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Calcium inhibition of adenylate cyclase: Studies in turkey erythrocyte and S49 CYC− cell membranes
ARCHIVESOF BIOCHEMISTRYAND BIOPHYSICS Vol. 216, No. 1, June, pp. 345-355, 1982
Calcium inhibition of Adenylate Cyciase: Studies in Turkey Erythrocyte and S49 CYC- Cell Membranes ROZ D. LASKER,l ROBERT W. DOWNS, JR., AND GERALD D. AURBACH Metabolic Disease Branch, National Institute of Arthritis, Diabetes, and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland, 20205 Received November 16, 1981, and in revised form February 12,1982
We studied the mechanism of calcium inhibition of adenylate cyclase using partially purified components of the enzyme complex and computer analysis of free metal and substrate concentrations. A sigmoidal relationship was observed between percentage maximal adenylate cyclase activity with l-isoproterenol/guanylyl-P,y-imidodiphosphate and the calculated free calcium. Fifty percent inhibition occurred at 2.5 X lop4 M free calcium. This inhibition appeared to be independent of calmodulin. Calcium inhibited the holocatalytic enzyme in a manner indentical to that of the native enzyme, but did not affect the ability of 1-isoproterenol and guanylyl-&y-imidodiphosphate to promote the formation of the holocatalytic state. There was no effect of calcium on the conformation of the activated G unit nor on the holocatalytic enzyme as determined by sedimentation velocity analysis. Calcium did not cause detectable dissociation of the activated G unit from the catalytic unit, nor convert activated G unit to an inactive form. Calcium inhibition of the catalytic unit of adenylate cyclase was studied in S49 CYC- lymphoma cell membranes. High concentrations of calcium inhibited manganesestimulated CYC- enzyme, but this could be explained by competition between calcium and manganese for ATP. With addition of forskolin, CYC- adenylate cyclase utilized MgATP2- as substrate and was shown to have a separate binding site for free magnesium. Calcium inhibited forskolin-stimulated CYC- enzyme by competing with free magnesium for its regulatory site. Calcium inhibition was noncompetitive with respect to MgATP2-. We conclude that calcium inhibits adenylate cyclase by direct competition with magnesium for a regulatory site on the catalytic unit.
Adenylate cyclase (EC 4.6.1.1) is a membrane-bound enzyme complex that translates extracellular signals into intracellular information through the generation of the second messenger, cyclic AMP. The enzyme complex consists of at least three subunits: a hormone receptor (R) on the external surface of the plasma membrane (l), and a catalytic unit (C) and guanine nucleotide binding protein (G), both located on the cytoplasmic surface of the plasma membrane (2, 3). Hormonal con-
trol of CAMP formation can be described by the following model (4-6): (1) binding of hormone (H) to R facilitates exchange of GTP for GDP on G, forming an activated G unit (G*); (2) G* interacts with C to enhance the formation of CAMP from ATP; (3) hydrolysis of GTP to GDP deactivates the enzyme. Certain analogs of GTP (i.e., G~~NHP)~ are resistant to hydrolysis, and in the presence of hormone ’ Abbreviations used: GppNHp, guanylyl-&y-imidodiphosphate; EGTA, ethylene glycol bis (@-aminoethyl etber)N,N,N’,N’-tetraacetic acid, PEP, phosphoenol pyruvate.
1Author to whom all correspondence should be addressed. 345
0003-9861/82/070345-11$02.00/O Copyright All righta
Q 1982 by Academic Pnss. Inc. of reproduction in any form reserved.
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LASKER, DOWNS, AND AURBACH
can form a persistently activated state of the enzyme, the holocatalytic state. Fluoride activates adenylate cyclase in vitro by activating G without participation of R or guanine nucleotide exchange (6). Calcium is an important modulator of the activity of adenylate cyclase. Several tissues, including brain and adrenal, show a biphasic response to calcium, with low concentrations stimulating and higher concentrations inhibiting enzyme activity (7,8). Most tissues, however, show only an inhibitory response to this ion (9-14). Previous work has shown that calcium inhibits maximal activation of the enzyme by hormone and fluoride (8, 10). However, since these studies utilized crude membrane preparations, neither the mechanism nor the locus of this inhibition has been fully elucidated. We used partially separated components of turkey erythrocyte adenylate cyclase (15, 16), as well as enzyme from S49 CYC- lymphoma cells which are deficient in functional G units, to define more clearly the site of calcium inhibition. Our results show that, unlike other inhibitors of adenylate cyclase activity, calcium inhibits the enzyme directly at the level of the catalytic unit. We investigated the mechanism of this inhibition with kinetic analysis and a computer program designed to evaluate the effects of calcium on substrate concentration. These studies demonstrate that calcium inhibits adenylate cyclase by direct competition with magnesium for a regulatory site on the catalytic unit. This inhibition is noncompetitive with respect to MgATP2-, and appears to be independent of calmodulin. EXPERIMENTAL
PROCEDURES
Membrane preparation Turkey erythrocyte membranes were prepared as previously described (17). Heparinized whole turkey blood, shipped on ice, was obtained from Pel-Freeze Biologicals, Inc. Nucleated ghosts, prepared by hypotonic lysis, were homogenized in a Waring blender. After centrifugation of the homogenate, membranes in the upper layer of the pellet were washed, resuspended in 10 mM Tris, pH 7.5, 0.25 M sucrose (Tris/sucrose), and stored in liquid nitrogen until use. Where indicated, calcium-depleted membranes were
utilized. Turkey membranes, prepared as described above, were incubated with 10 mM imidazole, pH 7.5, and 1 mM EGTA for 30 min at 4°C with frequent mixing. The pellets were washed by resuspension and centrifugation twice more in this buffer, and then washed three times in 10 mM Tris, pH 7.5,l mM DTT, 5 mM MgCl*, and 0.1 mM EGTA. The final membrane pellet was then suspended in Tris/sucrose and kept on ice until use. Medane solubilizatim Turkey erythrocyte membranes were incubated with 1 m&f MgC12,1 mM DTT, I-isoproterenol (5 X 10m5M), and either GMP (1 mM) or GppNHp (0.1 m&f) for 30 min at 3’7°C. After three washes in 2 mM Tris, pH 7.5, 0.1 mM MgCl*, 0.1 mM EGTA, 0.25 M sucrose, 1 mM DTT, the membrane pellet was resuspended (final protein 7-10 mg/ml) in this buffer with 0.5% Lubrol PX and incubated at 22°C for 30 min. The mixture was centrifuged at 50,000~for 60 min and the supernatant stored frozen at -20% (16). Partial purification of GppNHpactivated G. Partially purified G units were prepared using GTPagarose as previously described (16), except that EGTA was substituted for EDTA in buffer solutions. After GppNHp elution, G units were concentrated fivefold on an Amicon filter (No. CF25) and placed on ice until use. This preparation was free of catalytic unit activity (16). Preactivatim with &or&. Turkey membranes were suspended in 10 m&i Tris, pH 7.5,0.25 M sucrose, 5 mM Mg&, 1 mM DTT, 0.1 mM EGTA, with 10 mM NaF and incubated at 30°C for 20 min. The membranes were then washed three times with 10 mM Tris, pH 7.5, 1 mM DTT, 1 mM MgC12, and 0.1 mM EGTA (buffer A). The final pellet was resuspended in Tris/sucrose and kept on ice. Preactivation with l-isgproterenol/G~NHp. Turkey erythrocyte membranes were incubated in buffer A with 0.25 M sucrose, 50 PM I-isoproterenol, 0.1 mM GppNHp with or without calcium chloride in the concentrations indicated, for 30 min at 37°C. The reaction was terminated with the addition of 0.1 mM lpropranolol, and the mixture was placed on ice. The membranes were washed three times in buffer A with 0.1 mM I-propranolol, with or without calcium at the concentration shown. The final pellets were suspended in Tris/sucrose. Lknsity gradient u&acentrifugatiun. Aliquots (100 ~1)of either concentrated GppNHp-activated G units from the affinity eluate, or solubilized l-isoproterenol/GppNHp-activated turkey membranes prepared with or without 1 mM CaCl% were applied to a 5-201 sucrose density gradient in 10 mM Tris, pH 7.5, 10 mM MgCl*, 0.1 mM EGTA, 1 mM DIT, 0.1% Lubrol PX with or without 1 m?dCaCl,. The gradients were centrifuged at 150,OOOg for 16 h at 4°C. Cytochrome c, malate dehydrogenase, lactate dehydrogenase, and fumarase were used as calibrating enzymes and were
CALCIUM INHIBITION
OF TURKEY ERYTHROCYTE
assayed spectrophotometrically under conditions where changes in optical density were proportional to the quantity of protein (18). The sedimentation coefficients for these enzymes have been reported previously (19): cytochrome c 1.71 S, malate dehydrogenase 4.32 S, lactate dehydrogenase 7.3 S, and fumarase 9.09 S. Preparation of &9 CYC- cell membranes.These membranes were prepared as previously described @A 21). Adenylate cyclase assay. This assay was performed as previously described (22) with the following modifications. The assay mixture contained 0.1 mM EGTA, 0.1 m&i ATP, 5 mM MgCle, 5 mM phosphoenolpyruvate, and 50 units pyruvate kinase in a final volume of 100 ~1 unless otherwise indicated. PK/PEP was used as the regenerating system to avoid adding contaminating calmodulin (23). The assays were performed in triplicate at 30°C for 20 min. The assay was linear for at least 30 min with or without added calcium. Total and free metal ion and substrate umcentratims. An Orion Calcette 801A automatic calcium analyzer was used to check the calcium concentrations in solutions of calcium chloride. Calcium content of membranes and reagents was determined with a Perkin-Elmer 5000 atomic absorption spectrophotometer with 1 mM lanthanum. Free calcium concentrations were calculated using a computer program modified from that of Perrin and Sayce (24). The following logarithmic association constants were used (25): H’ to EGTA”- 9.46; H+ to HEGTA3- 8.85; H+ to HZEGTA-2 2.65; H+ to H3EGTA3- 2.0. H+ to EDTA”- 10.26;H+ to HEDTA3- 6.16;H+ to HaEDTA*2.67; H’ to HsEDTA’- 1.99. H+ to ATP’- 6.50; H+ to HATPs- 3.95. Ca2+to EGTA4- 10.97;Ca*+ to HEGTA35.3. Ca*+ to EDTA4- 10.59; Ca2+ to HEDTA’- 3.51. Ca2+ to ATP4- 3.99; Ca*’ to HATP3- 2.13 Mp to EGTA’- 5.20; MgZ+to HEGTA’- 3.4. M%’ to EDTA’8.69; Mg2’ to HEDTA+ 2.28. Mg2+to ATP’- 3.84; Mgs to HATPs- 2.09. Mns+ to EGTA4- 12.11; Mn2+ to HEGTA3- 6.59. Mns+ to EDTA’- 14.04, Mn*+ to HEDTA3- 6.90. Mns’ to ATP4- 4.52; Mn2+ to HATP3- 2.61. Protein determinations were performed by the method of Lowry et al (26). Forskolin was obtained from Calbiochem-Behring Corporation. Sources for all other reagents were as cited in (5) or were the best grade available from standard suppliers. Data are presented as the mean of triplicate determinations +/- the standard error of the mean unless otherwise indicated. RESULTS
Inhibition
of
l-isoproterenol-activated
enzyme. A sigmoidal relationship was observed between percentage maximal ade-
ADENYLATE
CALCULATED
CYCLASE
347
LOG FREE [W-i
FIG. 1. Effect of Ca2+concenration on the activity of I-isoproterenol/GppNHp-stimulated adenylate cyclase. Aliquots of turkey erythrocyte membranes containing approximately 0.075 mg protein were added to tubes containing 5 X 10m5M I-isoproterenol, 10s5M GppNHp, and varying concentrations of CaCls and assayed for adenylate cyclase activity. Free calcium concentrations were calculated as described under Experimental Procedures. Maximal adenylate cyclase activity was 206 pmol cAMP/min/mg protein in the absence of added calcium. A computer-generated curve (solid line) with 95% confidence limits (dotted lines) is presented. Each point represents a single experimental determination. 50% inhibition occurred at 2.5 X lo-’ M free calcium concentration (95% confidence limit 2.2-2.8 X 10m4M).
