain and other tissues, substantial purification of a Gz GAP from bovine brain, and several aspects of its mechanism of action and regulation.


EXPERIMENTAL PROCEDURES

Materials

Procedures for purification of Galpha z (20), Galpha i1 (24, 25), Galpha o (24, 26), Galpha s (24), Galpha q (6), Gbeta gamma (6), m2 muscarinic cholinergic receptor (23), and phospholipase C-beta 1 (6) have been described. Sf9 membranes that contain recombinant adenylyl cyclase isoforms (27) and purified Galpha 12 (20) were gifts from Drs. Carmen Dessauer and Tohru Kozasa (this department). Galpha z-agarose was prepared according to Mumby et al. (28) and phenyl-Sepharose was purchased from Pharmacia Biotech Inc. Cholic acid was purified as described (29), and other detergents were purchased from various suppliers. [gamma -32P]GTP was either purchased or synthesized (30) and purified as described (6).

Hydrolysis of Galpha z-bound [gamma -32P]GTP

[gamma -32P]GTP was bound to Galpha z by incubating 10-50 pmol of Galpha z for 20 min at 30 °C in 200 µl of 25 mM NaHepes (pH 7.5), 3 mM DTT, 0.1% Triton X-100, 1 mM EDTA, 2.5 µM [gamma -32P]GTP (20-80 cpm/fmol), and sufficient MgCl2 to provide 1 µM free Mg2+. After incubation, [gamma -32P]GTP and [32P]orthophosphate were removed by chromatography on a 2-ml column of Sephadex G25, which was run in the same buffer but without GTP or MgCl2. Galpha z-bound [gamma -32P]GTP was determined by nitrocellulose binding assay (31) and was usually 25% of the total Galpha z, with the remainder bound to GDP.

Hydrolysis of Galpha z-bound [gamma -32P]GTP in routine GAP assays was measured by incubation of the substrate (usually ~1-2 nM) in the buffer used for its preparation but including 1 mM free Mg2+, 10 µg/ml bovine albumin, and 5 mM nonradioactive GTP. Unlabeled GTP was added to inhibit nucleoside triphosphatase activity present in crude GAP fractions in the event that any [gamma -32P]GTP dissociated from Galpha z. Assays were carried out at 15 °C for times that varied from 30 s to 60 min. Hydrolysis of bound [gamma -32P]GTP was measured as release of [32P]orthophosphate (31). Hydrolysis followed a single exponential time course. Hydrolysis is expressed either as the amount of bound GTP hydrolyzed at early times (quasilinear time course) or as a first-order rate constant.2 GAP-independent hydrolysis is subtracted from all data except in Figs. 1, 6, and 8B.


Fig. 1. Gz GAP activity in bovine brain membranes. A, Galpha z-[gamma -32P]GTP was incubated at 15 °C with increasing amounts of bovine brain membranes (diamond , 5 µg; open circle , 10 µg; triangle , 15 µg; down-triangle, 30 µg; square , 15 µg of boiled membrane). Release of [32P]orthophosphate was assayed at the times shown. Data are normalized to the total amount of Galpha z-[gamma -32P]GTP at zero time (125 fmol) determined by nitrocellulose binding assay. At each time point, samples were also assayed for bound [gamma -32P]GTP (not shown). The sum of [32P]orthophosphate plus bound [gamma -32P]GTP was constant over 120 min and not altered by the presence of the membranes. Hydrolysis was not altered by substitution of buffer for boiled membrane protein (not shown). In this early experiment, the assay buffer contained only 1 µM free Mg2+ and 0.1% Lubrol in place of 0.1% Triton X-100, resulting in the relatively low activities shown. B, each data set from A was well fit by a single component first-order reaction scheme (not shown). Values of the rate constant kapp2 obtained from least-squares fits of these data are plotted against the amount of membrane protein added.
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Fig. 6. Effect of Mg2+ on Gz GAP activity. Gz GAP-stimulated (bullet ) and basal (open circle ) GTP hydrolysis by Galpha z were assayed at the concentrations of free Mg2+ shown on the abscissa. The GAP-stimulated data are the apparent hydrolytic rate constants, kapp, i.e. basal hydrolysis was not subtracted from that measured in the presence of GAP. The free Mg2+ concentrations shown on the abscissa were obtained by varying the concentration of MgCl2 in the presence of 1 mM EDTA and 5 mM GTP (see "Experimental Procedures") and were measured as described by Huskens and Sherry (36). The concentration of Galpha z-[gamma -32P]GTP was 1.5 nM, and the concentration of GAP was 50 pM.
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Fig. 8. Inhibition of Gz GAP activity by Gbeta gamma subunits. A, the activity of 40 pM Gz GAP was assayed at the concentrations of Galpha z-[gamma -32P]GTP shown on the abscissa either in the presence (triangle ) or absence (black-triangle) of 1.2 µM Gbeta 1gamma 2. B, GAP activity was assayed at 2.0 nM Galpha z-[gamma -32P]GTP in the presence of the concentrations of purified Gbeta 1gamma 2 shown on the abscissa (bullet ). Hydrolysis of Galpha z-[gamma -32P]GTP was also measured without GAP at the same concentrations of Gbeta gamma (open circle ).
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Other GTPase Assays

Steady-state GTPase assays were performed as described (31) at 30 °C in buffer that contained 20 mM NaHepes (pH 8.0), 25 mM NaCl, 0.1 mM EDTA, 1.1 mM MgCl2, 20 µg/ml bovine albumin, 50 nM GDP, 100 nM [gamma -32P]GTP, and either 100 µM carbachol or 5 µM atropine. The rate of hydrolysis of [gamma -32P]GTP bound to Galpha o and Galpha i1 was measured at 15 °C essentially as described previously (26), except that the buffer for the initial [gamma -32P]GTP binding reaction contained 5 mM EDTA, and hydrolysis was initiated by adding 6.0 mM MgCl2 plus 0.1 mM unlabeled GTP. The concentration of Galpha -bound [gamma -32P]GTP at initiation of the hydrolysis reaction is given as the total amount of [32P]orthophosphate released during the reaction.

