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Comparison of the effects of Ca2+ and Mg2+ on the adenyl cyclase of beef brain
434
BIOCHIMICAET BIOPHYSICAACTA
BBA 66650 COMPARISON OF T H E E F F E C T S OF Ca 2+ AND Mg~+ ON T H E A D E N Y L CYCLASE OF B E E F B R A I N
LAURENCE S. BRADHAM Brain Research Institute and Department of Biochemistry, University of Tennessee Medical Units and the Laboratories of Endocrinology and Metabolism, Research Division, Veterans Administration Hospital, 3/lemphis, Tenn. 38io 4 (U.S.A.)
(Received December 23rd, i971) (Revised nlanuscript received April I ith, 1972)
SUMMARY The adenyl cyclase of beef cerebral cortex required both Mg~+ and Ca =+ for maximal activity. The requirement for Mg=+ was absolute, and it was demonstrated that this cation was necessary for the interaction of the substrate with the enzyme. Enzymic activity was dependent upon the ratio of Mg 2+ to ATP, and maximal activity was obtained only when there was an excess of the divalent cation. Under these conditions, the apparent K m for ATP was 1.2-1. 3 mM and V was 45 nmoles/mg. The requirement for Ca 2+ was demonstrated by use of the chelating agent 1,2bis-(2-dicarboxymethylaminoethoxy)ethane (EGTA) which specifically chelates Ca 2+ in the presence of Mg 2+. Adenyl cyclase activity was inhibited non-competitively by this compound. The V and the initial velocity were reduced approx. 6o% in the presence of o.I mM EGTA. There was no significant effect on the apparent K m for ATP. The velocity of the reaction was returned to normal by the addition of Ca 2+ in concentrations significantly lower than the concentration of the chelating agent.
INTRODUCTION The activity of adenyl cyclase is affected by many factors, and among these are divalent cations. The addition of either Mg 2÷ or Mn =+ to adenyl cyclase preparations is required to elicit enzymic activity, and it is generally accepted that the Mg=+ is the natural co-factor 1. Tile addition of Ca 2+ in place of Mg2+ has no activating effect on the enzyme 2. It has been demonstrated, however, that the adenyl cyclase of beef cerebral cortex depends upon both Ca =+ and Mg2+ for maximal activity a. Maximal activity in the presence of Mg 2+ depends upon trace amounts of Ca z+ which appear to be bound to the particulate matter containing the adenyl cyclase. Tile chelation of these ions by the Abbreviation: EGTA, 1,2-bis-(2-dicarboxymethylaminoethoxy)ethane. Biochim. Biophys. Acta, 276 (1972) 434-443
ADENYL CYCLASE AND DIVALENT CATIONS
435
relatively Ca2+-specific chelator 1,2-bis-(2-dicarboxymethylaminoethoxy)ethane (EGTA) results in inhibition of the enzyme which is reversed b y the addition of Ca 2+. A number of recent reports have provided evidence that Ca ~+ m a y play a similar role in the control of the adenyl cyclase activity of other tissues as well *-9. In view of this evidence, the results presented here are of particular significance. A kinetic analysis of the effects of Ca ~+ and Mg ~+ on the adenyl cyclase activity of particulate fractions from beef cerebral cortex has been performed. The results show that both ions are required for activation of the enzyme, but the mechanisms by which they produce activation are quite dissimilar. EXPERIMENTAL PROCEDURE
Materials
Theophylline was purchased from Nutritional Biochemicals, Inc. ; EGTA from K and K Laboratories; and adenosine 3',5'-monophosphate (cyclic AMP) and the disodium salt of ATP from Sigma Chemical Co. Ion-exchange resins used in the chromatographic steps were Bio-Rad preparations obtained from Calbiochem. All other chemicals were reagent grade preparations obtained from various commercial sources. Methods
Particulate fractions containing adenyl cyclase were prepared from fresh beef cerebral cortex. The preparation of the 2000 × g fraction has been described elsewher@. The ioo ooo X g fraction was prepared b y a procedure which incorporated techniques developed by Fitzpatrick et al. TM. Cortical tissue was homogenized for 30 s in a Waring Blendor with 2 vol. of a solution composed of equal parts glycerol and io mM NaC1-KC1 solution. The homogenate was centrifuged at 6000 X g for 15 min, and the pellet was washed with IO mM NaC1-KC1 solution. The washed pellet was homogenized in I vol. of 2 M sucrose using a motor-driven Teflon pestle. This homogenate was centrifuged at 13 300 × g for IO min, and the residue was discarded. The supernatant suspension was diluted with 7 vol. of cold water, and the suspension was centrifuged at I I 900 × g for IO min. The pellet was extracted with I vol. of 0.25 M sucrose, and the combined suspension was centrifuged at IOO ooo × g for 30 rain. The resulting pellet was resuspended in a small volume of 50% glycerol solution. The protein concentration of suspensions prepared in this manner ranged from 15-2o mg/ml. The enzyme was stored in this suspension at --7 ° °C. When incubated with 2 mM ATP and 5 mM MgC12 under the conditions described below, I m g of this particulate fraction catalyzed the formation of 25 nmoles of cyclic AMP in 30 rain. The adenyl cyclase activity of the IOO ooo × g fraction was routinely assayed at 37 °C at a protein concentration of I mg/ml. The basic incubation solution contained 5 ° mM theophylline, IO mM NaF, and 40 mM Tris (pH 7-5). A T P and MgC12 were added as described in individual experiments. The same incubation solution was used in assaying the 2000 x g fraction, but the protein concentration was 2. 7 mg/ml and the temperature was 30 °C. Unless otherwise specified, the incubation time was 30 min. The reaction was terminated b y adjusting the p H of the samples to 3.5 and heating to IOO °C. Protein was removed b y centrifugation after readjustment of the pH to 7.5. Quantitative analysis for cyclic AMP was performed by a modification of proBiochim. Biophys, Acta, 276 (1972) 434-443
436
L.S. BRADHAM
cedures a l r e a d y described3, n. I n the modified version of this m e t h o d , the p r e l i m i n a r y purification of e n z y m e reaction m i x t u r e s was carried out on a xo-ml column of Dowex-2 resin ( B i o - R a d AG z-X, 2o0-4oo mesh) in the f o r m a t e form. The cyclic nucleotide was eluted from this column w i t h 5 ° ml of 2 M formic acid (flow r a t e not exceeding o.3 ml/min). An aliquot of this eluate was c h r o m a t o g r a p h e d on a cation exchange colunm ( B i o - R a d A G 5o-X8, 2oo-4o0 mesh) with the dimensions described in the original procedure. The cyclic nucleotide was e l u t e d from this column with o.o 5 M He1 a n d the fraction of e l u a t e (75-215 ml) c o n t a i n i n g cyclic A M P was used for the m e a s u r e m e n t of a b s o r b a n c e ~. The r e c o v e r y of cyclic A M P from the columns was essentially q u a n t i t a t i v e ( > 95%), a n d the results of assay of d u p l i c a t e samples agreed w i t h i n a few per cent. F o r the d e t e r m i n a t i o n of A T P a s e a c t i v i t y , the p a r t i c u l a t e fractions were incub a t e d with 4 m N M g A T P u n d e r conditions i d e n t i c a l to those used in the a d e n y l cyclase assay. The inorganic p h o s p h a t e released was d e t e r m i n e d b y the m e t h o d of F i s k e a n d S u b b a R o w 1~. P r o t e i n was a s s a y e d b y the m e t h o d of L o w r y et al? ~.
