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Epinephrine stimulation of fat cell adenylate cyclase: Regulation by guanosine-5′-triphosphate and magnesium ion
Molecular and Cellular Endocrinology
1 (1974) 89-98. @ North-Holland
Publ. Comp.
EPINEPHRINE STIMULATION OF FAT CELL ADENYLATE CYCLASE: REGULATION BY GUANOSINE-5’-TRIPHOSPHATE AND MAGNESIUM ION*
Marvin Department
I. SIEGEL
of Pharmacology
The Johns Hopkins
and Pedro CUATRECASAS
and Experimental
Therapeutics,
University School of Medicine,
Received 12 December
and Department
Baltimore,
Maryland
of Medicine,
2I205,
U.S.A.
1973
Guanosine-5’-triphosphate (GTP) affects the activity and responsiveness of the fat cell adenylate cyclase to epinephrine. In the absence of free magnesium ion, the presence of GTP is an absolute requirement for epinephrine stimulation. Half-maximal activation of both basal and stimulated adenylate cyclase activity occurs at a GTP concentration of 0.6 mM. In the presence of 5 mM MgCh, GTP is no longer required but enhances epinephrine stimulation. The half-maximal concentration for this effect occurs at approximately 10 uM GTP. At high (5 mM) magnesium ion concentrations GTP inhibits basal adenylate cyclase activity. GTP lowers the apparent affinity of adenylate cyclase for ATP while simultaneously increasing the velocity of the catalytic reaction, possibly by competing with ATP at the active site as well as by binding at a regulatory site. This effect is observed on both basal and epinephrine-stimulated activities. Epinephrine itself raises the apparent KM for ATP and the V,,, of adenylate cyclase. The interdependence of these effects suggests that transient changes in the levels of GTP, ATP, and magnesium ions in the fat cell may directly regulate the responsiveness of adenylate cyclase to epinephrine. Keywords:
epinephrine;
adenylate cyclase; fat cell; GTP; ATP.
* Supported by grants from (AMl4956) and The Kroc United States Public Health P.C. is recipient of United Award AM31464.
the National Institute of Arthritis and Metabolic Diseases Foundation, Santa Ynez, California. M.I.S. is recipient of Service Research Postdoctoral Fellowship Award AM53928. States Public Health Service Research Career Development
M. I. Siegel and P. Cuatresasas
90
The mechanisms by which hormones stimulate adenylate cyclase are not fully understood. The fat cell adenylate cyclase responds to numerous hormones including ACTH”, glucagon, and catecholamines (Birnbaumer and Rodbell, 1969; Rodbell et al., 1970). Each hormone appears to have its own independent receptor (Birnbaumer and Rodbell, 1969). The mechanism through which hormone-receptor complexes stimulate adenylate cyclase activity has yet to be defined. The observation that GTP” stimulates or is required for the response of cyclases from various cells to certain hormones (Bockaert et al., 1972; Goldfine et al., 1972; Krishna et al., 1972; Leray et al., 1972; Wolff and Cook, 1973) has shed some light on this activation phenomenon. This report describes the complexity of the regulation of the fat cell adenylate cyclase by GTP, metal ions, and epinephrine. The evidence presented suggests that cyclase responsiveness is a function of levels of nucleotides and magnesium ions. Moreover, the effects of these regulators appear to be interdependent.
EXPERIMENTAL
PROCEDURE
Materials
ATP, GTP, and phosphoenol pyruvate were purchased from Sigma. Pyruvate kinase was obtained from Boehringer. 32P-Pi was ordered from New England Nuclear. All other reagents were of the highest grade available. Preparation
of [a-32P]ATP [a-3ZP]ATP was synthesized from 32P-Pi according to the method of Symons (1972). Labeled ATP (150 Ci/mmole) was diluted with cold ATP immediately prior to use. The final specific activity was at least 500 cpm per pmole of ATP.
Preparation
of fat cell membranes Fat cell membranes were prepared essentially by previously described methods (Rodbell, 1964). Fat pads from six male Sprague-Dawley rats (120140 g) were digested with collagenase (1 mg perml) in Krebs-Ringer bicarbonate buffer, pH 7.4, containing 0.1% bovine serum albumin. After incubation for 30 min at 37 “C, the cells were harvested, passed through a nylon mesh screen, and washed three times with 25 mM Tris-HCl, pH 7.6. The cells were suspended
* The abbreviations used are ACTH, adrenocorticotropic hormone; ATP, adenosine5’-triphosphate; GTP, guanosine-5’-triphosphate; GDP, guanosine-5’-diphosphate; GMP, guanosine-5’-monophosphate; cGMP, guanosine-3’:5’-cyclic monophosphate; PEP, phosphoenol pyruvate; CAMP, adenosine-3’:5’-cyclic monophosphate.