Purification of Gz GAP

Gz GAP was purified from bovine cerebral cortical membranes prepared according to Sternweis and Pang (32). All procedures were performed at 0-4 °C except where explicitly noted. In practice, we pool active fractions from multiple runs of the Q-Sepharose column before gel filtration and pool several gel filtration peaks for Galpha z affinity chromatography. Combining preparations in this way improves both the yield and purification. However, Table II is an example of an early preparation that was completed exactly as described below.

Table II.

Purification of Galpha z GAP from bovine brain

Activities and amounts of protein refer to pooled fractions carried to the next step of the purification.
Total protein Specific activity Purification Yield

mg unit/mg -fold %
Triton X-100 extract 13,500 35 (1.0) (100)
DEAE-Sephacel 7,500 50 1.5 80
Q-Sepharose 1,000 130 3.7 27
Ultrogel AcA-34 60 1,000 29 12.5
Galpha z-agarose 0.6 35,000 1,000 4.4
Phenyl-Sepharose 0.045 280,000 8,000 2.7

Membranes were washed once in 50 mM Tris-Cl (pH 7.5), 1 mM DTT, 1 mM EDTA, 0.5 M NaCl, and 0.1 mM PMSF and resuspended to 2.5 mg/ml in 20 mM NaHepes (pH 7.5), 0.1 mM EDTA, 0.3 mM PMSF, and 2% Triton X-100. The suspension was sloshed for 25 min at 30 °C and centrifuged for 40 min at 150,000 × g. The supernatant was loaded on a DEAE-Sephacel column (700 ml) that was equilibrated with Buffer 1 (20 mM NaHepes (pH 7.5), 0.1 mM EDTA, 1% Triton X-100, 0.1 mM PMSF). The column was washed with Buffer 1 and eluted with a gradient of 0-300 mM NaCl in Buffer 1. Both Gz GAP activity and protein were eluted in parallel as a broad, asymmetric peak. Active fractions were pooled and diluted 3-fold with Buffer 2 (20 mM NaHepes (pH 7.5), 0.1 mM EDTA, 1 mM DTT, 1% cholate, 0.1 mM PMSF). DTT was added to a final concentration of 20 mM, and the solution was applied to a column of Q-Sepharose that had been equilibrated with Buffer 2. The column was washed sequentially with Buffer 2, Buffer 2 plus 0.1 M NaCl, and Buffer 2 plus 0.25 M NaCl, and then eluted with a gradient of 0.25-0.55 M NaCl in Buffer 2. Protein and GAP activity again eluted as a broad peak. Active fractions were concentrated on an Amicon PM30 membrane and chromatographed on a column of Ultrogel AcA-34 equilibrated with Buffer 2 plus 0.1 M NaCl. A typical elution profile is shown in Fig. 2. The second peak of GAP activity was pooled and concentrated by adsorption to Mono Q and elution with a gradient of 0.1-0.55 M NaCl in Buffer 2. 


Fig. 2. Ultrogel AcA-34 chromatography of Gz GAP. Pooled fractions from Q-Sepharose were concentrated by ultrafiltration and chromatographed on Ultrogel AcA-34 as described under "Experimental Procedures." The first peak is at the void volume.
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For affinity chromatography, pooled Mono Q fractions were diluted 20-fold with Buffer 3 (25 mM NaHepes (pH 7.5), 1 mM EDTA, 1 mM DTT, 0.1% Triton X-100, 0.25% cholate, 0.1 mM PMSF, 10 µM GDP, 30 µM AlCl3, 5 mM MgCl2, 10 mM NaF) and applied to a column of Galpha z-agarose that was equilibrated with Buffer 3. The column was washed with Buffer 3 plus 25 mM NaCl, and GAP activity was eluted with a gradient of 25-500 mM NaCl in Buffer 4 (Buffer 3 but containing 0.5% cholate and without AlCl3, MgCl2, or NaF). Peak fractions were pooled, concentrated by adsorption and elution from Q Sepharose as described above, and diluted 8-fold with Buffer 5 (20 mM NaHepes (pH 7.5), 1 mM DTT, 0.1 mM EDTA, 1 M NaCl, 0.1 mM PMSF). The pool was applied to a column of phenyl-Sepharose equilibrated with Buffer 5 plus 0.2% cholate and washed with 5 volumes of the same buffer. GAP activity was eluted with a discontinuous gradient of 0.2-1.0% cholate (0.2% steps) in Buffer 5 but without NaCl (see Fig. 3).


Fig. 3. Phenyl-Sepharose chromatography of Gz GAP. Pooled affinity chromatography fractions were concentrated by adsorption to Q-Sepharose and chromatographed on phenyl-Sepharose essentially as described under "Experimental Procedures." A, elution profile. Fraction 1 begins after the high salt wash. Flow-through (FT) and wash fractions accounted for 19% of the activity and 46% of the protein originally applied. About 40% of applied protein was not eluted from the column, but all GAP activity was accounted for in the eluted fractions. This profile is from a micro-scale fractionation performed during development of the method, and the profile of the step gradient was modified subsequently. B, samples of each fraction (8.3% of total), of the load and flow-through (0.37%), and of the wash (0.83%) were analyzed by SDS-gel electrophoresis followed by silver staining. Positions of molecular weight markers are shown at the left.
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Miscellaneous Methods

Gz GAP was co-reconstituted with Galpha zbeta gamma and m2 muscarinic cholinergic receptor into unilamellar phospholipid vesicles (phosphatidylserine:phosphatidylethanolamine:cholesteryl hemisuccinate, 5:8:1) according to the method of Parker et al. (23). Trimeric Gz was prepared by mixing GDP-bound Galpha z with Gbeta 1gamma 2 (alpha :beta gamma  = 0.4) before reconstitution (26). Galpha z was routinely quantitated according to the binding of 10 µM [35S]GTPgamma S for 1.5 h at 30  °C (31). Binding of [gamma -32P] and [alpha -32P]GTP to Gz was also measured by the nitrocellulose binding assay (31). Protein was measured by amido black binding (33).

