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Adenosine 3′,5′-monophosphate
Printed
inc&reat BT1La1119'
Part II,
ADENOSIN1i
pp . 861-868, 1970.
Pergamon Press
3',5'-MONOPHOSPHPLTE
Demonstration of a Binding Site Specific for the Cyclic Nucleotide Wai Yiu Cheung Laboratory of Biochemistry, St . Jude Children's Research Hospital and Department of Biochemistry, University of Tennessee Medical Units, Memphis, Tennessee .
(Received 21 April 1970 ; in final form 10 June 1970)
Summary An extract of bovine brain exhibits a binding site specific for cyclic AMP . Binding of labeled cyclic AMP was competitive with respect to carrier cyclic AMP and its dibutyryl derivative, but essentially not with other nucleo tides . The binding component was sensitive to proteolytic enzymes and appeared to be associated with a protein. The amount of cyclic AMP bound was proportional to the concentration of protein and to that of cyclic AMP . Binding was most efficient around pH 6 .5 . The binding protein was found in all the brain areas examined . Subcellular fractionation of the cortex showed that the mitochondrial and microsomal fractions had the most activity and the high speed supernatant fluid the highest specific activity . Introduction Cyclic AMP is involved in a variety of different cellular functions (1) . Perhaps one common thread of all these different manifestations is that cyclic AMP interacts with proteins .
The interaction probably involves binding of the
cyclic nucleotide to the protein .
An analogy is the binding of an effector to
the regulatory site of an allosteric enzyme, as in the case of the binding of cytidine triphosphate to aspartate transcarbamylase (2) .
Another well known
example is the binding of 2,3-diphosphoglycerate to hemoglobin (3) . The brain is the most active tissue both in adenyl cyclase and cyclic 3', 5'-nucleotide phosphodiesterase (1) .
Also, the level of cyclic AMP in the brain
changed dramatically within seconds after decapitation (4) .
We have, therefore,
searched in this tissue for the existence of proteins with a binding site specifi .; for cyclic AMP . This note demonstrates the existence of such a binding site, which is associated with a protein extracted from the bovine brain . binding site are presented .
861
Some properties of the
BINDING OF CYCLIC AMP
862
Vol . 9, No . 15
Methods Preparation of Binding Protein . stored at -20 0 until use .
Bovine brain cortices were used fresh or
The binding protein was prepared according to a pro-
cedure developed for the purification of a cyclic 3',5'-nucleotide phospho diesterase (5) .
The procedure consisted of extracting the tissue with water,
followed by pH fractionation, differential centrifugation, (NH4)2S04 precipitation and calcium phosphate gel adsorption .
Subcellular fractions were prepared
according to De Robertis et al . (6), using fresh brain cortex . Binding of Cyclic AMP .
A sample for binding study was placed in a small
cellulose tubing and was dialyzed for 16 to 20 hours at 4 0 against a buffer about 100 times the volume of the sample .
The buffer contained 4 mM EDTA, 20
mM Tris-C1, pH 7 .5, 25-50 nM cyclic AMP - 8-C14 , with a specific activity of 41 uCi/umole .
EDTA was added to inhibit cyclic 3',5'-nucleotide phosphodies-
terase activity present in these preparations (7) .
At the end of the dialysis,
an aliquot of the dialyzed sample was counted in a liquid scintillation Spectrometer .
An equal aliquot of the buffer was also counted and the radioactivity
in the buffer was subtracted from that of the sample to give the amount of radioactivity bound to the sample .
Binding is expressed as the amount of cyclic
AMP bound per mg protein or per aliquot of sample . Protein was determined using the spectrophotometric technique of Warburg and Christian (8) and the biuret method with bovine serum albumin as a standard . At the end of equilibrium dialysis, protein was routinely determined again to correct for a possible slight change of protein concentration due to dialysis . Results and Discussion Bound cyclic AMP is defined as that amount in an aliquot of sample inside the dialysis tubing over and above that in a similar aliquot of the buffer after equilibrium is attained .
In the absence of a sample, the concentration of cy-
clic AMP inside and outside the dialysis tubing was equal at equilibrium .
Table
I shows the effect of protein concentration on the amount of cyclic AMP bound to an aliquot of the sample .
The amount of cyclic AMP bound was proportional up to
Vol. 9, No . 15
889
BINDING OF CYCLIC AMP
6 mg protein per ml, reaching a plateau shortly thereafter .