nylate cyclase activity with 5 X lop5 M l-isoproterenol/10-5 M GppNHp and the calculated value for free calcium in the assay (Fig. 1). No stimulation of adenylate cyclase was observed as free calcium concentrations were varied from 4.0 nM to 5.0 mM. With 5 mM magnesium, 50% inhibition of the enzyme occurred at 2.5 X 10e4 M free calcium. Calculated concentrations of MgATP2- varied from 9.7 X 1O-5M with 4.0 nM free calcium, to 8.7 X lo-‘, 7.7 X 10m5and 4.0 X 10e5M with 0.40, 0.90, and 5.0 mM free calcium in the assay respectively. Inhibition of holocatalytic enzyme. Since turkey erythrocyte adenylate cyclase is dependent upon hormone for guanine nucleotide activation of the G unit, it is possible to evaluate the effect of calcium on this activation step and on the activity of the holocatalytic enzyme independently. The following experimental protocol was utilized for this purpose. Membranes were activated during an incubation with l-iso-
348
LASKER, DOWNS, AND AURBACH
)x10-‘M[Ca’+]
5.0x10-M
I
[Ca**]
1
AA+I
FIG. 2. Effect of Ca” on the activation of adenylate cyclase by I-isoproterenol/GppNHp, and on the activity of the holocatalytic enzyme. Turkey erythrocyte membranes were preactivated with l-isoproterenol/GppNHp as described under Experimental Procedures and aliquots of holocatalytic enzyme containing approximately 0.075 mg protein were assayed for adenylate cyclase activity. Maximal adenylate cyclase activity was 200 pmol cAMP/min/mg protein in the absence of added calcium. Calcium at three different free ion concentrations (4.0 X lo-‘, 9.0 X lo-“, 5.0 X lo-’ M) was added to the incubation alone (I), the assay alone (A), or during both incubation and assay (A + I). Activity in (A) and in (A + I) was significantly different from maximal (P < 0.001). Activity in (I) was not significantly different from maximal. The experiment was repeated with lo-’ M trifluoperazine present in both the incubation and assay (solid bars). Maximal adenylate cyclase activity in the presence of trifluoperazine was 170 pmol CAMP/ min/mg protein.
proterenol/GppNHp as described under Experimental Procedures. The holocatalytic enzyme was then assayed for adenylate cyclase activity (Fig. 2). Maximal activity was achieved without added calcium in either the incubation or assay. Effects of calcium at three different free ion concentrations (4.0 X 10m4,9.0 X 10W4,and 5.0 X 10m3M) were tested in the assay alone, the incubation alone, or during both incubation and assay. Addition of calcium in the assay alone caused inhibition of the holocatalytic enzyme in a manner identical to that observed in Fig. 1. Inhibition was similar with calcium added both in the incubation and assay. However, calcium added only during the activation step did not cause significant inhibition. Since activation depends on hormone-dependent exchange of GppNHp for GDP, it therefore appears that the inhibitory effect of
calcium is independent of hormone-receptor interactions and hormone-dependent guanine nucleotide exchange. Trifluoperazine, at concentrations that inhibit calmodulin in other preparations (10m4M), did not affect adenylate cyclase activity (27). Inhibition of turkey erythrocyte adenylate cyclase thus may be mediated through a calmodulin-independent mechanism. Inhibit&m of$uoride-stimulated enzyme. Fluoride stimulates adenylate cyclase by activating the G unit without guanine nucleotide exchange. Turkey erythrocyte adenylate cyclase preactivated by fluoride was inhibited by calcium in a manner identical to that of hormone-stimulated enzyme (Fig. 3). This response was also identical in membranes that had been incubated and washed in imidazole/EGTA, a procedure that depletes other membrane preparations of calcium and calmodulin (28). Calcium, then, appears to inhibit the holocatalytic state of the enzyme. This could be accomplished by one of several mechanisms: (i) a change in conformation of G* to a less active state; (ii) interference
-5 CALCULATED
4
-2
LOG FREE [Ca-]
FIG. 3. Effect of Gas’ on the activity of fluoridestimulated adenylate cyclase. Turkey erythrocyte membranes were preactivated with fluoride as described under Experimental Procedures and aliquots of membranes containing approximately 0.075 mg protein were assayed for adenylate cyclase activity with varying concentrations of CaClz (open circles). The experiment was repeated with membranes that had been incubated and washed in imidazole/EGTA (solid circles) as described under Experimental Procedures. Maximal adenylate cyclase activity was ‘77 pmol cAMP/min/mg protein without added calcium for both membrane preparations.
CALCIUM INHIBITION
OF TURKEY ERYTHROCYTE ADENYLATE
with the interaction of G* and C; (iii) inhibition of C directly; or more than one of these effects. Efects of calcium on G* Corlfomzation. One can distinguish between the active and inactive states of the G unit using density gradient ultracentrifugation (29, 30). In our laboratory, turkey erythrocyte G units activated with either GppNHp or fluoride sediment with a coefficient of 2.7 S whereas unactivated G units sediment with a coefficient of 4.0 S (unpublished data). GppNHp-activated turkey erythrocyte G units were prepared from an affinity eluate as described under Experimental Procedures. Assay of these activated G units with catalytic units from turkey erythrocyte membranes (16) showed an inhibitory response to calcium identical to that observed in Fig. 1 (data not shown). Aliquots of the activated G units were equilibrated with 1 mM calcium and applied to a sucrose density gradient that also contained 1 mM calcium and 0.1 mM EGTA. Control gradients containing no added calcium in either the G-unit preparation or in the gradient were analyzed simultaneously. Fractions from each gradient were assayed with S49 CYC membranes in media containing 1.5 mM EGTA. The activated G unit sedimented at 2.7 S with or without added calcium (Fig. 4). Thus, calcium does not appear to alter the active conformational state of the G unit, as determined by sucrose density gradient ultracentrifugation. Efect.s of calcium on G*-C interacticm Since calcium does not inhibit the formation of the holocatalytic state of the enzyme, it is unlikely that it interferes with G*-C interaction. We studied this question in more detail with the use of density gradient ultracentrifugation. In our laboratory, holocatalytic enzyme sediments with a coefficient of 6.0 S (unpublished data). Excess activated G units sediment at 2.7 S. We examined the effect of calcium on the conformation of the holoactive enzyme with this method. Holoactive enzyme was prepared with I-isoproterenol (50 PM) and GppNHp (0.1 mM) with or without 1 mM CaC& as des-
CYCLASE
349
FRACTION
FIG. 4. Effect of Ca” on density gradient ultracentrifugation of GppNHp-activated G unit. lOO-~1aliquots of GppNHp-activated turkey erythrocyte G units were prepared from an affinity eluate and applied to a 520% sucrose density gradient as described under Experimental Procedures. Activated G units in 1 mM CaCla were applied to a density gradient containing 1 mhl CaClr and centrifuged simultaneously. Enzyme markers for the determination of S,,, were as follows: cytochrome c 1.71 S, malate dehydrogenase 4.32 S, lactate dehydrogenase 7.3 S, and fumarase 9.09 S. 20-~1aliquots from control fractions (solid line) and calcium fractions (dotted line) were assayed for G* activity with 20 pl of S49 CYC membranes in the presence of 1.5 mM EGTA.
cribed under Experimental Procedures. Aliquots of this preparation were applied to sucrose density gradients prepared with or without 1 mM CaClz and assayed with 1.5 mM EGTA. Fractions were assayed alone (to detect holocatalytic enzyme), with S49 CYC- acceptor (to detect G*) and with S49 CYC- plus fluoride (to detect G). The results are presented in Fig. 5. Holocatalytic enzyme was detected at 6.0 S with or without 1 mM CaClz. The yield of holocatalytic enzyme was greater than 65% in both gradients. Excess G* was detected at 2.7 S in each gradient. The ratio of holocatalytic enzyme to total G* (calculated from the area under the appropriate peaks in Fig. 5) was the same with or withut calcium. In neither gradient was unactivated G unit (G) detected at 4.0 S. These results suggest that calcium does not change the conformation of the holocatalytic enzyme. Further, calcium does not appear to cause detectable dissociation of G* from C, nor convert G* to G. Since the addition of excess C in the form of
350
LASKER, DOWNS, AND AURBACH
5
10
15
20
FRACTION
s
5
10
15
20
FRACTION
FIG. 5. Effect of Ca2+on density gradient ultracentrifugation of holoactive adenylate cyclase. Holoactive solubilized turkey erythrocyte adenylate cyclase was prepared with or without 1 mM CaCls as described under Experimental Procedures. loo-al aliquota of these preparations were applied to 5-20s sucrose density gradients prepared with or without 1 mM CaClc. 20-~1aliquots from control fractions (A) and calcium fractions (B) were assayed for holocatalytic activity (dotted line), for G* activity with S49 CYC membranes (solid lines), and for G activity with S49 CYC membranes and 10-s M NaF (dashed lines) in the presence of 1.5 mM EGTA.