Eflect of GTP on adenylate
cyclase
91
in 30 ml of 25 mM Tris-HCl, pH 7.6, and homogenized with a Brinkman polytron (setting#3, 30 set). The homogenate was centrifuged at 40,000 g for 20 min (4 “C, Sorvall RC-2B) and the pellet was resuspended Tris-HCl, pH 7.6, at 4 “C. These resuspended membranes assayed under the conditions described below.
in 2 ml of 25 mM were immediately
Assay of adenylate cyclase Adenylate cyclase was assayed using a modification of the method of Pohl et al. (1971). The standard assay medium contained 25 mM Tris-HCl, pH 7.6, 0.1% bovine serum albumin, 10 mM aminophylline, 5 mM phosphoenol pyruvate, 5 pg of pyruvate kinase, and various concentrations of [a-32P]ATP, GTP, and MgCl, in a volume of 0.1 ml. The specific activity of the added [a-3ZP]ATP was never less than 500 cpm per pmole (approximately 3 pCi per assay). The reaction was stopped after 10 min by heating for 2 min in a boiling water bath. After cooling, 1.0 ml of a recovery mixture containing cyclic [3H]adenosine monophosphate was added to each sample. Cyclic AMP was isolated on a column containing 1 g of alumina (Ramachandran, 1971; White and Zenser, 1972) which is eluted with 2.0 ml of 25 mM Tris-HCl, pH 7.6. Assay blanks (boiled membranes) were 300 cpm per 3 pCi of [a-32ATP]ATP added; basal activity was approximately 500 cpm above blank values.
RESULTS GTP requirement for epinephrine stimulation Available data on the fat cell adenylate cyclase indicates that the apparent KM for ATP is approximately 40 pM (Birnbaumer et al., 1969). Therefore, in order to investigate the mechanisms by which adenylate cyclase activity is controlled, it was useful to assay the enzyme at this substrate concentration. Since divalent metal ions have also been shown to play a role in the regulation of adenylate cyclase (Birnbaumer et al., 1969), the magnesium ion concentration was arbitrarily set as equal to the nucleotide concentration (that is, no free MgZ f). Under these conditions, stimulation of cyclase activity by epinephrine exhibits an absolute dependence on the presence of GTP (figs. 1 and 2). While no epinephrine stimulation is observed in the absence of GTP, in the presence of 2.5 mM GTP there is an approximately 5-fold epinephrine stimulation over baseline activity which, in itself, is stimulated several-fold by GTP (fig. 1). The half-maximal effect of this nucleotide on epinephrine stimulation occurs at a GTP concentration of 0.6 mM. GDP, GMP, and cGMP at the same concentration have no effect (not shown).
92
M. I. Siegel and P. Cuatresasas
J
5 GTP.
mM
Fig. 1. Effect of GTP concentration on basal and epinephrine-stimulated adenylate cyclase activity on fat cell membranes. The assay medium (0.1 ml) consisted of 25 mM Tris-HCI, pH 7.6, O.l’A bovine serum albumin, 10 mM aminophylline, 5 mM phosphoenol pyruvate, 5 ug pyruvate kinase, 40 uM [a-““P]ATP: Mg *+ (1 : 1 metal-nucleotide mixture, the specific activity of which was 1500 cpm per pmole), and various concentrations of GTP added as a 1:l GTP : Mg”+ mixture. The assay was initiated by the addition of 40 ug of fat cell membranes. The reaction was stopped after 10 min at 37 ‘C by heating for 2 min in a boiling water bath. All assays were performed in triplicate. Labelled CAMP was isolated as described in Methods. Basal activity -0; plus 15 uM epinephrine O--O-O.
The absolute dependence of epinephrine stimulation of adenylate cyclase activity on the presence of GTP is further illustrated in fig. 2. In the absence of GTP, no hormonal stimulation of adenylate cyclase occurs when the epinephrine concentration is varied from 1 x 1O-7 M to 3 x 1O-5 M. Furthermore, the apparent affinity of the cyclase response for epinephrine does not appear to be increased by GTP, since, at 1 mM GTP, half-maximal stimulation occurs at an epinephrine concentration of approximately 5 PM. Under very different assay conditions, including high magnesium ion concentrations, the concentration of epinephrine required for half-maximal activation is nearly the same (G.P.E. Tell and P. Cuatrecasas, unpublished). EfJect
qf magnesium
Divalent stimulated Therefore,
ion on adenylate
cyclase afJinity for GTP
metal ions have been shown to profoundly influence hormonally adenylate cyclase activity from various tissues (Birnbaumer, 1973). it was of interest to determine the effect of free magnesium ion on
Eflect of GTP on adenylate
93
cyclase
-i
.
4-w..
I
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3
6
9
I
I
I2 15 Epinephrlne
I
18 ,pM
I
I
21
24
I
27
A 3(
Fig. 2. Effect of epinephrine concentration on adenylate cyclase activity in the presence and absence of GTP. Adenylate cyclase activity was assayed as in fig. 1 except that epinephrine concentrations were varied in the presence (O-0-O) or absence (O-O--O ) of 1 mM GTP (added as 1 : 1 GTP : Mg2+).
cyclase activity in the presence of GTP. As illustrated in fig. 3, in the presence of 5 mM free Mg2+ (that is, Mg2+ in excess of added ATP and GTP), the adenylate cyclase response to epinephrine and to GTP is markedly altered. With this high metal ion concentration, epinephrine is capable of stimulating cyclase activity in the absence of GTP, in contrast to the absolute requirement for GTP when there is no free magnesium ion (see fig. 2). In addition, in the presence of 5 mM MgCl, basal and epinephrine stimulated cyclase activities are approximately IO-fold greater than in the absence of free metal ion (compare the enzyme activities in figs. 1 and 3). The concentration at which GTP is effective is dramatically decreased by magnesium ion. In the presence of 5 mM Mg2+ (fig. 3), the half-maximal stimulatory effect occurs at a GTP concentration of approximately 10 PM, while in the absence of free metal ion this value is 600 pM (fig. 1). Moreover, at very low GTP concentrations (10 PM), baseline adenylate cyclase activity is markedly inhibited (fig. 3), an effect not easily observed in the absence of high metal ion concentrations. A further effect of Mg2+ is that at high (5 mM) metal ion concentrations GTP is inhibitory when its concentration exceeds 0.4 mM (fig. 3) while no GTP inhibition is seen in the absence of free Mg2+- (fig. 1). This inhibitory effect of GTP is apparent whether epinephrine-stimulated or basal adenylate cyclase activity is measured.