The last column in
the table shows the amount of cyclic AMP bound per mg protein, which varied from 11 to 15 p moles, depending on the concentration of proteins in the dialysis tubing .
Cyclic AMP retained inside the dialysis tubing was as high as six
times that in the buffer (compare Sample Nos . O. and 9), indicating that binding was accomplished against a considerable concentration gradient . TABLE I EFFECT OF PROTEIN CONCENTRATION ON THE AMOUNT OF BOUND CYCLIC AMP
Sample No .
Protein conc .
cAMP/200 ul
cAMP bound/200 ul cAMP bound/mg protein
mg/ml
(p moles)
(p moles)
(p moles)
0
0
5 .1
-
1
1.4
9.2
4 .1
15 .1
2
2 .5
12 .1
7 .0
14 .2
3
3 .3
14 .7
9 .6
14 .6
4
4 .3
17 .1
12 .0
13 .8
5
5 .3
19 .7
14 .6
13 .7
6
6 .3
22 .2
17 .1
13 .6
7
8.5
26 .8
21 .7
12 .8
8
8 .8
27 .9
22 .8
13 .0
9
11 .0
30 .1
25 .0
11 .4
-
An aliquot of 0.5 ml of a 30 to 50% (NH4)2S04 fraction (4) was dialyzed overnight in a small cellulose tubing against an excess Trio-EDTA buffer, containing 25 nM cyclic AMP-8-C14. At the end of the equilibrium dialysis, 200 ul of the dialyzed protein sample and 200 ul of the buffer were counted in a scintillation spectrometer . The amount of cyclic AMP bound was a function of the concentration of cyclic AMP present in the buffer .
Half maximal binding was obtained when the
buffer contained approximately 40 nM cyclic AMP.
In this experiment, the con
centration of protein was 8 mg per ml, and the maximal amount of cyclic AMP
864
BINDING OF CYCLIC AMP
Vol . 9, No. 15
bound was 25 p moles/mg protein . Binding of cyclic AMP was also a function of the pH of the buffer . Binding was most efficient around pH 6 .5 .
Between pH 4 .0 and pH 5 .0, the so
lution became turpid at the end of the equilibrium dialysis .
The sample used
in this as well as in other experiments was heterogeneous in its protein components .
The possibility existed that the decrease in binding at the acid side
might be due to a masking of available binding sites by some of the denatured proteins . An aliquot of the binding material (2 .4 mg protein) was exposed either to 25 ug of trypsin, pronase, RNase or DNase at 30 0 for 3 hours . not so treated served as a control .
A similar sample
At the end of the incubation, these sam
ples were examined for its ability to bind cyclic AMP .
Table II shows that the
samples treated with trypsin or pronase were virtually incapable of binding cyclic AMP, but the ones treated with RNase or DNase were as efficient as the control .
It was concluded that the binding site for cyclic AMP was associated
with a protein or some proteins, and not with a nucleic acid .
Exposure of the
protein to 8M urea sharply reduced, while exposure to 100 0 abolished all the binding ability . TABLE II EFFECT OF PROTEOLYTIC ENZYMES, RNase OR DNase ON THE BINDING OF CYCLIC AMP
No .
Treatment
cAMP bound/100 ul (p moles)
1
None
3 .92
2
Trypsin
0 .15
3
Pronase
0 .30
4
RNase
4 .17
5
DNase
3 .61
BINDING OF CYCLIC AMP
Vol . 9, No . 15
885
An aliquot of 0 .6 ml of a calcium phosphate gel eluate (4) was incubated with 25 ug of trypsin, pronase, Mesa or DNase for 3 hours at 30 0. At the end of the incubation, the sample was transferred to a cellulose tubing for equili brium dialysis as in Table I. Counting was done in duplicates of 100 ul aliquots . The figures have been rrected for radioactivity due to the random distribution of cyclic AMP-8-J4 in the buffer . The effect of other nucleotides on the binding of cyclic AMP was examined in Table III.
The sample was dialyzed against Trio-EDTA buffer containing 50
nM cyclic AMP-8-Cl 4 and another nucleotide at 10 uM, a concentration about 200 times that of cyclic AMP.
The other nucleotide was allowed to compete with cy-
clic AMP for the binding site .