CYC- membranes enhanced activity of the holocatalytic (6.0 S) peak in the calcium gradient, it is possible that calcium increases the lability of the catalytic unit under the conditions of the gradient. Inhibition of catalytic unit. Since calcium inhibition of adenylate cyclase appears to be independent of hormone-receptor ineractions, hormone-dependent guanine nucleotide exchange, G* conformation, and G*-C interactions, it is possible that inhibition is effected at the level of the catalytic unit. We tested this possibility with the -- enzyme from S49 CYClymphoma cells which are deficient in
functional G units. Catalytic activity of the enzyme from these cells can be detected with manganese and ATP. Calcium inhibition curves were generated at varying concentrations of manganese present in the assay (Fig. 6). The enzyme activity of CYC- membranes with manganese was more resistant to inhibition by calcium than was turkey erythrocyte enzyme assayed with 5 mM magnesium. This resistance became more pronounced as the concentration of manganese in the assay was increased. Plots of reciprocal velocity versus reciprocal free manganese concentration at several calcium concentrations (Fig. 7) indicated competitive inhibition by calcium with manganese. The Km for manganese, calculated from linear regression analysis of reciprocal data with no added calcium in Fig. 7, was 2.1 X 10e4M. The Ki for calcium, calculated according to Dixon (31), was 3.6 X 10m4M. Computer analysis of MnATP2- concentrations with or without added calcium (Fig. 8) suggested that this competition was at the level of ATP, and that, within the range of calculated substrate concentration, in-
CALCULATED
LOG FREE [Cd+]
FIG. 6. Effect of Cax+ on manganese-stimulated adenylate cyclase in S49 CYC- membranes. Aliquots of S49 CYC membranes containing approximately 0.04 mg protein were assayed for adenylate cyclase activity with 1.0 X lo-so Mn*+ (open circles), 5.0 X low3 M Mn2+ (solid circles), and 5.0 X low4 M Mn2+ (open triangles) at varying Ca2+concentrations. Maxima! adenylate cyclase activity without added calcium was 6.8,10.1, and 4.3 pmol cAMP/min/mg protein with 10-s, 5 X lo-*, or 5 X 10e4M Mns+.
CALCIUM INHIBITION
OF TURKEY ERYTHROCYTE ADENYLATE
hibition of enzyme activity with calcium could be explained by depletion of the active substrate, MnATP2-. Recently, forskolin, a cardioactive diterpene, has been shown to activate adenylate cyclase from S49 CYC lymphoma cells, and to convert this variant enzyme to a form that can utilize MgATP2- as substrate (32). Forskolin-stimulated adenylate cyclase in S49 CYC- membranes, with magnesium/O.1 mM ATP as substrate, was inhibited by calcium in a manner similar to that of hormone-stimulated turkey erythrocyte enzyme (data not shown). This inhibition became less pronounced as the concentration of magnesium in the assay was increased. We studied the relationship of free magnesium concentration to calcium inhibition in experiments designed to keep the concentration of MgATP2- constant at each concentration of calcium. As shown in Fig. 9, free magnesium, in concentrations up to 4.4 mM, stimulated enzyme activity independently of MgATP2concentration. This suggests the existence of a free magnesium regulatory site in forskolin-stimulated CYC. Plots of recipro-
I
L
I
I
1234567 l/free iMnz+l ImM-‘I
FIG. 7. Effect of Ca” on the activity of adenylate cyclase with Mn2+ in S49 CYC- membranes. Aliquots of S49 CYC- membranes containing approximately 0.04 mg protein were assayed for adenylate cyclase activity. Assays were performed with no added calcium (solid circles), 5.0 X lo-* M calcium (open circles), 1.0 X 1O-3 M calcium (open triangles), or 5.0 X lo+M CakiUm (open Squares). Lines were generated by analysis of reciprocal data using linear regression analvsis.
Calculated
CYCLASE
lMnATP2-1
351
10-5M
FIG. 8. Effect of Ca*+ on the relationship between MnATP’- concentration and adenylate cyclase activity in S49 CYC- membranes. Data from Fig. 7 was analyzed using computer-generated MnATP’- concentrations at each concentration of calcium. Assays were performed with no added calcium (open circles), 5.0 X 1O-4M Ca’+ (solid circles), 1.0 X 10W3M Ca2’ (open triangles), or 5.0 X lo-’ M Ca2+(solid triangles). The concentration of ATP in the assay was 0.1 mM.
cal velocity versus reciprocal free magnesium concentration at several calcium concentrations (Fig. 9) showed competitive inhibition of magnesium by calcium. The Km for magnesium was 2.6 X low4 M, and the Ki for calcium was 0.70 X 10e4M. We next studied the relationship of MgATP2- concentration to calcium inhibition in experiments designed to keep the concentration of free magnesium relatively constant at each concentration of calcium. Plots of reciprocal velocity versus reciprocal MgATP2- concentration at several calcium concentrations (Fig. 10) showed noncompetitive inhibition of calcium with substrate. The Kmfor MgATP2in these experiments was 2.8 X 10e5M, and the Ki for calcium was 0.90 X low4 M. DISCUSSION
The current work demonstrates that calcium inhibits adenylate cyclase by direct competition with magnesium for a regulatory site on the catalytic unit. Earlier studies have shown that calcium inhibits the maximal activation of adenylate cyclase by agonist or fluoride (8, 10). The apparent Ki for calcium varies in different reports: 0.86 X 10e4 M for turkey erythro-
352
LASKER, DOWNS, AND AURBACH
l/free
wdg*+1
ImM-‘I
FIG. 9. Effect of Ca*+ on magnesium-stimulated adenylate cyclase activity in 549 CYC- membranes in the presence of forskolin. Aliquots of S49 CYC membranes containing approximately 0.08 mg protein were assayed for adenylate cyclase activity with 0.1 mM forskolin, 0.1 mM ATP and no added calcium (open circles), 5.0 X lo-‘Id Ca*+ (solid circles), and 1.0 X lo-’ M Ca*+ (solid triangles) at varying magnesium concentrations. Enzyme activity was inbibited at magnesium concentrations greater than 4.4 mM. MgATP2- concentrations were 9.3 X lo-’ M with no added calcium, 8.4 X 10e6M with 5.0 X lo-” M Ca2’, and 6.9 X lo-’ M with 1.0 X 10m3bf Ca’+. Linear regression analysis was carried out on data for reciprocal enzyme velocity versus reciprocal Mgs+ concentration.
cyte (34), 0.5-1.0 X 10m4M for parathyroid tissue (35,36) and 2.0 X 10e4M for adrenal cortex (8). These several results are not strictly comparable because EGTA buffered systems were not used in some experiments, and in others free calcium concentrations were neither measured nor calculated. In addition, magnesium concentration varied considerably. We have calculated free calcium concentrations from the data in several previous reports. Fifty percent inhibition of adenylate cyclase was achieved with calculated free calcium concentrations of 1.4 X 10c4 and 4.8 X 10e4M in brain (6,37), and 2.0 X 10e4 M in parathyroid (36). These analyses agree well with our own.3 3 One exception is a report that guinea pig brain adenylate cyclase is half-maximally inhibited by 35 X lo-’ M free calcium (43). Free calcium concentrations were calculated by a computer program similar
Previous work has suggested that calmodulin does not mediate calcium inhibition of adenylate cyclase (38, 39). Our results support these observations by experiments with trifluoperazine, EGTA-imidazole washed membranes, and tests of enzyme sensitivity to calcium and magnesium. In brain, which shows a biphasic response to calcium, calmodulin does appear to mediate the stimulatory action of calcium, and an activated G unit may be required for calmodulin sensitivity of the enzyme (7, 34, 35, 37, 44). In the adrenal, calcium stimulates activation of enzyme by guanine nucleotides, although it is not known whether calmodulin is involved in this stimulation (8). The stimulation of adenylate cyclase by calcium in brain and adrenal appears to be dependent on G. The lack of effect of calcium on G and G-C interactions that we have demonstrated in the turkey erythrocyte is consistent with the lack of stimulation by calcium in this system. Studies in the turkey erythrocyte and in the adrenal have shown that calcium does not influence hormone binding to receptor, and that calcium can inhibit the holocatalytic state of the enzyme (8,33,34, 37). However, these studies did not establish which subunit is primarily affected by calcium, nor the mechanism of this inhibition. We have utilized recent information on the components of adenylate cyclase to analyze further the interaction of calcium with this system. By studying the effects of calcium on hormone activation and holocatalytic activity of the enzyme independently, we have shown that calcium does not influence hormone-receptor interactions or hormone-dependent guanine nucleotide exchange. We evaluated to that employed in our studies. The original data were not reported so that we cannot recalculate the data. Our calculated free calcium concentrations could be invalid if the membrane preparations bound enough calcium to cause a significant reduction in the free calcium of the assay medium. This is unlikely, however, since the calcium inhibition curves were unaffected by varying the amount of membrane protein in the medium.