M. 1. Siegel and P. Cuatresasas
94
I
01
I
05
I
I
0.9
13 GTP
,
1
17
2 I
2 5
mM
Fig. 3. Effect of GTP concentration on basal and epinephrine-stimulated adenylate cyclase activities in the presence of excess magnesium ion. Adenylate cyclase was assayed exactly as in fig. 1 with the addition of 5 mM M~CIZ. Basal activity *O--O; plus 15 uM epinephrine X-X-X
ATP , p’M
Fig. 4. Effect of ATP concentration on basal and epinephrine-stimulated adenylate cyclase activities. Adenylate cyclase was assayed as in fig. 1 except that the concentration of ATP : Mg2+ (1 : 1) was varied. In the presence of 0.5 mM GTP : Mgz-’ (I : 1): basal activity w; plus 15 uM epinephrine O--i?-0. In the presence of 1.0 mM GTP : Mg2+ (1 : 1): basal activity X-->: -- Y , plus 15 uM epinephrine [?-_O-_O.
Eflect of GTP on adenylate
cyclase
9.5
Effect of GTP on the apparent afinity of adenylate cyclase for ATP The inhibitory effect of high concentrations of GTP on adenylate cyclase activity in the presence of free magnesium ion suggests that, in addition to its stimulatory effect on epinephrine activation, GTP competes with ATP for the catalytic site of the enzyme. If this were the case, then the apparent affinity of adenylate cyclase for its substrate, ATP, should decrease with increasing concentrations of GTP. In order to eliminate the additional complication of free metal ion activation (compare the enzyme activities in figs. 1 and 3) the affinity of the cyclase for ATP was measured in the absence of free magnesium ions. As shown in fig. 4, at each concentration of ATP, epinephrine-stimulated and basal cyclase activities are greater at 1 mM than 0.5 mM GTP. This is the expected result since the apparent K, for the GTP effect on epinephrine stimulation under these conditions is 0.6 mM GTP (see fig. 1). The effect of GTP upon the affinity of adenylate cyclase for ATP is better illustrated in double-reciprocal plots (fig. 5). At 0.5 mM GTP, the apparent KM’S for ATP for basal and epinephrine-stimulated cyclase are 35 and 61 uM respectively. In the presence of 1 mM GTP, the apparent affinity for ATP is further decreased to 100 uM for basal activity and 180 pM for the epinephrine-stimulated cyclase activity. It is therefore apparent that GTP is capable of affecting the observed
‘/ATp x lO+M Fig. 5. Double-reciprocal plot of the effect of GTP on basal and epinephrine stimulated adenylate cyclase activities. Assay conditions and symbols are identical to those in fig. 4.
96
M. I. Siegel and P. Cuatrecasas
affinity of the cyclase for its substrate, ATP, possibly by competing with ATP for the enzymatically active site. The results illustrated in fig, 5 indicate that, under these conditions, epinephrine increases the apparent V,,, of adenylate cyclase, as has been previously reported (Birnbaumer, 1973), and that in addition it decreases the apparent affinity of the cyclase for its substrate, ATP. The increased adenylate cyclase activity observed in the presence of epinephrine must therefore reflect increased catalytic activity which predominates in spite of an apparent fall in affinity for the substrate.
DISCUSSION The present study reveals that fat cell adenylate cyclase activity is regulated in a complex manner by GTP, magnesium ion, and epinephrine. The regulatory effects of each of these stimulants are interdependent. At low magnesium ion concentrations, GTP is required for epinephrine stimulation of adenylate cyclase activity (fig. l), while in the presence of free metal ion this is no longer the case (fig. 3). The adenylate cyclases from amphibian red blood cells (G. V. Bennett and P. Cuatrecasas, unpublished) and mouse mammary cells (E. 0’ Keefe and P. Cuatrecasas, unpublished) also exhibit similar dependence on GTP. Moreover, the presence of magnesium ion dramatically decreases the concentration of GTP required to achieve effective stimulation. The concentration at which the GTP effect on epinephrine stimulation of adenylate cyclase is halfmaximal decreases from 600 uM in absence of free Mg2 + (fig. 1) to approximately 10 uM at 5 mM MgCl, (fig. 3). This 60-fold increase in the effectiveness of GTP may be due to either of two factors: a) an increased affinity of a regulatory binding site of adenylate cyclase for GTP, or b) an increased ability of GTP to magnify the stimulatory effect of epinephrine. In the absence of free magnesium ion, GTP modulates adenylate cyclase by increasing basal as well as epinephrine stimulated catalytic activity (fig. 1). However, in the presence of free Mg’ I, the effect of GTP becomes very complex. Basal adenylate cyclase activity is inhibited at both very low and high concentrations of GTP (fig. 3). This result is in agreement with the reports of Harwood et al. (1973) and Cryer et al. (1969) that GTP at concentrations ranging from 0.1 uM to 0.5 mM inhibit basal adenylate cyclase activity. The mechanism by which this inhibition occurs is not known, but it appears that the terminal phosphate of GTP may be involved in some chemical reaction (Harwood et al., 1973). The effect of GTP on epinephrine-stimulated adenylate cyclase activity in