The control sample was dialyzed in a Tris-EDTA
buffer containing cyclic AMP-8-C 14 , but no other nucleotides .
2'-AMP, 3'-AMP
and cyclic 2',3'-AMP were without effect ; 5'-AMP, ATP and cyclic GMP were slightly inhibitory . be most effective,
At the same concentration, unlabeled cyclic AMP proved to
followed by its N6,2'-0-dibutyryl derivative .
This experi-
ment showed that the binding site was specific for cyclic AMP. TABLE III EFFECT OF NUCLEOTIDES ON THE BINDING OF CYCLIC AMP
No .
Other nucleotides
cAMP bound (p moles/mg protein)
1
None
21 .7
2
2'-AMP
20 .0
3
3'-AMP
20 .0
4
Cyclic 2',3'-AMP
20 .0
5
5'-AMP
15 .8
6
Unlabeled cAMP
0 .5
7
dBCAMP
10 .0
8
ATP
18 .1
9
CGMP
16 .6
An aliquot of 0 .5 ml of a 30 to 507 (NH4)2S0~, fraction was dialysed in Trio-EDTA buffer containing 50 nM cyclic AMP-8-C14 and another competing
86 6
BINDING OF CYCLIC AMP
Vol. 9, No. 15
nucleotide at 10 uM. The control contained cyclic AMP-8-Cl4 and no other nucleotide . At the end of equilibrium dialysis, counting was done in duplicates of 100 ul aliquots . Abbreviations : dBcAMP - N6 ,2'-0-dibutyryl cyclic AMP ; cGMP - cyclic 3',5'-GMP ; others are standard abbreviations . The binding protein was detected in all areas examined in the bovine brain . They were :
cerebellum, cortex, hypothalamus, medulla, olfactory bulb, pineal
gland, pons and thalamus, with the highest specific activity in the gray matter of the cortex .
Subcellular fractionation of the cortex showed that with the
possible exception of the nuclear °_raction, the binding protein was present in all the different fractions . the particulate fractions .
Most of the binding activity was associated with Approximately half of the particulate activity was
present in the mitochondria and the other half in the microsome .
The superna-
tant fluid appeared to have the highest specific activity . The present work did not determine whether the protein possessed one or more binding sites, or whether the different brain areas contained one or more binding proteins .
It should be noted that not all proteins bind cyclic AMP .
For example, bovine serum albumin at 10 mg protein per ml was ineffective under comparable conditions . Experiments designed to identify the fate of the bound cyclic AMP indicated that the nucleotide was recovered in a form indistinguishable chromatographically from an authentic sample of cyclic AMP.
Thus, binding to the protein did
not alter its molecular structure .
.
The damonatration of a protein with a binding site specific for cyclic AMP may shed light on the mechanism of action of the nucleotide . about a conformational change on a protein .
In the case of an enzyme, a change
of conformation would lead to a change in enzymic activity . may be cited as being particularly pertinent . AMP, and it binds the nucleotide (9) .
Binding may bring
Phosphofructokinase
The enzyme is activated by cyclic
Other enzymes that are activated by cy-
clic AMP include lipase (10), protein kinase (11,12,13), and glycogen synthetase (14) . Cyclic AMP also affects systems possibly involving alteration of some phy-
HINDING OF CYCLIC AMP
Vol . 9, No. 15
867
sical properties of a non-enzymic protein, leading to a change of some cellular processes .
Examples may include the increase of membrane permeability of toad
bladder to water (15), the increase of amylase secretion in the parotid gland (16) and the increase of fluid secretion in the salivary gland (17) . In view of the binding of effectors to allosteric enzymes, it seems reasonable that cyclic AMP, through its interaction with proteins, would be bound to such proteins .
Binding of cyclic AMP maybe a common denominator in all the
apparently different manifestations attributed to this nucleotide .
Different
effects of cyclic AMP in different tissues are thus explainable on the kinds of proteins interacting with the nucleotide . If the binding of cyclic AMP to a protein were indeed related to its mechanism of action, such a protein or proteins would be expected to be distributed widely in various tissues .
Indeed, Gill and Garren (18) have demonstrated a
protein with a specific site for cyclic AMP in the adrenal cortex .
Also,
Solomon and Schramm (19) reported a binding site for cyclic AMP in the smooth membranes of the rat parotid microsomes .