2( <
[o
ATP (raM)
Fig. i. Effect of variation of ATP concentration on the adeny] cyclase activity of the ioo ooo x g fraction. In each experiment, the Mg2+ concentration was kept constant and the concentration of ATP was varied over the range indicated. ID---ID, i mM Mg~+; ©--(2), 2 mM Mg2+; &--A, 5 mM Mg 2+.
RESULTS
Effect of variation of A T P and Mg ~+ on adenyl cyclase activity T h e a c t i v i t y of t h e a d e n y l cyclase of the I00 000 × g fraction was sensitive to v a r i a t i o n s in the r e l a t i v e c o n c e n t r a t i o n s of A T P a n d Mg 2+. As i l l u s t r a t e d b y the curves in Fig. I, m a x i m a l a c t i v i t y was o b t a i n e d o n l y when the c o n c e n t r a t i o n of Mg 2+ exceeded the c o n c e n t r a t i o n of A T P . Excess A T P i n h i b i t e d the e n z y m e c o m p e t i t i v e l y . The i n t e r r e l a t i o n s h i p between A T P a n d Mg z+ c o n c e n t r a t i o n s is f u r t h e r illus-
Biochim. Biophys. Acta, 276 (1972) 434-443
437
ADENYL CYCLASE AND DIVALENT CATIONS
500 B
I
i
i
l
400
6oo u -6
zoo Io
too
o
I
2
3
M g A T P (TriM)
4
5 0
10(30
1500
2000
I,/Mg ATP
Fig. 2. Effect of v a r i a t i o n of t h e relative c o n c e n t r a t i o n s of A T P a n d Mg 2+ on t h e a d e n y l cyclase a c t i v i t y o f t h e i o o ooo × g fraction. (A) E n z y m e a c t i v i t y o b t a i n e d u n d e r t h e c o n d i t i o n s listed below p l o t t e d as a f u n c t i o n of M g A T P c o n c e n t r a t i o n . O---O, A T P m a i n t a i n e d c o n s t a n t (4 raM) a n d Mg 2+ c o n c e n t r a t i o n v a r i e d f r o m i to 4 raM; © - - C ) , A T P a n d Mg 2+ c o n c e n t r a t i o n s m a i n t a i n e d e q u i m o l a r over t h e c o n c e n t r a t i o n r a n g e ; A - - A , Mg 2+ c o n c e n t r a t i o n m a i n t a i n e d c o n s t a n t (5 mM) a n d A T P c o n c e n t r a t i o n v a r i e d f r o m I to 4 raM. (B) Reciprocal p l o t s of t h e d a t a o f (A). T h e m e t h o d of least s q u a r e s w a s u s e d to fit t h e b o t t o m curve. T h e d o t t e d line d e n o t e s e x t r a p o l a t i o n to t h e y-axis. Refer a b o v e for t h e definition of s y m b o l s .
trated by the experiments summarized in Fig. 2. These experiments were performed under conditions in which there was either an excess of ATP (bottom curve of Fig. 2A), an excess of Mg*÷ (top curve of Fig. 2A), or equimolar concentrations of ATP and Mg~+ (middle curve of Fig. 2A). Approximately the same maximal velocity was approached under the three sets of conditions, but the initial velocities were controlled by the relative concentrations of ATP and Mg*+. The reciprocal plots of these data (Fig. 2B) indicated that this was due to a progressive decrease in the Km for the substrate (MgATP) as the concentration of Mg2+ increased in relation to the concentration of ATP. This plot was linear only when there was an excess of Mg*+. The apparent Km for MgATP calculated from this data was 1.27 mM.
Inhibition of adenyl cyclase activity by EGTA The pattern of inhibition of the adenyl cyclase activity of the IOO ooo × g fraction by EGTA was identical to that obtained earlier using the 2000 / g fraction as the source of enzyme 3. As indicated by the data of Fig. 3, there was no effect of the chelating agent at concentrations of o.o125 mM or less. Maximal inhibition (approx. 60%) was obtained at an EGTA concentration of 0.025 mM, and there was no further effect of increasing the concentration up to o.I raM. In all of the subsequent experiments, an EGTA concentration of o.I mM was used routinely. The inhibition by EGTA was independent of substrate concentration. As shown by the data in Fig. 4A, the degree of inhibition remained the same when the concentration of ATP was varied over the range of 0.5 to 4 mM (in presence of 5 mM Mg*+). Biochim. Biophys. Acta, 276 (1972) 434-443
438
]~. s. BRAI)HAM
,oo
90
E
0o z~ 7o
60
5O
4O
30
0
004
0.06
008
0, 0
EGTA ('m M)
Fig. 3. I n h i b i t i o n of adenyl cyclase activity b y EGTA. The adenyl cyclase activity of the ioo ooo × g fraction was determined in the presence of various concentrations of EGTA. The s t a n d a r d i n c u b a t i o n m i x t u r e contained 2 mM A T P and 5 mM Mg 2+. E a c h p o i n t is the m e a n of the values o b t a i n e d in three experiments.
The reciprocal plot of these data (Fig. 4 B) indicated that this inhibition was noncompetitive. The m a x i m u m velocity of the reaction was reduced by approx. 6o%, but there was no significant effect on the apparent Km for the substrate. The V for the reaction (as calculated by the method of least squares) in the presence of EGTA was A
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i
i
I
El
i
~
i
30o
2oo
, I
2C
5
IOO
I
~o
%.I
,
L
ATP(mM)
ATP
('mM)
Fig. 4. Effect of variation of tile s u b s t r a t e c o n c e n t r a t i o n on the inhibition of adenyl cyclase b y E G T A . The IOO ooo X g fraction was used as the source of enzyme. (A) The concentration of A T I ~ was varied in the presence and in the absence of EGTA. The c o n c e n t r a t i o n of Mg 2+ in all samples was 5 raM. (B) Reciprocal plots of the d a t a of (A). The lines were fit to the points b y the m e t h o d of least squares. O---O, Control, no E G T A ; © - - O , o.I mM EGTA.
I~iochim. Biophys. Acta, 276 (i972) 434-443
439
ADENYL CYCLASE AND DIVALENT CATIONS
18.1 nmoles/mg, whereas in the control samples the value for this constant was 45.2 nmoles/mg. The apparent Km values in the presence and in the absence of the chelator were calculated to be 0.94 and 1.27 mM, respectively.
