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The effect of Ca2+ on the adenyl cyclase of calf brain
250
BIOCHIMICA ET BIOPHYSICA ACTA
BBA 26282 T H E E F F E C T OF Ca 2+ ON T H E A D E N Y L CYCLASE OF CALF B R A I N L A U R E N C E S. BRADHAM, D A R L E N E A. HOLT AND MARILYNNE SIMS
Department of Medical Research, Veterans Administration Hospital, Little Rock, Ark. 72 206 ( U.S.A .) (Received August 7th, 1969) (Revised manuscript received November 4th, 1969)
SUMMARY
A study was made of the effect of various substances on the formation of 3',5'-AMP in the presence of a particulate fraction from homogenates of calf cerebral cortex. The activity of adenyl cyclase in these preparations was inhibited by the chelating agent 1,2-bis-(2-dicarboxymethylaminoethoxy)ethane. This inhibition was reversed by equimolar concentrations of Ca *+. Sr 2+ was almost as effective as Ca 2+ in reversing the inhibition by the chelating agent, while Mg *+ and Ba 2+ were ineffective. Higher concentrations of Ca *+ inhibited the formation of the nucleotide.
INTRODUCTION
The enzyme adenyl cyclase catalyzes the formation of 3',5'-AMP from adenosine triphosphate. SUTHERLANDet al. x have discussed the physiological significance of this enzyme and its role in the mediation of hormonal effects in the intact cell. An early report by SUTHERLAND et al. 2 described the properties of adenyl cyclase in a particulate fraction derived from brain and other tissues. These preparations required Mg *+ or Mn *+ for optimal activity. Ca 2+ and ions of other divalent metals did not replace Mg 2+. In this paper are described the results of investigation of the properties of adenyl cyclase in a particulate fraction from calf brain cortex. Evidence is presented that the activity of the enzyme in these preparations is dependent upon metal ions which contaminate the reaction mixture. The results indicate that this metal ion is Ca 2+. The possible relationship of these findings to the physiological function of adenyl cyclase and 3',5'-AMP is discussed. EXPERIMENTAL PROCEDURE Materials
The adenyl cyclase used in these experiments was contained in a 2000 × g particulate fraction isolated from homogenates of calf cerebral cortex. The brains were obtained soon after slaughter of the animal and were cooled with ice. All subsequent operations were performed in the cold room. The cortex was homogenized for 30 sec in a Waring Blender with 2 vol. of a solution containing IO mM NaC1 and Abbreviation: EGTA, 1,2-bis-(2-dicarboxymethylaminoethoxy)ethane.
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251
IO mM KC1. Some particulate fractions were obtained from homogenates which were prepared in the presence of glycerol. The tissue in this case was homogenized in a mixture composed of I vol. of glycerol and I vol. of the salt solution. Unless they were to be used immediately, the homogenates were frozen and stored at - 7 o°. Immediately before use, the homogenates were thawed and were centrifuged at 2000 × g for 30 min. The supernatant suspension was decanted, and the residue was resuspended to its original volume in a Io-mM NaC1-KC1 solution. Each ml of the suspension contained the particulate fraction derived from 0.3 g of brain tissue. Ion-exchange resins were Baker Analyzed reagent grade. 3',5'-AMP and the disodium salt of ATP were obtained from Sigma Chemical Co. According to the manufacturer, the ATP had been specially purified to reduce the concentration of trace metals. It was used without further purification. The chelating agent 1,2-bis-(2dicarboxymethylaminoethoxy)ethane (EGTA) was obtained from K and K Laboratories. Caffeine was purchased from Eastman Organic Chemicals. All other chemicals were reagent grade. Glass-distilled-deionized water was used to prepare the solutions used in the preparative procedures and the experiments. All glassware was rinsed before use with I mM EDTA followed by glass-distilled-deionized water.
Methods The conditions for the enzymic formation of 3',5'-AMP and the assay procedure have been described in an earlier publication a. Minor modifications have been made in this procedure so that some description is necessary here. The standard reaction mixture contained 2 mM ATP, 5 mM MgC12, IO mM NaF, 6. 7 mM caffeine, and 4 ° mM Tris (pH 7.5). Other reagents were added in small volumes. The final volume of the reaction mixture was adjusted to 220 ml b y the addition of a Io-mM NaC1-KC1 solution. The concentration of protein in these samples was 1.2 mg/ml. In the experiments described in Table III, the caffeine was replaced b y 50 mM theophylline. In these samples, a 5-ml aliquot of the brain suspension was used as the source of adenyl cyclase, and the final volume of the reaction mixture was 55 ml. The concentration of protein in these samples was 2. 4 mg/ml. The concentration of all other reagents remained the same. The samples were incubated with swirling at 3 °0 . The usual incubation time was 30 rain. The reaction was stopped b y the addition of sufficient o.I M HC1 to drop the p H to 3 (indicator paper), and the temperature of the suspension was brought to IOO° by heating in a boiling-water bath. The p H was then adjusted to 7 with I M NaOH, and the samples were heated at IOO° for 15 min. After the samples were cooled, they were again acidified to p H 3 and were filtered under gravity. Filtration occurred more rapidly under these conditions. The filtrates were prepared for chromatography by readjusting the p H to 7. The assay procedure utilized sequential chromatography of enzyme reaction mixtures on an anionic exchange resin (Dowex 2-X8) and a cationic exchange resin (Dowex 5oW-X8). As pointed out in the original description of the method, this sequence effectively separated 3',5'-AMP from the other adenine-containing nucleotides and resulted in quantitative recovery of the cyclic nucleotide. The concentration of 3',5'-AMP in the eluate from the Dowex-5o column was determined from the absorbance of this solution at 2600 •. The measurement of absorbance was perBiochim. Biophys. Acta, 2o'r (:t97o) 250-260
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L.S. BRADHAMet al.
formed with a Cary-I 4 spectrophotometer using a cell with an optical path length of IO cm. This modification permitted the elimination of the terminal evaporation of the Dowex-5o eluate as described in the original procedure, but it also made it necessary to modify slightly the procedure for chromatography on the Dowex-5o columns. These columns were washed in the same manner as before except that the final washing was carried out with 0.05 M HC1 rather than with 0.3 M formic acid. The sample was transferred to the column in 0.05 M HC1, and the column was eluted with 230 ml of this solution. The first 9 ° nil of the eluate was discarded. The next 14o ml was collected and was used for the measurement of absorbance. Correction was made for the column blank as described in the original procedure. Measurements with the Cary 14 were made at 2500, 2600, and 2668/~ to determine the numerical values of the terms El, Ep, Es, and r. In this communication, enzymic activity is expressed as nmoles of 3',5'-AMP per mg of protein. This means of expression was used rather than the units proposed b y SUTHERLANDet al.* because the rate of accumulation of 3',5'-AMP in these reaction mixtures was non-linear (see Fig. 2). Analysis for Ca 2+ and Mn *+ was performed by the atomic absorption technique using a Perkin-Elmer Model 303 spectrophotometer. Particulate fractions were prepared for analysis using the method of SPARROW AND JOHNSTON4. Protein was determined b y the method of LOWRY et al. 5. RESULTS
General properties of adenyl cyclase preparations The adenyl cyclase preparations used in these experiments had, in general, the properties described by SUTHERLANDet al. ~. A Mg *+ concentration of 5 mM produced m a x i m u m accumulation of 3',5'-AMP, and this effect could not be duplicated by Ca 2+. N a F at a concentration of IO mM produced a 5-fold increase in enzymic activity. Enzyme preparations were more stable when stored at - 7 o°. This was especially true of the response of these preparations to EGTA and Ca *+. The homogenization of brain tissue in the presence of glycerol increased the activity of the enzyme by about 50 %. The increase was observed in preparations which were used immediately as well as in those which were used after storage at - - 7 o°. This effect is illustrated by the difference between the activities of enzyme preparations I I and I I I shown in Table I. Maximum enzymic activity was observed in samples in which glycerol-treated preparations were incubated with IO mM N a F and 50 mM theophylline. Under these conditions, I m g of protein catalyzed the formation of 9 nmoles of 3',5'-AMP in 30 rain. This represented the conversion of 1 % of the ATP to the cyclic nucleotide. EBect of EGTA on adenyl cyclase The effect of the inclusion of EGTA in enzyme reaction mixtures is illustrated by Fig. I. This figure summarizes the data obtained from five experiments. Because of the variation in the amount of 3',5'-AMP produced in individual experiments, the results are expressed as percent of the concentration of nucleotide produced in the control samples. I t can be seen from these data that the inclusion of EGTA at a concentration of Biochim. Biophys..4cta, 2Ol (197 o) 250-260
ADENYL CYCLASE AND Ca 2+
253
TABLE I EFFECT
OF Ca $+ ON
THE
INHIBITION
OF BRAIN
ADENYL
CYCLASE
BY
EGTA
Additions to the enzyme suspension were m a d e i m m e d i a t e l y before the reaction was started. The final concentration of E G T A and Ca ~+ in the e x p e r i m e n t s w i t h e n z y m e p r e p a r a t i o n I was o.o25 raM. The concentration of these reagents in the e x p e r i m e n t s w i t h e n z y m e p r e p a r a t i o n s I I a n d I I I was 0.05 mM. The n u m b e r s in p a r e n t h e s e s represent the n u m b e r of samples assayed.