Standard procedures were used for SDS-gel electrophoresis (34) and staining with Coomassie Blue or silver (35). For samples in which GAP activity was to be measured after electrophoresis, samples were denatured in sample buffer (34) that contained 1% SDS and 10 mM DTT. Gz GAP activity was extracted from slices of SDS-polyacrylamide gels and renatured by homogenizing the gel in 5-10 volumes of renaturation buffer (20 mM NaHepes (pH 7.5), 1 mM DTT, 0.1 mM EDTA, 1% Triton X-100) and shaking overnight at 0 °C.

The concentration of free Mg2+ in assay buffers that contained significant concentrations of both EDTA and GTP was calibrated as described by Huskens and Sherry (36).


RESULTS

Identification of Gz-GAP Activity

We used the Galpha z-[gamma -32P]GTP complex as substrate to test for the presence of proteins that can increase the rate of hydrolysis of Galpha z-bound GTP. Purified Galpha z hydrolyzes bound GTP very slowly (16), such that Galpha z-[gamma -32P]GTP can be prepared and purified with good yield. About 25% of total Galpha z is bound to [gamma -32P]GTP after gel filtration, with the rest bound to unlabeled GDP. Under standard assay conditions at 15 °C, GTP bound to Galpha z is hydrolyzed with a rate constant, khydrol, of about 0.014 min-1, which corresponds to a t1/2 of about 50 min (Fig. 1).

Addition of a crude membrane fraction from bovine brain increased the rate of hydrolysis of Galpha z-bound GTP dramatically (Fig. 1A). Hydrolysis was a single component, first-order reaction over a 10-fold range of rates, and rate constants increased linearly with increasing amounts of membrane protein (Fig. 1B). These data indicate the existence of a GTPase-accelerating activity in bovine brain, i.e. a Gz GAP. Based on the linearity of GTP hydrolysis with added membrane protein, we defined a unit of Gz GAP activity as an increment in the hydrolytic rate constant of 1.0 min-1. Both the basal rate of GTP hydrolysis by Galpha z and the GAP activities of several tissues were quite reproducible in this assay. The hydrolysis-accelerating activity is evidently that of a protein. In membranes, activity was destroyed by incubation with 6.7 µg/ml trypsin, 20 µg/ml chymotrypsin plus detergent or 0.5 mM N-ethylmaleimide. Added detergent markedly sensitized the GAP to proteolysis.

Release of [32P]orthophosphate in GAP assays such as shown in Fig. 1 reflects only hydrolysis of [gamma -32P]GTP bound to Galpha z rather than dissociation followed by hydrolysis catalyzed by other nucleoside triphosphatases in the membranes. Dissociation of GTP from Galpha z was unmeasurably slow, as estimated either by comparing the loss of bound [gamma -32P]GTP with the appearance of [32P]orthophosphate or by monitoring the dissociation of [alpha -32P]GTP. The rate of formation of [32P]orthophosphate was equal both to the rate of loss of Galpha z-bound [gamma -32P]GTP and to the rate of conversion of bound [alpha -32P]GTP to bound [alpha -32P]GDP (not shown), either with or without added membrane protein. Furthermore, the reaction is carried out in the presence of 5 mM free GTP to block any other nucleoside triphosphatase activity. In control experiments, no hydrolysis was observed when free [gamma -32P]GTP was substituted for Galpha z-[gamma -32P]GTP (not shown).

Homogenates of several mammalian tissues and cultured cells were screened for Gz GAP activity (Table I), and its distribution was found to be similar to that of Galpha z itself (16-19; confirmed qualitatively by anti-alpha z immunoblot durning this study). Activity was highest in cerebral cortex, although there was considerable activity in membranes of spleen and retina. Peripheral fat, lung, platelets, and testis also displayed readily measurable activity. Several other tissues displayed low activity, which may represent contamination by adipose, neuronal, or vascular tissue or the action of other GAPs with low activity toward Gz. We do not know how many proteins in these tissues display Gz GAP activity. Among the cultured cells tested, C6 glioma cells displayed activity similar to that of brain, 20-50 units/mg depending upon the source of the cells and the culture conditions. Several other cell lines displayed more modest activities. Gz GAP activity was low in S49 murine lymphoma cells and was barely detected in Sf9 cells. Because activity was highest in brain and bovine brain is readily obtainable, we concentrated on this source and have not attempted to determine the multiplicity of Gz GAPs in other tissues.

Table I.

Distribution of Gz GAP activity

Gz GAP activity was measured in total homogenates of various rat tissues, bovine retina, or cultured cells using 0.4-120 µg of homogenate protein as needed. Data are representative of assays on at least two animals or plates. Cerebral cortex membranes from rat, rabbit, and steer have similar activities.
GAP activity

units/mg protein
Cerebral cortex 38
Spleen 12
Retina 10
Fat 2.1
Lung 1.8
Platelet 1
Kidney, heart, liver, testis, pancreas, erythrocyte 0.1-0.5
Skeletal muscle 0.04
C6 glioma 22-47
CHO 11
Sf9, S49, Ins-1 0.2-0.65
Other cell lines
  293, 8PP15, 7RP16, L, 1321N1, Cos, MDCK,   NG108, MA104 2.5-6.5

Purification of Gz GAP

There is no soluble Gz GAP activity detectable in brain homogenates (<2%). Gz GAP behaves as an integral membrane protein and is difficult to solubilize. It is not extracted by washing at high or low ionic strength and is not solubilized by many detergents. At 0 °C, neither cholate, deoxycholate, Lubrol PX, Triton X-100, CHAPS, digitonin, nor several other detergents solubilized any Gz GAP activity. Small and irreproducible amounts of activity were solubilized by dodecyl maltoside and lauroyl sucrose. Fortunately, incubation of membranes with 2% Triton X-100 at 30 °C released considerable Gz GAP activity into a 200,000 × g supernatant. This apparently soluble GAP was still badly aggregated, however, and substantial increases in specific activity were not obtained by chromatography in multiple systems. Two sequential rounds of anion exchange chromatography, which included transfer from Triton X-100 to cholate, provided little purification and substantial loss of activity (Table II) but did allow subsequent purification. Although about half of the Gz GAP activity remained aggregated, the other half behaved as an apparently monodisperse species of reasonable molecular size and was thus purified about 20-fold by gel filtration in cholate (Fig. 2). This soluble material was used for further purification.