CALCIUM INHIBITION
OF TURKEY ERYTHROCYTE
the effect of calcium on the G unit and on G-C interactions with the use of density gradient ultracentrifugation. This technique can distinguish between the active and inactive states of the G unit. Regardless of the mechanism accounting for the difference in sedimentation coefficients for these two states (i.e., changes in conformation or true changes in size due to dissociation of subunits), calcium does not appear to influence this process. Calcium does not change the sedimentation coefficient of G* nor of the holocatalytic enzyme. In addition, calcium does not appear to cause measurable dissociation of the G*-C complex, nor convert G* to G. We have established that calcium can directly inhibit the catalytic unit of adenylate cyclase through the use of enzyme from S49 CYC- lymphoma cells which are deficient in functional G units. Although high concentrations of calcium can inhibit manganese-stimulated CYC- adenylate cyclase, this appears to be secondary to competition between calcium and manganese for ATP. This finding stresses the importance of evaluating the effects of calcium on substrate concentrations in experiments where other cations are present. Calcium inhibition of forskolinstimulated CYC enzyme with magnesium and ATP, however, cannot be explained by depletion of substrate. Under these conditions, calcium competes with magnesium for a metal binding site on the enzyme itself. This finding is in agreement with prior work in bone and cardiac adenylate cyclase systems (11, 41), but not in the turkey erythrocyte (10). Whereas previous studies have demonstrated the existence of sites for free magnesium on adenylate cyclase (14), the location of these sites has not been fully clarified. Since nucleotide or nucleotide plus hormone increases the apparent affinity of free magnesium for its binding sites in wild-type enzyme, and since native CYC- enzyme is not stimulated by magnesium, it has been postulated that free magnesium interacts with the G unit of adenylate cyclase. However, since forskolin-stimulated CYC enzyme can interact with magnesium both
ADENYLATE 0.5
CYCLASE
353
r
1 I lMgATP*-1
(mM-‘1
FIG. 10. Effect of Ca*+ on the relationship between enzyme velocity and MgATP*- concentration in forskolin-stimulated S49 CYC- membranes. Aliquots of S49 CYC- membranes containing approximately 0.08 mg protein were assayed for adenylate cyclase activity with no added calcium (open circles), 5.0 X 10e4 M Ca2+(solid circles), or 1.0 X 10e3M Ca’+ (solid triangles), at varying MgATP2- concentrations. Free M$+ concentrations were 2.2 X lo-” M with no added calcium, 3.6 X lo-’ M with 5.0 X lo-’ M calcium, and 4.4 X 10M4M with 1.0 X 10m3M calcium. Lines were generated by linear regression analysis of reciprocal enzyme velocity versus reciprocal MgATP2- concentration.
in complex with substrate and, as we have shown, at an independent binding site, it is likely that one such independent magnesium binding site is on the catalytic unit of the enzyme. Since calcium competes with magnesium for this regulatory site, and since calcium does not appear to influence R, G*, or G-C interactions of adenylate cyclase, it appears tha calcium inhibits the catalytic unit of the enzyme directly. The possibility remains, however, that CYC contains an aberrant subunit of G which can be stimulated in the presence of forskolin, and that the metal binding site is on this protein, rather than on the catalytic unit. Clarification of this problem must await further purification of c. Cyclic AMP is an important intracellular messenger. Thus, the regulation of adenylate cyclase has been of considerable interest. With the exception of forskolin, known activators of adenylate cyclase ei-
354
LASKER,
DOWNS, AND AURBACH
ther require the presence of, or directly interact with, the guanine nucleotide binding protein. Calcium is one of the few inhibitors of the enzyme. Calcium inhibition of adenylate cyclase may be of particular importance in the parathyroid cell where CAMP regulates parathyroid hormone secretion and the amount of CAMP produced is controlled in part by calcium. Indeed, two recent reports (36,42) have shown reduced sensitivity of adenylate cyclase to inhibition by calcium in tissue from adenomatous and hyperplastic parathyroid glands, suggesting the possibility that abnormal calcium regulation of adenylate cyclase might be part of the etiology of hyperparathyroidism. The identification in the current work of direct calcium competition for a magnesium binding site on C is the first demonstration of inhibition of adenylate cyclase at the level of the catalytic unit. This illuminates a further mechanism for possible physiologic control of the enzyme. ACKNOWLEDGMENTS We wish to thank Dr. John Fakunding for his aid with computer calculations, Dr. Allen Spiegel for his helpful discussions, Mr. Charles Woodard and Mrs. Sharon Reen for expert technical assistance, and Mrs. Lillian Perry for expert secretarial assistance. REFERENCES 1. CARON, M. G., LIMBIRD, L. E., AND LEFKOWITZ, R. J. (1979) MoL CelL Biochem 28,45. 2. NEER, E. J. (1978) J. Bid Chem 253,1498-X562. 3. KASLOW, H. R., JOHNSON, G. L., BROTHERS, V. M., AND BOURNE, H. R. (1980) J. BioL Chem. 255, 3736-3741. 4. CASSEL, D., AND SELINGER, Z. (1978) Proc Nat.
Acad Sci USA 75.4155-4159. 5. DOWNS, R. W., JR., SPIEGEL, A. M., SINGER, M., REEN, S., AND AURBACH, G. D. (1980) J. BioL Chem 255, 949-954. 6. SPIEGEL, A. M., AND DOWNS, R. W. (1981) Endocrine Rev. 2, 275-305. 7. BROSTROM, M. A., BROSTROM, C. O., BRECKENRIDGE, B. M., AND WOLFF, D. J. (1976) J. Bid Chem 251,4744-4750. 8. MAHAFFEE, D. D., AND ONTJES, D. A. (1980) J. BioL Chem 255, 1565-1571.
9. BIRNBAUMER, L. (1973) B&him. Biophys. Actn 300,129-158. 10. STEER, M. L., AND LEVITZKI, A. (1975) J. Biol Chem 250, 2080-2084. 11. DRUMMOND, G. I., AND DUNCAN, L. (1970) J. Bid
Chem 245,976-983. 12. LONDOS, C., AND RODBELL, M. (1977) in Drug Action at the Molecular Level (Roberts, G. C. K., ed.), pp. 235-247, University Park Press, Baltimore. 13. PERKINS, J. P. (1973) in Advances in Cyclic Nucleotide Research (Greengard, P., and Robison, G. A., eds), p. 21, Raven Press, New York. 14. CECH, S. Y., BROADDUS, W. C., AND MAGUIRE, M. E. (1980) MoL Cell. B&hem 33, 67-92. 15. PFEUFFER, T. (1977) J. BioL Chem 252.7224-7234. 16. SPIEGEL, A. M., DOWNS, R. W., JR., AND AURBACH, G. D. (1979) J. Cyclic Nucleotide Res. 5, 3-17. 17. BILEZIKIAN, J. P., AND AURBACH, G. D. (1973)
J. BioL Chem 248, 5577-5583. 18. WORTHINGTON BIOCHEMICAL COMPANY (1977) Worthington Enzyme Manual, Freehold, N. J. 19. HAGA, T., HAGA, K., AND GILMAN, A. G. (1977)
J. BioL Chem 252, 5776-5782. 20. Ross, E. M., HOWLEW, A. C., FERGUSON, K. M., AND GILMAN, A. G. (1978) J. BabL Chem 253, 64016412. 21. BOURNE, H. R., COFFINO, P., AND TOMKINS, G. M. (1975) Science 187, 750-752. 22. SPIEGEL, A. M., DOWNS, R. W., JR., AND AURBACH, G. D. (1977) Biochem Biophys. Res. Cemmun 76.758-764. 23. GOLDHAMMER, A. R., WOLFF, J., COOK, G. H., BERKOWITZ. S. A., KLEE. C. B.. MANCLARK. C. R., AND. HEWLETT, El L. (1981) Eur. i Biochem 115,605-609. 24. PERRIN, D. D., AND SAYCE, I. G. (1967) Taluntu 14, 833-842: 25. SILLEN, L. G., AND MARTELL, A. E. (1971) Stability Constants of Metal Ion Complexes, Suppl. 1. Special Publication 25, The Chemical Society, Burlington House, London. 26. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J. (1951) J. Bid Chem 193, 265-275. 27. WOLFF, J., COOK, G. H., GOLDHAMMER, A. R., AND BERKOWITZ, S. A. (1980) Proc, Nat. Acad Sci
USA 77,3841-3844. 28. KLEE, C. B., CROUCH, T. H., AND RICHMAN, P. G. (1980) Annu. Rev, Biochem 49,489-515. 29. PFEUFFER, J. (1979) FEBS L.&f. 101.85-89. 36. HOWLETT, A. C., AND GILMAN, A. G. (1980) J. BioL Chem 265,2861-2866. 31. DIXON, M., AND WEBB, E. (1964) Enzymes, Academic Press, New York. 32. SEAMON, K., PADGETT, W., AND DALY, J. W. (1981)
CALCIUM
INHIBITION
OF TURKEY
ERYTHROCYTE
P~OGNat Acad Sci USA 78, 3363-3367, and personal communication. 33. HANSKI, E., SEVILLA, N., AND LEVITZKI, A. (1977)
Eur. J. Biochem 76,513-520. 34. LEVITZKI, A., SEVILLA, N., AND ATLAS, D. (1975) J. IUOL BioL 97, 35-53. 35. TOSCANO, W. A., JR., WESTCOTT, K. R., LAPORTE, D. C., AND STORM, D. R. (1979) Proc Nat Acud
Ski USA 76.5582-5586. 36. BELLORIN-FONT, E., MARTIN, K. J., FREITAG, J. J., ANDERSON, C., SICARD, G., SLATOPOLSKY, E., AND KLAHR, S. (1981) J. clin Endorrind Metab. 52, 499-507. 37. SEVILLA, N., STEER, M. L., AND LEVITZKI, A. (1976) Biochemist?y 15.4393-3499. 38. WESTCOTP, K. R., LAPORTE, D. C., AND STORM,
ADENYLATE
CYCLASE
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D. R. (1979) Proc Nat. Acad. Sci USA 76,204208. 39. BROSTROM, M. A., BROSTROM, C. O., BRECKENRIDGE, B. McL., AND WOLFF, D. J. (1978) Ad-
van Cyclic Nuckotide Ran 9,85-99. 40. NEER, E. J. (1979) J. Bid Chem 254,2089-2096. 41. RODAN, S. B., GOLUB, E. E., EGAN, J. J., AND RoDAN, G. A. (1980) Biochem. J. 185.629-637. 42. ONTJES, D. A., MAHAFFEE, D. D., AND WELLS, S. A. (1981) Metabolism 30,406-411. 43. PIASCIK, M. T., WISLER, P. L., JOHNSON, C. L., AND POTTER, J. D. (1980) J. BioL Chem 255,41764181. 44. CHEUNG, W. Y., BRADHAM, L. S., LYNCH, T. J., LIN, Y. M., AND TALLANT, E. A. (1975) Biochem Biophys Res. Commun 66, 1055-1062.