Enzyme prep.
Additions
3",5"-AMP (nmoles/mg
Activity (%)
(± S.E.)) I
None (9) E G T A (8) E G T A + Ca 2+ (4)
3.42 ± 0.08 1.47 i 0.06 3.34 :J2 o.oi
ioo 43 98
II
None (6) E G T A (6) E G T A + Ca ~+ (6)
4.34 4- o . i o 1.77 ~ 0.o6 3.98 ± o.o6
IOO 41 92
III*
N o n e (5) E G T A (5) E G T A + Ca 2+ (6)
6.11 ~ o.o 7 3.23 ± 0-05 5.37 • o-07
ioo 53 88
* This p r e p a r a t i o n was obtained f r o m the same tissue as e n z y m e p r e p a r a t i o n I I b u t was treated w i t h glycerol as described u n d e r EXPERIMENTAL PROCEDURE.
t 0.02 EGTA
I 0.04
I I I 0.06 0.08 O.lO CONCENTRATION ( m M)
Fig. i. Effect of E G T A on the f o r m a t i o n of 3',5'-AMP. V a r y i n g concentrations of E G T A were included in the usual i n c u b a t i o n m i x t u r e , a n d the i n c u b a t i o n was performed as described in the text. E a c h p o i n t is the m e a n of the p e r c e n t activity obtained in the n u m b e r of samples indicated b y the n u m b e r s in parentheses. The vertical bars represent t h e S.E. No d a t a were o b t a i n e d for concentrations of E G T A less t h a n o.oi25 mM.
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o.I mM reduced by more than 6o % the amount of 3',5'-AMP forlned in the reaction mixture. This inhibition was little more than that obtained in the presence of 0.05 mM EGTA. The presence of the chelating agent at a concentration of 12.5 #M had very little effect. Addition of EGTA to samples after the reaction was stopped had no effect on the 3',5'-AMP measured. This experiment was done to eliminate the possibility that the EGTA interfered with the assay. The inhibitory effect of EGTA on brain adenyl cyclase was reproduced on many different enzyme preparations. Occasionally samples were not affected by the chelating agent. In these instances, however, the enzyme preparations were partially inactive, the enzyme activity having fallen already to the level obtained in the presence of EGTA.
Reversal of EGTA inhibition by Ca 2+ The inhibitory effect of EGTA on adenyl cyclase preparations was prevented by the inclusion of Ca 2+ in the enzyme reaction mixture. The results of several such experiments are summarized in Table I. In these experiments, EGTA and CaC1z were added to the mixture before the reaction was started. In each experiment, the lowest concentration of EGTA which produced maximal inhibition of the particular enzyme preparation was used. This concentration varied from one preparation to another, but the maximum percent inhibition was roughly the same. As indicated by the table, the inclusion of CaC12 at a concentration equivalent to the concentration of EGTA almost completely prevented the inhibition by the chelating agent. Under these conditions, the mean concentration of 3',5'-AMP accumulated in the reaction mixture was 92 % of the control value. The effect of the inclusion of other alkaline earth metal ions in the reaction mixture is shown in Table II. It can be seen that Sr 2+ were almost as effective as Ca z+ in preventing the inhibition by EGTA. Ba ~+ had no effect. The effects of EGTA and EGTA plus Ca ~+ on the time course of the reaction are illustrated in Fig. 2. Each part of this figure summarizes the results of two individual experiments using the same preparations of glycerol-treated adenyl cyclase. Fig. 2A TABLE II RELATIVE EFFECTS OF ALKALINE EARTH METALS ON THE INHIBITION OF BRAIN ADI~NYL CYCLASE BY E G T A The reaction was carried o u t in the presence of glycerol-treated 2o00 × g particulate suspension. Additions to the reaction m i x t u r e were m a d e i m m e d i a t e l y before the reaction was started. The final concentration of E G T A and the various salts was 0.05 mM. The n u m b e r of samples in each e x p e r i m e n t a l g r o u p is indicated b y the n u m b e r in parentheses.
Additions
3",5"-A M P (nmoles /mg
A ctivity (%)
6.15 3.66 3.87 5.16 5.60
ioo 60 63 84 91
(~S.E.))
None (5) E G T A (5) E G T A + BaCI~ (4) E G T A + SrC12 (8) E G T A + CaClz (12)
-~ i ± ± ±
o.ii 0-05 0.04 0.04 o.09
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shows the effect of the inclusion of EGTA in the reaction mixture, while Fig. 2B shows the effect of the inclusion of EGTA and an equimolar concentration of Ca ~+. It can be seen that the rate of accumulation of the cyclic nucleotide in these reaction mixtures was non-linear. Because of the small number of points on these curves, it was impossible to determine if EGTA had any effect on the initial rate of the reaction. It was clear, however, that the maximum accumulation of 3',5'-AMP was reduced in the presence of EGTA, and that this inhibition was prevented by the inclusion of an equimolar concentration of Ca 2+. These results indicated that the inclusion of Ca ~+ prevented the inhibitory effect of EGTA, but they did not provide definite proof that the effect of Ca 2+ was a true reversal of EGTA inhibition. This was demonstrated by modifying the experimental conditions so that Ca 2+ were added to the reaction mixtures containing EGTA at a time when the accumulation of 3',5'-AMP was approaching the maximum. Samples containing EGTA at a concentration of 0.05 mM were incubated for 30 min. At this time CaC12 was added to duplicate samples to give a final concentration of 0.05 mM. Incubation of the Ca2+-treated samples and the untreated controls was continued for another 15 rain. i
A
i
i
B
S
i
i
o /' /s
i
/
E
i
/
t' tJ
i
2
0
0
ll5
I 50 0 MINUTES
I 15
i 50
0 0
i 15
310
i 45
MINUTES
Fig. 2. The effect of E G T A and E G T A plus Ca n+ on the time course of the a c c u m u l a t i o n of 3',5' AMP. The concentration of E G T A and Ca 2+ in these e x p e r i m e n t s was o.o 5 raM. 3',5'-AMP concentrations at zero time were obtained b y acidifying the reaction m i x t u r e s before t h e addition of ATP. E a c h p o i n t is t h e m e a n c o n c e n t r a t i o n o b t a i n e d in t w o individual experiments. The vertical bars represent the range of the values. A. 0 - - 0 , i n c u b a t e d w i t h E G T A ; ( D - - O , incubated w i t h o u t EGTA. 13. 0 - - 0 , incubated w i t h E G T A ; O - - ( D , incubated w i t h equimolar E G T A and Ca 2+. Fig. 3- Reversal of E G T A inhibition b y Ca ~+. E G T A was included in t h e usual i n c u b a t i o n m i x t u r e at a concentration of o.o 5 raM. An equimolar concentration of Ca ~+ was added to samples at the t i m e indicated b y the arrow. 3',5'-AMP concentration at zero time was o b t a i n e d b y acidifying reaction m i x t u r e s before the addition of ATP. 0 - - 0 , incubated w i t h E G T A ; O, incubated with equimolar E G T A and Ca n+.