After gel filtration, Gz GAP activity was appreciably purified by affinity chromatography on Galpha z-agarose. GAP activity bound to the column when the covalently coupled Galpha z was activated with either GTPgamma S or with GDP/AlF4 but not when the Galpha z was in the nonactivated, GDP-bound form (not shown). For purification, peak fractions from gel filtration were applied to Al3+/F4--activated Galpha z-agarose, and after extensive washing, GAP activity was eluted by removal of Al3+, F-, and Mg2+ and increasing the concentration of detergent and salt.

Further purification of Gz GAP was achieved by phenyl-Sepharose chromatography (Fig. 3). Gz GAP activity was consistently eluted from phenyl-Sepharose in two peaks. This was true for multiple elution protocols that included increasing the concentration of detergent and/or decreasing the concentration of salt, although it was possible to alter the peak shapes and the total and specific activities in each peak. Typically, both peaks were purified about 5000-12,000-fold relative to the Triton extract (Table II shows data for pools). Neither peak contained homogeneous protein, as shown by SDS-gel electrophoresis (Fig. 3B). The peak fractions contained a large but reproducible group of proteins, the only identifiable one of which was the G protein beta  subunit. Gbeta gamma has no GAP activity (see below), and GAP activity was not co-immunoprecipitated by anti-Gbeta antibodies (not shown). Its appearance is apparently an artifact of Galpha z affinity chromatography. No single polypeptide obviously co-fractionated exactly with Gz GAP activity. The phenyl-Sepharose pools contained no GTPgamma S binding activity (<0.015 mol/mol GAP).

We attempted to identify the polypeptide(s) that accounts for Gz GAP activity by further fractionating phenyl-Sepharose peak fractions on SDS-polyacrylamide gels and then renaturing the GAP protein eluted from individual gel slices (see "Experimental Procedures"). About 50-75% of Gz GAP activity was recovered from the gel. As shown in Fig. 4, activity was broadly distributed on SDS gels between 20 and 30 kDa, with two peaks of activity reproducibly appearing at about 22 and 28 kDa. Smearing during electrophoresis is an unlikely cause of this behavior because both activity and discrete protein bands eluted from individual gel slices retain their distinct electrophoretic mobilities upon extraction and a second round of gel electrophoresis (Fig. 4B). The 22-kDa peak of activity corresponds to a relatively blurry band, but we have not assigned the 28-kDa activity to a specific stained band. In addition to the two peaks of activity, Gz GAP activity is readily detected throughout the region between 22 and 28 kDa, indicating considerable heterogeneity. It is unclear whether the 22-kDa band and intermediate forms are proteolytic products of the 28-kDa form or whether they are unique Gz GAP species. SDS gel analysis of GAP activity in membranes and earlier fractions during the purification have not clarified this question, although they indicate the presence of GAP activity with molecular size up to 40 kDa. Thus, the data of Fig. 4 indicate that Gz GAP activity results from monomeric proteins in the size range 22-28 kDa, but the number of species remains uncertain.


Fig. 4. SDS-polyacrylamide gel electrophoresis of partially purified Gz GAP. A sample of the second peak from phenyl-Sepharose chromatography (fraction 6 in Fig. 3) was diluted into SDS sample buffer and electrophoresed on a 15% polyacrylamide gel. The gel lane was sliced, and protein was eluted from each slice as described under "Experimental Procedures." A, Gz GAP activity was assayed in the eluate from each slice. B, an aliquot of each eluate (and of phenyl-Sepharose fraction 6, denoted c) was denatured in SDS and electrophoresed again. The gel was silver-stained. The molecular mass markers were carbonic anhydrase (29 kDa) and beta -lactoglobulin (18.4 kDa).
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In addition to indicating which molecular weight species contribute to Gz GAP activity, SDS-gel electrophoresis and renaturation provide substantial purification of GAP activity. The specific activity of Gz GAP extracted from SDS gels is increased more than 50,000-fold relative to the Triton extract. It is likely that these fractions are essentially pure. Such extensive purification indicates that Gz GAP is of low abundance even in brain.

Mechanism of Action of Gz GAP

A single GAP molecule can turn over multiple molecules of Galpha z-GTP (Fig. 5). Its behavior is most readily analyzed when it is considered as an enzyme that acts upon the substrate Galpha z-GTP and converts it to the products Galpha z-GDP plus orthophosphate. Its Km for Galpha z-GTP is about 2 nM, which represents sufficiently high affinity binding to be physiologically reasonable. The maximum GAP-stimulated hydrolysis rate can be estimated two ways. Because velocity increases linearly with the amount of GAP at low GAP concentrations (Figs. 1 and 5B), the maximum GAP-stimulated hydrolytic rate constant (kgap2) can be calculated by dividing Vmax by the molar concentration of GAP, which is calculated according to its estimated purity and approximate molecular weight. This calculation yields a kgap of 3 min-1 at 15 °C. The other estimate of kgap derives from a titration of GAP when the concentration of Galpha z-GTP is maintained at or above the Km (Fig. 5B). The maximum in such an experiment, 1.8 min-1, can be corrected for the subsaturating concentration of Galpha z-GTP to yield a true maximum kgap of 3.1 min-1. Thus, both determinations of kgap are about 3 min-1, more than a 200-fold stimulation over khydrol for Galpha z-GTP. We estimate that kgap at 30 °C is over 20 min-1, which corresponds to an average lifetime for activated Gz of <2 s. Thus, because kgap is fast and because Gz GAP has a high affinity for its Galpha z-GTP substrate (Km ~2 nM), its action is sufficient to allow Gz-mediated signal transduction with physiologically appropriate rates.