The bound nucleotide in both systems
was recovered as cyclic AMP . Acunowledgments This work was supported by grants NB08059 and CA08480 from the U .S . Public Health Service .
It was also supported by ALSAC .
It is a pleasure to acknow-
ledge the valuable help of Dr . Seung-Yil Song in dissecting brain areas and the excellent technical assistance of Mrs . Sandra Patrick . References 1.
Robison, G .A ., Butcher, R .W ., and Sutherland, E .W ., Arm . Rev . Biochem. , 37 ,149 (1968) .
2.
Changeux, J .P ., Gerhart, J .C ., and Schachman, H .K ., Biochemistry , 7,531 (1968) .
3.
Bsnesch, R ., Bsnesch, R.E ., and Yu, C .I ., Proc . Natl . Acad . Sei ., 59,526 (1968) .
4.
Kakiuchi, S ., and Rall, T .W ., Mol . Pharmacol ., 4,379 (1968) .
5.
Cheung, W .Y ., Biochim . Biophys . Acta , 191,303 (1969) .
BINDING OF CYCLIC AMP
868
Vol . 9, No. 15
6.
De Robertis, E., end Salganicoff,
Pellegrino de Iraldi, A., Rodrignez de Lores Arnait, G., L ., J . Neurochem., 9,23 (1962) .
7.
Cheung, W.Y ., Biochemistry , 6,1079 (1967) .
8.
Warburg, 0 ., and Christian, W., Biochem . Z. , 310,384 (1941) .
9.
Kemp, R.G ., and Krebs, E.G ., Biochemistry , 6,423 (1967) .
10 .
Rizack, M.A., J. Biol . Chem., 239,392 (1964) .
11 .
Walsh, D.A ., Perkins, J.P ., and Krebs, (1968) .
12 .
Langea, T.A ., Science , 162,579 (1968) .
13 .
Kuo, J.F ., and Greengard, P., Proc . Natl . Acad . Sci., 64,1349 (1970) .
14 .
Rosell-Perez, M., and Larner, J ., Biochemistry , 3,81
15 .
Orloff, J ., and Handler, J .S ., J. Clin . Invest ., 41,702 (1962) .
16 .
Bdolah, A., and Schramm, M., Biochem. Biophys . Res . Commun.,
17 .
Berridge, M.J ., and Patel,
18 .
Gill, G.N ., and Garren, L.D ., Proc . Natl . Acad . Sci., 63,512
19 .
Solomon, Y ., and Schramm, M., Biochem. Biophys. Res. Commun ., 38,106
N.G .,
E.G ., J . Biol . Chem . , 243,3763 _
(1964) .
18,452 (1965) .
Science , 162,462 (1968) . (1969) . (1970) .
inc&reat BT1La1119'
Part II,
ADENOSIN1i
pp . 861-868, 1970.
Pergamon Press
3',5'-MONOPHOSPHPLTE
Demonstration of a Binding Site Specific for the Cyclic Nucleotide Wai Yiu Cheung Laboratory of Biochemistry, St . Jude Children's Research Hospital and Department of Biochemistry, University of Tennessee Medical Units, Memphis, Tennessee .
(Received 21 April 1970 ; in final form 10 June 1970)
Summary An extract of bovine brain exhibits a binding site specific for cyclic AMP . Binding of labeled cyclic AMP was competitive with respect to carrier cyclic AMP and its dibutyryl derivative, but essentially not with other nucleo tides . The binding component was sensitive to proteolytic enzymes and appeared to be associated with a protein. The amount of cyclic AMP bound was proportional to the concentration of protein and to that of cyclic AMP . Binding was most efficient around pH 6 .5 . The binding protein was found in all the brain areas examined . Subcellular fractionation of the cortex showed that the mitochondrial and microsomal fractions had the most activity and the high speed supernatant fluid the highest specific activity . Introduction Cyclic AMP is involved in a variety of different cellular functions (1) . Perhaps one common thread of all these different manifestations is that cyclic AMP interacts with proteins .
The interaction probably involves binding of the
cyclic nucleotide to the protein .
An analogy is the binding of an effector to
the regulatory site of an allosteric enzyme, as in the case of the binding of cytidine triphosphate to aspartate transcarbamylase (2) .
Another well known
example is the binding of 2,3-diphosphoglycerate to hemoglobin (3) . The brain is the most active tissue both in adenyl cyclase and cyclic 3', 5'-nucleotide phosphodiesterase (1) .