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The results of such an experiment are shown in Fig. 3. These results were typical of those obtained in four experiments using the same glycerol-treated adenyl cyclase preparation. It can be seen that, in the presence of EGTA, the concentration of 3',5'-AMP had almost reached a maximum at 30 min and extension of the incubation period to 45 min did not result in the accumulation of appreciably more of the cyclic nucleotide. The addition of Ca 2+ at 30 min, however, reversed the effects of EGTA and about 7° % more 3',5'-AMP was formed during the subsequent i5-min incubation. The final concentration of the nucleotide in the Ca2+-treated samples was close to that normally obtained in the absence of EGTA.
Effect of EGTA on the recovery of 3',5'-AMP When commercial 3',5'-AMP was incubated with the 2000 × g particulate fraction in the absence of ATP, there was a very rapid destruction of the nucleotide. This loss was attributed to the presence of high concentrations of nucleotide 3',5'phosphodiesterase. Caffeine at a concentration of 6. 7 mM had no effect on this loss. It was possible, however, to partially inhibit the destruction of the nucleotide by carrying out the incubation in a medium containing theophylline at a concentration of 50 mM. A detailed description of the conditions for these incubations is presented in Methods. Using these conditions, experiments were performed to determine if the inhibitory effect of EGTA were due to the inhibition of adenyl cyclase or to the stimulation of the phosphodiesterase. The recovery of added 3',5'-AMP was used as a measure of phosphodiesterase activity. A known amount of the nucleotide was added to the enzyme reaction mixtures in the absence of ATP. EGTA at a concentration of o.I mM was included in some of the samples. The samples were incubated in the usual manner. Control samples containing ATP and o.I mM EGTA were incubated at the same time. The results of these experiments are presented in Table III. It can be seen that the mean recovery of added 3',5'-AMP in these experiments was 35 %, and that this recovery was not affected by the inclusion of EGTA. In fact there appeared to be a small increase in the amount recovered when EGTA was pres-
TABLE III EFFECT OF E G T A ON THE RECOVERY OF 3',5'-AMP Samples of brain adenyl cyclase were incubated as described in the text. The samples in G r o u p A were incubated with added 3',5'-AMP b u t no ATP. The a m o u n t of added 3',5'-AMP was 1.155 mg. The concentration of A T P in t h e samples of G r o u p B was 2 raM. I n b o t h groups, the concentration of E G T A was o.i mM. The n u m b e r s in p a r e n t h e s e s indicate the n u m b e r of samples incubated u n d e r each set of conditions.
Group
Additions
Total 3",5'-A M P #g (±S.E.)
A
3',5 '-AMP (7) 3',5'-AMP + E G T A (16)
390 ~ 14 441 ± 14
B
ATP (7) ATP + E G T A (7)
399 -I:: 17 2o6 -+- i i
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Activity
Recovery
(%1
(%) 34 38
ioo 52
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ADENYL CYCLASE AND C a 2+
ent. On the other hand, addition of EGTA to the control samples produced a 5o % inhibition of the formation of 3',5'-AMP from ATP. It appeared, therefore, that the effect of EGTA was due to the inhibition of the formation of the nucleotide rather than to the stimulation of its destruction.
Inhibition of the formation of 3',5'-AMP by Ca 2+ When Ca 2+ was added to reaction mixtures in the absence of Mg2+, no 3',5'AMP was produced. Nor was there any additive effect of Ca2+ when it was included in complete reaction mixtures at a concentration of o.I mM or less. Higher concentrations of Ca 2+, however, were found to inhibit the formation of the nucleotide in the complete reaction mixture. With different enzyme preparations, some variability in the precise concentration of Ca 2+ required to produce maximum inhibition was encountered. In general, however, almost 80 % inhibition was obtained when Ca ~+ included in complete reaction mixtures at concentrations ranging between 0.5 and i raM. The addition of Ca 2+ to samples at the end of the incubation had no effect, indicating that Ca 2+ was not interfering with the assay.
Assay of reaction ,mixtures for Ca ~+ and Mn 2+ Four samples of the 2000 × g particulate fraction and a sample of the complete reaction mixture less the particulate fraction were analyzed for Ca 2+ and Mn ~+. The results of these analyses are recorded in Table IV. These results indicate that the mean concentration of Ca ~+ in a typical complete reaction mixture was in the neighborhood of 0.033 raM. About 60 % of this Ca 2+ was contributed by the particulate suspension while the remainder was contributed by the other components of the reaction mixture. Contamination from this latter source was due largely to Ca ~+ which contaminated the ATP. Most of the Mn 2+ which contaminated the final reaction mixtures was contributed by the reagent solutions. The mean concentration of Mn ~+ in a typical reaction mixture was 2.2/~M. T A B L E IV METAL CONTENT OF COMPONENTS OF ENZYME REACTION MIXTURES F o u r samples of the glycerol-treated particulate fractions suspended in o.o1 M NaC1-KC1 solution and four samples of the incubation solution containing all the c o m p o n e n t s of the reaction m i x t u r e except the particulate fraction were assayed for Ca ~+ and Mn 2+. The concentrations of the metals were corrected to give the concentration c o n t r i b u t e d to the final reaction mixture. The n u m b e r s in p a r e n t h e s e s are the s t a n d a r d deviations for each determination.
Reaction mixture component
Particulate fraction I n c u b a t i o n solution Total
Metal conch. (l~M) Ca~+
Mn2+
19.3 (2.8) 14.o (2.5) 33.3
0.38 (0.026) 1.8 (o.I5) 2.18
DISCUSSION
The experiments described in this communication provide evidence that the formation of 3',5'-AMP in the presence of a 2000 × g particulate fraction of calf brain Biochim. Biophys. Acta, 2Ol (197o) 25o-26o
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L.S. BRADHAMet al.
cortex is dependent upon metal ions which are present in the reaction mixture. This dependency becomes apparent when the enzyme preparation is treated with the chelating agent EGTA. This compound causes inhibition of the formation of the cyclic nucleotide when it is included in enzyme reaction mixtures at very low concentrations. It has been established previously2 that the formation of 3',5'-AMP in the presence of enzyme preparations of this type is dependent upon Mg2+. The inhibitory effect of EGTA, however, is observed in samples that contain Mg2+ at an optimal concentration. This concentration of Mg2+ is IOO times the concentration of chelating agent which produces maximal inhibition. The effect, therefore, cannot be attributed to the chelation of Mg2+, since the amount of Mg2+ chelated by inhibitory concentrations of EGTA would not significantly affect adenyl cyclase activity. It is apparent, therefore, that some metal other than Mg2+ is present in the reaction mixture and is required for the accumulation of 3',5'-AMP. One likely candidate for this role is Ca 2+. In the presence of equimolar concentrations of Ca 2+, the effect of EGTA is reversed. This reversal suggests that the inhibition by EGTA is due to the chelation of Ca2+ present in the reaction mixture. These Ca 2+ are necessary for maximum accumulation of 3',5'-AMP, and the chelation of these ions by EGTA results in inhibition of the formation of the nucleotide. It is recognized that the data do not rule out completely the participation of a metal other than Ca 2+. The nature of the chelation by EGTA in the reaction mixture has not been clarified. It is possible that the metal ions responsible for the formation of 3',5'-AMP are bound to some component of the particulate fraction, and that EGTA inhibits the reaction by forming a chelate with this metal without actually removing it from its binding site. The addition of Ca2+ to the reaction mixture then reverses the inhibition by competing with the unknown metal chelate for the chelating agent. This model would be possible only if the unknown metal chelate were more stable than the Ca 2+ chelate. The stability constants of a number of EGTA-metal chelates have been determined 6-s. Mn 2+ is a possible candidate since the stability constant of its EGTA