Effect of Ca z+ on the inhibition by EGTA Fig. 5 shows the effect of the inclusion of various concentrations of Ca 2+ in reaction mixtures containing o. I mM EGTA. These curves have the appearance of titration curves with the enzymic activity as the end-point. It should be noted that there was significant restoration of activity at concentrations of Ca 2+ lower than the concentration of EGTA. With both enzyme preparations, full activity was restored at 0.075 mM Ca 2+, whereas half the inhibited activity was restored at approx. 0.06 mM. t
I
I
I
I
IOG
90
x
E
,o
80
£
"5 7O
= u
5G
40
I
I
I
I
002
004
006
008
Ca CIz ('raM)
0
/
I
,o
I0
I ~
30
"rime (min)
Fig. 5. Effect of i n c l u s i o n of Ca 2+ on t h e i n h i b i t i o n of a d e n y l c y c l a s e b y E G T A . V a r y i n g concent r a t i o n s of CaC1, we re a d d e d to s a m p l e s of t h e 20oo × g f r a c t i o n a n d t h e IOO ooo × g f r a c t i o n i n c u b a t e d w i t h o . i mM E G T A . The c o n c e n t r a t i o n of Mg 2+ in all s a m p l e s w a s 5 mM. T h e concent r a t i o n of A T P w a s e i t h e r 2 mM (2ooo × g fraction) or 4 m M ( i o o ooo × g fraction). T h e d o t t e d line r e p r e s e n t s t h e a c t i v i t y of s i m i l a r s a m p l e s i n c u b a t e d in t h e a b s e n c e of E G T A . t ~ - - Q , 2 0 0 0 × g fraction; 0--0, IOO ooo X g f r a c t i o n . Fig. 6. Effect of E G T A a n d Ca *+ on t h e r a t e of f o r m a t i o n of cyclic AMP. S a m p l e s of t h e 2000 × g f r a c t i o n were i n c u b a t e d w i t h E G T A a n d Ca ~+ for v a r i o u s t i m e i n t e r v a l s in t h e p r e s e n c e of 2 mM A T P a n d 5 mM Mg a+. × - - X , control, no E G T A ; A - - A , o,i mM E G T A , no Ca*+; O - - O , o.I mM E G T A , 0.06 mM Ca2+; O---O, o.I mM E G T A , o.08 mM Ca *+.
The effect of these concentrations of Ca *+ on the rate of formation of cyclic AMP in the presence of the 2000 × g fraction, is illustrated by the data of Fig. 6. The concentrations of ATP and Mg~+ in these samples were 2 and 5 mM, respectively. In the control samples (no EGTA), cyclic AMP was formed at the rate of 383 pmoles/min per rag. The inclusion of o.I mM EGTA reduced this rate by 50% to 192 pmoles/min per mg. The rate was restored to normal (406 pmoles/min per mg) by 0.08 mM Ca 2+, Biochim. Biophys. Acta, 276 (1972) 4 3 4 - 4 4 3
440
L. S. B R A D H A M
whereas the inclusion of Ca 2+ at a concentration of o.o6 mM resulted in a rate (3o8 pmoles/min per rag) which was approximately halfway between the maximally inhibited rate and the control rate. The effect of Ca 2+ on the reduction of maximal velocity by EGTA is illustrated by the data in Table I. The concentration of Mg2+ in these samples was 5 raM. The data show that the 60% reduction in V caused by EGTA was reversed by the inclusion of 0.075 mM Ca 2+. Again, there was no significant effect of either EGTA or Ca 2+ on the apparent Km for ATP. TABLE
I
EFFECT OF E G T A
AND G a s+ ON KINETIC PARAMETERS OF BRAIN ADENYL CYCLASE
S a m p l e s o f t h e i o o o o o × g f r a c t i o n w e r e i n c u b a t e d w i t h E G T A a n d C a 2+ a s d e s i g n a t e d in t h e p r e s e n c e o f c o n c e n t r a t i o n s o f A T P r a n g i n g f r o m o. 5 t o 4 m M . T h e c o n c e n t r a t i o n o f M g ~+ in a l l s a m p l e s w a s 5 m M . T h e v a l u e s f o r B2m a n d V w e r e c a l c u l a t e d b y tile m e t h o d o f l e a s t s q u a r e s f r o n l r e c i p r o c a l p l o t s o f t h e s e d a t a . E a c h v a l u e r e p o r t e d is t h e n l e a n o b t a i n e d f r o m five e x p e r i m e n t s .
Expt
EGTA (mYI)
CaCl~ (~nM)
I£m × Io a
V (nrnoles[rng ~z S.E.)
I 2 3
o o.I o.I
o o 0.075
1.19 ~. o . 1 4 1.11 ± o.2 1.17 ± o . o i
4 5 . 8 -~ I. 7 18.1 ~z 1.2 4 2 . 9 ~ 1.6
( ± S.E.)
Effect of EGTA on cyclic nucleotide phosphodiesterase and A TPase activities The particulate fractions used in these experiments contained very high activities of cyclic nucleotide phosphodiesterase and ATPase. The effect of EGTA on the rate of appearance of the cyclic nucleotide could be attributed to the stimulation of either or both of these enzymes. In order to eliminate this possibility, the effect of EGTA on the activity of these enzymes was determined. The activity of ATPase in both enzyme preparations was inhibited 75-80% by IO mM NaF, a routine component of the adenyl cyclase assay medium. In the absence of F-, there was no effect of o.i mM EGTA on the ATPase activity of the 200o × g fraction, and the only effect on the IOO ooo × g fraction was a slight inhibition (io20%). These results eliminated the possibility that stimulation of ATPase activity accounted for the decreased formation of cyclic AMP in the presence of EGTA. The effect of EGTA on cyclic nucleotide phosphodiesterase activity was assayed by the measurement of the rate of disappearance of a known amount of cyclic AMP incubated with adenyl cyclase in the absence of ATP. In all other respects, the incubation conditions were identical to those used for the adenyl cyclase assay. As shown in Fig. 7, there was a loss of about 40% of the cyclic nucleotide, at an almost linear rate, during the 3o-min incubation period. This loss was sustained in spite of the high concentration (50 mM) of theophylline in the reaction medium. Whether this loss actually occurred under the conditions of the adenyl cyclase assay is open to question, since Cheung 14 has shown that brain cyclic nucleotide phosphodiesterase is inhibited by ATP at concentrations comparable to those used in the adenyl cyclase assay. In any case, there was no significant effect of EGTA on the rate of loss of the cyclic nucleotide. The observed effect of EGTA on adenyl cyclase could not be ascribed, therefore, to the stimulation of phosphodiesterase activity. Biochim. Biophys. Acta, 2 7 6 (1972) 4 3 4 - 4 4 3
ADENYL CYCLASE AND DIVALENT CATIONS
441
CE
I I0
I 2.0
I 30
Time (rnm)
Fig. 7. Effect o f E G T A on cyclic n u c l e o t i d e p h o s p h o d i e s t e r a s e a c t i v i t y . Cyclic A M P (0. 5 mg) w a s i n c u b a t e d w i t h s a m p l e s of t h e 200o × g f r a c t i o n in t h e a b s e n c e of A T P . All o t h e r c o n d i t i o n s were identical to t h e u s u a l a d e n y l cyclase assay. S a m p l e s were r e m o v e d at t i m e d i n t e r v a l s a n d a n a l y z e d for t h e r e m a i n i n g cyclic A M P . T h e p o i n t s are t h e m e a n s o f v a l u e s o b t a i n e d in t h r e e e x p e r i m e n t s . C)--C), control, no E G T A ; O---Q, o.I m M E G T A .