Fig. 5. GAP activity at increasing concentrations of Gz GAP and of Galpha z-[gamma -32P]GTP. A, GAP activity was assayed at the concentrations of Galpha z-[gamma -32P]GTP shown on the abscissa, as determined in a [gamma -32P]GTP binding assay. The concentration of GAP was 30 pM (7.5 ng/ml, ~10% pure; Mr~25,000; purified through affinity chromatography). Inset, Hanes replot. B, GAP activity was assayed at the GAP concentrations shown on the abscissa using 2.5 nM Galpha z-[gamma -32P]GTP as substrate. Assay times in both experiments were adjusted to obtain accurate measurements of either the initial reaction rate or of the first-order rate constant kapp.
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Galpha z can hydrolyze bound GTP at very low concentrations of free Mg2+, and its intrinsic khydrol is independent of the concentration of Mg2+ up to 10 mM (Fig. 6). In contrast, Gz GAP activity displays a marked Mg2+ optimum at ~1 mM. Although the GAP is active over a wide range of Mg2+ concentrations, stimulation is ~4-fold higher at the optimum. Neither Ca2+ nor Mn2+ exerted a unique regulatory effect on Gz GAP, although either can replace Mg2+ over approximately the same range of concentrations (not shown).

Gz GAP binds tightly to Galpha z, but only in its GTP-activated form. Both Galpha z-GTPgamma S and Galpha z-GDP/AlF4 thus inhibited Gz GAP activity, with a Ki of ~1.5 nM for either nucleotide (Fig. 7). This value of Ki is similar to the Km for the substrate Galpha z-GTP, which suggests that Galpha z-GTP, Galpha z-GTPgamma S, and Galpha z-GDP/AlF4 all bind the GAP at a common site and with similar affinities. Galpha z-GDP did not inhibit at concentrations up to 20 nM. Note that in the experiment of Fig. 7, Galpha z-GDP/AlF4 was formed in the reaction mixture by the addition of Al3+ plus F-, which appear to inhibit GAP activity by themselves. This inhibition results from the presence of Galpha z-GDP in the preparation of Galpha z-GTP substrate, such that about 6 nM Galpha z-GDP/AlF4 is present in the assay even when no excess Galpha z-GDP was added. Selectivity of the GAP for the active conformation of Galpha z is confirmed by the selective binding of the GAP to Galpha z-agarose when it is activated by either GTPgamma S or GDP/AlF4.


Fig. 7. Inhibition of Gz GAP activity by Galpha z or Galpha o bound to different guanine nucleotides. GAP activity was measured using 1.35 nM Galpha z-[gamma -32P]GTP as substrate in the presence of increasing concentrations of Galpha z-GDP (open circle ), Galpha z-GTPgamma S (black-triangle), or bovine brain Galpha o-GTPgamma S (black-square), or in the presence of 30 µM AlCl3, 10 mM NaF, and increasing amounts of Galpha z-GDP (bullet ). Assays were carried out for 10 min. The concentrations of Galpha z-GTPgamma S and Galpha o-GTPgamma S were measured by direct binding assays using tracer amounts of [35S]GTPgamma S. The concentration of Galpha z-GDP was measured in a parallel [35S]GTPgamma S binding assay. Apparent inhibition by Al3+/F- was caused by its binding to Galpha z-GDP added with the substrate.
[View Larger Version of this Image (26K GIF file)]


The following small molecules had no effect upon Gz GAP activity: inositol trisphosphate, cyclic AMP, cyclic GMP, GTP, GTPgamma S, and ATP (not shown).

Selectivity of Gz GAP

The selectivity of brain Gz GAP among different Galpha subunits was tested initially by comparing their abilities to compete with Galpha z-GTP in the standard assay (Fig. 7, Table III). Both myristoylated, recombinant Galpha i1 and bovine brain Galpha o inhibited competitively, Galpha o-GTPgamma S with a Ki of ~5 nM (Fig. 7) and Galpha i1-GTPgamma S with a Ki of about 20 nM. Their GDP-bound forms did not inhibit (not shown). Inhibition by Galpha o or Galpha i required that they be myristoylated; nonmyristoylated Galpha subunits inhibited weakly or not at all. Based on these data, we measured the ability of the Gz GAP to accelerate hydrolysis of Galpha o-GTP and Galpha i1-GTP, using the single turnover assay of Higashijima et al. (26) to accommodate the faster basal hydrolytic rates of these Galpha subunits. Although both Galpha o and Galpha i1 hydrolyze bound GTP much faster than does Galpha z, the relative effect of Gz GAP on both Galpha subunits was minimal when compared with Galpha z (Table IV): 30% and 7% compared with more than a 30-fold effect on Galpha z. The GTPgamma S-bound forms of Galpha s, Galpha q, and Galpha 12 did not compete significantly in the Gz GAP assay (Table III), and their activities as GAP substrates were not tested.

Table III.

Inhibition of Gz GAP by GTPgamma S-bound Galpha subunits

Assays contained 1.35 nM Galpha z-[gamma -32P]GTP and 15.6 nM competing alpha  subunit purified from the sources shown. Concentrations of competing Galpha subunits were measured according to bound [35S]GTPgamma S. E. coli, myr, co-expressed in E. coli with yeast N-myristoyltransferase (25). 100% activity was 60 milliunits. GDP-bound forms of these Galpha subunits did not inhibit (not shown).
Galpha GAP activity

%
None (100)
Galpha z (Sf9) 15
Galpha o (brain) 30
Galpha o (Escherichia coli) 85
Galpha i1 (E. coli, myr) 59
Galpha i1 (E. coli) 88
Galpha q (Sf9) 93
Galpha s (E. coli) 99
Galpha 12 (Sf9) 100

Table IV.