chelate is only about 25 times the stability constant for the Ca 2+ chelate. SUTHERLAND e~ al. 2 have shown, moreover, that Mn 2+ can replace Mg2+ as an activator of adenyl cyclase. Mn 2+ is a contaminant of the reaction mixtures used in the experiments reported here. The ratio of the concentrations of Ca 2+ to Mn 2+ in the reaction mixture was about 15 to I. It is felt that at this concentration ratio, Ca~+ would not compete effectively with Mn 2+ for the EGTA, and thus would not be effective in reversing the EGTA inhibition. It is even less likely that Sr ~+ would compete effectively with Mn ~+ for the chelating agent since the Mn 2+ chelate with EGTA is about 20 oo0 times more stable than the Sr 2+ chelate. The data of Table II show, however, that Sr 2~ is almost as effective as Ca 2+ in reversing the inhibition by EGTA. It is felt, therefore, that it is extremely unlikely that the effects of EGTA demonstrated in these experiments are due to the chelation of Mn 2+. The participation of other transition metals such as Fe 2+, Cu 2+ or Zn 2+ is also improbable, since the stability constants of the EGTA chelates of these metals are considerably higher than the stability constant of Ca 2+ chelate. These possibilities, however, cannot be completely ruled out until experiments of the type described here are carried out with enzyme preparations and reagents which are completely free of metal contamination. Biochim. Biophys. Mcta, 2Ol (197o) 25o-26o
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Other evidence supports the conclusion that the effects of EGTA are due to chelation of Ca *+. The concentration of EGTA which produces m a x i m u m inhibition of the formation of 3',5'-AMP is roughly equivalent to the concentration of Ca *+ which is found in the reaction mixture. This inhibition is reversed by the addition of Ca 2+ in a concentration equivalent to the concentration of EGTA. Sr*+ produces partial reversal of the inhibition while Ba *+ is ineffective. The similarity of the chemical properties of Ca *+ and Sr 2÷ is well-known, and the ionic radii of the two metals are quite close. It is reasonable to conclude, therefore, that the formation of 3',5'-AMP in the presence of the particulate fraction from brain used in these experiments is dependent upon the presence of both Mg *+ and Ca*÷. This dual requirement for Mg*+ and Ca *+ has parallels in other biological systems. The ATPase of myofibrils has been shown to require both cations for activity*. This property accounts for the fact that the ATPase and the contraction of muscle are both inhibited by EGTA 1°, 11. Another example is the ATPase found in the vesicular elements of the sarcoplasmic reticulum which possess relaxing factor activity. The activity of the ATPase and the inhibitory effect of the vesicular granules on the myofibrillar ATPase have been shown to depend upon both Ca 2÷ and Mg*+ (ref. 12). Similar enzymes have been found recently in fractions of brain tissue 13,14. Two possible explanations must be considered in assigning a mechanism to the effect of Ca *+ on the accumulation of 3',5'-AMP. One possibility is that Ca *+ is necessary for maximal activity of adenyl cyclase and is, therefore, involved in the formation of the nucleotide. The alternative explanation is that Ca *+ inhibit the destruction of the nucleotide by the enzyme nucleotide 3',5'-phosphodiesterase. Previous work has shown that the latter enzyme is associated with particulate fractions isolated from brain and other tissues15, TM. Phosphodiesterase activity was most certainly present in our reaction mixtures since the accumulation of 3',5'-AMP required the presence of caffeine or theophylline. These compounds have been shown to be competitive inhibitors of phosphodiesterase activity 15,17. The data in Table I I I indicate that it was impossible to eliminate all of the phosphodiesterase activity in our reaction mixtures. In the presence of 50 mM theophylline, the mean recovery of added 3',5'-AMP was about 35 %. This recovery was used as a measure of phosphodiesterase activity in order to determine if the effect of EGTA was due to inhibition of adenyl cyclase or to stimulation of the phosphodiesterase. The data show that the recovery of added 3',5'-AMP was not decreased at all by the inclusion of o.I mM EGTA. The same concentration of EGTA, however, inhibited by 50 % the formation of the nucleotide from ATP in the control experiments performed under identical conditions. These results eliminate the possibility that the inhibitory effect of EGTA on the accumulation of 3',5'-AMP is due to the stimulation of the phosphodiesterase. It is concluded, therefore, that the effect of the chelating agent is due to inhibition of adenyl cyclase, and that this inhibition is caused by the chelation of Ca 2+. This Ca ~+ m a y be bound to the enzyme or some structure associated with the enzyme in the 2000 × g particulate fraction. DAVOREN AND SUTHERLANDTM have shown that adenyl cyclase isolated from a number of tissues is associated with the plasma membrane, and DE ROBERTIS et al. TM have demonstrated that part of the adenyl cyclase of rat cerebral cortex is associated with synaptic membranes which sediment in centrifugal fields between 900 and i i 500 × g. These cell components were probably Biochim. Biophys. Acta, 2Ol (197 o) 25o-26o
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contained in the particulate fractions used in our experiments. It is possible, therefore, that the Ca ~+ required for adenyl cyclase activity is bound to membrane components. There is ample physiological evidence to suggest that the stimulatory effect of Ca ~+ on adenyl cyclase has functional significance. Some of this evidence has been reviewed by RASMUSSEN AND TENENHOUSE 20, who have proposed that the function of adenyl cyclase is to either alter the permeability of the plasma membrane to Ca 2+ or to effect the intracellular release of Ca2+ from the membrane. In particular, they cite experiments which demonstrate a rise in the intracellnlar concentration of both 3',5'-AMP and Ca *+ in kidney and bone when these tissues are stimulated by parathormone. Furthermore, the adenyl cyclase activity of fat cells .1 and of isolated fat cell membranes 22 has been shown to be dependent upon Ca2+. In view of this evidence, the finding that the adenyl cyclase of brain is dependent upon Ca ~+ for its activity is highly significant. ACKNOWLEDGMENTS
The authors express their appreciation to Dr. Edwin R. Hughes for performance of the metal analyses, to Dr. Charles L. Wadkins for constructive criticism of the manuscript, and to Dr. Eugene J. Towbin for continued encouragement and support of the work. REFERENCES i E. W. SUTHERLAND, I. OYE AND ~R. W. BUTCHER, Recent Progr. Hormone Res., 21 (1965) 623. 2 E. W. SUTHERLAND, T. W. HALL AND T. MENON, J. Biol. Chem., 237 (1962) 122o. 3 L. S. BRADHAM AND D. W. WOOLLEY, Biochim. Biophys. Acta, 93 (1964) 475. 4 M . T . SPARROW AND B. M. JOHNSTON, Biochim. Biophys. Acta, 90 (1964) 425 • 5 0 . H. LowRY, N. J. ROSEBROUGH, A. L. FARR AND H. J. RANDALL, J. Biol. Chem., 193 (1951) 265. 6 S. CHABEREK AND A. E. MARTELL, Organic Sequestering Agents, John Wiley, New York, 1959, p. 577. 7 J. H. HOLLOWAY AND C- N. RE1LLEY, U.S. Department of Commerce, O~ce of Technical Services, Hept. No. PB 114499 (1959); Chem. Abstr., 55 (1961) I6IO5a. 8 K. H. SCHROEDER, Acta Chem. Scand., 17 (1963) 15o9, 9 J. C. SEIDEL AND J. GERGELY, J. Biol. Chem., 238 (1963) 3648. io A. WEBER AND R. HERZ, J. Biol. Chem., 238 (1963) 599. i i S. EBASHI, J. Biochem. Tokyo, 5° (I961) 236. 12 Y. NAGAI, J. Biochem. Tokyo, 58 (1965) 429. 13 K. KADOTA, S. MORI AND R. IVIAIZUMI,J. Biochem. Tokyo, 61 (1967) 424 . 14 Y. NAKAMARU, J. Biochem. Tokyo, 63 (1968) 626. 15 H. W. BUTCHER AND E. W. SUTHERLAND, J. Biol. Chem., 237 (1962) 1244. 16 E. DE ROBERTIS, G. H. D. ARNAIZ, M. ALBERICI, R. W. BUTCHER AND E. W. SUTHERLAND, J. Biol. Chem., 242 (1967) 3487 . 17 W. Y. CHEUNG, Biochemistry, 6 (1967) lO79. I8 G. I. DRUMMOND AND S. PERROTT-YEE, J. Biol. Chem., 236 (1961) 1126. 19 P. R. DAVOREN AND E. W. SUTHERLAND, J. Biol. Chem., 238 (1963) 3o16. 20 ]-I. RASMUSSEN AND A. TENENHOUSE, Proc. Natl. Acad. Sci. U.S., 59 (1968) 1364. 21 1~. H. WILLIAMS, S. A. WALSH AND J. W. ENSlNCK, Proc. Soe. Exptl. Biol. Med., 128 (1968) 279. 22 H. P. BAR AND O. HECHTER, Federation Proc., 28 (1969) 571.