DISCUSSION
The data presented here demonstrate that Ca z÷ and Mg*+ activate the adenyl cyclase of beef cerebral cortex by distinct mechanisms. Both cations are necessary for maximal enzymic activity. Mg~+ must be added to the enzyme reaction mixtures, whereas the requirement for Ca 2+ is demonstrated by inhibition of the adenyl cyclase activity with the relatively Ca 2+ specific chelator EGTA. Since the early reports of this property of adenyl cyclase*,15,16, EGTA has been shown to inhibit the adenyl cyclase prepared from a number of tissues. In most instances, EGTA inhibits only the hormonal response of the enzymeS,7,s. This effect has been attributed to interference with the binding of the hormone to its receptor 7. Other reports indicate, however, that, like the adenyl cyclase of brain 3, the enzyme from fat cells4 and liver 6 is inhibited by EGTA in the absence of hormones. The activation by Mg*+ is undoubtedly due to its effect on the affinity of the enzyme for its substrate. The sensitivity of adenyl cyclase to variations in the relative concentrations of Mg*+ and ATP has been observed repeatedly 17-19, and it has been proposed that the substrate for the enzyme is the MgATP chelate 17. Free ATP competes with this substrate for the catalytic site on the enzyme. The enhancement of activity by excess free Mg2+ has also been observed with adenyl cyclase preparations from fat cells17 and cardiac tissue 18. Birnbaumer et al. 17 have proposed that the binding of Mg~+ to a non-catalytic site influences activity at the catalytic site. It would appear that the same mechanism is operative in the brain enzyme (see Fig. 2), although the data do not permit a quantitation of the number of binding sites. The results of the kinetic analysis of the inhibitory effect of EGTA on the brain Biochim. Biophys. Acta, 276 (1972) 434-443
442
L.S. BRADHAM
enzyme, a n d the reversal of this effect hy Ca "~, indicate t h a t these ions control the velocity of the reaction w i t h o u t affecting the f o r m a t i o n of the e n z y m e - s u b s t r a t e con> plex. The Km for M g A T P is not affected b y tile chelating agent, whereas the initial velocity a n d V are significantly reduced. W i t h M g A T P as the s u b s t r a t e , the e n z y m e is able to synthesize cyclic A M P in the absence of Ca 2 ~ b u t does so at a r e d u c e d rate. The degree of inhibition b y E G T A is i n d e p e n d e n t of the c o n c e n t r a t i o n of both lVIgATP a n d free 1rige+ (see Vig. 4). The effect of the chelating agent is o b s e r v e d in the presence of a large excess of Mg 2+. Since the s t a b i l i t y c o n s t a n t of the M g A T P chelate is higher t h a n t h a t of the C a A T P chelate 2°, it is most likely t h a t Ca 2 + r a t h e r t h a n the chelate are i n v o l v e d in the a c t i v a t i o n . The b i n d i n g of these Ca 2+ to the p a r t i c u l a t e fractions c o n t a i n i n g the e n z y m e is a p p a r e n t l y v e r y stable The s t a b i l i t y of this binding is d e m o n s t r a t e d b y the fact t h a t the inhibition b y E G T A is reversed b y Ca 2+ at conc e n t r a t i o n s significantly lower t h a n the c o n c e n t r a t i o n of the chelating agent (see Fig 5). The d a t a of B a r a n d H e c h t e r 5 indicate a c o m p a r a b l e b i n d i n g of the Ca 2~ i n v o l v e d in the a c t i v a t i o n of the a d e n y l cyclase of the fat cell m e m b r a n e a n d a d r e n a l microsomes. I t seems certain t h a t Ca ~+ p l a y s a significant role in controlling the a c t i v i t y of a d e n y l cyclase in b r o k e n cell p r e p a r a t i o n s isolated from various tissues. W h e t h e r these ions control the a c t i v i t y of the e n z y m e in i n t a c t cells has not been d e t e r m i n e d . As p o i n t e d o u t b y R a s m u s s e n 2., the a c t i v a t i o n of a d e n y l cyclase in i n t a c t cells does not require Ca 2+ in the e x t e r n a l m e d i u m . This does not preclude the possibility t h a t i n t r a cellular Ca ~ ~ or m e m b r a n e - b o u n d Ca 2+ are i n v o l v e d in the control of a d e n y l cyclase a c t i v i t y in the i n t a c t cell,
ACKNOWLEDGMENTS The e x p e r i m e n t a l p o r t i o n of this w o r k was c o m p l e t e d in the D e p a r t m e n t of Medical Research, V e t e r a n s A d m i n i s t r a t i o n H o s p i t a l , L i t t l e Rock, Ark., a n d was s u p p o r t e d b y V e t e r a n s A d m i n i s t r a t i o n d e s i g n a t e d research funds. F i n a n c i a l assistance for p u b l i c a t i o n was s u p p l i e d b y the Tennessee D e p a r t m e n t of Mental H e a l t h . The technical assistance of Victoria D r a k e a n d M a r i l y n n e Sims is g r a t e f u l l y acknowledged. The a u t h o r also expresses his sincere a p p r e c i a t i o n to D r W i l l i a m L. B y r n e a n d D r A b b a s E. K i t a b c h i for t h e i r e n c o u r a g e m e n t a n d support. REFERENCES I G. A. Robison, M. J. Schmidt and E. X.V. Sutherland, Adv. Biochem. Psychopharmacol., 3 (197o) ii. 2 E. W. Sutherland, T. W. Rail and T. Menon, J. Biol. Chem., 237 (1962) 122o. 3 L. S. Bradham, D. A. Holt and M. Sims, Biochim. Biophys. Acta, 2Ol (197o) 25o. 4 R. H. Williams, S. A. Walsh and J. w. Ensinck, Proc. Soc. Exp. Biol. Med., 128 (1968) 279. 5 H. P. Bar and O. Hechter, Bioehem. Biophys. Res. Commun., 35 (1969) 681. 6 K. D. Hepp, R. Edel and O. Wieland, Eur. J. Biochem., I7 (197o) 171. 7 L. Birnbaumer and M. Rodbell, J. Biol. Chem., 244 (1969) 3477. 8 H. P. Bar and O. Hechter, Pro& Natl. Acad. Sci. U.S., 63 (1969) 350. 9 T. K. Ray, V. Tomasi and G. V. Marinetti, Biochim. Biophys. Acta, 211 (I97 o) 20. io D. F. Fitzpatrick, G. R. Davenport, L. Forte and E. J. Landon, J. Biol. Chem., 244 (1969) 3561. i i L. S. Bradham and D. W. Woolley, Biochim. Biophys. Aria, 93 (1964) 475. 12 C. H. Fiske and Y. SubbaRow, J. Biol. Chem., 66 (1925) 375. Biochim. Biophys. Acta, 276 (I972) 434-443
ADENYL CYCLASE AND DIVALENT CATIONS 13 14 15 16 17 18 19 2o 21
443
O. H. Lowry, N. J. R o s e b r o u g h , A. L. F a r r a n d R. J. R a n d a l l , J. Biol. Chem., I93 (I95 I) 265. W. Y. C h e u n g , Biochemistry, 6 (1967) lO79. L. S. B r a d h a m a n d D. A. Holt, Fed. Proc., 28 (1969) 474. H. P. B a r a n d O. H e c h t e r , Fed. Proc., 28 (1969) 571. L. B i r n b a u m e r , S. L. P o h l a n d M. Rodbell, J. Biol. Chem., 244 (1969) 3468. G. 1. D r u m m o n d , D. L. S e v e r s o n a n d L. D u n c a n , J. Biol. Chem., 246 (1971) 4166. S. L. Pohl, L. B i r n b a u m e r a n d M. Rodbell, J. Biol. Chem., 246 (1971) 1849. W. J. ( ) ' S u l l i v a n a n d D. D. Perrin, Biochemistry, 3 (1964) 18. H. R a s m u s s e n , Science, 17o (197 o) 4o4 .