Eflect of GTP on adenylate
cyclase
97
the presence of high magnesium ion concentrations is also very complex. Low concentrations of GTP dramatically increase the epinephrine stimulated activity (fig. 3). However, as the concentration of GTP exceeds 0.4 mM, inhibition becomes pronounced. While it is difficult to interpret the inhibition of basal cyclase activity at low GTP concentrations, a possible explanation of the inhibitory effect of high concentrations of GTP on both basal and epinephrine-stimulatedactivitiesis thatGTPcompetes withATPfortheactivesiteoftheenzyme. That GTP does affect the apparent affinity with which the cyclase binds its substrate, ATP, is suggested by the data presented in fig. 5. In the absence of free magnesium ion, the apparent K M of basal adenylate cyclase for ATP increases from 35 uM at 0.5 mM GTP to 100 uM at 1 mM GTP. At the same time, however, there is a marked increase in V,,, (about 3-fold). GTP has the same effect on epinephrine-stimulated cyclase. The apparent K, for ATP increases from 61 uM at 0.5 mM GTP to 180 uM at I mM GTP, while the V,,, increases. Therefore, the increased cyclase activity observed in the presence of increasing concentrations of GTP (fig. 1) occurs as a result of an elevation which overcomes an apparently decreased affinity for the substrate. in V,,, Upon stimulation of adenylate cyclase by epinephrine, the enzyme’s affinity for ATP similarly decreases. In the absence of free magnesium ion but in the presence of 0.5 mM GTP, epinephrine stimulation results in an increase in the KM for ATP from 35 to 61 uM; at 1 mM GTP, the increase is from 100 to 180 uM (fig. 5). Therefore, increased activity in the presence of epinephrine is a result of an elevated catalytic velocity which is observed in spite of a decreased affinity for substrate. It has become increasingly apparent that the fat cell hormone-receptor adenylate cyclase system is exceedingly complex. In addition to responding to numerous hormones (Birnbaumer and Rodbell, 1969) this system contains regulatory binding sites for GTP that enhance the responsiveness of the system to epinephrine. The fact that at high magnesium ion concentrations GTP also inhibits basal cyclase activity magnifies the apparent degree of stimulation of the enzyme by hormone. Similarly, in the frog bladder oxytocin-sensitive adenylate cyclase, the activation ratio (oxytocin to basal activity) is nearly constant at various substrate concentrations (Bockaert et al., 1972), but it is increased by GTP at low ATP concentrations. While some adenylate cyclase systems show additional dependence on ATP concentration for hormonal activation (Krishna et al., 1968; Birnbaumer et al., 1972) the fat cell adenylate cyclase shows no such dependence at low ATP concentrations (Harwood et al., 1973). Presumably, then, the predominant effect of GTP on regulatory sites is to enhance the velocity of the cyclase to a greater extent in the presence of hormone than in its absence (fig. 1).
98
M. I. Siegel and P. Cuatrecasas
Hormonal responsiveness can thus be regulated by combinations of varying substrate, GTP, and magnesium ion concentrations. GTP is involved in relatively few enzymatic reactions in the fat cell (predominantly biosynthetic ones) and may well reflect the metabolic viability of the cell. Since GTP affects the apparent affinity of the cyclase for ATP, since magnesium ion influences the responsiveness of the cyclase system to the levels of GTP, and since hormonal sensitivity is dependent upon the presence of GTP, it is probable that the metabolic state of the cell (that is, the transient levels of GTP, ATP, and metal ions) directly regulates the fat cell’s response to the presence of the lipolytic hormone, epinephrine.
REFERENCES Birnbaumer, L. (1973) Biochim. Biophys. Acta 300, 129. Birnbaumer, L., Pohl, S. L. and Rodbell, M. (1969) J. Biol. Chem. 244, 3468. Birnbaumer, L., Pohl, S. L., Rodbell, M. and Sundby, F. (1972) J. Biol. Chem. 247,2038. Birnbaumer, L. and Rodbell, M. (1969) J. Biol. Chem. 244, 3477. Bockaert, S., Roy, C. and Jurd, S. (1972) J. Biol. Chem. 247, 7073. Cryer, P. E., Jarett, L. and Kipnis, D. M. (1969) Biochim. Biophys. Acta 177, 586. Goldfine, I. D., Roth, J. and Birnbaumer, L. (1972) J. Biol. Chem 247, 1211. Harwood, J. P., Low, H. and Rodbell, M. (1973) J. Biol. Chem. 248, 6239. Krishna, G., Harwood, J. P., Barber, A. J. and Jamieson, G. A. (1972) J. Biol. Chem. 247, 2253. Krishna, G., Weiss, B. and Brodie, B. B. (1968) J. Pharmacol. Exptl. Therap. 163, 379. Leray, F., Chambaut, A. M. and Hanoune, J. (1972) Biochem. Biophys. Res. Commun. 48, 1385. Pohl, S. L., Birnbaumer, L. and Rodbell, M. (1971) J. Biol. Chem. 246, 1849. Ramachandran, J. (1971) Anal. Biochem. 43, 227. Rodbell, M. (1964) J. Biol. Chem. 239, 375. Rodbell, M., Birnbaumer, L. and Pohl, S. L. (1970) J. Biol. Chem. 245, 718. Symons, R. H. (1972) pers. commun. White, A. A. and Zenser, T. V. (1972) Anal. Biochem. 41, 372. Wolff, J. and Cook, G. H. (1973) J. Biol. Chem. 248, 350.