Biochim. Biophys. Acta, 2ol (197 o) 250-260
BIOCHIMICA ET BIOPHYSICA ACTA
BBA 26282 T H E E F F E C T OF Ca 2+ ON T H E A D E N Y L CYCLASE OF CALF B R A I N L A U R E N C E S. BRADHAM, D A R L E N E A. HOLT AND MARILYNNE SIMS
Department of Medical Research, Veterans Administration Hospital, Little Rock, Ark. 72 206 ( U.S.A .) (Received August 7th, 1969) (Revised manuscript received November 4th, 1969)
SUMMARY
A study was made of the effect of various substances on the formation of 3',5'-AMP in the presence of a particulate fraction from homogenates of calf cerebral cortex. The activity of adenyl cyclase in these preparations was inhibited by the chelating agent 1,2-bis-(2-dicarboxymethylaminoethoxy)ethane. This inhibition was reversed by equimolar concentrations of Ca *+. Sr 2+ was almost as effective as Ca 2+ in reversing the inhibition by the chelating agent, while Mg *+ and Ba 2+ were ineffective. Higher concentrations of Ca *+ inhibited the formation of the nucleotide.
INTRODUCTION
The enzyme adenyl cyclase catalyzes the formation of 3',5'-AMP from adenosine triphosphate. SUTHERLANDet al. x have discussed the physiological significance of this enzyme and its role in the mediation of hormonal effects in the intact cell. An early report by SUTHERLAND et al. 2 described the properties of adenyl cyclase in a particulate fraction derived from brain and other tissues. These preparations required Mg *+ or Mn *+ for optimal activity. Ca 2+ and ions of other divalent metals did not replace Mg 2+. In this paper are described the results of investigation of the properties of adenyl cyclase in a particulate fraction from calf brain cortex. Evidence is presented that the activity of the enzyme in these preparations is dependent upon metal ions which contaminate the reaction mixture. The results indicate that this metal ion is Ca 2+. The possible relationship of these findings to the physiological function of adenyl cyclase and 3',5'-AMP is discussed. EXPERIMENTAL PROCEDURE Materials
The adenyl cyclase used in these experiments was contained in a 2000 × g particulate fraction isolated from homogenates of calf cerebral cortex. The brains were obtained soon after slaughter of the animal and were cooled with ice. All subsequent operations were performed in the cold room. The cortex was homogenized for 30 sec in a Waring Blender with 2 vol. of a solution containing IO mM NaC1 and Abbreviation: EGTA, 1,2-bis-(2-dicarboxymethylaminoethoxy)ethane.
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251
IO mM KC1. Some particulate fractions were obtained from homogenates which were prepared in the presence of glycerol. The tissue in this case was homogenized in a mixture composed of I vol. of glycerol and I vol. of the salt solution. Unless they were to be used immediately, the homogenates were frozen and stored at - 7 o°. Immediately before use, the homogenates were thawed and were centrifuged at 2000 × g for 30 min. The supernatant suspension was decanted, and the residue was resuspended to its original volume in a Io-mM NaC1-KC1 solution. Each ml of the suspension contained the particulate fraction derived from 0.3 g of brain tissue. Ion-exchange resins were Baker Analyzed reagent grade. 3',5'-AMP and the disodium salt of ATP were obtained from Sigma Chemical Co. According to the manufacturer, the ATP had been specially purified to reduce the concentration of trace metals. It was used without further purification. The chelating agent 1,2-bis-(2dicarboxymethylaminoethoxy)ethane (EGTA) was obtained from K and K Laboratories. Caffeine was purchased from Eastman Organic Chemicals. All other chemicals were reagent grade. Glass-distilled-deionized water was used to prepare the solutions used in the preparative procedures and the experiments. All glassware was rinsed before use with I mM EDTA followed by glass-distilled-deionized water.
Methods The conditions for the enzymic formation of 3',5'-AMP and the assay procedure have been described in an earlier publication a. Minor modifications have been made in this procedure so that some description is necessary here. The standard reaction mixture contained 2 mM ATP, 5 mM MgC12, IO mM NaF, 6. 7 mM caffeine, and 4 ° mM Tris (pH 7.5). Other reagents were added in small volumes. The final volume of the reaction mixture was adjusted to 220 ml b y the addition of a Io-mM NaC1-KC1 solution. The concentration of protein in these samples was 1.2 mg/ml. In the experiments described in Table III, the caffeine was replaced b y 50 mM theophylline. In these samples, a 5-ml aliquot of the brain suspension was used as the source of adenyl cyclase, and the final volume of the reaction mixture was 55 ml. The concentration of protein in these samples was 2. 4 mg/ml. The concentration of all other reagents remained the same. The samples were incubated with swirling at 3 °0 . The usual incubation time was 30 rain. The reaction was stopped b y the addition of sufficient o.I M HC1 to drop the p H to 3 (indicator paper), and the temperature of the suspension was brought to IOO° by heating in a boiling-water bath. The p H was then adjusted to 7 with I M NaOH, and the samples were heated at IOO° for 15 min. After the samples were cooled, they were again acidified to p H 3 and were filtered under gravity. Filtration occurred more rapidly under these conditions. The filtrates were prepared for chromatography by readjusting the p H to 7. The assay procedure utilized sequential chromatography of enzyme reaction mixtures on an anionic exchange resin (Dowex 2-X8) and a cationic exchange resin (Dowex 5oW-X8). As pointed out in the original description of the method, this sequence effectively separated 3',5'-AMP from the other adenine-containing nucleotides and resulted in quantitative recovery of the cyclic nucleotide. The concentration of 3',5'-AMP in the eluate from the Dowex-5o column was determined from the absorbance of this solution at 2600 •. The measurement of absorbance was perBiochim. Biophys. Acta, 2o'r (:t97o) 250-260
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L.S. BRADHAMet al.
formed with a Cary-I 4 spectrophotometer using a cell with an optical path length of IO cm. This modification permitted the elimination of the terminal evaporation of the Dowex-5o eluate as described in the original procedure, but it also made it necessary to modify slightly the procedure for chromatography on the Dowex-5o columns. These columns were washed in the same manner as before except that the final washing was carried out with 0.05 M HC1 rather than with 0.3 M formic acid. The sample was transferred to the column in 0.05 M HC1, and the column was eluted with 230 ml of this solution. The first 9 ° nil of the eluate was discarded. The next 14o ml was collected and was used for the measurement of absorbance. Correction was made for the column blank as described in the original procedure. Measurements with the Cary 14 were made at 2500, 2600, and 2668/~ to determine the numerical values of the terms El, Ep, Es, and r. In this communication, enzymic activity is expressed as nmoles of 3',5'-AMP per mg of protein. This means of expression was used rather than the units proposed b y SUTHERLANDet al.* because the rate of accumulation of 3',5'-AMP in these reaction mixtures was non-linear (see Fig. 2). Analysis for Ca 2+ and Mn *+ was performed by the atomic absorption technique using a Perkin-Elmer Model 303 spectrophotometer. Particulate fractions were prepared for analysis using the method of SPARROW AND JOHNSTON4. Protein was determined b y the method of LOWRY et al. 5. RESULTS
General properties of adenyl cyclase preparations The adenyl cyclase preparations used in these experiments had, in general, the properties described by SUTHERLANDet al. ~. A Mg *+ concentration of 5 mM produced m a x i m u m accumulation of 3',5'-AMP, and this effect could not be duplicated by Ca 2+. N a F at a concentration of IO mM produced a 5-fold increase in enzymic activity. Enzyme preparations were more stable when stored at - 7 o°. This was especially true of the response of these preparations to EGTA and Ca *+. The homogenization of brain tissue in the presence of glycerol increased the activity of the enzyme by about 50 %. The increase was observed in preparations which were used immediately as well as in those which were used after storage at - - 7 o°. This effect is illustrated by the difference between the activities of enzyme preparations I I and I I I shown in Table I. Maximum enzymic activity was observed in samples in which glycerol-treated preparations were incubated with IO mM N a F and 50 mM theophylline. Under these conditions, I m g of protein catalyzed the formation of 9 nmoles of 3',5'-AMP in 30 rain. This represented the conversion of 1 % of the ATP to the cyclic nucleotide. EBect of EGTA on adenyl cyclase The effect of the inclusion of EGTA in enzyme reaction mixtures is illustrated by Fig. I. This figure summarizes the data obtained from five experiments. Because of the variation in the amount of 3',5'-AMP produced in individual experiments, the results are expressed as percent of the concentration of nucleotide produced in the control samples. I t can be seen from these data that the inclusion of EGTA at a concentration of Biochim. Biophys..4cta, 2Ol (197 o) 250-260
ADENYL CYCLASE AND Ca 2+
253
TABLE I EFFECT
OF Ca $+ ON
THE
INHIBITION
OF BRAIN
ADENYL
CYCLASE
BY
EGTA
Additions to the enzyme suspension were m a d e i m m e d i a t e l y before the reaction was started. The final concentration of E G T A and Ca ~+ in the e x p e r i m e n t s w i t h e n z y m e p r e p a r a t i o n I was o.o25 raM. The concentration of these reagents in the e x p e r i m e n t s w i t h e n z y m e p r e p a r a t i o n s I I a n d I I I was 0.05 mM. The n u m b e r s in p a r e n t h e s e s represent the n u m b e r of samples assayed.