Activity of Gz GAP toward Galpha o and Galpha i1

Hydrolytic rate constants were determined at 15 °C as described under "Experimental Procedures," either in the presence or absence of 0.5 nM purified bovine brain Gz GAP. Concentrations of substrate were 2.7 nM Galpha z-GTP, 5.5 nM Galpha o-GTP, and 35 nM Galpha i1-GTP (about the Km for Galpha z and the Ki for Galpha o and Galpha i1). Data are the average of duplicate determinations in two separate assays. The results for Galpha o were confirmed qualitatively at 20 °C. Galpha o was purified from bovine brain, and Galpha i1 was the recombinant myristoylated form purified from E. coli (25).
Substrate khydrol (min-1)
Fold stimulation
 -GAP +GAP

Galpha z-GTP 0.0135 0.455 33.7
Galpha o-GTP 0.488 0.635 1.3
Galpha i1-GTP 1.10 1.18 1.07

Regulation of Gz GAP by G Protein beta gamma Subunits

Although G protein beta gamma subunits had little if any effect on the rate of hydrolysis of Galpha z-bound GTP, Gbeta gamma inhibited Gz GAP activity up to 80% (Fig. 8). Inhibition was most marked at low concentrations of Galpha z and is caused by an increase in Km of at least 5-fold (Fig. 8A). No effect of Gbeta gamma on Vmax was detected, but we were unable to achieve saturation with Galpha z-GTP at high Gbeta gamma concentrations, and we may have failed to observe a slight decrease in Vmax. Gz GAP was inhibited approximately equally by Gbeta 1gamma 2, Gbeta 2gamma 2, and Gbeta 2gamma 3 (not shown). The increase in Km caused by Gbeta gamma apparently reflects formation of the GTP-bound Galpha zbeta gamma heterotrimer. The IC50 for Gbeta 1gamma 2 (~400 nM, Fig. 8B) agrees well with its affinity for GTP-bound Galpha z (37), and we have found no evidence for Gbeta gamma binding directly to the GAP (which would yield classical competitive inhibition). Galpha zbeta gamma -GTP may be a low affinity (high Km) GAP substrate or it may simply block GAP binding to Galpha z-GTP. These alternatives are potentially distinguishable according to the dependence of the apparent Km on the concentration of Gbeta gamma , but we have been unable to determine Km accurately over a high enough range of Gbeta gamma concentrations to answer this question.

In addition to inhibiting the GAP, Gbeta gamma decreased the rate of dissociation of GDP, but not GTP, from Galpha z, as is true for other Galpha subunits (38). We observed no other effects of Gbeta gamma in this system.

Gz GAP Activity During Receptor-stimulated Steady-state GTP Hydrolysis

To study the effect of Gz GAP on the receptor-stimulated steady-state GTPase cycle, we co-reconstituted Gz GAP with m2 muscarinic cholinergic receptor and heterotrimeric Gz into unilamellar phospholipid vesicles. When the muscarinic agonist carbachol was added to promote receptor-catalyzed exchange (23), Gz GAP increased the steady-state GTPase rate by about 2.5-fold (Fig. 9A). This effect seems small in comparison to the 200-fold maximum effect of the GAP on hydrolysis of preformed Galpha z-GTP, but the steady-state concentration of Galpha z-GTP in the vesicles is in significant molar excess over that of the GAP. The effect of the GAP on the steady-state GTPase rate is consistent with its observed activity in the single turnover assay. The effect of GAP on steady-state GTPase rates is evidently exerted only at the hydrolytic step. In the absence of agonist, where steady-state GTPase activity is limited by the GDP/GTP exchange rate, GAP had no effect, and GAP also had no effect on the rates of nucleotide binding (Fig. 9B) or release (not shown).


Fig. 9. Gz GAP amplifies agonist-stimulated steady-state GTPase activity in phospholipid vesicles that contain trimeric Gz and m2 muscarinic cholinergic receptor. M2 muscarinic receptor and trimeric Gz were co-reconstituted into phospholipid vesicles, with (open circle , bullet ) or without (triangle , black-triangle) Gz GAP, essentially as described previously (23) and under "Experimental Procedures". A, steady-state GTPase activity was measured in the presence of either 100 µM carbachol (bullet , black-triangle]) or 5 µM atropine (open circle , triangle ). In parallel with the measurement of GTP hydrolysis, the steady-state concentration of Gz-[gamma -32P]GTP in the assays with carbachol was assayed both at 5 and 15 min. The concentration of Gz-[gamma -32P]GTP at both times was 460 pM in vesicles with GAP and 640 pM in vesicles without GAP. The concentration of GAP in the assay, when present, was about 30 pM. B, GTPgamma S binding was measured in the same vesicles.
[View Larger Version of this Image (18K GIF file)]


Consistent with its effect on hydrolysis of bound GTP, Gz GAP decreased the steady-state concentration of the active Gz-GTP complex during stimulation by agonist. When assayed at either 5 or 15 min in the system shown in Fig. 9A, the addition of Gz GAP decreased the concentration of Gz-GTP by about 30%. Both of these sample times are after steady-state was reached, as indicated by the constant concentration of bound GTP in this interval and by the completion of agonist-stimulated GTPgamma S binding in about 2 min (Fig. 9B). Simple kinetic models predict that the relative effect of GAP on the accumulation of Gz-GTP would be greater in the absence of agonist, but we were unable to measure accurately the small amount of binding of [gamma -32P]GTP binding that occurred without agonist.

The results of the experiments shown in Fig. 9 indicate that purified Gz GAP can reassociate with membrane lipids and regulate Gz appropriately in (or on the surface of) a phospholipid bilayer. The addition of detergent-soluble Gz GAP to preformed vesicles had no effect on the steady-state GTPase rate (not shown), which is consistent with the idea that the GAP is an integral membrane protein.


DISCUSSION

Gz hydrolyzes bound GTP (deactivates) extremely slowly. Although it is activated at a normal rate in response to receptors (23), its active state decays with an average lifetime of about 7 min at physiological temperature (16). With these kinetics, it would be hard to understand how Gz can mediate signaling responses in a reasonable way, although it clearly does.

