Eur. J. Biochem. 83. 411 -417 (1978)

Mechanism of Activation of Protein Kinase I from Rabbit Skeletal Muscle Mapping of the CAMP Site by Spin-Labeled Cyclic Nucleotides Jurgen HOPPE, Erwin RIEKE, and Karl G. WAGNER Gesellschaft fur Biotechnologische Forschung, Abteilung Molekularbiologie, Braunschweig-Stockheim (Received August 22, 1977)

Binding of adenosine 3’ :5’-monophosphate (CAMP)to protein kinase (type I) from rabbit skeletal muscle has been investigated using spin-labeled CAMP derivatives. Different compounds were synthesized with the spin label attached by spacer chains of different length at different positions on the adenine base. Immobilization of the spin label, determined by comparing the electron-spin resonance spectra recorded in the presence of the kinase with those of the free ligand in solutions of different viscosities, gave information about the geometry of the CAMP site. Strong immobilization of the N-6 substituents up to a spacer length of seven atoms indicates a rather deep cleft of the cAMP site. The depth of this cleft differs, however, when the spin label is attached to the different positions at the adenine (N-6, C-2 and C-8). Whereas the N-6 derivatives indicate a rather deep site, the C-2 derivatives reveal a significantly smaller depth and C-8 substituents (syn conformation) obviously occupy a very shallow surface with almost no immobilization. In addition the binding affinities of the spin-labeled cAMP derivatives have been determined, together with those of a series of (diamagnetic) C-2 derivatives bearing hydrophobic alkyl chains of different length. The latter results helped to clarify the differences between the regions near to C-2 and N-6, respectively, of the CAMPsite. N-6 spin-labeled derivatives have also been investigated in the presence of ATP and protein kinase. These results are interpreted as indicative of a conformational change at the CAMPsite upon formation of the holoenzyme, due to binding of ATP, leaving cAMP less strongly immobilized.

The cleft provided for the binding of cyclic AMP on a protein kinase must be constructed to meet the following requirements: (a) it must provide the affinity for specific recognition and binding of the effector; (b) it must signal the effector binding towards the interface between the subunits, in order to initiate dissociation of the holoenzyme and activation of the catalytic subunit. As an approach to the elucidation of the spatial dimensions of the cAMP cleft near the adenine binding site, an investigation was performed with CAMP derivatives bearing a spin label reporter group attached to the adenine at different positions by a spacer arm of different lengths. CAMP-dependent protein kinase type I from rabbit skeletal muscle was chosen, because it is This is paper 2 of a series; paper 1 appeared earlier in this journal [51. Part of this work was presented at the 10th International Congress of Biochemistry, Hamburg 1976. Abbreviations. CAMP, adenosine 3’:5’-monophosphate; ESR, electron spin resonance; R and C, regulatory and catalytic subunit of CAMP-dependent protein kinase. Enzyme. Protein kinase (EC 2.7.1.37).

the best studied protein kinase of this type [1,2] and because it offers an approach for studying the ‘signalling’ of bound effector. ATP, besides acting as a substrate of the free catalytic subunit of this enzyme, also binds to the holoenzyme with high affinity [2- 51; the affinity towards the holoenzyme is much higher than that which corresponds to the K, value of the free catalytic subunit (C). This is the reason why ATP drives the equilibrium: R,C,+R, + C, towards the holoenzyme. On the other hand, cAMP binds with high affinity to the dimeric regulatory subunit (R) promoting dissociation of the holoenzyme. Assuming that the holoenzyme has also binding sites for CAMP, these must be of lower affinity than those of the free R, when CAMP has to promote enzyme dissociation. Hence ‘signalling’ of effector binding, i. e. dissociation of the holoenzyme, would be accompanied by a transformation of the weak cAMP sites at the holoenzyme to strong sites at free R,. Such differences should be detected by differences in the immobilization of appropriate spin-labeled cAMP derivatives, provided

412

Interaction between Protein Kinase and Spin-Labeled CAMP

holoenzyme-bound CAMP can be expected in a high enough concentration. To achieve this goal studies should be performed in the presence of ATP, because ATP shifts the equilibrium towards the holoenzyme. The presented work offers results to the two approachs outlined and is a continuation of previous work on the properties of some of the spin-labeled CAMP derivatives used [6].