Enzyme prep.
Additions
3",5"-AMP (nmoles/mg
Activity (%)
(± S.E.)) I
None (9) E G T A (8) E G T A + Ca 2+ (4)
3.42 ± 0.08 1.47 i 0.06 3.34 :J2 o.oi
ioo 43 98
II
None (6) E G T A (6) E G T A + Ca ~+ (6)
4.34 4- o . i o 1.77 ~ 0.o6 3.98 ± o.o6
IOO 41 92
III*
N o n e (5) E G T A (5) E G T A + Ca 2+ (6)
6.11 ~ o.o 7 3.23 ± 0-05 5.37 • o-07
ioo 53 88
* This p r e p a r a t i o n was obtained f r o m the same tissue as e n z y m e p r e p a r a t i o n I I b u t was treated w i t h glycerol as described u n d e r EXPERIMENTAL PROCEDURE.
t 0.02 EGTA
I 0.04
I I I 0.06 0.08 O.lO CONCENTRATION ( m M)
Fig. i. Effect of E G T A on the f o r m a t i o n of 3',5'-AMP. V a r y i n g concentrations of E G T A were included in the usual i n c u b a t i o n m i x t u r e , a n d the i n c u b a t i o n was performed as described in the text. E a c h p o i n t is the m e a n of the p e r c e n t activity obtained in the n u m b e r of samples indicated b y the n u m b e r s in parentheses. The vertical bars represent t h e S.E. No d a t a were o b t a i n e d for concentrations of E G T A less t h a n o.oi25 mM.
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254
o.I mM reduced by more than 6o % the amount of 3',5'-AMP forlned in the reaction mixture. This inhibition was little more than that obtained in the presence of 0.05 mM EGTA. The presence of the chelating agent at a concentration of 12.5 #M had very little effect. Addition of EGTA to samples after the reaction was stopped had no effect on the 3',5'-AMP measured. This experiment was done to eliminate the possibility that the EGTA interfered with the assay. The inhibitory effect of EGTA on brain adenyl cyclase was reproduced on many different enzyme preparations. Occasionally samples were not affected by the chelating agent. In these instances, however, the enzyme preparations were partially inactive, the enzyme activity having fallen already to the level obtained in the presence of EGTA.
Reversal of EGTA inhibition by Ca 2+ The inhibitory effect of EGTA on adenyl cyclase preparations was prevented by the inclusion of Ca 2+ in the enzyme reaction mixture. The results of several such experiments are summarized in Table I. In these experiments, EGTA and CaC1z were added to the mixture before the reaction was started. In each experiment, the lowest concentration of EGTA which produced maximal inhibition of the particular enzyme preparation was used. This concentration varied from one preparation to another, but the maximum percent inhibition was roughly the same. As indicated by the table, the inclusion of CaC12 at a concentration equivalent to the concentration of EGTA almost completely prevented the inhibition by the chelating agent. Under these conditions, the mean concentration of 3',5'-AMP accumulated in the reaction mixture was 92 % of the control value. The effect of the inclusion of other alkaline earth metal ions in the reaction mixture is shown in Table II. It can be seen that Sr 2+ were almost as effective as Ca z+ in preventing the inhibition by EGTA. Ba ~+ had no effect. The effects of EGTA and EGTA plus Ca ~+ on the time course of the reaction are illustrated in Fig. 2. Each part of this figure summarizes the results of two individual experiments using the same preparations of glycerol-treated adenyl cyclase. Fig. 2A TABLE II RELATIVE EFFECTS OF ALKALINE EARTH METALS ON THE INHIBITION OF BRAIN ADI~NYL CYCLASE BY E G T A The reaction was carried o u t in the presence of glycerol-treated 2o00 × g particulate suspension. Additions to the reaction m i x t u r e were m a d e i m m e d i a t e l y before the reaction was started. The final concentration of E G T A and the various salts was 0.05 mM. The n u m b e r of samples in each e x p e r i m e n t a l g r o u p is indicated b y the n u m b e r in parentheses.
Additions
3",5"-A M P (nmoles /mg
A ctivity (%)
6.15 3.66 3.87 5.16 5.60
ioo 60 63 84 91
(~S.E.))
None (5) E G T A (5) E G T A + BaCI~ (4) E G T A + SrC12 (8) E G T A + CaClz (12)
-~ i ± ± ±
o.ii 0-05 0.04 0.04 o.09
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ADENYL CYCLASE AND C a 2+
shows the effect of the inclusion of EGTA in the reaction mixture, while Fig. 2B shows the effect of the inclusion of EGTA and an equimolar concentration of Ca ~+. It can be seen that the rate of accumulation of the cyclic nucleotide in these reaction mixtures was non-linear. Because of the small number of points on these curves, it was impossible to determine if EGTA had any effect on the initial rate of the reaction. It was clear, however, that the maximum accumulation of 3',5'-AMP was reduced in the presence of EGTA, and that this inhibition was prevented by the inclusion of an equimolar concentration of Ca 2+. These results indicated that the inclusion of Ca ~+ prevented the inhibitory effect of EGTA, but they did not provide definite proof that the effect of Ca 2+ was a true reversal of EGTA inhibition. This was demonstrated by modifying the experimental conditions so that Ca 2+ were added to the reaction mixtures containing EGTA at a time when the accumulation of 3',5'-AMP was approaching the maximum. Samples containing EGTA at a concentration of 0.05 mM were incubated for 30 min. At this time CaC12 was added to duplicate samples to give a final concentration of 0.05 mM. Incubation of the Ca2+-treated samples and the untreated controls was continued for another 15 rain. i
A
i
i
B
S
i
i
o /' /s
i
/
E
i
/
t' tJ
i
2
0
0
ll5
I 50 0 MINUTES
I 15
i 50
0 0
i 15
310
i 45
MINUTES
Fig. 2. The effect of E G T A and E G T A plus Ca n+ on the time course of the a c c u m u l a t i o n of 3',5' AMP. The concentration of E G T A and Ca 2+ in these e x p e r i m e n t s was o.o 5 raM. 3',5'-AMP concentrations at zero time were obtained b y acidifying the reaction m i x t u r e s before t h e addition of ATP. E a c h p o i n t is t h e m e a n c o n c e n t r a t i o n o b t a i n e d in t w o individual experiments. The vertical bars represent the range of the values. A. 0 - - 0 , i n c u b a t e d w i t h E G T A ; ( D - - O , incubated w i t h o u t EGTA. 13. 0 - - 0 , incubated w i t h E G T A ; O - - ( D , incubated w i t h equimolar E G T A and Ca 2+. Fig. 3- Reversal of E G T A inhibition b y Ca ~+. E G T A was included in t h e usual i n c u b a t i o n m i x t u r e at a concentration of o.o 5 raM. An equimolar concentration of Ca ~+ was added to samples at the t i m e indicated b y the arrow. 3',5'-AMP concentration at zero time was o b t a i n e d b y acidifying reaction m i x t u r e s before the addition of ATP. 0 - - 0 , incubated w i t h E G T A ; O, incubated with equimolar E G T A and Ca n+.