1 (1974) 89-98. @ North-Holland
Publ. Comp.
EPINEPHRINE STIMULATION OF FAT CELL ADENYLATE CYCLASE: REGULATION BY GUANOSINE-5’-TRIPHOSPHATE AND MAGNESIUM ION*
Marvin Department
I. SIEGEL
of Pharmacology
The Johns Hopkins
and Pedro CUATRECASAS
and Experimental
Therapeutics,
University School of Medicine,
Received 12 December
and Department
Baltimore,
Maryland
of Medicine,
2I205,
U.S.A.
1973
Guanosine-5’-triphosphate (GTP) affects the activity and responsiveness of the fat cell adenylate cyclase to epinephrine. In the absence of free magnesium ion, the presence of GTP is an absolute requirement for epinephrine stimulation. Half-maximal activation of both basal and stimulated adenylate cyclase activity occurs at a GTP concentration of 0.6 mM. In the presence of 5 mM MgCh, GTP is no longer required but enhances epinephrine stimulation. The half-maximal concentration for this effect occurs at approximately 10 uM GTP. At high (5 mM) magnesium ion concentrations GTP inhibits basal adenylate cyclase activity. GTP lowers the apparent affinity of adenylate cyclase for ATP while simultaneously increasing the velocity of the catalytic reaction, possibly by competing with ATP at the active site as well as by binding at a regulatory site. This effect is observed on both basal and epinephrine-stimulated activities. Epinephrine itself raises the apparent KM for ATP and the V,,, of adenylate cyclase. The interdependence of these effects suggests that transient changes in the levels of GTP, ATP, and magnesium ions in the fat cell may directly regulate the responsiveness of adenylate cyclase to epinephrine. Keywords:
epinephrine;
adenylate cyclase; fat cell; GTP; ATP.
* Supported by grants from (AMl4956) and The Kroc United States Public Health P.C. is recipient of United Award AM31464.
the National Institute of Arthritis and Metabolic Diseases Foundation, Santa Ynez, California. M.I.S. is recipient of Service Research Postdoctoral Fellowship Award AM53928. States Public Health Service Research Career Development
M. I. Siegel and P. Cuatresasas
90
The mechanisms by which hormones stimulate adenylate cyclase are not fully understood. The fat cell adenylate cyclase responds to numerous hormones including ACTH”, glucagon, and catecholamines (Birnbaumer and Rodbell, 1969; Rodbell et al., 1970). Each hormone appears to have its own independent receptor (Birnbaumer and Rodbell, 1969). The mechanism through which hormone-receptor complexes stimulate adenylate cyclase activity has yet to be defined. The observation that GTP” stimulates or is required for the response of cyclases from various cells to certain hormones (Bockaert et al., 1972; Goldfine et al., 1972; Krishna et al., 1972; Leray et al., 1972; Wolff and Cook, 1973) has shed some light on this activation phenomenon. This report describes the complexity of the regulation of the fat cell adenylate cyclase by GTP, metal ions, and epinephrine. The evidence presented suggests that cyclase responsiveness is a function of levels of nucleotides and magnesium ions. Moreover, the effects of these regulators appear to be interdependent.
EXPERIMENTAL
PROCEDURE
Materials
ATP, GTP, and phosphoenol pyruvate were purchased from Sigma. Pyruvate kinase was obtained from Boehringer. 32P-Pi was ordered from New England Nuclear. All other reagents were of the highest grade available. Preparation
of [a-32P]ATP [a-3ZP]ATP was synthesized from 32P-Pi according to the method of Symons (1972). Labeled ATP (150 Ci/mmole) was diluted with cold ATP immediately prior to use. The final specific activity was at least 500 cpm per pmole of ATP.
Preparation
of fat cell membranes Fat cell membranes were prepared essentially by previously described methods (Rodbell, 1964). Fat pads from six male Sprague-Dawley rats (120140 g) were digested with collagenase (1 mg perml) in Krebs-Ringer bicarbonate buffer, pH 7.4, containing 0.1% bovine serum albumin. After incubation for 30 min at 37 “C, the cells were harvested, passed through a nylon mesh screen, and washed three times with 25 mM Tris-HCl, pH 7.6. The cells were suspended
* The abbreviations used are ACTH, adrenocorticotropic hormone; ATP, adenosine5’-triphosphate; GTP, guanosine-5’-triphosphate; GDP, guanosine-5’-diphosphate; GMP, guanosine-5’-monophosphate; cGMP, guanosine-3’:5’-cyclic monophosphate; PEP, phosphoenol pyruvate; CAMP, adenosine-3’:5’-cyclic monophosphate.