Biochim. Biophys. Acta, 276 (1972) 434-443
BIOCHIMICAET BIOPHYSICAACTA
BBA 66650 COMPARISON OF T H E E F F E C T S OF Ca 2+ AND Mg~+ ON T H E A D E N Y L CYCLASE OF B E E F B R A I N
LAURENCE S. BRADHAM Brain Research Institute and Department of Biochemistry, University of Tennessee Medical Units and the Laboratories of Endocrinology and Metabolism, Research Division, Veterans Administration Hospital, 3/lemphis, Tenn. 38io 4 (U.S.A.)
(Received December 23rd, i971) (Revised nlanuscript received April I ith, 1972)
SUMMARY The adenyl cyclase of beef cerebral cortex required both Mg~+ and Ca =+ for maximal activity. The requirement for Mg=+ was absolute, and it was demonstrated that this cation was necessary for the interaction of the substrate with the enzyme. Enzymic activity was dependent upon the ratio of Mg 2+ to ATP, and maximal activity was obtained only when there was an excess of the divalent cation. Under these conditions, the apparent K m for ATP was 1.2-1. 3 mM and V was 45 nmoles/mg. The requirement for Ca 2+ was demonstrated by use of the chelating agent 1,2bis-(2-dicarboxymethylaminoethoxy)ethane (EGTA) which specifically chelates Ca 2+ in the presence of Mg 2+. Adenyl cyclase activity was inhibited non-competitively by this compound. The V and the initial velocity were reduced approx. 6o% in the presence of o.I mM EGTA. There was no significant effect on the apparent K m for ATP. The velocity of the reaction was returned to normal by the addition of Ca 2+ in concentrations significantly lower than the concentration of the chelating agent.
INTRODUCTION The activity of adenyl cyclase is affected by many factors, and among these are divalent cations. The addition of either Mg 2÷ or Mn =+ to adenyl cyclase preparations is required to elicit enzymic activity, and it is generally accepted that the Mg=+ is the natural co-factor 1. Tile addition of Ca 2+ in place of Mg2+ has no activating effect on the enzyme 2. It has been demonstrated, however, that the adenyl cyclase of beef cerebral cortex depends upon both Ca =+ and Mg2+ for maximal activity a. Maximal activity in the presence of Mg 2+ depends upon trace amounts of Ca z+ which appear to be bound to the particulate matter containing the adenyl cyclase. Tile chelation of these ions by the Abbreviation: EGTA, 1,2-bis-(2-dicarboxymethylaminoethoxy)ethane. Biochim. Biophys. Acta, 276 (1972) 434-443
ADENYL CYCLASE AND DIVALENT CATIONS
435
relatively Ca2+-specific chelator 1,2-bis-(2-dicarboxymethylaminoethoxy)ethane (EGTA) results in inhibition of the enzyme which is reversed b y the addition of Ca 2+. A number of recent reports have provided evidence that Ca ~+ m a y play a similar role in the control of the adenyl cyclase activity of other tissues as well *-9. In view of this evidence, the results presented here are of particular significance. A kinetic analysis of the effects of Ca ~+ and Mg ~+ on the adenyl cyclase activity of particulate fractions from beef cerebral cortex has been performed. The results show that both ions are required for activation of the enzyme, but the mechanisms by which they produce activation are quite dissimilar. EXPERIMENTAL PROCEDURE
Materials
Theophylline was purchased from Nutritional Biochemicals, Inc. ; EGTA from K and K Laboratories; and adenosine 3',5'-monophosphate (cyclic AMP) and the disodium salt of ATP from Sigma Chemical Co. Ion-exchange resins used in the chromatographic steps were Bio-Rad preparations obtained from Calbiochem. All other chemicals were reagent grade preparations obtained from various commercial sources. Methods
Particulate fractions containing adenyl cyclase were prepared from fresh beef cerebral cortex. The preparation of the 2000 × g fraction has been described elsewher@. The ioo ooo X g fraction was prepared b y a procedure which incorporated techniques developed by Fitzpatrick et al. TM. Cortical tissue was homogenized for 30 s in a Waring Blendor with 2 vol. of a solution composed of equal parts glycerol and io mM NaC1-KC1 solution. The homogenate was centrifuged at 6000 X g for 15 min, and the pellet was washed with IO mM NaC1-KC1 solution. The washed pellet was homogenized in I vol. of 2 M sucrose using a motor-driven Teflon pestle. This homogenate was centrifuged at 13 300 × g for IO min, and the residue was discarded. The supernatant suspension was diluted with 7 vol. of cold water, and the suspension was centrifuged at I I 900 × g for IO min. The pellet was extracted with I vol. of 0.25 M sucrose, and the combined suspension was centrifuged at IOO ooo × g for 30 rain. The resulting pellet was resuspended in a small volume of 50% glycerol solution. The protein concentration of suspensions prepared in this manner ranged from 15-2o mg/ml. The enzyme was stored in this suspension at --7 ° °C. When incubated with 2 mM ATP and 5 mM MgC12 under the conditions described below, I m g of this particulate fraction catalyzed the formation of 25 nmoles of cyclic AMP in 30 rain. The adenyl cyclase activity of the IOO ooo × g fraction was routinely assayed at 37 °C at a protein concentration of I mg/ml. The basic incubation solution contained 5 ° mM theophylline, IO mM NaF, and 40 mM Tris (pH 7-5). A T P and MgC12 were added as described in individual experiments. The same incubation solution was used in assaying the 2000 x g fraction, but the protein concentration was 2. 7 mg/ml and the temperature was 30 °C. Unless otherwise specified, the incubation time was 30 min. The reaction was terminated b y adjusting the p H of the samples to 3.5 and heating to IOO °C. Protein was removed b y centrifugation after readjustment of the pH to 7.5. Quantitative analysis for cyclic AMP was performed by a modification of proBiochim. Biophys, Acta, 276 (1972) 434-443
436
L.S. BRADHAM
cedures a l r e a d y described3, n. I n the modified version of this m e t h o d , the p r e l i m i n a r y purification of e n z y m e reaction m i x t u r e s was carried out on a xo-ml column of Dowex-2 resin ( B i o - R a d AG z-X, 2o0-4oo mesh) in the f o r m a t e form. The cyclic nucleotide was eluted from this column w i t h 5 ° ml of 2 M formic acid (flow r a t e not exceeding o.3 ml/min). An aliquot of this eluate was c h r o m a t o g r a p h e d on a cation exchange colunm ( B i o - R a d A G 5o-X8, 2oo-4o0 mesh) with the dimensions described in the original procedure. The cyclic nucleotide was e l u t e d from this column with o.o 5 M He1 a n d the fraction of e l u a t e (75-215 ml) c o n t a i n i n g cyclic A M P was used for the m e a s u r e m e n t of a b s o r b a n c e ~. The r e c o v e r y of cyclic A M P from the columns was essentially q u a n t i t a t i v e ( > 95%), a n d the results of assay of d u p l i c a t e samples agreed w i t h i n a few per cent. F o r the d e t e r m i n a t i o n of A T P a s e a c t i v i t y , the p a r t i c u l a t e fractions were incub a t e d with 4 m N M g A T P u n d e r conditions i d e n t i c a l to those used in the a d e n y l cyclase assay. The inorganic p h o s p h a t e released was d e t e r m i n e d b y the m e t h o d of F i s k e a n d S u b b a R o w 1~. P r o t e i n was a s s a y e d b y the m e t h o d of L o w r y et al? ~.