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The results of such an experiment are shown in Fig. 3. These results were typical of those obtained in four experiments using the same glycerol-treated adenyl cyclase preparation. It can be seen that, in the presence of EGTA, the concentration of 3',5'-AMP had almost reached a maximum at 30 min and extension of the incubation period to 45 min did not result in the accumulation of appreciably more of the cyclic nucleotide. The addition of Ca 2+ at 30 min, however, reversed the effects of EGTA and about 7° % more 3',5'-AMP was formed during the subsequent i5-min incubation. The final concentration of the nucleotide in the Ca2+-treated samples was close to that normally obtained in the absence of EGTA.
Effect of EGTA on the recovery of 3',5'-AMP When commercial 3',5'-AMP was incubated with the 2000 × g particulate fraction in the absence of ATP, there was a very rapid destruction of the nucleotide. This loss was attributed to the presence of high concentrations of nucleotide 3',5'phosphodiesterase. Caffeine at a concentration of 6. 7 mM had no effect on this loss. It was possible, however, to partially inhibit the destruction of the nucleotide by carrying out the incubation in a medium containing theophylline at a concentration of 50 mM. A detailed description of the conditions for these incubations is presented in Methods. Using these conditions, experiments were performed to determine if the inhibitory effect of EGTA were due to the inhibition of adenyl cyclase or to the stimulation of the phosphodiesterase. The recovery of added 3',5'-AMP was used as a measure of phosphodiesterase activity. A known amount of the nucleotide was added to the enzyme reaction mixtures in the absence of ATP. EGTA at a concentration of o.I mM was included in some of the samples. The samples were incubated in the usual manner. Control samples containing ATP and o.I mM EGTA were incubated at the same time. The results of these experiments are presented in Table III. It can be seen that the mean recovery of added 3',5'-AMP in these experiments was 35 %, and that this recovery was not affected by the inclusion of EGTA. In fact there appeared to be a small increase in the amount recovered when EGTA was pres-
TABLE III EFFECT OF E G T A ON THE RECOVERY OF 3',5'-AMP Samples of brain adenyl cyclase were incubated as described in the text. The samples in G r o u p A were incubated with added 3',5'-AMP b u t no ATP. The a m o u n t of added 3',5'-AMP was 1.155 mg. The concentration of A T P in t h e samples of G r o u p B was 2 raM. I n b o t h groups, the concentration of E G T A was o.i mM. The n u m b e r s in p a r e n t h e s e s indicate the n u m b e r of samples incubated u n d e r each set of conditions.
Group
Additions
Total 3",5'-A M P #g (±S.E.)
A
3',5 '-AMP (7) 3',5'-AMP + E G T A (16)
390 ~ 14 441 ± 14
B
ATP (7) ATP + E G T A (7)
399 -I:: 17 2o6 -+- i i
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Activity
Recovery
(%1
(%) 34 38
ioo 52
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ADENYL CYCLASE AND C a 2+
ent. On the other hand, addition of EGTA to the control samples produced a 5o % inhibition of the formation of 3',5'-AMP from ATP. It appeared, therefore, that the effect of EGTA was due to the inhibition of the formation of the nucleotide rather than to the stimulation of its destruction.
Inhibition of the formation of 3',5'-AMP by Ca 2+ When Ca 2+ was added to reaction mixtures in the absence of Mg2+, no 3',5'AMP was produced. Nor was there any additive effect of Ca2+ when it was included in complete reaction mixtures at a concentration of o.I mM or less. Higher concentrations of Ca 2+, however, were found to inhibit the formation of the nucleotide in the complete reaction mixture. With different enzyme preparations, some variability in the precise concentration of Ca 2+ required to produce maximum inhibition was encountered. In general, however, almost 80 % inhibition was obtained when Ca ~+ included in complete reaction mixtures at concentrations ranging between 0.5 and i raM. The addition of Ca 2+ to samples at the end of the incubation had no effect, indicating that Ca 2+ was not interfering with the assay.
Assay of reaction ,mixtures for Ca ~+ and Mn 2+ Four samples of the 2000 × g particulate fraction and a sample of the complete reaction mixture less the particulate fraction were analyzed for Ca 2+ and Mn ~+. The results of these analyses are recorded in Table IV. These results indicate that the mean concentration of Ca ~+ in a typical complete reaction mixture was in the neighborhood of 0.033 raM. About 60 % of this Ca 2+ was contributed by the particulate suspension while the remainder was contributed by the other components of the reaction mixture. Contamination from this latter source was due largely to Ca ~+ which contaminated the ATP. Most of the Mn 2+ which contaminated the final reaction mixtures was contributed by the reagent solutions. The mean concentration of Mn ~+ in a typical reaction mixture was 2.2/~M. T A B L E IV METAL CONTENT OF COMPONENTS OF ENZYME REACTION MIXTURES F o u r samples of the glycerol-treated particulate fractions suspended in o.o1 M NaC1-KC1 solution and four samples of the incubation solution containing all the c o m p o n e n t s of the reaction m i x t u r e except the particulate fraction were assayed for Ca ~+ and Mn 2+. The concentrations of the metals were corrected to give the concentration c o n t r i b u t e d to the final reaction mixture. The n u m b e r s in p a r e n t h e s e s are the s t a n d a r d deviations for each determination.
Reaction mixture component
Particulate fraction I n c u b a t i o n solution Total
Metal conch. (l~M) Ca~+
Mn2+
19.3 (2.8) 14.o (2.5) 33.3
0.38 (0.026) 1.8 (o.I5) 2.18
DISCUSSION
The experiments described in this communication provide evidence that the formation of 3',5'-AMP in the presence of a 2000 × g particulate fraction of calf brain Biochim. Biophys. Acta, 2Ol (197o) 25o-26o
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L.S. BRADHAMet al.
cortex is dependent upon metal ions which are present in the reaction mixture. This dependency becomes apparent when the enzyme preparation is treated with the chelating agent EGTA. This compound causes inhibition of the formation of the cyclic nucleotide when it is included in enzyme reaction mixtures at very low concentrations. It has been established previously2 that the formation of 3',5'-AMP in the presence of enzyme preparations of this type is dependent upon Mg2+. The inhibitory effect of EGTA, however, is observed in samples that contain Mg2+ at an optimal concentration. This concentration of Mg2+ is IOO times the concentration of chelating agent which produces maximal inhibition. The effect, therefore, cannot be attributed to the chelation of Mg2+, since the amount of Mg2+ chelated by inhibitory concentrations of EGTA would not significantly affect adenyl cyclase activity. It is apparent, therefore, that some metal other than Mg2+ is present in the reaction mixture and is required for the accumulation of 3',5'-AMP. One likely candidate for this role is Ca 2+. In the presence of equimolar concentrations of Ca 2+, the effect of EGTA is reversed. This reversal suggests that the inhibition by EGTA is due to the chelation of Ca2+ present in the reaction mixture. These Ca 2+ are necessary for maximum accumulation of 3',5'-AMP, and the chelation of these ions by EGTA results in inhibition of the formation of the nucleotide. It is recognized that the data do not rule out completely the participation of a metal other than Ca 2+. The nature of the chelation by EGTA in the reaction mixture has not been clarified. It is possible that the metal ions responsible for the formation of 3',5'-AMP are bound to some component of the particulate fraction, and that EGTA inhibits the reaction by forming a chelate with this metal without actually removing it from its binding site. The addition of Ca2+ to the reaction mixture then reverses the inhibition by competing with the unknown metal chelate for the chelating agent. This model would be possible only if the unknown metal chelate were more stable than the Ca 2+ chelate. The stability constants of a number of EGTA-metal chelates have been determined 6-s. Mn 2+ is a possible candidate since the stability constant of its EGTA chelate is only about 25 