Eflect of GTP on adenylate
cyclase
91
in 30 ml of 25 mM Tris-HCl, pH 7.6, and homogenized with a Brinkman polytron (setting#3, 30 set). The homogenate was centrifuged at 40,000 g for 20 min (4 “C, Sorvall RC-2B) and the pellet was resuspended Tris-HCl, pH 7.6, at 4 “C. These resuspended membranes assayed under the conditions described below.
in 2 ml of 25 mM were immediately
Assay of adenylate cyclase Adenylate cyclase was assayed using a modification of the method of Pohl et al. (1971). The standard assay medium contained 25 mM Tris-HCl, pH 7.6, 0.1% bovine serum albumin, 10 mM aminophylline, 5 mM phosphoenol pyruvate, 5 pg of pyruvate kinase, and various concentrations of [a-32P]ATP, GTP, and MgCl, in a volume of 0.1 ml. The specific activity of the added [a-3ZP]ATP was never less than 500 cpm per pmole (approximately 3 pCi per assay). The reaction was stopped after 10 min by heating for 2 min in a boiling water bath. After cooling, 1.0 ml of a recovery mixture containing cyclic [3H]adenosine monophosphate was added to each sample. Cyclic AMP was isolated on a column containing 1 g of alumina (Ramachandran, 1971; White and Zenser, 1972) which is eluted with 2.0 ml of 25 mM Tris-HCl, pH 7.6. Assay blanks (boiled membranes) were 300 cpm per 3 pCi of [a-32ATP]ATP added; basal activity was approximately 500 cpm above blank values.
RESULTS GTP requirement for epinephrine stimulation Available data on the fat cell adenylate cyclase indicates that the apparent KM for ATP is approximately 40 pM (Birnbaumer et al., 1969). Therefore, in order to investigate the mechanisms by which adenylate cyclase activity is controlled, it was useful to assay the enzyme at this substrate concentration. Since divalent metal ions have also been shown to play a role in the regulation of adenylate cyclase (Birnbaumer et al., 1969), the magnesium ion concentration was arbitrarily set as equal to the nucleotide concentration (that is, no free MgZ f). Under these conditions, stimulation of cyclase activity by epinephrine exhibits an absolute dependence on the presence of GTP (figs. 1 and 2). While no epinephrine stimulation is observed in the absence of GTP, in the presence of 2.5 mM GTP there is an approximately 5-fold epinephrine stimulation over baseline activity which, in itself, is stimulated several-fold by GTP (fig. 1). The half-maximal effect of this nucleotide on epinephrine stimulation occurs at a GTP concentration of 0.6 mM. GDP, GMP, and cGMP at the same concentration have no effect (not shown).
92
M. I. Siegel and P. Cuatresasas
J
5 GTP.
mM
Fig. 1. Effect of GTP concentration on basal and epinephrine-stimulated adenylate cyclase activity on fat cell membranes. The assay medium (0.1 ml) consisted of 25 mM Tris-HCI, pH 7.6, O.l’A bovine serum albumin, 10 mM aminophylline, 5 mM phosphoenol pyruvate, 5 ug pyruvate kinase, 40 uM [a-““P]ATP: Mg *+ (1 : 1 metal-nucleotide mixture, the specific activity of which was 1500 cpm per pmole), and various concentrations of GTP added as a 1:l GTP : Mg”+ mixture. The assay was initiated by the addition of 40 ug of fat cell membranes. The reaction was stopped after 10 min at 37 ‘C by heating for 2 min in a boiling water bath. All assays were performed in triplicate. Labelled CAMP was isolated as described in Methods. Basal activity -0; plus 15 uM epinephrine O--O-O.
The absolute dependence of epinephrine stimulation of adenylate cyclase activity on the presence of GTP is further illustrated in fig. 2. In the absence of GTP, no hormonal stimulation of adenylate cyclase occurs when the epinephrine concentration is varied from 1 x 1O-7 M to 3 x 1O-5 M. Furthermore, the apparent affinity of the cyclase response for epinephrine does not appear to be increased by GTP, since, at 1 mM GTP, half-maximal stimulation occurs at an epinephrine concentration of approximately 5 PM. Under very different assay conditions, including high magnesium ion concentrations, the concentration of epinephrine required for half-maximal activation is nearly the same (G.P.E. Tell and P. Cuatrecasas, unpublished). EfJect
qf magnesium
Divalent stimulated Therefore,
ion on adenylate
cyclase afJinity for GTP
metal ions have been shown to profoundly influence hormonally adenylate cyclase activity from various tissues (Birnbaumer, 1973). it was of interest to determine the effect of free magnesium ion on
Eflect of GTP on adenylate
93
cyclase
-i
.
4-w..
I
I
I
3
6
9
I
I
I2 15 Epinephrlne
I
18 ,pM
I
I
21
24
I
27
A 3(
Fig. 2. Effect of epinephrine concentration on adenylate cyclase activity in the presence and absence of GTP. Adenylate cyclase activity was assayed as in fig. 1 except that epinephrine concentrations were varied in the presence (O-0-O) or absence (O-O--O ) of 1 mM GTP (added as 1 : 1 GTP : Mg2+).
cyclase activity in the presence of GTP. As illustrated in fig. 3, in the presence of 5 mM free Mg2+ (that is, Mg2+ in excess of added ATP and GTP), the adenylate cyclase response to epinephrine and to GTP is markedly altered. With this high metal ion concentration, epinephrine is capable of stimulating cyclase activity in the absence of GTP, in contrast to the absolute requirement for GTP when there is no free magnesium ion (see fig. 2). In addition, in the presence of 5 mM MgCl, basal and epinephrine stimulated cyclase activities are approximately IO-fold greater than in the absence of free metal ion (compare the enzyme activities in figs. 1 and 3). The concentration at which GTP is effective is dramatically decreased by magnesium ion. In the presence of 5 mM Mg2+ (fig. 3), the half-maximal stimulatory effect occurs at a GTP concentration of approximately 10 PM, while in the absence of free metal ion this value is 600 pM (fig. 1). Moreover, at very low GTP concentrations (10 PM), baseline adenylate cyclase activity is markedly inhibited (fig. 3), an effect not easily observed in the absence of high metal ion concentrations. A further effect of Mg2+ is that at high (5 mM) metal ion concentrations GTP is inhibitory when its concentration exceeds 0.4 mM (fig. 3) while no GTP inhibition is seen in the absence of free Mg2+- (fig. 1). This inhibitory effect of GTP is apparent whether epinephrine-stimulated or basal adenylate cyclase activity is measured.