The data presented here describe the identification of a GAP for Gz in brain membranes, its purification, and mechanistic behavior. Similar activity was identified in membranes of several other tissues and cultured cells. By accelerating GTP hydrolysis, a Gz GAP reconciles aberrant deactivation kinetics with normal signaling functions. However, its precise role in signaling physiology remains unclear. Gz GAP may be a Gz-regulated effector protein, in analogy with phospholipase C-beta and cyclic GMP phosphodiesterase. These effectors are both regulated by G proteins and have GAP activity specific for their G protein regulators, Gq and Gt (5, 7, 8). The low Km of the Gz GAP, about 2 nM, is in the same range of affinities as that displayed by Galpha q, Galpha s, or Galpha t for their effectors (5, 6, 39, 40), and its selectivity for the activated form of Galpha z is also consistent with this role. Alternatively, the GAP may be a negative regulatory component of the Gz pathway, involved either in desensitization or in mediating negative input from another signaling pathway. The model for such regulation could be either the GAPs for p21RAS and related small, monomeric G proteins (2) or the RGS proteins, a large family of related proteins that inhibit signaling (4, 9, 10) and whose prototypes are GAPs (12). Whether the cerebral Gz GAP is an effector or a modulator of inhibition will probably be elucidated when its cDNA can be used to manipulate its expression in cells.

Purification

Gz GAP was initially purified about 12,000-fold according to the specific activity of phenyl-Sepharose fractions, and the specific activity of the purest fractions from SDS gels is about 4-fold higher (Fig. 4). Gz GAP is thus a rare protein in brain, its richest source, and is perhaps expressed in only a few cell types. By rough comparison with published data (16, 41) and with immunoblots performed during this study (not shown), Gz GAP is about 5-10-fold less abundant than is Galpha z. This is not surprising, however, whether the GAP is an effector or purely a negative regulator. G proteins are generally in molar excess over their effector proteins. Alternatively, because Gz GAP acts catalytically, it could readily function as an efficient inhibitor of Gz signaling.

Despite extensive purification, preparations of Gz GAP remain heterogeneous. GAP activity in peak fractions from phenyl-Sepharose is distributed bimodally between 22 and 28 kDa. We do not know if the 22-kDa GAP is distinct from the 28-kDa GAP or if it is a proteolytic product, although we have been unable to proteolyze the larger form to the smaller form. There is also obvious GAP activity and protein between the two major peaks. Because the ratio of GAP activity to silver-stained protein is low between the peaks, we suspect that major silver-stained bands in this region are contaminants and that the activity represents distribution of active proteolytic fragments of the 28-kDa GAP. We approached the question of proteolysis during purification by analyzing unfractionated brain membranes by SDS-gel electrophoresis. The principal peak of activity was at about 28 kDa, with tailing to about 20 kDa, but we could also detect small peaks of activity higher in the gel. These larger forms are not observed in purified preparation; peptides of Mr > 30,000 in the phenyl-Sepharose fractions have no GAP activity.

Mechanism, Selectivity, and Regulation of Gz GAP

Purified cerebral Gz GAP is highly specific in its action on Galpha z. It displayed only slight activity with either Galpha i1-GTP or Galpha o-GTP as substrates under conditions where hydrolysis of Galpha z-GTP was accelerated over 30-fold (Table IV). According to competitive inhibition, however, the affinity of the GAP for Galpha z is only about 3-fold greater than that for Galpha o and about 10-fold higher than for Galpha i1 (Table III). Evidently, Gz GAP can bind these other G proteins with high affinity but cannot efficiently promote their deactivation. This unusual pattern of selectivity suggests that other members of the Gi family may inhibit Gz GAP in cells, where they are much more abundant than is Gz. Given the selectivity of the purified GAP for Galpha z, it was initially surprising that there is significant activity in cells and tissues that express little if any Galpha z (Table I). It is likely that this activity is that of GAPs for other Gi family members but which act on Gz with low efficiency (47, 48).

Cerebral Gz GAP behaves generally as an integral membrane protein, although it was unusually refractory to solubilization by nondenaturing detergents. This behavior is reminiscent of caveolar proteins (42-44). However, caveolae are reported to be solubilized by octyl glucoside (Gz GAP was not) and, in one experiment, Gz GAP activity did not co-fractionate with caveolin in lysates of MA104 cells. We suspect that Gz GAP is a markedly hydrophobic protein because of its resistance to solubilization and its tendency to aggregate. This conclusion is supported by its functional co-reconstitution with m2 muscarinic receptors and trimeric Gz into phospholipid vesicles (Fig. 9), in contrast to its inactivity when added to preformed receptor-Gz vesicles. We have been unable to perform the standard tests for monomeric solubility of purified Gz GAP because removing detergent by dilution or chromatography before assay led to loss of GAP activity. This was true even though the assay was performed in the presence of Triton X-100. Some of this behavior is similar to difficulties encountered in solubilizing adenylyl cyclase, a G protein-regulated effector that is a much larger, multi-span membrane protein. There is inadequate information to compare this aspect of Gz GAP with RGS proteins, although RGS4 and GAIP are both water-soluble (11, 12) and Sst2p binding to membranes is sensitive to ionic strength (15).

The enzymologic mechanism of Gz GAP action is apparently straightforward.2 Galpha z-GTP is essentially stable over the usual assay interval. The GAP binds the GTP-bound form of Galpha z with nanomolar affinity (Figs. 5 and 7), and the GAP-Galpha z-GTP complex then hydrolyzes GTP fairly quickly (t1/2 ~15 s at 15 °C; t1/2 <2 s at 30 °C). The GAP binds Galpha z-GDP weakly if at all, such that the complex rapidly dissociates after hydrolysis. This mechanism allows Gz GAP to act catalytically; i.e. one GAP molecule can cycle among multiple molecules of Galpha z-GTP. A corollary to this behavior is that the rate-limiting step in the GAP-mediated GTPase reaction is hydrolysis of the GAP-Galpha z-GTP complex. This conclusion is supported by the finding that the maximum reaction rate at saturating and super-stoichiometric concentrations of GAP (Fig. 5B) is the same as the maximum specific activity of the GAP at saturating Galpha z-GTP (Fig. 5A). In apparent contrast, the effect of RGS4 on Go and Gi seems to be limited by substrate binding (12).