times the stability constant for the Ca 2+ chelate. SUTHERLAND e~ al. 2 have shown, moreover, that Mn 2+ can replace Mg2+ as an activator of adenyl cyclase. Mn 2+ is a contaminant of the reaction mixtures used in the experiments reported here. The ratio of the concentrations of Ca 2+ to Mn 2+ in the reaction mixture was about 15 to I. It is felt that at this concentration ratio, Ca~+ would not compete effectively with Mn 2+ for the EGTA, and thus would not be effective in reversing the EGTA inhibition. It is even less likely that Sr ~+ would compete effectively with Mn ~+ for the chelating agent since the Mn 2+ chelate with EGTA is about 20 oo0 times more stable than the Sr 2+ chelate. The data of Table II show, however, that Sr 2~ is almost as effective as Ca 2+ in reversing the inhibition by EGTA. It is felt, therefore, that it is extremely unlikely that the effects of EGTA demonstrated in these experiments are due to the chelation of Mn 2+. The participation of other transition metals such as Fe 2+, Cu 2+ or Zn 2+ is also improbable, since the stability constants of the EGTA chelates of these metals are considerably higher than the stability constant of Ca 2+ chelate. These possibilities, however, cannot be completely ruled out until experiments of the type described here are carried out with enzyme preparations and reagents which are completely free of metal contamination. Biochim. Biophys. Mcta, 2Ol (197o) 25o-26o
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Other evidence supports the conclusion that the effects of EGTA are due to chelation of Ca *+. The concentration of EGTA which produces m a x i m u m inhibition of the formation of 3',5'-AMP is roughly equivalent to the concentration of Ca *+ which is found in the reaction mixture. This inhibition is reversed by the addition of Ca 2+ in a concentration equivalent to the concentration of EGTA. Sr*+ produces partial reversal of the inhibition while Ba *+ is ineffective. The similarity of the chemical properties of Ca *+ and Sr 2÷ is well-known, and the ionic radii of the two metals are quite close. It is reasonable to conclude, therefore, that the formation of 3',5'-AMP in the presence of the particulate fraction from brain used in these experiments is dependent upon the presence of both Mg *+ and Ca*÷. This dual requirement for Mg*+ and Ca *+ has parallels in other biological systems. The ATPase of myofibrils has been shown to require both cations for activity*. This property accounts for the fact that the ATPase and the contraction of muscle are both inhibited by EGTA 1°, 11. Another example is the ATPase found in the vesicular elements of the sarcoplasmic reticulum which possess relaxing factor activity. The activity of the ATPase and the inhibitory effect of the vesicular granules on the myofibrillar ATPase have been shown to depend upon both Ca 2÷ and Mg*+ (ref. 12). Similar enzymes have been found recently in fractions of brain tissue 13,14. Two possible explanations must be considered in assigning a mechanism to the effect of Ca *+ on the accumulation of 3',5'-AMP. One possibility is that Ca *+ is necessary for maximal activity of adenyl cyclase and is, therefore, involved in the formation of the nucleotide. The alternative explanation is that Ca *+ inhibit the destruction of the nucleotide by the enzyme nucleotide 3',5'-phosphodiesterase. Previous work has shown that the latter enzyme is associated with particulate fractions isolated from brain and other tissues15, TM. Phosphodiesterase activity was most certainly present in our reaction mixtures since the accumulation of 3',5'-AMP required the presence of caffeine or theophylline. These compounds have been shown to be competitive inhibitors of phosphodiesterase activity 15,17. The data in Table I I I indicate that it was impossible to eliminate all of the phosphodiesterase activity in our reaction mixtures. In the presence of 50 mM theophylline, the mean recovery of added 3',5'-AMP was about 35 %. This recovery was used as a measure of phosphodiesterase activity in order to determine if the effect of EGTA was due to inhibition of adenyl cyclase or to stimulation of the phosphodiesterase. The data show that the recovery of added 3',5'-AMP was not decreased at all by the inclusion of o.I mM EGTA. The same concentration of EGTA, however, inhibited by 50 % the formation of the nucleotide from ATP in the control experiments performed under identical conditions. These results eliminate the possibility that the inhibitory effect of EGTA on the accumulation of 3',5'-AMP is due to the stimulation of the phosphodiesterase. It is concluded, therefore, that the effect of the chelating agent is due to inhibition of adenyl cyclase, and that this inhibition is caused by the chelation of Ca 2+. This Ca ~+ m a y be bound to the enzyme or some structure associated with the enzyme in the 2000 × g particulate fraction. DAVOREN AND SUTHERLANDTM have shown that adenyl cyclase isolated from a number of tissues is associated with the plasma membrane, and DE ROBERTIS et al. TM have demonstrated that part of the adenyl cyclase of rat cerebral cortex is associated with synaptic membranes which sediment in centrifugal fields between 900 and i i 500 × g. These cell components were probably Biochim. Biophys. Acta, 2Ol (197 o) 25o-26o
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contained in the particulate fractions used in our experiments. It is possible, therefore, that the Ca ~+ required for adenyl cyclase activity is bound to membrane components. There is ample physiological evidence to suggest that the stimulatory effect of Ca ~+ on adenyl cyclase has functional significance. Some of this evidence has been reviewed by RASMUSSEN AND TENENHOUSE 20, who have proposed that the function of adenyl cyclase is to either alter the permeability of the plasma membrane to Ca 2+ or to effect the intracellular release of Ca2+ from the membrane. In particular, they cite experiments which demonstrate a rise in the intracellnlar concentration of both 3',5'-AMP and Ca *+ in kidney and bone when these tissues are stimulated by parathormone. Furthermore, the adenyl cyclase activity of fat cells .1 and of isolated fat cell membranes 22 has been shown to be dependent upon Ca2+. In view of this evidence, the finding that the adenyl cyclase of brain is dependent upon Ca ~+ for its activity is highly significant. ACKNOWLEDGMENTS
The authors express their appreciation to Dr. Edwin R. Hughes for performance of the metal analyses, to Dr. Charles L. Wadkins for constructive criticism of the manuscript, and to Dr. Eugene J. Towbin for continued encouragement and support of the work. REFERENCES i E. W. SUTHERLAND, I. OYE AND ~R. W. BUTCHER, Recent Progr. Hormone Res., 21 (1965) 623. 2 E. W. SUTHERLAND, T. W. HALL AND T. MENON, J. Biol. Chem., 237 (1962) 122o. 3 L. S. BRADHAM AND D. W. WOOLLEY, Biochim. Biophys. Acta, 93 (1964) 475. 4 M . T . SPARROW AND B. M. JOHNSTON, Biochim. Biophys. Acta, 90 (1964) 425 • 5 0 . H. LowRY, N. J. ROSEBROUGH, A. L. FARR AND H. J. RANDALL, J. Biol. Chem., 193 (1951) 265. 6 S. CHABEREK AND A. E. MARTELL, Organic Sequestering Agents, John Wiley, New York, 1959, p. 577. 7 J. H. HOLLOWAY AND C- N. RE1LLEY, U.S. Department of Commerce, O~ce of Technical Services, Hept. No. PB 114499 (1959); Chem. Abstr., 55 (1961) I6IO5a. 8 K. H. SCHROEDER, Acta Chem. Scand., 17 (1963) 15o9, 9 J. C. SEIDEL AND J. GERGELY, J. Biol. Chem., 238 (1963) 3648. io A. WEBER AND R. HERZ, J. Biol. Chem., 238 (1963) 599. i i S. EBASHI, J. Biochem. Tokyo, 5° (I961) 236. 12 Y. NAGAI, J. Biochem. Tokyo, 58 (1965) 429. 13 K. KADOTA, S. MORI AND R. IVIAIZUMI,J. Biochem. Tokyo, 61 (1967) 424 . 14 Y. NAKAMARU, J. Biochem. Tokyo, 63 (1968) 626. 15 H. W. BUTCHER AND E. W. SUTHERLAND, J. Biol. Chem., 237 (1962) 1244. 16 E. DE ROBERTIS, G. H. D. ARNAIZ, M. ALBERICI, R. W. BUTCHER AND E. W. SUTHERLAND, J. Biol. Chem., 242 (1967) 3487 . 17 W. Y. CHEUNG, Biochemistry, 6 (1967) lO79. I8 G. I. DRUMMOND AND S. PERROTT-YEE, J. Biol. Chem., 236 (1961) 1126. 19 P. R. DAVOREN AND E. W. SUTHERLAND, J. Biol. Chem., 238 (1963) 3o16. 20 ]-I. RASMUSSEN AND A. TENENHOUSE, Proc. Natl. Acad. Sci. U.S., 59 (1968) 1364. 21 1~. H. WILLIAMS, S. A. WALSH AND J. W. ENSlNCK, Proc. Soe. Exptl. Biol. Med., 128 (1968) 279. 22 H. P. BAR AND O. HECHTER, Federation Proc., 28 (1969) 571.
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