2( <
[o
ATP (raM)
Fig. i. Effect of variation of ATP concentration on the adeny] cyclase activity of the ioo ooo x g fraction. In each experiment, the Mg2+ concentration was kept constant and the concentration of ATP was varied over the range indicated. ID---ID, i mM Mg~+; ©--(2), 2 mM Mg2+; &--A, 5 mM Mg 2+.
RESULTS
Effect of variation of A T P and Mg ~+ on adenyl cyclase activity T h e a c t i v i t y of t h e a d e n y l cyclase of the I00 000 × g fraction was sensitive to v a r i a t i o n s in the r e l a t i v e c o n c e n t r a t i o n s of A T P a n d Mg 2+. As i l l u s t r a t e d b y the curves in Fig. I, m a x i m a l a c t i v i t y was o b t a i n e d o n l y when the c o n c e n t r a t i o n of Mg 2+ exceeded the c o n c e n t r a t i o n of A T P . Excess A T P i n h i b i t e d the e n z y m e c o m p e t i t i v e l y . The i n t e r r e l a t i o n s h i p between A T P a n d Mg z+ c o n c e n t r a t i o n s is f u r t h e r illus-
Biochim. Biophys. Acta, 276 (1972) 434-443
437
ADENYL CYCLASE AND DIVALENT CATIONS
500 B
I
i
i
l
400
6oo u -6
zoo Io
too
o
I
2
3
M g A T P (TriM)
4
5 0
10(30
1500
2000
I,/Mg ATP
Fig. 2. Effect of v a r i a t i o n of t h e relative c o n c e n t r a t i o n s of A T P a n d Mg 2+ on t h e a d e n y l cyclase a c t i v i t y o f t h e i o o ooo × g fraction. (A) E n z y m e a c t i v i t y o b t a i n e d u n d e r t h e c o n d i t i o n s listed below p l o t t e d as a f u n c t i o n of M g A T P c o n c e n t r a t i o n . O---O, A T P m a i n t a i n e d c o n s t a n t (4 raM) a n d Mg 2+ c o n c e n t r a t i o n v a r i e d f r o m i to 4 raM; © - - C ) , A T P a n d Mg 2+ c o n c e n t r a t i o n s m a i n t a i n e d e q u i m o l a r over t h e c o n c e n t r a t i o n r a n g e ; A - - A , Mg 2+ c o n c e n t r a t i o n m a i n t a i n e d c o n s t a n t (5 mM) a n d A T P c o n c e n t r a t i o n v a r i e d f r o m I to 4 raM. (B) Reciprocal p l o t s of t h e d a t a o f (A). T h e m e t h o d of least s q u a r e s w a s u s e d to fit t h e b o t t o m curve. T h e d o t t e d line d e n o t e s e x t r a p o l a t i o n to t h e y-axis. Refer a b o v e for t h e definition of s y m b o l s .
trated by the experiments summarized in Fig. 2. These experiments were performed under conditions in which there was either an excess of ATP (bottom curve of Fig. 2A), an excess of Mg*÷ (top curve of Fig. 2A), or equimolar concentrations of ATP and Mg~+ (middle curve of Fig. 2A). Approximately the same maximal velocity was approached under the three sets of conditions, but the initial velocities were controlled by the relative concentrations of ATP and Mg*+. The reciprocal plots of these data (Fig. 2B) indicated that this was due to a progressive decrease in the Km for the substrate (MgATP) as the concentration of Mg2+ increased in relation to the concentration of ATP. This plot was linear only when there was an excess of Mg*+. The apparent Km for MgATP calculated from this data was 1.27 mM.
Inhibition of adenyl cyclase activity by EGTA The pattern of inhibition of the adenyl cyclase activity of the IOO ooo × g fraction by EGTA was identical to that obtained earlier using the 2000 / g fraction as the source of enzyme 3. As indicated by the data of Fig. 3, there was no effect of the chelating agent at concentrations of o.o125 mM or less. Maximal inhibition (approx. 60%) was obtained at an EGTA concentration of 0.025 mM, and there was no further effect of increasing the concentration up to o.I raM. In all of the subsequent experiments, an EGTA concentration of o.I mM was used routinely. The inhibition by EGTA was independent of substrate concentration. As shown by the data in Fig. 4A, the degree of inhibition remained the same when the concentration of ATP was varied over the range of 0.5 to 4 mM (in presence of 5 mM Mg*+). Biochim. Biophys. Acta, 276 (1972) 434-443
438
]~. s. BRAI)HAM
,oo
90
E
0o z~ 7o
60
5O
4O
30
0
004
0.06
008
0, 0
EGTA ('m M)
Fig. 3. I n h i b i t i o n of adenyl cyclase activity b y EGTA. The adenyl cyclase activity of the ioo ooo × g fraction was determined in the presence of various concentrations of EGTA. The s t a n d a r d i n c u b a t i o n m i x t u r e contained 2 mM A T P and 5 mM Mg 2+. E a c h p o i n t is the m e a n of the values o b t a i n e d in three experiments.
The reciprocal plot of these data (Fig. 4 B) indicated that this inhibition was noncompetitive. The m a x i m u m velocity of the reaction was reduced by approx. 6o%, but there was no significant effect on the apparent Km for the substrate. The V for the reaction (as calculated by the method of least squares) in the presence of EGTA was A
i
i
i
I
El
i
~
i
30o
2oo
, I
2C
5
IOO
I
~o
%.I
,
L
ATP(mM)
ATP
('mM)
Fig. 4. Effect of variation of tile s u b s t r a t e c o n c e n t r a t i o n on the inhibition of adenyl cyclase b y E G T A . The IOO ooo X g fraction was used as the source of enzyme. (A) The concentration of A T I ~ was varied in the presence and in the absence of EGTA. The c o n c e n t r a t i o n of Mg 2+ in all samples was 5 raM. (B) Reciprocal plots of the d a t a of (A). The lines were fit to the points b y the m e t h o d of least squares. O---O, Control, no E G T A ; © - - O , o.I mM EGTA.
I~iochim. Biophys. Acta, 276 (i972) 434-443
439
ADENYL CYCLASE AND DIVALENT CATIONS
18.1 nmoles/mg, whereas in the control samples the value for this constant was 45.2 nmoles/mg. The apparent Km values in the presence and in the absence of the chelator were calculated to be 0.94 and 1.27 mM, respectively.