The activity of Gz GAP during receptor-stimulated steady-state GTP hydrolysis is evident when it is co-reconstituted in phospholipid vesicles with Gz and m2 muscarinic receptor (Fig. 9). The relative effect of the GAP activity was limited by its concentration and/or by the ratio of its concentration to that of Gz. Gz was in molar excess over GAP in the vesicles, the probable physiologic condition, and stimulation of steady-state hydrolysis increased if either more GAP or less Gz was used. We have not yet pursued this relationship quantitatively because the availability of GAP and its concentration in stock solutions were both limiting. The need for Gbeta gamma in the vesicles to permit receptor-Galpha z coupling probably diminished the effect of the GAP (Fig. 8), and we also suspect that there was less than one molecule of GAP per vesicle. It is important to note that GAP does not alter the rates of dissociation of either GDP or GTP from Galpha z. It will not, therefore, influence activation rates and will only accelerate the deactivation limb of the GTPase cycle.

Gz GAP binds the activated form of Galpha z with about the same affinity when it is bound to GTP (according to Km), to GTPgamma S, or to GDP/AlF4 (according to Ki). In contrast, RGS4, a GAP for the Gi family and Gq, is inhibited much more potently by an GDP/AlF4-bound Galpha than by the same Galpha bound to GTPgamma S (47, 48). Because GDP/AlF4 binds to Galpha i1 and Galpha t as a transition state analog (45, 46), these authors suggested that RGS4 acts as a GAP by favoring the transition state structure of a Galpha over its GTP-bound form. This mechanism would presumably differentiate the GAP activity of RGS proteins from that of effectors, which are activated both by GTPgamma S- and GDP/AlF4-liganded G proteins, and would thus suggest that the cerebral Gz GAP is an effector. This distinction may not be generally valid, however, or may perhaps not extend to Gz. In preliminary experiments, we found that RGS4 is potently inhibited by the GTPgamma S-bound form of Galpha z, a behavior similar to that of the cerebral Gz GAP. The active site and enzymatic properties of Gz differ markedly from those of other Gi family members (16-18), and it is possible that GDP/AlF4 is not a transition state analog at the active site of Galpha z. If true, however, this argument would favor an effector function for the Gz GAP.

Regardless of any yet unknown cellular roles of the Gz GAP, its presence and regulation will influence Gz-modulated signaling. The first mode of regulation so far observed is inhibition of the GAP by Gbeta gamma subunits. Inhibition of GAP activity by Gbeta gamma over a reasonable range of concentrations allows modulation of Gz signaling by other G protein pathways, where activation will release Gbeta gamma in large excess over Galpha z. Other controls of GAP activity are also likely, and their understanding should help us understand the cellular pathways uniquely regulated by Gz.


FOOTNOTES

*   This work was supported by U. S. Public Health Service Grant R37GM30355, R. A. Welch Foundation Grant I-0982, and a postdoctoral fellowship (to J. W.) from Cadus Pharmaceutical Corp. The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Dagger    To whom correspondence should be addressed: Dept. of Pharmacology, University of Texas Southwestern Medical Center, Dallas, TX 75235-9041. E-mail: ross{at}utsw.swmed.edu.
1    The abbreviations used are: GAP, GTPase-activating protein; PMSF, phenylmethylsulfonyl fluoride; DTT, dithiothreitol; GTPgamma S, guanosine 5'-O-thiotriphosphate; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid.
2    For a GAP in rapid equilibrium with the GTP-bound form of Galpha z and for kgap > khydrol, the scheme below predicts a single exponential time course for total GTP hydrolysis. The apparent hydrolytic rate constant, kapp, is an average of kgap and khydrol, weighted by the ratio k-1/k1 and by the concentration of GAP. If background hydrolysis (assayed in the absence of GAP) is subtracted from total hydrolysis, then GAP activity can also be described by analogy to steady-state enzyme kinetics: Galpha z-GTP is the substrate, Km = ((k-1 + kgap)/k1) and, for [GAP] <<  [Galpha z-GTP], Vmax = (kgap)·[GAP]. GAP presumably dissociates from Galpha z-GDP immediately after hydrolysis (Fig. 7).
<AR><R><C><UP>G&agr;<SUB>z</SUB>-GTP </UP><LIM><OP><ARROW>→</ARROW></OP><UL>k<SUB><UP>hydrol</UP></SUB></UL></LIM> <UP>G&agr;<SUB>z</SUB>-GDP</UP>+<UP>P<SUB>i</SUB></UP></C></R><R><C>k<SUB>1</SUB>⥯k<SUB>−1</SUB></C></R><R><C><UP>GAP-G&agr;<SUB>z</SUB>-GTP </UP><LIM><OP><ARROW>→</ARROW></OP><UL>k<SUB><UP>gap</UP></SUB></UL></LIM> <UP>GAP-G&agr;<SUB>z</SUB>-GDP</UP>+<UP>P<SUB>i</SUB></UP></C></R></AR> (Eq. 1)


Acknowledgments

We thank Juriaan Huskens and A. Dean Sherry (University of Texas, Dallas) for generously providing the reagents and NMR facilities for measuring free Mg2+ and for help with the measurements themselves. We also thank Gloria Biddlecome for extensive and insightful discussion of the manuscript, Karen Chapman for help performing Sf9 cell culture, Carmen Dessauer (UT-Southwestern) for samples of adenylyl cyclase isoforms, Tohru Kozasa (UT-Southwestern) for a sample of Galpha 12 and for advice on the expression and purification of Galpha z, and other members of this department for various cultured cells. Pilot measurements of Km for Gz-GTP were performed by Yoon-Hang Kim.


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