H

2

N

G

spin label I,

N -0

H,N--CH,

spin label

Hfl-CH,-NH

I,

-C spin label 1,

EXPERIMENTAL PROCEDURE Matcriu1.t

4-Amino-2.2,6,6-tetramethyl-piperidine and 3-carbamido-I -oxyl-2,2,S,S-tetramethyl-pyrrolidine were obtained froin Aldrich Chemical Co. Cyclic AMP, 6-chloropurinc riboside 3’ :5’-monophosphate and 8-bromoadenosine 3’ :5’-monophosphate were purchased from Boehringer Mannheim and 2-chloroadenosine was obtained from Sigma Chemical Co. 2Chloroadenosine 3’ :5’-monophosphate was prepared according to Jastorff and Freist [7].Cyclic [fb3H]AMP was purchased from Ainersham Buchler GmbH, while the series of C-2 substituted derivatives of CAMP were a gift from Dr J . P. Miller, ICN Nucleic Acid Research Institute (c;f. [XI). Clzeniical S j ~tlit~.si.s i o f Spin-Labeled Derivatives The spin-labeled CAMP derivatives, substituted at N-6, C-8 or c‘-2. were synthesized by reacting the respective chloro ((1-6 and C-2) or bromo (C-8) compounds of purine riboside 3’: 5’-monophosphate (N-6) or adenosine 3’ : 5’-monophosphate (C-2 and C-8) with the spin label bearing a primary amino group. Different spin label coinpounds were chosen so as to produce spacers of different length between the purine base and the aliphatic cyclic group bearing the nitroxide radical. In Fig. 1 the spin label amino compounds are shown together with the abbreviations used in the tables and figures of this work. CAMPSsubstituted at N-6 by spin label I, or II,, at C-8 by I , or TI,, and at C-2 by I , , respectively, were prepared as previously described [6]. CAMPderivatives bearing the spin labels 11,-11, were synthesized according to the following general procedure which is described in more detail elsewhere [9] : 3-carbamido-loxyl-2,2,S.5-tetramethyl-pyrrolidinewas hydrolyzed with a boiling aqueous solution of Ba(OH), to the corresponding acid, which was subsequently esterified with N-hydro.xysuccinimide in dry ethyl acetate using dicyclohexyl mrbodiimide (I;$ [ 101). The active ester was aminolyred with a considerable excess of the respective diainino alkane. Purification was achieved by adsorption of the products onto SP-Sephadex A25 and elution m ith 150 mM potassium phosphate buffer of pH 7. The fractions containing the spin label were

Fig. I . Spin lcihrl urnino cornpouncis. uwtl to suhstitiitf tlic r e . s l ~ ( ~ i ~ t i v c ~ h u h deriivrtiivs o f c A M P , crrid rhe cor.re.\pontlitig cihhr.ei1iLition.s.I and I1 refer to the type of the spin label (piperidinc or pyrrolidine) and 1.2 etc. refer to the number of atoms in the spacer, i.e. between the aliphatic cyclic group and the purine nucleus. after substitution of CAMP

Table I . Priiprrties o f the .vpitr-lubeled C A M P tkwiiarive..s The spectroscopic data have been determined in water at p H 7 . Solvent A is isopropanol’ammonialwater (711 2). solvent B butan-lollacelic acid,’water (51’2’3).For explanation of the abbreviations of the derivatives see legend of Fig. 1

CAMP substituted at the indicated position by the spin label

N-6 N-6 N-6 N-6 N-6 c-8

c-7 c-2

115 11, 11, 11,

11, 11,

11, 11,

i.,,,

R, values in solvent

I:

B

0.78 0.81 0.83 0.86 0.87 0.80 0.86 0.85

0.36 0.36 0.39 0.41 0.48 0.47 0.35 0.45

’ cni

m i

mM

266 266 266 266 266 214 282. 2% 281. 259

13 5 13 5 13 I 13 8 13 8 18 8 16 5 (258) 16 2 (259)

~

A

collected, adjusted to pH 13 with I M NaOH, and thoroughly extracted with butan- 1-01, which was subsequently removed under reduced pressure. The residue was dissolved in a small volume of ethanol. The reaction of the spin-label compounds with the halogen derivatives of CAMP and their purification were carried out as previously described [6]. The purity of the derivatives was tested by thin-layer chromatography on cellulose plates using isopropanol/ammonialwater (7/1,’2. v!v:v) and butan1-ol/acetic acid/water (5/2/3, v/v/v) as eluant. The molar absorption coefficients were obtained on the basis of quantitative organic phosphate determination [ l l ] . Ultraviolet spectra were recorded with a Zeiss DMR 21 spectrometer. Table 1 shows the spectroscopic data and the R, values deterrnincd for the different spin-labeled CAMPderivatives. The derivatives proved to be homogeneous by these two thin-layer chromatographic assays.

41 3

J . Hoppe, E. Rieke, and K. G . Wagner

ESR Measurements

ESR measurements were made on a Bruker BER 414s operating at the X band with 100-kHz field modulation. A flat quartz cell was used and the measurements were performed at room temperature. A modulation amplitude of 10 G was used throughout. Binding to Protein Kinuse CAMP-dependent protein kinase from rabbit skeletal muscle was purified until homogeneous as described previously [ 121. The binding affinity of the derivatives was determined by competition with tritiated CAMP using a modified binding assay according to Gill and Garren [13]. The incubation buffer contained 0.15 pM protein kinase holoenzyme, 20 mM potassium phosphate pH 6.9 and 4 mM magnesium acetate in a total volume of 200 p1. The incubations were carried out for 15 min. The data were plotted according to Dixon [ 141.