Effect of Ca z+ on the inhibition by EGTA Fig. 5 shows the effect of the inclusion of various concentrations of Ca 2+ in reaction mixtures containing o. I mM EGTA. These curves have the appearance of titration curves with the enzymic activity as the end-point. It should be noted that there was significant restoration of activity at concentrations of Ca 2+ lower than the concentration of EGTA. With both enzyme preparations, full activity was restored at 0.075 mM Ca 2+, whereas half the inhibited activity was restored at approx. 0.06 mM. t
I
I
I
I
IOG
90
x
E
,o
80
£
"5 7O
= u
5G
40
I
I
I
I
002
004
006
008
Ca CIz ('raM)
0
/
I
,o
I0
I ~
30
"rime (min)
Fig. 5. Effect of i n c l u s i o n of Ca 2+ on t h e i n h i b i t i o n of a d e n y l c y c l a s e b y E G T A . V a r y i n g concent r a t i o n s of CaC1, we re a d d e d to s a m p l e s of t h e 20oo × g f r a c t i o n a n d t h e IOO ooo × g f r a c t i o n i n c u b a t e d w i t h o . i mM E G T A . The c o n c e n t r a t i o n of Mg 2+ in all s a m p l e s w a s 5 mM. T h e concent r a t i o n of A T P w a s e i t h e r 2 mM (2ooo × g fraction) or 4 m M ( i o o ooo × g fraction). T h e d o t t e d line r e p r e s e n t s t h e a c t i v i t y of s i m i l a r s a m p l e s i n c u b a t e d in t h e a b s e n c e of E G T A . t ~ - - Q , 2 0 0 0 × g fraction; 0--0, IOO ooo X g f r a c t i o n . Fig. 6. Effect of E G T A a n d Ca *+ on t h e r a t e of f o r m a t i o n of cyclic AMP. S a m p l e s of t h e 2000 × g f r a c t i o n were i n c u b a t e d w i t h E G T A a n d Ca ~+ for v a r i o u s t i m e i n t e r v a l s in t h e p r e s e n c e of 2 mM A T P a n d 5 mM Mg a+. × - - X , control, no E G T A ; A - - A , o,i mM E G T A , no Ca*+; O - - O , o.I mM E G T A , 0.06 mM Ca2+; O---O, o.I mM E G T A , o.08 mM Ca *+.
The effect of these concentrations of Ca *+ on the rate of formation of cyclic AMP in the presence of the 2000 × g fraction, is illustrated by the data of Fig. 6. The concentrations of ATP and Mg~+ in these samples were 2 and 5 mM, respectively. In the control samples (no EGTA), cyclic AMP was formed at the rate of 383 pmoles/min per rag. The inclusion of o.I mM EGTA reduced this rate by 50% to 192 pmoles/min per mg. The rate was restored to normal (406 pmoles/min per mg) by 0.08 mM Ca 2+, Biochim. Biophys. Acta, 276 (1972) 4 3 4 - 4 4 3
440
L. S. B R A D H A M
whereas the inclusion of Ca 2+ at a concentration of o.o6 mM resulted in a rate (3o8 pmoles/min per rag) which was approximately halfway between the maximally inhibited rate and the control rate. The effect of Ca 2+ on the reduction of maximal velocity by EGTA is illustrated by the data in Table I. The concentration of Mg2+ in these samples was 5 raM. The data show that the 60% reduction in V caused by EGTA was reversed by the inclusion of 0.075 mM Ca 2+. Again, there was no significant effect of either EGTA or Ca 2+ on the apparent Km for ATP. TABLE
I
EFFECT OF E G T A
AND G a s+ ON KINETIC PARAMETERS OF BRAIN ADENYL CYCLASE
S a m p l e s o f t h e i o o o o o × g f r a c t i o n w e r e i n c u b a t e d w i t h E G T A a n d C a 2+ a s d e s i g n a t e d in t h e p r e s e n c e o f c o n c e n t r a t i o n s o f A T P r a n g i n g f r o m o. 5 t o 4 m M . T h e c o n c e n t r a t i o n o f M g ~+ in a l l s a m p l e s w a s 5 m M . T h e v a l u e s f o r B2m a n d V w e r e c a l c u l a t e d b y tile m e t h o d o f l e a s t s q u a r e s f r o n l r e c i p r o c a l p l o t s o f t h e s e d a t a . E a c h v a l u e r e p o r t e d is t h e n l e a n o b t a i n e d f r o m five e x p e r i m e n t s .
Expt
EGTA (mYI)
CaCl~ (~nM)
I£m × Io a
V (nrnoles[rng ~z S.E.)
I 2 3
o o.I o.I
o o 0.075
1.19 ~. o . 1 4 1.11 ± o.2 1.17 ± o . o i
4 5 . 8 -~ I. 7 18.1 ~z 1.2 4 2 . 9 ~ 1.6
( ± S.E.)
Effect of EGTA on cyclic nucleotide phosphodiesterase and A TPase activities The particulate fractions used in these experiments contained very high activities of cyclic nucleotide phosphodiesterase and ATPase. The effect of EGTA on the rate of appearance of the cyclic nucleotide could be attributed to the stimulation of either or both of these enzymes. In order to eliminate this possibility, the effect of EGTA on the activity of these enzymes was determined. The activity of ATPase in both enzyme preparations was inhibited 75-80% by IO mM NaF, a routine component of the adenyl cyclase assay medium. In the absence of F-, there was no effect of o.i mM EGTA on the ATPase activity of the 200o × g fraction, and the only effect on the IOO ooo × g fraction was a slight inhibition (io20%). These results eliminated the possibility that stimulation of ATPase activity accounted for the decreased formation of cyclic AMP in the presence of EGTA. The effect of EGTA on cyclic nucleotide phosphodiesterase activity was assayed by the measurement of the rate of disappearance of a known amount of cyclic AMP incubated with adenyl cyclase in the absence of ATP. In all other respects, the incubation conditions were identical to those used for the adenyl cyclase assay. As shown in Fig. 7, there was a loss of about 40% of the cyclic nucleotide, at an almost linear rate, during the 3o-min incubation period. This loss was sustained in spite of the high concentration (50 mM) of theophylline in the reaction medium. Whether this loss actually occurred under the conditions of the adenyl cyclase assay is open to question, since Cheung 14 has shown that brain cyclic nucleotide phosphodiesterase is inhibited by ATP at concentrations comparable to those used in the adenyl cyclase assay. In any case, there was no significant effect of EGTA on the rate of loss of the cyclic nucleotide. The observed effect of EGTA on adenyl cyclase could not be ascribed, therefore, to the stimulation of phosphodiesterase activity. Biochim. Biophys. Acta, 2 7 6 (1972) 4 3 4 - 4 4 3
ADENYL CYCLASE AND DIVALENT CATIONS
441
CE
I I0
I 2.0
I 30
Time (rnm)
Fig. 7. Effect o f E G T A on cyclic n u c l e o t i d e p h o s p h o d i e s t e r a s e a c t i v i t y . Cyclic A M P (0. 5 mg) w a s i n c u b a t e d w i t h s a m p l e s of t h e 200o × g f r a c t i o n in t h e a b s e n c e of A T P . All o t h e r c o n d i t i o n s were identical to t h e u s u a l a d e n y l cyclase assay. S a m p l e s were r e m o v e d at t i m e d i n t e r v a l s a n d a n a l y z e d for t h e r e m a i n i n g cyclic A M P . T h e p o i n t s are t h e m e a n s o f v a l u e s o b t a i n e d in t h r e e e x p e r i m e n t s . C)--C), control, no E G T A ; O---Q, o.I m M E G T A .