Also, the level of cyclic AMP in the brain
changed dramatically within seconds after decapitation (4) .
We have, therefore,
searched in this tissue for the existence of proteins with a binding site specifi .; for cyclic AMP . This note demonstrates the existence of such a binding site, which is associated with a protein extracted from the bovine brain . binding site are presented .
861
Some properties of the
BINDING OF CYCLIC AMP
862
Vol . 9, No . 15
Methods Preparation of Binding Protein . stored at -20 0 until use .
Bovine brain cortices were used fresh or
The binding protein was prepared according to a pro-
cedure developed for the purification of a cyclic 3',5'-nucleotide phospho diesterase (5) .
The procedure consisted of extracting the tissue with water,
followed by pH fractionation, differential centrifugation, (NH4)2S04 precipitation and calcium phosphate gel adsorption .
Subcellular fractions were prepared
according to De Robertis et al . (6), using fresh brain cortex . Binding of Cyclic AMP .
A sample for binding study was placed in a small
cellulose tubing and was dialyzed for 16 to 20 hours at 4 0 against a buffer about 100 times the volume of the sample .
The buffer contained 4 mM EDTA, 20
mM Tris-C1, pH 7 .5, 25-50 nM cyclic AMP - 8-C14 , with a specific activity of 41 uCi/umole .
EDTA was added to inhibit cyclic 3',5'-nucleotide phosphodies-
terase activity present in these preparations (7) .
At the end of the dialysis,
an aliquot of the dialyzed sample was counted in a liquid scintillation Spectrometer .
An equal aliquot of the buffer was also counted and the radioactivity
in the buffer was subtracted from that of the sample to give the amount of radioactivity bound to the sample .
Binding is expressed as the amount of cyclic
AMP bound per mg protein or per aliquot of sample . Protein was determined using the spectrophotometric technique of Warburg and Christian (8) and the biuret method with bovine serum albumin as a standard . At the end of equilibrium dialysis, protein was routinely determined again to correct for a possible slight change of protein concentration due to dialysis . Results and Discussion Bound cyclic AMP is defined as that amount in an aliquot of sample inside the dialysis tubing over and above that in a similar aliquot of the buffer after equilibrium is attained .
In the absence of a sample, the concentration of cy-
clic AMP inside and outside the dialysis tubing was equal at equilibrium .
Table
I shows the effect of protein concentration on the amount of cyclic AMP bound to an aliquot of the sample .
The amount of cyclic AMP bound was proportional up to
Vol. 9, No . 15
889
BINDING OF CYCLIC AMP
6 mg protein per ml, reaching a plateau shortly thereafter .
The last column in
the table shows the amount of cyclic AMP bound per mg protein, which varied from 11 to 15 p moles, depending on the concentration of proteins in the dialysis tubing .
Cyclic AMP retained inside the dialysis tubing was as high as six
times that in the buffer (compare Sample Nos . O. and 9), indicating that binding was accomplished against a considerable concentration gradient . TABLE I EFFECT OF PROTEIN CONCENTRATION ON THE AMOUNT OF BOUND CYCLIC AMP
Sample No .
Protein conc .
cAMP/200 ul
cAMP bound/200 ul cAMP bound/mg protein
mg/ml
(p moles)
(p moles)
(p moles)
0
0
5 .1
-
1
1.4
9.2
4 .1
15 .1
2
2 .5
12 .1
7 .0
14 .2
3
3 .3
14 .7
9 .6
14 .6
4
4 .3
17 .1
12 .0
13 .8
5
5 .3
19 .7
14 .6
13 .7
6
6 .3
22 .2
17 .1
13 .6
7
8.5
26 .8
21 .7
12 .8
8
8 .8
27 .9
22 .8
13 .0
9
11 .0
30 .1
25 .0
11 .4
-
An aliquot of 0.5 ml of a 30 to 50% (NH4)2S04 fraction (4) was dialyzed overnight in a small cellulose tubing against an excess Trio-EDTA buffer, containing 25 nM cyclic AMP-8-C14. At the end of the equilibrium dialysis, 200 ul of the dialyzed protein sample and 200 ul of the buffer were counted in a scintillation spectrometer . The amount of cyclic AMP bound was a function of the concentration of cyclic AMP present in the buffer .
Half maximal binding was obtained when the
buffer contained approximately 40 nM cyclic AMP.