M. 1. Siegel and P. Cuatresasas
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I
01
I
05
I
I
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13 GTP
,
1
17
2 I
2 5
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Fig. 3. Effect of GTP concentration on basal and epinephrine-stimulated adenylate cyclase activities in the presence of excess magnesium ion. Adenylate cyclase was assayed exactly as in fig. 1 with the addition of 5 mM M~CIZ. Basal activity *O--O; plus 15 uM epinephrine X-X-X
ATP , p’M
Fig. 4. Effect of ATP concentration on basal and epinephrine-stimulated adenylate cyclase activities. Adenylate cyclase was assayed as in fig. 1 except that the concentration of ATP : Mg2+ (1 : 1) was varied. In the presence of 0.5 mM GTP : Mgz-’ (I : 1): basal activity w; plus 15 uM epinephrine O--i?-0. In the presence of 1.0 mM GTP : Mg2+ (1 : 1): basal activity X-->: -- Y , plus 15 uM epinephrine [?-_O-_O.
Eflect of GTP on adenylate
cyclase
9.5
Effect of GTP on the apparent afinity of adenylate cyclase for ATP The inhibitory effect of high concentrations of GTP on adenylate cyclase activity in the presence of free magnesium ion suggests that, in addition to its stimulatory effect on epinephrine activation, GTP competes with ATP for the catalytic site of the enzyme. If this were the case, then the apparent affinity of adenylate cyclase for its substrate, ATP, should decrease with increasing concentrations of GTP. In order to eliminate the additional complication of free metal ion activation (compare the enzyme activities in figs. 1 and 3) the affinity of the cyclase for ATP was measured in the absence of free magnesium ions. As shown in fig. 4, at each concentration of ATP, epinephrine-stimulated and basal cyclase activities are greater at 1 mM than 0.5 mM GTP. This is the expected result since the apparent K, for the GTP effect on epinephrine stimulation under these conditions is 0.6 mM GTP (see fig. 1). The effect of GTP upon the affinity of adenylate cyclase for ATP is better illustrated in double-reciprocal plots (fig. 5). At 0.5 mM GTP, the apparent KM’S for ATP for basal and epinephrine-stimulated cyclase are 35 and 61 uM respectively. In the presence of 1 mM GTP, the apparent affinity for ATP is further decreased to 100 uM for basal activity and 180 pM for the epinephrine-stimulated cyclase activity. It is therefore apparent that GTP is capable of affecting the observed
‘/ATp x lO+M Fig. 5. Double-reciprocal plot of the effect of GTP on basal and epinephrine stimulated adenylate cyclase activities. Assay conditions and symbols are identical to those in fig. 4.
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affinity of the cyclase for its substrate, ATP, possibly by competing with ATP for the enzymatically active site. The results illustrated in fig, 5 indicate that, under these conditions, epinephrine increases the apparent V,,, of adenylate cyclase, as has been previously reported (Birnbaumer, 1973), and that in addition it decreases the apparent affinity of the cyclase for its substrate, ATP. The increased adenylate cyclase activity observed in the presence of epinephrine must therefore reflect increased catalytic activity which predominates in spite of an apparent fall in affinity for the substrate.
DISCUSSION The present study reveals that fat cell adenylate cyclase activity is regulated in a complex manner by GTP, magnesium ion, and epinephrine. The regulatory effects of each of these stimulants are interdependent. At low magnesium ion concentrations, GTP is required for epinephrine stimulation of adenylate cyclase activity (fig. l), while in the presence of free metal ion this is no longer the case (fig. 3). The adenylate cyclases from amphibian red blood cells (G. V. Bennett and P. Cuatrecasas, unpublished) and mouse mammary cells (E. 0’ Keefe and P. Cuatrecasas, unpublished) also exhibit similar dependence on GTP. Moreover, the presence of magnesium ion dramatically decreases the concentration of GTP required to achieve effective stimulation. The concentration at which the GTP effect on epinephrine stimulation of adenylate cyclase is halfmaximal decreases from 600 uM in absence of free Mg2 + (fig. 1) to approximately 10 uM at 5 mM MgCl, (fig. 3). This 60-fold increase in the effectiveness of GTP may be due to either of two factors: a) an increased affinity of a regulatory binding site of adenylate cyclase for GTP, or b) an increased ability of GTP to magnify the stimulatory effect of epinephrine. In the absence of free magnesium ion, GTP modulates adenylate cyclase by increasing basal as well as epinephrine stimulated catalytic activity (fig. 1). However, in the presence of free Mg’ I, the effect of GTP becomes very complex. Basal adenylate cyclase activity is inhibited at both very low and high concentrations of GTP (fig. 3). This result is in agreement with the reports of Harwood et al. (1973) and Cryer et al. (1969) that GTP at concentrations ranging from 0.1 uM to 0.5 mM inhibit basal adenylate cyclase activity. The mechanism by which this inhibition occurs is not known, but it appears that the terminal phosphate of GTP may be involved in some chemical reaction (Harwood et al., 1973). The effect of GTP on epinephrine-stimulated adenylate cyclase activity in