Calcium inhibition of Adenylate Cyciase: Studies in Turkey Erythrocyte and S49 CYC- Cell Membranes ROZ D. LASKER,l ROBERT W. DOWNS, JR., AND GERALD D. AURBACH Metabolic Disease Branch, National Institute of Arthritis, Diabetes, and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland, 20205 Received November 16, 1981, and in revised form February 12,1982
We studied the mechanism of calcium inhibition of adenylate cyclase using partially purified components of the enzyme complex and computer analysis of free metal and substrate concentrations. A sigmoidal relationship was observed between percentage maximal adenylate cyclase activity with l-isoproterenol/guanylyl-P,y-imidodiphosphate and the calculated free calcium. Fifty percent inhibition occurred at 2.5 X lop4 M free calcium. This inhibition appeared to be independent of calmodulin. Calcium inhibited the holocatalytic enzyme in a manner indentical to that of the native enzyme, but did not affect the ability of 1-isoproterenol and guanylyl-&y-imidodiphosphate to promote the formation of the holocatalytic state. There was no effect of calcium on the conformation of the activated G unit nor on the holocatalytic enzyme as determined by sedimentation velocity analysis. Calcium did not cause detectable dissociation of the activated G unit from the catalytic unit, nor convert activated G unit to an inactive form. Calcium inhibition of the catalytic unit of adenylate cyclase was studied in S49 CYC- lymphoma cell membranes. High concentrations of calcium inhibited manganesestimulated CYC- enzyme, but this could be explained by competition between calcium and manganese for ATP. With addition of forskolin, CYC- adenylate cyclase utilized MgATP2- as substrate and was shown to have a separate binding site for free magnesium. Calcium inhibited forskolin-stimulated CYC- enzyme by competing with free magnesium for its regulatory site. Calcium inhibition was noncompetitive with respect to MgATP2-. We conclude that calcium inhibits adenylate cyclase by direct competition with magnesium for a regulatory site on the catalytic unit.
Adenylate cyclase (EC 4.6.1.1) is a membrane-bound enzyme complex that translates extracellular signals into intracellular information through the generation of the second messenger, cyclic AMP. The enzyme complex consists of at least three subunits: a hormone receptor (R) on the external surface of the plasma membrane (l), and a catalytic unit (C) and guanine nucleotide binding protein (G), both located on the cytoplasmic surface of the plasma membrane (2, 3). Hormonal con-
trol of CAMP formation can be described by the following model (4-6): (1) binding of hormone (H) to R facilitates exchange of GTP for GDP on G, forming an activated G unit (G*); (2) G* interacts with C to enhance the formation of CAMP from ATP; (3) hydrolysis of GTP to GDP deactivates the enzyme. Certain analogs of GTP (i.e., G~~NHP)~ are resistant to hydrolysis, and in the presence of hormone ’ Abbreviations used: GppNHp, guanylyl-&y-imidodiphosphate; EGTA, ethylene glycol bis (@-aminoethyl etber)N,N,N’,N’-tetraacetic acid, PEP, phosphoenol pyruvate.
1Author to whom all correspondence should be addressed. 345
0003-9861/82/070345-11$02.00/O Copyright All righta
Q 1982 by Academic Pnss. Inc. of reproduction in any form reserved.
346
LASKER, DOWNS, AND AURBACH
can form a persistently activated state of the enzyme, the holocatalytic state. Fluoride activates adenylate cyclase in vitro by activating G without participation of R or guanine nucleotide exchange (6). Calcium is an important modulator of the activity of adenylate cyclase. Several tissues, including brain and adrenal, show a biphasic response to calcium, with low concentrations stimulating and higher concentrations inhibiting enzyme activity (7,8). Most tissues, however, show only an inhibitory response to this ion (9-14). Previous work has shown that calcium inhibits maximal activation of the enzyme by hormone and fluoride (8, 10). However, since these studies utilized crude membrane preparations, neither the mechanism nor the locus of this inhibition has been fully elucidated. We used partially separated components of turkey erythrocyte adenylate cyclase (15, 16), as well as enzyme from S49 CYC- lymphoma cells which are deficient in functional G units, to define more clearly the site of calcium inhibition. Our results show that, unlike other inhibitors of adenylate cyclase activity, calcium inhibits the enzyme directly at the level of the catalytic unit. We investigated the mechanism of this inhibition with kinetic analysis and a computer program designed to evaluate the effects of calcium on substrate concentration. These studies demonstrate that calcium inhibits adenylate cyclase by direct competition with magnesium for a regulatory site on the catalytic unit. This inhibition is noncompetitive with respect to MgATP2-, and appears to be independent of calmodulin. EXPERIMENTAL
PROCEDURES
Membrane preparation Turkey erythrocyte membranes were prepared as previously described (17). Heparinized whole turkey blood, shipped on ice, was obtained from Pel-Freeze Biologicals, Inc. Nucleated ghosts, prepared by hypotonic lysis, were homogenized in a Waring blender. After centrifugation of the homogenate, membranes in the upper layer of the pellet were washed, resuspended in 10 mM Tris, pH 7.5, 0.25 M sucrose (Tris/sucrose), and stored in liquid nitrogen until use. Where indicated, calcium-depleted membranes were
utilized. Turkey membranes, prepared as described above, were incubated with 10 mM imidazole, pH 7.5, and 1 mM EGTA for 30 min at 4°C with frequent mixing. The pellets were washed by resuspension and centrifugation twice more in this buffer, and then washed three times in 10 mM Tris, pH 7.5,l mM DTT, 5 mM MgCl*, and 0.1 mM EGTA. The final membrane pellet was then suspended in Tris/sucrose and kept on ice until use. Medane solubilizatim Turkey erythrocyte membranes were incubated with 1 m&f MgC12,1 mM DTT, I-isoproterenol (5 X 10m5M), and either GMP (1 mM) or GppNHp (0.1 m&f) for 30 min at 3’7°C. After three washes in 2 mM Tris, pH 7.5, 0.1 mM MgCl*, 0.1 mM EGTA, 0.25 M sucrose, 1 mM DTT, the membrane pellet was resuspended (final protein 7-10 mg/ml) in this buffer with 0.5% Lubrol PX and incubated at 22°C for 30 min. The mixture was centrifuged at 50,000~for 60 min and the supernatant stored frozen at -20% (16). Partial purification of GppNHpactivated G. Partially purified G units were prepared using GTPagarose as previously described (16), except that EGTA was substituted for EDTA in buffer solutions. After GppNHp elution, G units were concentrated fivefold on an Amicon filter (No. CF25) and placed on ice until use. This preparation was free of catalytic unit activity (16). Preactivatim with &or&. Turkey membranes were suspended in 10 m&i Tris, pH 7.5,0.25 M sucrose, 5 mM Mg&, 1 mM DTT, 0.1 mM EGTA, with 10 mM NaF and incubated at 30°C for 20 min. The membranes were then washed three times with 10 mM Tris, pH 7.5, 1 mM DTT, 1 mM MgC12, and 0.1 mM EGTA (buffer A). The final pellet was resuspended in Tris/sucrose and kept on ice. Preactivation with l-isgproterenol/G~NHp. Turkey erythrocyte membranes were incubated in buffer A with 0.25 M sucrose, 50 PM I-isoproterenol, 0.1 mM GppNHp with or without calcium chloride in the concentrations indicated, for 30 min at 37°C. The reaction was terminated with the addition of 0.1 mM lpropranolol, and the mixture was placed on ice. The membranes were washed three times in buffer A with 0.1 mM I-propranolol, with or without calcium at the concentration shown. The final pellets were suspended in Tris/sucrose. Lknsity gradient u&acentrifugatiun. Aliquots (100 ~1)of either concentrated GppNHp-activated G units from the affinity eluate, or solubilized l-isoproterenol/GppNHp-activated turkey membranes prepared with or without 1 mM CaCl% were applied to a 5-201 sucrose density gradient in 10 mM Tris, pH 7.5, 10 mM MgCl*, 0.1 mM EGTA, 1 mM DIT, 0.1% Lubrol PX with or without 1 m?dCaCl,. The gradients were centrifuged at 150,OOOg for 16 h at 4°C. Cytochrome c, malate dehydrogenase, lactate dehydrogenase, and fumarase were used as calibrating enzymes and were
CALCIUM INHIBITION
OF TURKEY ERYTHROCYTE
assayed spectrophotometrically under conditions where changes in optical density were proportional to the quantity of protein (18). The sedimentation coefficients for these enzymes have been reported previously (19): cytochrome c 1.71 S, malate dehydrogenase 4.32 S, lactate dehydrogenase 7.3 S, and fumarase 9.09 S. Preparation of &9 CYC- cell membranes.These membranes were prepared as previously described @A 21). Adenylate cyclase assay. This assay was performed as previously described (22) with the following modifications. The assay mixture contained 0.1 mM EGTA, 0.1 m&i ATP, 5 mM MgCle, 5 mM phosphoenolpyruvate, and 50 units pyruvate kinase in a final volume of 100 ~1 unless otherwise indicated. PK/PEP was used as the regenerating system to avoid adding contaminating calmodulin (23). The assays were performed in triplicate at 30°C for 20 min. The assay was linear for at least 30 min with or without added calcium. Total and free metal ion and substrate umcentratims. An Orion Calcette 801A automatic calcium analyzer was used to check the calcium concentrations in solutions of calcium chloride. Calcium content of membranes and reagents was determined with a Perkin-Elmer 5000 atomic absorption spectrophotometer with 1 mM lanthanum. Free calcium concentrations were calculated using a computer program modified from that of Perrin and Sayce (24). The following logarithmic association constants were used (25): H’ to EGTA”- 9.46; H+ to HEGTA3- 8.85; H+ to HZEGTA-2 2.65; H+ to H3EGTA3- 2.0. H+ to EDTA”- 10.26;H+ to HEDTA3- 6.16;H+ to HaEDTA*2.67; H’ to HsEDTA’- 1.99. H+ to ATP’- 6.50; H+ to HATPs- 3.95. Ca2+to EGTA4- 10.97;Ca*+ to HEGTA35.3. Ca*+ to EDTA4- 10.59; Ca2+ to HEDTA’- 3.51. Ca2+ to ATP4- 3.99; Ca*’ to HATP3- 2.13 Mp to EGTA’- 5.20; MgZ+to HEGTA’- 3.4. M%’ to EDTA’8.69; Mg2’ to HEDTA+ 2.28. Mg2+to ATP’- 3.84; Mgs to HATPs- 2.09. Mns+ to EGTA4- 12.11; Mn2+ to HEGTA3- 6.59. Mns+ to EDTA’- 14.04, Mn*+ to HEDTA3- 6.90. Mns’ to ATP4- 4.52; Mn2+ to HATP3- 2.61. Protein determinations were performed by the method of Lowry et al (26). Forskolin was obtained from Calbiochem-Behring Corporation. Sources for all other reagents were as cited in (5) or were the best grade available from standard suppliers. Data are presented as the mean of triplicate determinations +/- the standard error of the mean unless otherwise indicated. RESULTS
Inhibition
of
l-isoproterenol-activated
enzyme. A sigmoidal relationship was observed between percentage maximal ade-
ADENYLATE
CALCULATED
CYCLASE
347
LOG FREE [W-i
FIG. 1. Effect of Ca2+concenration on the activity of I-isoproterenol/GppNHp-stimulated adenylate cyclase. Aliquots of turkey erythrocyte membranes containing approximately 0.075 mg protein were added to tubes containing 5 X 10m5M I-isoproterenol, 10s5M GppNHp, and varying concentrations of CaCls and assayed for adenylate cyclase activity. Free calcium concentrations were calculated as described under Experimental Procedures. Maximal adenylate cyclase activity was 206 pmol cAMP/min/mg protein in the absence of added calcium. A computer-generated curve (solid line) with 95% confidence limits (dotted lines) is presented. Each point represents a single experimental determination. 50% inhibition occurred at 2.5 X lo-’ M free calcium concentration (95% confidence limit 2.2-2.8 X 10m4M).