DISCUSSION
The data presented here demonstrate that Ca z÷ and Mg*+ activate the adenyl cyclase of beef cerebral cortex by distinct mechanisms. Both cations are necessary for maximal enzymic activity. Mg~+ must be added to the enzyme reaction mixtures, whereas the requirement for Ca 2+ is demonstrated by inhibition of the adenyl cyclase activity with the relatively Ca 2+ specific chelator EGTA. Since the early reports of this property of adenyl cyclase*,15,16, EGTA has been shown to inhibit the adenyl cyclase prepared from a number of tissues. In most instances, EGTA inhibits only the hormonal response of the enzymeS,7,s. This effect has been attributed to interference with the binding of the hormone to its receptor 7. Other reports indicate, however, that, like the adenyl cyclase of brain 3, the enzyme from fat cells4 and liver 6 is inhibited by EGTA in the absence of hormones. The activation by Mg*+ is undoubtedly due to its effect on the affinity of the enzyme for its substrate. The sensitivity of adenyl cyclase to variations in the relative concentrations of Mg*+ and ATP has been observed repeatedly 17-19, and it has been proposed that the substrate for the enzyme is the MgATP chelate 17. Free ATP competes with this substrate for the catalytic site on the enzyme. The enhancement of activity by excess free Mg2+ has also been observed with adenyl cyclase preparations from fat cells17 and cardiac tissue 18. Birnbaumer et al. 17 have proposed that the binding of Mg~+ to a non-catalytic site influences activity at the catalytic site. It would appear that the same mechanism is operative in the brain enzyme (see Fig. 2), although the data do not permit a quantitation of the number of binding sites. The results of the kinetic analysis of the inhibitory effect of EGTA on the brain Biochim. Biophys. Acta, 276 (1972) 434-443
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L.S. BRADHAM
enzyme, a n d the reversal of this effect hy Ca "~, indicate t h a t these ions control the velocity of the reaction w i t h o u t affecting the f o r m a t i o n of the e n z y m e - s u b s t r a t e con> plex. The Km for M g A T P is not affected b y tile chelating agent, whereas the initial velocity a n d V are significantly reduced. W i t h M g A T P as the s u b s t r a t e , the e n z y m e is able to synthesize cyclic A M P in the absence of Ca 2 ~ b u t does so at a r e d u c e d rate. The degree of inhibition b y E G T A is i n d e p e n d e n t of the c o n c e n t r a t i o n of both lVIgATP a n d free 1rige+ (see Vig. 4). The effect of the chelating agent is o b s e r v e d in the presence of a large excess of Mg 2+. Since the s t a b i l i t y c o n s t a n t of the M g A T P chelate is higher t h a n t h a t of the C a A T P chelate 2°, it is most likely t h a t Ca 2 + r a t h e r t h a n the chelate are i n v o l v e d in the a c t i v a t i o n . The b i n d i n g of these Ca 2+ to the p a r t i c u l a t e fractions c o n t a i n i n g the e n z y m e is a p p a r e n t l y v e r y stable The s t a b i l i t y of this binding is d e m o n s t r a t e d b y the fact t h a t the inhibition b y E G T A is reversed b y Ca 2+ at conc e n t r a t i o n s significantly lower t h a n the c o n c e n t r a t i o n of the chelating agent (see Fig 5). The d a t a of B a r a n d H e c h t e r 5 indicate a c o m p a r a b l e b i n d i n g of the Ca 2~ i n v o l v e d in the a c t i v a t i o n of the a d e n y l cyclase of the fat cell m e m b r a n e a n d a d r e n a l microsomes. I t seems certain t h a t Ca ~+ p l a y s a significant role in controlling the a c t i v i t y of a d e n y l cyclase in b r o k e n cell p r e p a r a t i o n s isolated from various tissues. W h e t h e r these ions control the a c t i v i t y of the e n z y m e in i n t a c t cells has not been d e t e r m i n e d . As p o i n t e d o u t b y R a s m u s s e n 2., the a c t i v a t i o n of a d e n y l cyclase in i n t a c t cells does not require Ca 2+ in the e x t e r n a l m e d i u m . This does not preclude the possibility t h a t i n t r a cellular Ca ~ ~ or m e m b r a n e - b o u n d Ca 2+ are i n v o l v e d in the control of a d e n y l cyclase a c t i v i t y in the i n t a c t cell,
ACKNOWLEDGMENTS The e x p e r i m e n t a l p o r t i o n of this w o r k was c o m p l e t e d in the D e p a r t m e n t of Medical Research, V e t e r a n s A d m i n i s t r a t i o n H o s p i t a l , L i t t l e Rock, Ark., a n d was s u p p o r t e d b y V e t e r a n s A d m i n i s t r a t i o n d e s i g n a t e d research funds. F i n a n c i a l assistance for p u b l i c a t i o n was s u p p l i e d b y the Tennessee D e p a r t m e n t of Mental H e a l t h . The technical assistance of Victoria D r a k e a n d M a r i l y n n e Sims is g r a t e f u l l y acknowledged. The a u t h o r also expresses his sincere a p p r e c i a t i o n to D r W i l l i a m L. B y r n e a n d D r A b b a s E. K i t a b c h i for t h e i r e n c o u r a g e m e n t a n d support. REFERENCES I G. A. Robison, M. J. Schmidt and E. X.V. Sutherland, Adv. Biochem. Psychopharmacol., 3 (197o) ii. 2 E. W. Sutherland, T. W. Rail and T. Menon, J. Biol. Chem., 237 (1962) 122o. 3 L. S. Bradham, D. A. Holt and M. Sims, Biochim. Biophys. Acta, 2Ol (197o) 25o. 4 R. H. Williams, S. A. Walsh and J. w. Ensinck, Proc. Soc. Exp. Biol. Med., 128 (1968) 279. 5 H. P. Bar and O. Hechter, Bioehem. Biophys. Res. Commun., 35 (1969) 681. 6 K. D. Hepp, R. Edel and O. Wieland, Eur. J. Biochem., I7 (197o) 171. 7 L. Birnbaumer and M. Rodbell, J. Biol. Chem., 244 (1969) 3477. 8 H. P. Bar and O. Hechter, Pro& Natl. Acad. Sci. U.S., 63 (1969) 350. 9 T. K. Ray, V. Tomasi and G. V. Marinetti, Biochim. Biophys. Acta, 211 (I97 o) 20. io D. F. Fitzpatrick, G. R. Davenport, L. Forte and E. J. Landon, J. Biol. Chem., 244 (1969) 3561. i i L. S. Bradham and D. W. Woolley, Biochim. Biophys. Aria, 93 (1964) 475. 12 C. H. Fiske and Y. SubbaRow, J. Biol. Chem., 66 (1925) 375. Biochim. Biophys. Acta, 276 (I972) 434-443
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