Eflect of GTP on adenylate
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97
the presence of high magnesium ion concentrations is also very complex. Low concentrations of GTP dramatically increase the epinephrine stimulated activity (fig. 3). However, as the concentration of GTP exceeds 0.4 mM, inhibition becomes pronounced. While it is difficult to interpret the inhibition of basal cyclase activity at low GTP concentrations, a possible explanation of the inhibitory effect of high concentrations of GTP on both basal and epinephrine-stimulatedactivitiesis thatGTPcompetes withATPfortheactivesiteoftheenzyme. That GTP does affect the apparent affinity with which the cyclase binds its substrate, ATP, is suggested by the data presented in fig. 5. In the absence of free magnesium ion, the apparent K M of basal adenylate cyclase for ATP increases from 35 uM at 0.5 mM GTP to 100 uM at 1 mM GTP. At the same time, however, there is a marked increase in V,,, (about 3-fold). GTP has the same effect on epinephrine-stimulated cyclase. The apparent K, for ATP increases from 61 uM at 0.5 mM GTP to 180 uM at I mM GTP, while the V,,, increases. Therefore, the increased cyclase activity observed in the presence of increasing concentrations of GTP (fig. 1) occurs as a result of an elevation which overcomes an apparently decreased affinity for the substrate. in V,,, Upon stimulation of adenylate cyclase by epinephrine, the enzyme’s affinity for ATP similarly decreases. In the absence of free magnesium ion but in the presence of 0.5 mM GTP, epinephrine stimulation results in an increase in the KM for ATP from 35 to 61 uM; at 1 mM GTP, the increase is from 100 to 180 uM (fig. 5). Therefore, increased activity in the presence of epinephrine is a result of an elevated catalytic velocity which is observed in spite of a decreased affinity for substrate. It has become increasingly apparent that the fat cell hormone-receptor adenylate cyclase system is exceedingly complex. In addition to responding to numerous hormones (Birnbaumer and Rodbell, 1969) this system contains regulatory binding sites for GTP that enhance the responsiveness of the system to epinephrine. The fact that at high magnesium ion concentrations GTP also inhibits basal cyclase activity magnifies the apparent degree of stimulation of the enzyme by hormone. Similarly, in the frog bladder oxytocin-sensitive adenylate cyclase, the activation ratio (oxytocin to basal activity) is nearly constant at various substrate concentrations (Bockaert et al., 1972), but it is increased by GTP at low ATP concentrations. While some adenylate cyclase systems show additional dependence on ATP concentration for hormonal activation (Krishna et al., 1968; Birnbaumer et al., 1972) the fat cell adenylate cyclase shows no such dependence at low ATP concentrations (Harwood et al., 1973). Presumably, then, the predominant effect of GTP on regulatory sites is to enhance the velocity of the cyclase to a greater extent in the presence of hormone than in its absence (fig. 1).
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Hormonal responsiveness can thus be regulated by combinations of varying substrate, GTP, and magnesium ion concentrations. GTP is involved in relatively few enzymatic reactions in the fat cell (predominantly biosynthetic ones) and may well reflect the metabolic viability of the cell. Since GTP affects the apparent affinity of the cyclase for ATP, since magnesium ion influences the responsiveness of the cyclase system to the levels of GTP, and since hormonal sensitivity is dependent upon the presence of GTP, it is probable that the metabolic state of the cell (that is, the transient levels of GTP, ATP, and metal ions) directly regulates the fat cell’s response to the presence of the lipolytic hormone, epinephrine.
REFERENCES Birnbaumer, L. (1973) Biochim. Biophys. Acta 300, 129. Birnbaumer, L., Pohl, S. L. and Rodbell, M. (1969) J. Biol. Chem. 244, 3468. Birnbaumer, L., Pohl, S. L., Rodbell, M. and Sundby, F. (1972) J. Biol. Chem. 247,2038. Birnbaumer, L. and Rodbell, M. (1969) J. Biol. Chem. 244, 3477. Bockaert, S., Roy, C. and Jurd, S. (1972) J. Biol. Chem. 247, 7073. Cryer, P. E., Jarett, L. and Kipnis, D. M. (1969) Biochim. Biophys. Acta 177, 586. Goldfine, I. D., Roth, J. and Birnbaumer, L. (1972) J. Biol. Chem 247, 1211. Harwood, J. P., Low, H. and Rodbell, M. (1973) J. Biol. Chem. 248, 6239. Krishna, G., Harwood, J. P., Barber, A. J. and Jamieson, G. A. (1972) J. Biol. Chem. 247, 2253. Krishna, G., Weiss, B. and Brodie, B. B. (1968) J. Pharmacol. Exptl. Therap. 163, 379. Leray, F., Chambaut, A. M. and Hanoune, J. (1972) Biochem. Biophys. Res. Commun. 48, 1385. Pohl, S. L., Birnbaumer, L. and Rodbell, M. (1971) J. Biol. Chem. 246, 1849. Ramachandran, J. (1971) Anal. Biochem. 43, 227. Rodbell, M. (1964) J. Biol. Chem. 239, 375. Rodbell, M., Birnbaumer, L. and Pohl, S. L. (1970) J. Biol. Chem. 245, 718. Symons, R. H. (1972) pers. commun. White, A. A. and Zenser, T. V. (1972) Anal. Biochem. 41, 372. Wolff, J. and Cook, G. H. (1973) J. Biol. Chem. 248, 350.