nylate cyclase activity with 5 X lop5 M l-isoproterenol/10-5 M GppNHp and the calculated value for free calcium in the assay (Fig. 1). No stimulation of adenylate cyclase was observed as free calcium concentrations were varied from 4.0 nM to 5.0 mM. With 5 mM magnesium, 50% inhibition of the enzyme occurred at 2.5 X 10e4 M free calcium. Calculated concentrations of MgATP2- varied from 9.7 X 1O-5M with 4.0 nM free calcium, to 8.7 X lo-‘, 7.7 X 10m5and 4.0 X 10e5M with 0.40, 0.90, and 5.0 mM free calcium in the assay respectively. Inhibition of holocatalytic enzyme. Since turkey erythrocyte adenylate cyclase is dependent upon hormone for guanine nucleotide activation of the G unit, it is possible to evaluate the effect of calcium on this activation step and on the activity of the holocatalytic enzyme independently. The following experimental protocol was utilized for this purpose. Membranes were activated during an incubation with l-iso-
348
LASKER, DOWNS, AND AURBACH
)x10-‘M[Ca’+]
5.0x10-M
I
[Ca**]
1
AA+I
FIG. 2. Effect of Ca” on the activation of adenylate cyclase by I-isoproterenol/GppNHp, and on the activity of the holocatalytic enzyme. Turkey erythrocyte membranes were preactivated with l-isoproterenol/GppNHp as described under Experimental Procedures and aliquots of holocatalytic enzyme containing approximately 0.075 mg protein were assayed for adenylate cyclase activity. Maximal adenylate cyclase activity was 200 pmol cAMP/min/mg protein in the absence of added calcium. Calcium at three different free ion concentrations (4.0 X lo-‘, 9.0 X lo-“, 5.0 X lo-’ M) was added to the incubation alone (I), the assay alone (A), or during both incubation and assay (A + I). Activity in (A) and in (A + I) was significantly different from maximal (P < 0.001). Activity in (I) was not significantly different from maximal. The experiment was repeated with lo-’ M trifluoperazine present in both the incubation and assay (solid bars). Maximal adenylate cyclase activity in the presence of trifluoperazine was 170 pmol CAMP/ min/mg protein.
proterenol/GppNHp as described under Experimental Procedures. The holocatalytic enzyme was then assayed for adenylate cyclase activity (Fig. 2). Maximal activity was achieved without added calcium in either the incubation or assay. Effects of calcium at three different free ion concentrations (4.0 X 10m4,9.0 X 10W4,and 5.0 X 10m3M) were tested in the assay alone, the incubation alone, or during both incubation and assay. Addition of calcium in the assay alone caused inhibition of the holocatalytic enzyme in a manner identical to that observed in Fig. 1. Inhibition was similar with calcium added both in the incubation and assay. However, calcium added only during the activation step did not cause significant inhibition. Since activation depends on hormone-dependent exchange of GppNHp for GDP, it therefore appears that the inhibitory effect of
calcium is independent of hormone-receptor interactions and hormone-dependent guanine nucleotide exchange. Trifluoperazine, at concentrations that inhibit calmodulin in other preparations (10m4M), did not affect adenylate cyclase activity (27). Inhibition of turkey erythrocyte adenylate cyclase thus may be mediated through a calmodulin-independent mechanism. Inhibit&m of$uoride-stimulated enzyme. Fluoride stimulates adenylate cyclase by activating the G unit without guanine nucleotide exchange. Turkey erythrocyte adenylate cyclase preactivated by fluoride was inhibited by calcium in a manner identical to that of hormone-stimulated enzyme (Fig. 3). This response was also identical in membranes that had been incubated and washed in imidazole/EGTA, a procedure that depletes other membrane preparations of calcium and calmodulin (28). Calcium, then, appears to inhibit the holocatalytic state of the enzyme. This could be accomplished by one of several mechanisms: (i) a change in conformation of G* to a less active state; (ii) interference
-5 CALCULATED
4
-2
LOG FREE [Ca-]
FIG. 3. Effect of Gas’ on the activity of fluoridestimulated adenylate cyclase. Turkey erythrocyte membranes were preactivated with fluoride as described under Experimental Procedures and aliquots of membranes containing approximately 0.075 mg protein were assayed for adenylate cyclase activity with varying concentrations of CaClz (open circles). The experiment was repeated with membranes that had been incubated and washed in imidazole/EGTA (solid circles) as described under Experimental Procedures. Maximal adenylate cyclase activity was ‘77 pmol cAMP/min/mg protein without added calcium for both membrane preparations.
CALCIUM INHIBITION
OF TURKEY ERYTHROCYTE ADENYLATE
with the interaction of G* and C; (iii) inhibition of C directly; or more than one of these effects. Efects of calcium on G* Corlfomzation. One can distinguish between the active and inactive states of the G unit using density gradient ultracentrifugation (29, 30). In our laboratory, turkey erythrocyte G units activated with either GppNHp or fluoride sediment with a coefficient of 2.7 S whereas unactivated G units sediment with a coefficient of 4.0 S (unpublished data). GppNHp-activated turkey erythrocyte G units were prepared from an affinity eluate as described under Experimental Procedures. Assay of these activated G units with catalytic units from turkey erythrocyte membranes (16) showed an inhibitory response to calcium identical to that observed in Fig. 1 (data not shown). Aliquots of the activated G units were equilibrated with 1 mM calcium and applied to a sucrose density gradient that also contained 1 mM calcium and 0.1 mM EGTA. Control gradients containing no added calcium in either the G-unit preparation or in the gradient were analyzed simultaneously. Fractions from each gradient were assayed with S49 CYC membranes in media containing 1.5 mM EGTA. The activated G unit sedimented at 2.7 S with or without added calcium (Fig. 4). Thus, calcium does not appear to alter the active conformational state of the G unit, as determined by sucrose density gradient ultracentrifugation. Efect.s of calcium on G*-C interacticm Since calcium does not inhibit the formation of the holocatalytic state of the enzyme, it is unlikely that it interferes with G*-C interaction. We studied this question in more detail with the use of density gradient ultracentrifugation. In our laboratory, holocatalytic enzyme sediments with a coefficient of 6.0 S (unpublished data). Excess activated G units sediment at 2.7 S. We examined the effect of calcium on the conformation of the holoactive enzyme with this method. Holoactive enzyme was prepared with I-isoproterenol (50 PM) and GppNHp (0.1 mM) with or without 1 mM CaC& as des-
CYCLASE
349
FRACTION
FIG. 4. Effect of Ca” on density gradient ultracentrifugation of GppNHp-activated G unit. lOO-~1aliquots of GppNHp-activated turkey erythrocyte G units were prepared from an affinity eluate and applied to a 520% sucrose density gradient as described under Experimental Procedures. Activated G units in 1 mM CaCla were applied to a density gradient containing 1 mhl CaClr and centrifuged simultaneously. Enzyme markers for the determination of S,,, were as follows: cytochrome c 1.71 S, malate dehydrogenase 4.32 S, lactate dehydrogenase 7.3 S, and fumarase 9.09 S. 20-~1aliquots from control fractions (solid line) and calcium fractions (dotted line) were assayed for G* activity with 20 pl of S49 CYC membranes in the presence of 1.5 mM EGTA.
cribed under Experimental Procedures. Aliquots of this preparation were applied to sucrose density gradients prepared with or without 1 mM CaClz and assayed with 1.5 mM EGTA. Fractions were assayed alone (to detect holocatalytic enzyme), with S49 CYC- acceptor (to detect G*) and with S49 CYC- plus fluoride (to detect G). The results are presented in Fig. 5. Holocatalytic enzyme was detected at 6.0 S with or without 1 mM CaClz. The yield of holocatalytic enzyme was greater than 65% in both gradients. Excess G* was detected at 2.7 S in each gradient. The ratio of holocatalytic enzyme to total G* (calculated from the area under the appropriate peaks in Fig. 5) was the same with or withut calcium. In neither gradient was unactivated G unit (G) detected at 4.0 S. These results suggest that calcium does not change the conformation of the holocatalytic enzyme. Further, calcium does not appear to cause detectable dissociation of G* from C, nor convert G* to G. Since the addition of excess C in the form of
350
LASKER, DOWNS, AND AURBACH
5
10
15
20
FRACTION
s
5
10
15
20
FRACTION
FIG. 5. Effect of Ca2+on density gradient ultracentrifugation of holoactive adenylate cyclase. Holoactive solubilized turkey erythrocyte adenylate cyclase was prepared with or without 1 mM CaCls as described under Experimental Procedures. loo-al aliquota of these preparations were applied to 5-20s sucrose density gradients prepared with or without 1 mM CaClc. 20-~1aliquots from control fractions (A) and calcium fractions (B) were assayed for holocatalytic activity (dotted line), for G* activity with S49 CYC membranes (solid lines), and for G activity with S49 CYC membranes and 10-s M NaF (dashed lines) in the presence of 1.5 mM EGTA.