In this experiment, the con
centration of protein was 8 mg per ml, and the maximal amount of cyclic AMP
864
BINDING OF CYCLIC AMP
Vol . 9, No. 15
bound was 25 p moles/mg protein . Binding of cyclic AMP was also a function of the pH of the buffer . Binding was most efficient around pH 6 .5 .
Between pH 4 .0 and pH 5 .0, the so
lution became turpid at the end of the equilibrium dialysis .
The sample used
in this as well as in other experiments was heterogeneous in its protein components .
The possibility existed that the decrease in binding at the acid side
might be due to a masking of available binding sites by some of the denatured proteins . An aliquot of the binding material (2 .4 mg protein) was exposed either to 25 ug of trypsin, pronase, RNase or DNase at 30 0 for 3 hours . not so treated served as a control .
A similar sample
At the end of the incubation, these sam
ples were examined for its ability to bind cyclic AMP .
Table II shows that the
samples treated with trypsin or pronase were virtually incapable of binding cyclic AMP, but the ones treated with RNase or DNase were as efficient as the control .
It was concluded that the binding site for cyclic AMP was associated
with a protein or some proteins, and not with a nucleic acid .
Exposure of the
protein to 8M urea sharply reduced, while exposure to 100 0 abolished all the binding ability . TABLE II EFFECT OF PROTEOLYTIC ENZYMES, RNase OR DNase ON THE BINDING OF CYCLIC AMP
No .
Treatment
cAMP bound/100 ul (p moles)
1
None
3 .92
2
Trypsin
0 .15
3
Pronase
0 .30
4
RNase
4 .17
5
DNase
3 .61
BINDING OF CYCLIC AMP
Vol . 9, No . 15
885
An aliquot of 0 .6 ml of a calcium phosphate gel eluate (4) was incubated with 25 ug of trypsin, pronase, Mesa or DNase for 3 hours at 30 0. At the end of the incubation, the sample was transferred to a cellulose tubing for equili brium dialysis as in Table I. Counting was done in duplicates of 100 ul aliquots . The figures have been rrected for radioactivity due to the random distribution of cyclic AMP-8-J4 in the buffer . The effect of other nucleotides on the binding of cyclic AMP was examined in Table III.
The sample was dialyzed against Trio-EDTA buffer containing 50
nM cyclic AMP-8-Cl 4 and another nucleotide at 10 uM, a concentration about 200 times that of cyclic AMP.
The other nucleotide was allowed to compete with cy-
clic AMP for the binding site .
The control sample was dialyzed in a Tris-EDTA
buffer containing cyclic AMP-8-C 14 , but no other nucleotides .
2'-AMP, 3'-AMP
and cyclic 2',3'-AMP were without effect ; 5'-AMP, ATP and cyclic GMP were slightly inhibitory . be most effective,
At the same concentration, unlabeled cyclic AMP proved to
followed by its N6,2'-0-dibutyryl derivative .
This experi-
ment showed that the binding site was specific for cyclic AMP. TABLE III EFFECT OF NUCLEOTIDES ON THE BINDING OF CYCLIC AMP
No .
Other nucleotides
cAMP bound (p moles/mg protein)
1
None
21 .7
2
2'-AMP
20 .0
3
3'-AMP
20 .0
4
Cyclic 2',3'-AMP
20 .0
5
5'-AMP
15 .8
6
Unlabeled cAMP
0 .5
7
dBCAMP
10 .0
8
ATP
18 .1
9
CGMP
16 .6
An aliquot of 0 .5 ml of a 30 to 507 (NH4)2S0~, fraction was dialysed in Trio-EDTA buffer containing 50 nM cyclic AMP-8-C14 and another competing
86 6
BINDING OF CYCLIC AMP
Vol. 9, No. 15
nucleotide at 10 uM. The control contained cyclic AMP-8-Cl4 and no other nucleotide . At the end of equilibrium dialysis, counting was done in duplicates of 100 ul aliquots . Abbreviations : dBcAMP - N6 ,2'-0-dibutyryl cyclic AMP ; cGMP - cyclic 3',5'-GMP ; others are standard abbreviations . The binding protein was detected in all areas examined in the bovine brain . They were :
cerebellum, cortex, hypothalamus, medulla, olfactory bulb, pineal
gland, pons and thalamus, with the highest specific activity in the gray matter of the cortex .