CYC- membranes enhanced activity of the holocatalytic (6.0 S) peak in the calcium gradient, it is possible that calcium increases the lability of the catalytic unit under the conditions of the gradient. Inhibition of catalytic unit. Since calcium inhibition of adenylate cyclase appears to be independent of hormone-receptor ineractions, hormone-dependent guanine nucleotide exchange, G* conformation, and G*-C interactions, it is possible that inhibition is effected at the level of the catalytic unit. We tested this possibility with the -- enzyme from S49 CYClymphoma cells which are deficient in
functional G units. Catalytic activity of the enzyme from these cells can be detected with manganese and ATP. Calcium inhibition curves were generated at varying concentrations of manganese present in the assay (Fig. 6). The enzyme activity of CYC- membranes with manganese was more resistant to inhibition by calcium than was turkey erythrocyte enzyme assayed with 5 mM magnesium. This resistance became more pronounced as the concentration of manganese in the assay was increased. Plots of reciprocal velocity versus reciprocal free manganese concentration at several calcium concentrations (Fig. 7) indicated competitive inhibition by calcium with manganese. The Km for manganese, calculated from linear regression analysis of reciprocal data with no added calcium in Fig. 7, was 2.1 X 10e4M. The Ki for calcium, calculated according to Dixon (31), was 3.6 X 10m4M. Computer analysis of MnATP2- concentrations with or without added calcium (Fig. 8) suggested that this competition was at the level of ATP, and that, within the range of calculated substrate concentration, in-
CALCULATED
LOG FREE [Cd+]
FIG. 6. Effect of Cax+ on manganese-stimulated adenylate cyclase in S49 CYC- membranes. Aliquots of S49 CYC membranes containing approximately 0.04 mg protein were assayed for adenylate cyclase activity with 1.0 X lo-so Mn*+ (open circles), 5.0 X low3 M Mn2+ (solid circles), and 5.0 X low4 M Mn2+ (open triangles) at varying Ca2+concentrations. Maxima! adenylate cyclase activity without added calcium was 6.8,10.1, and 4.3 pmol cAMP/min/mg protein with 10-s, 5 X lo-*, or 5 X 10e4M Mns+.
CALCIUM INHIBITION
OF TURKEY ERYTHROCYTE ADENYLATE
hibition of enzyme activity with calcium could be explained by depletion of the active substrate, MnATP2-. Recently, forskolin, a cardioactive diterpene, has been shown to activate adenylate cyclase from S49 CYC lymphoma cells, and to convert this variant enzyme to a form that can utilize MgATP2- as substrate (32). Forskolin-stimulated adenylate cyclase in S49 CYC- membranes, with magnesium/O.1 mM ATP as substrate, was inhibited by calcium in a manner similar to that of hormone-stimulated turkey erythrocyte enzyme (data not shown). This inhibition became less pronounced as the concentration of magnesium in the assay was increased. We studied the relationship of free magnesium concentration to calcium inhibition in experiments designed to keep the concentration of MgATP2- constant at each concentration of calcium. As shown in Fig. 9, free magnesium, in concentrations up to 4.4 mM, stimulated enzyme activity independently of MgATP2concentration. This suggests the existence of a free magnesium regulatory site in forskolin-stimulated CYC. Plots of recipro-
I
L
I
I
1234567 l/free iMnz+l ImM-‘I
FIG. 7. Effect of Ca” on the activity of adenylate cyclase with Mn2+ in S49 CYC- membranes. Aliquots of S49 CYC- membranes containing approximately 0.04 mg protein were assayed for adenylate cyclase activity. Assays were performed with no added calcium (solid circles), 5.0 X lo-* M calcium (open circles), 1.0 X 1O-3 M calcium (open triangles), or 5.0 X lo+M CakiUm (open Squares). Lines were generated by analysis of reciprocal data using linear regression analvsis.
Calculated
CYCLASE
lMnATP2-1
351
10-5M
FIG. 8. Effect of Ca*+ on the relationship between MnATP’- concentration and adenylate cyclase activity in S49 CYC- membranes. Data from Fig. 7 was analyzed using computer-generated MnATP’- concentrations at each concentration of calcium. Assays were performed with no added calcium (open circles), 5.0 X 1O-4M Ca’+ (solid circles), 1.0 X 10W3M Ca2’ (open triangles), or 5.0 X lo-’ M Ca2+(solid triangles). The concentration of ATP in the assay was 0.1 mM.
cal velocity versus reciprocal free magnesium concentration at several calcium concentrations (Fig. 9) showed competitive inhibition of magnesium by calcium. The Km for magnesium was 2.6 X low4 M, and the Ki for calcium was 0.70 X 10e4M. We next studied the relationship of MgATP2- concentration to calcium inhibition in experiments designed to keep the concentration of free magnesium relatively constant at each concentration of calcium. Plots of reciprocal velocity versus reciprocal MgATP2- concentration at several calcium concentrations (Fig. 10) showed noncompetitive inhibition of calcium with substrate. The Kmfor MgATP2in these experiments was 2.8 X 10e5M, and the Ki for calcium was 0.90 X low4 M. DISCUSSION
The current work demonstrates that calcium inhibits adenylate cyclase by direct competition with magnesium for a regulatory site on the catalytic unit. Earlier studies have shown that calcium inhibits the maximal activation of adenylate cyclase by agonist or fluoride (8, 10). The apparent Ki for calcium varies in different reports: 0.86 X 10e4 M for turkey erythro-
352
LASKER, DOWNS, AND AURBACH
l/free
wdg*+1
ImM-‘I
FIG. 9. Effect of Ca*+ on magnesium-stimulated adenylate cyclase activity in 549 CYC- membranes in the presence of forskolin. Aliquots of S49 CYC membranes containing approximately 0.08 mg protein were assayed for adenylate cyclase activity with 0.1 mM forskolin, 0.1 mM ATP and no added calcium (open circles), 5.0 X lo-‘Id Ca*+ (solid circles), and 1.0 X lo-’ M Ca*+ (solid triangles) at varying magnesium concentrations. Enzyme activity was inbibited at magnesium concentrations greater than 4.4 mM. MgATP2- concentrations were 9.3 X lo-’ M with no added calcium, 8.4 X 10e6M with 5.0 X lo-” M Ca2’, and 6.9 X lo-’ M with 1.0 X 10m3bf Ca’+. Linear regression analysis was carried out on data for reciprocal enzyme velocity versus reciprocal Mgs+ concentration.
cyte (34), 0.5-1.0 X 10m4M for parathyroid tissue (35,36) and 2.0 X 10e4M for adrenal cortex (8). These several results are not strictly comparable because EGTA buffered systems were not used in some experiments, and in others free calcium concentrations were neither measured nor calculated. In addition, magnesium concentration varied considerably. We have calculated free calcium concentrations from the data in several previous reports. Fifty percent inhibition of adenylate cyclase was achieved with calculated free calcium concentrations of 1.4 X 10c4 and 4.8 X 10e4M in brain (6,37), and 2.0 X 10e4 M in parathyroid (36). These analyses agree well with our own.3 3 One exception is a report that guinea pig brain adenylate cyclase is half-maximally inhibited by 35 X lo-’ M free calcium (43). Free calcium concentrations were calculated by a computer program similar
Previous work has suggested that calmodulin does not mediate calcium inhibition of adenylate cyclase (38, 39). Our results support these observations by experiments with trifluoperazine, EGTA-imidazole washed membranes, and tests of enzyme sensitivity to calcium and magnesium. In brain, which shows a biphasic response to calcium, calmodulin does appear to mediate the stimulatory action of calcium, and an activated G unit may be required for calmodulin sensitivity of the enzyme (7, 34, 35, 37, 44). In the adrenal, calcium stimulates activation of enzyme by guanine nucleotides, although it is not known whether calmodulin is involved in this stimulation (8). The stimulation of adenylate cyclase by calcium in brain and adrenal appears to be dependent on G. The lack of effect of calcium on G and G-C interactions that we have demonstrated in the turkey erythrocyte is consistent with the lack of stimulation by calcium in this system. Studies in the turkey erythrocyte and in the adrenal have shown that calcium does not influence hormone binding to receptor, and that calcium can inhibit the holocatalytic state of the enzyme (8,33,34, 37). However, these studies did not establish which subunit is primarily affected by calcium, nor the mechanism of this inhibition. We have utilized recent information on the components of adenylate cyclase to analyze further the interaction of calcium with this system. By studying the effects of calcium on hormone activation and holocatalytic activity of the enzyme independently, we have shown that calcium does not influence hormone-receptor interactions or hormone-dependent guanine nucleotide exchange. We evaluated to that employed in our studies. The original data were not reported so that we cannot recalculate the data. Our calculated free calcium concentrations could be invalid if the membrane preparations bound enough calcium to cause a significant reduction in the free calcium of the assay medium. This is unlikely, however, since the calcium inhibition curves were unaffected by varying the amount of membrane protein in the medium.
CALCIUM INHIBITION
OF TURKEY ERYTHROCYTE
the effect of calcium on the G unit and on G-C interactions with the use of density gradient ultracentrifugation. This technique can distinguish between the active and inactive states of the G unit. Regardless of the mechanism accounting for the difference in sedimentation coefficients for these two states (i.e., changes in conformation or true changes in size due to dissociation of subunits), calcium does not appear to influence this process. Calcium does not change the sedimentation coefficient of G* nor of the holocatalytic enzyme. In addition, calcium does not appear to cause measurable dissociation of the G*-C complex, nor convert G* to G. We have established that calcium can directly inhibit the catalytic unit of adenylate cyclase through the use of enzyme from S49 CYC- lymphoma cells which are deficient in functional G units. Although high concentrations of calcium can inhibit manganese-stimulated CYC- adenylate cyclase, this appears to be secondary to competition between calcium and manganese for ATP. This finding stresses the importance of evaluating the effects of calcium on substrate concentrations in experiments where other cations are present. Calcium inhibition of forskolinstimulated CYC enzyme with magnesium and ATP, however, cannot be explained by depletion of substrate. Under these conditions, calcium competes with magnesium for a metal binding site on the enzyme itself. This finding is in agreement with prior work in bone and cardiac adenylate cyclase systems (11, 41), but not in the turkey erythrocyte (10). Whereas previous studies have demonstrated the existence of sites for free magnesium on adenylate cyclase (14), the location of these sites has not been fully clarified. Since nucleotide or nucleotide plus hormone increases the apparent affinity of free magnesium for its binding sites in wild-type enzyme, and since native CYC- enzyme is not stimulated by magnesium, it has been postulated that free magnesium interacts with the G unit of adenylate cyclase. However, since forskolin-stimulated CYC enzyme can interact with magnesium both
ADENYLATE 0.5
CYCLASE
353
r
1 I lMgATP*-1
(mM-‘1
FIG. 10. Effect of Ca*+ on the relationship between enzyme velocity and MgATP*- concentration in forskolin-stimulated S49 CYC- membranes. Aliquots of S49 CYC- membranes containing approximately 0.08 mg protein were assayed for adenylate cyclase activity with no added calcium (open circles), 5.0 X 10e4 M Ca2+(solid circles), or 1.0 X 10e3M Ca’+ (solid triangles), at varying MgATP2- concentrations. Free M$+ concentrations were 2.2 X lo-” M with no added calcium, 3.6 X lo-’ M with 5.0 X lo-’ M calcium, and 4.4 X 10M4M with 1.0 X 10m3M calcium. Lines were generated by linear regression analysis of reciprocal enzyme velocity versus reciprocal MgATP2- concentration.
in complex with substrate and, as we have shown, at an independent binding site, it is likely that one such independent magnesium binding site is on the catalytic unit of the enzyme. Since calcium competes with magnesium for this regulatory site, and since calcium does not appear to influence R, G*, or G-C interactions of adenylate cyclase, it appears tha calcium inhibits the catalytic unit of the enzyme directly. The possibility remains, however, that CYC contains an aberrant subunit of G which can be stimulated in the presence of forskolin, and that the metal binding site is on this protein, rather than on the catalytic unit. Clarification of this problem must await further purification of c. Cyclic AMP is an important intracellular messenger. Thus, the regulation of adenylate cyclase has been of considerable interest. With the exception of forskolin, known activators of adenylate cyclase ei-
354
LASKER,
DOWNS, AND AURBACH
ther require the presence of, or directly interact with, the guanine nucleotide binding protein. Calcium is one of the few inhibitors of the enzyme. Calcium inhibition of adenylate cyclase may be of particular importance in the parathyroid cell where CAMP regulates parathyroid hormone secretion and the amount of CAMP produced is controlled in part by calcium. Indeed, two recent reports (36,42) have shown reduced sensitivity of adenylate cyclase to inhibition by calcium in tissue from adenomatous and hyperplastic parathyroid glands, suggesting the possibility that abnormal calcium regulation of adenylate cyclase might be part of the etiology of hyperparathyroidism. The identification in the current work of direct calcium competition for a magnesium binding site on C is the first demonstration of inhibition of adenylate cyclase at the level of the catalytic unit. This illuminates a further mechanism for possible physiologic control of the enzyme. ACKNOWLEDGMENTS We wish to thank Dr. John Fakunding for his aid with computer calculations, Dr. Allen Spiegel for his helpful discussions, Mr. Charles Woodard and Mrs. Sharon Reen for expert technical assistance, and Mrs. Lillian Perry for expert secretarial assistance. REFERENCES 1. CARON, M. G., LIMBIRD, L. E., AND LEFKOWITZ, R. J. (1979) MoL CelL Biochem 28,45. 2. NEER, E. J. (1978) J. Bid Chem 253,1498-X562. 3. KASLOW, H. R., JOHNSON, G. L., BROTHERS, V. M., AND BOURNE, H. R. (1980) J. BioL Chem. 255, 3736-3741. 4. CASSEL, D., AND SELINGER, Z. (1978) Proc Nat.
Acad Sci USA 75.4155-4159. 5. DOWNS, R. W., JR., SPIEGEL, A. M., SINGER, M., REEN, S., AND AURBACH, G. D. (1980) J. BioL Chem 255, 949-954. 6. SPIEGEL, A. M., AND DOWNS, R. W. (1981) Endocrine Rev. 2, 275-305. 7. BROSTROM, M. A., BROSTROM, C. O., BRECKENRIDGE, B. M., AND WOLFF, D. J. (1976) J. Bid Chem 251,4744-4750. 8. MAHAFFEE, D. D., AND ONTJES, D. A. (1980) J. BioL Chem 255, 1565-1571.
9. BIRNBAUMER, L. (1973) B&him. Biophys. Actn 300,129-158. 10. STEER, M. L., AND LEVITZKI, A. (1975) J. Biol Chem 250, 2080-2084. 11. DRUMMOND, G. I., AND DUNCAN, L. (1970) J. Bid
Chem 245,976-983. 12. LONDOS, C., AND RODBELL, M. (1977) in Drug Action at the Molecular Level (Roberts, G. C. K., ed.), pp. 235-247, University Park Press, Baltimore. 13. PERKINS, J. P. (1973) in Advances in Cyclic Nucleotide Research (Greengard, P., and Robison, G. A., eds), p. 21, Raven Press, New York. 14. CECH, S. Y., BROADDUS, W. C., AND MAGUIRE, M. E. (1980) MoL Cell. B&hem 33, 67-92. 15. PFEUFFER, T. (1977) J. BioL Chem 252.7224-7234. 16. SPIEGEL, A. M., DOWNS, R. W., JR., AND AURBACH, G. D. (1979) J. Cyclic Nucleotide Res. 5, 3-17. 17. BILEZIKIAN, J. P., AND AURBACH, G. D. (1973)
J. BioL Chem 248, 5577-5583. 18. WORTHINGTON BIOCHEMICAL COMPANY (1977) Worthington Enzyme Manual, Freehold, N. J. 19. HAGA, T., HAGA, K., AND GILMAN, A. G. (1977)
J. BioL Chem 252, 5776-5782. 20. Ross, E. M., HOWLEW, A. C., FERGUSON, K. M., AND GILMAN, A. G. (1978) J. BabL Chem 253, 64016412. 21. BOURNE, H. R., COFFINO, P., AND TOMKINS, G. M. (1975) Science 187, 750-752. 22. SPIEGEL, A. M., DOWNS, R. W., JR., AND AURBACH, G. D. (1977) Biochem Biophys. Res. Cemmun 76.758-764. 23. GOLDHAMMER, A. R., WOLFF, J., COOK, G. H., BERKOWITZ. S. A., KLEE. C. B.. MANCLARK. C. R., AND. HEWLETT, El L. (1981) Eur. i Biochem 115,605-609. 24. PERRIN, D. D., AND SAYCE, I. G. (1967) Taluntu 14, 833-842: 25. SILLEN, L. G., AND MARTELL, A. E. (1971) Stability Constants of Metal Ion Complexes, Suppl. 1. Special Publication 25, The Chemical Society, Burlington House, London. 26. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J. (1951) J. Bid Chem 193, 265-275. 27. WOLFF, J., COOK, G. H., GOLDHAMMER, A. R., AND BERKOWITZ, S. A. (1980) Proc, Nat. Acad Sci
USA 77,3841-3844. 28. KLEE, C. B., CROUCH, T. H., AND RICHMAN, P. G. (1980) Annu. Rev, Biochem 49,489-515. 29. PFEUFFER, J. (1979) FEBS L.&f. 101.85-89. 36. HOWLETT, A. C., AND GILMAN, A. G. (1980) J. BioL Chem 265,2861-2866. 31. DIXON, M., AND WEBB, E. (1964) Enzymes, Academic Press, New York. 32. SEAMON, K., PADGETT, W., AND DALY, J. W. (1981)
CALCIUM
INHIBITION
OF TURKEY
ERYTHROCYTE
P~OGNat Acad Sci USA 78, 3363-3367, and personal communication. 33. HANSKI, E., SEVILLA, N., AND LEVITZKI, A. (1977)
Eur. J. Biochem 76,513-520. 34. LEVITZKI, A., SEVILLA, N., AND ATLAS, D. (1975) J. IUOL BioL 97, 35-53. 35. TOSCANO, W. A., JR., WESTCOTT, K. R., LAPORTE, D. C., AND STORM, D. R. (1979) Proc Nat Acud
Ski USA 76.5582-5586. 36. BELLORIN-FONT, E., MARTIN, K. J., FREITAG, J. J., ANDERSON, C., SICARD, G., SLATOPOLSKY, E., AND KLAHR, S. (1981) J. clin Endorrind Metab. 52, 499-507. 37. SEVILLA, N., STEER, M. L., AND LEVITZKI, A. (1976) Biochemist?y 15.4393-3499. 38. WESTCOTP, K. R., LAPORTE, D. C., AND STORM,
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D. R. (1979) Proc Nat. Acad. Sci USA 76,204208. 39. BROSTROM, M. A., BROSTROM, C. O., BRECKENRIDGE, B. McL., AND WOLFF, D. J. (1978) Ad-
van Cyclic Nuckotide Ran 9,85-99. 40. NEER, E. J. (1979) J. Bid Chem 254,2089-2096. 41. RODAN, S. B., GOLUB, E. E., EGAN, J. J., AND RoDAN, G. A. (1980) Biochem. J. 185.629-637. 42. ONTJES, D. A., MAHAFFEE, D. D., AND WELLS, S. A. (1981) Metabolism 30,406-411. 43. PIASCIK, M. T., WISLER, P. L., JOHNSON, C. L., AND POTTER, J. D. (1980) J. BioL Chem 255,41764181. 44. CHEUNG, W. Y., BRADHAM, L. S., LYNCH, T. J., LIN, Y. M., AND TALLANT, E. A. (1975) Biochem Biophys Res. Commun 66, 1055-1062.