Subcellular fractionation of the cortex showed that with the
possible exception of the nuclear °_raction, the binding protein was present in all the different fractions . the particulate fractions .
Most of the binding activity was associated with Approximately half of the particulate activity was
present in the mitochondria and the other half in the microsome .
The superna-
tant fluid appeared to have the highest specific activity . The present work did not determine whether the protein possessed one or more binding sites, or whether the different brain areas contained one or more binding proteins .
It should be noted that not all proteins bind cyclic AMP .
For example, bovine serum albumin at 10 mg protein per ml was ineffective under comparable conditions . Experiments designed to identify the fate of the bound cyclic AMP indicated that the nucleotide was recovered in a form indistinguishable chromatographically from an authentic sample of cyclic AMP.
Thus, binding to the protein did
not alter its molecular structure .
.
The damonatration of a protein with a binding site specific for cyclic AMP may shed light on the mechanism of action of the nucleotide . about a conformational change on a protein .
In the case of an enzyme, a change
of conformation would lead to a change in enzymic activity . may be cited as being particularly pertinent . AMP, and it binds the nucleotide (9) .
Binding may bring
Phosphofructokinase
The enzyme is activated by cyclic
Other enzymes that are activated by cy-
clic AMP include lipase (10), protein kinase (11,12,13), and glycogen synthetase (14) . Cyclic AMP also affects systems possibly involving alteration of some phy-
HINDING OF CYCLIC AMP
Vol . 9, No. 15
867
sical properties of a non-enzymic protein, leading to a change of some cellular processes .
Examples may include the increase of membrane permeability of toad
bladder to water (15), the increase of amylase secretion in the parotid gland (16) and the increase of fluid secretion in the salivary gland (17) . In view of the binding of effectors to allosteric enzymes, it seems reasonable that cyclic AMP, through its interaction with proteins, would be bound to such proteins .
Binding of cyclic AMP maybe a common denominator in all the
apparently different manifestations attributed to this nucleotide .
Different
effects of cyclic AMP in different tissues are thus explainable on the kinds of proteins interacting with the nucleotide . If the binding of cyclic AMP to a protein were indeed related to its mechanism of action, such a protein or proteins would be expected to be distributed widely in various tissues .
Indeed, Gill and Garren (18) have demonstrated a
protein with a specific site for cyclic AMP in the adrenal cortex .
Also,
Solomon and Schramm (19) reported a binding site for cyclic AMP in the smooth membranes of the rat parotid microsomes .
The bound nucleotide in both systems
was recovered as cyclic AMP . Acunowledgments This work was supported by grants NB08059 and CA08480 from the U .S . Public Health Service .
It was also supported by ALSAC .
It is a pleasure to acknow-
ledge the valuable help of Dr . Seung-Yil Song in dissecting brain areas and the excellent technical assistance of Mrs . Sandra Patrick . References 1.
Robison, G .A ., Butcher, R .W ., and Sutherland, E .W ., Arm . Rev . Biochem. , 37 ,149 (1968) .
2.
Changeux, J .P ., Gerhart, J .C ., and Schachman, H .K ., Biochemistry , 7,531 (1968) .
3.
Bsnesch, R ., Bsnesch, R.E ., and Yu, C .I ., Proc . Natl . Acad . Sei ., 59,526 (1968) .
4.
Kakiuchi, S ., and Rall, T .W ., Mol . Pharmacol ., 4,379 (1968) .
5.
Cheung, W .Y ., Biochim . Biophys . Acta , 191,303 (1969) .
BINDING OF CYCLIC AMP
868
Vol . 9, No. 15
6.
De Robertis, E., end Salganicoff,
Pellegrino de Iraldi, A., Rodrignez de Lores Arnait, G., L ., J . Neurochem., 9,23 (1962) .
7.
Cheung, W.Y ., Biochemistry , 6,1079 (1967) .
8.
Warburg, 0 ., and Christian, W., Biochem . Z. , 310,384 (1941) .
9.
Kemp, R.G ., and Krebs, E.G ., Biochemistry , 6,423 (1967) .
10 .
Rizack, M.A., J. Biol . Chem., 239,392 (1964) .
11 .
Walsh, D.A ., Perkins, J.P ., and Krebs, (1968) .
12 .
Langea, T.A ., Science , 162,579 (1968) .
13 .
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