RESULTS AND DISCUSSION Binditzg Affinities Table 2 contains the results from competitive binding of the spin-labeled derivatives with tritiated CAMP. Because of the underlying enzyme dissociation, equilibrium binding constants are dependent upon enzyme concentration. This is the reason why all binding experiments were performed at constant (0.15 pM) protein kinase concentration. The data have been transformed into 6 d C values which indicate the reduction in the free enthalpy due to substitution. Substitution at the N-6 position causes a reduction in the binding affinity of less than 4 kJ/mol. There is no significant dependence upon the length of the spacer arm between the base and the spin-label component. The rather bulky substituents do not seriously affect binding affinity which is in accord with results from several other substitutions at the N-6 position [5,15]. Even replacement of the 6-NH2 group with C1, SH or OH hardly influences the binding properties [ 151. The 6-amino group is obviously not essential for the specific recognation of CAMP by the receptor site of the protein kinase. It is apparently not involved in the formation of a hydrogen bond in contrast to evidence from the ATP site of other enzymes and the highaffinity ATP site of the holoenzyme of the protein kinase [5].Furthermore steric hindrance does not seem to be critical at the N-6 position. On the other hand, substitution at C-8 by the spin label reduces binding affinity up to 15 kJ/mol (Table 2). As with the N-6 derivatives no dependence upon the spacer length could be observed. Bulky substituents should force C-8 derivatives into the syn conformation while the predominant isomer for the N-6 compounds

Table 2. Binding uflkities of' the .spili-!ahe/td C A M P d w i s a t i w ~ to war.d,rprotein kinase ,front rabbit skelc2tal m i i s c f c , By competitive binding with tritiated cAMP K , values were determined; division by the Kvalue of unsubstituted CAMPlead to K ' , values; these were transformed into the free enthalpy scale. Hence. the 6AC values indicate the reduction in the free enthalpy of binding due to the respective substitution. Under the conditions applied and described in Experimental Procedures a AC value of - 35.2 kJ/mol was determined for cyclic AMP. The abbreviations used are illustrated in Fig. 1 Spin label sub\tituent

Position of cAMP substitutcd

N-6

C-8

c-2

13.0 14.7 12.6

10.0 8.0 4.2

-

-

-

-

kJ/mol

2.7 2.3 3.1 3.4 3.3 3.9 3.6

-

-

-

-

Table 3. Binding uffinitie.s uf CAMP derivntivrs suhstiiuird at C-2 h j , d(ffererit hydr.ophohic. residues The 6AG values indicate the reduction in the free enthalpy of binding relative to unsubstituted cAMP (cf legend of Table 2) Substituent

6AC kJimol

6.7 5.9 5.1 5.2 3.7 1.5 2.1 10.9 4.2

is the urzticonformation [6]. The significant reduction in binding affinity in the case of the C-8 derivatives should therefore partly originate in the false conformation around the glycoside bond, provided unsubstituted cAMP binds to its receptor site without a significant change in the glycosidic angle. Substitution at C-2 (Table 2) by spin label I, leads to reduction in binding affinity of 10 kJ/mol, the reduction is diminished by elongation of the spacer arm between spin label and purine ring. Meyer et ul. [8] have recently explored the region of the cAMP site facing the C-2 position by measuring the activation of bovine brain protein kinase in the presence of different C-2 derivatives. Through the courtesy of D r J. P. Miller we could investigate the same binding region at the rabbit muscle enzyme by determination of Ki values. Table 3 indicates the 6AG values derived therefrom.

414

Interaction between Protein Kinase and Spin-Labeled CAMP

The results of Table 3 show that a bulky group at C-2 like a phenyl residue strongly decreases binding affinities, while the effect is significantly diminished by insertion of a spacer (ethyl). Steric hindrance is already obvious through the action of a methyl group; however, elongation of the linear aliphatic substituent gradually improves binding affinity. These results agree very well with those found for the activation of bovine brain protein kinase [8] and emphasize the similarity of the CAMP site. at least near the C-2 region, for these two enzymes and also the validity of comparing activation and binding data. Meyer et al. [8] interpreted their results in terms of a strong steric hindrance generated essentially by the first two atoms of the C-2 substituents and a ‘hydrophobic slot’ which endows the hydrophobic substituents with gradually enhanced binding affinities. This is confirmed by our results with the rabbit muscle enzyme. On the other hand, the CAMPsite near to the N-6 and C-8 position of adenine behaves quite differently; there is no marked dependency upon the length of the substituent (Table 2).

. 0

Nitroxide radicals are sensitive monitors for molecular motion i n the range of tumbling times between 0.1 and 100 ns. a range covered by most of the biological macromolecules. Small effectors bearing a spin label like those ofthe present work adopt the slow motion of an cnzyme if they are bound tightly into their binding site. Gradual clongation of spacers between effector and uitroxidc moiety usually results in a gradual increase in the motion of the side chain with the result that the label is no longer affected by the enzyme cleft [16]. Thus information about the geometry of the enzyme binding site can be obtained. In Fig. 2 tumbling times ( r values) estimated by visual comparison of spectra, which were calibrated by solutions of 3-amino-2,2,6,6-tetramethyl piperidine- 1oxyl of different viscosities (c-f: [6,16]), were plotted against the total number of atoms between the adenine base and the nitroxide-radical-bearing ring. i.r. the length of the spacer arm (qf: Fig. 1). In previous work [6] the mobility of the spin-label substituents at the positions N-6, C-8 and C-2 of free cyclic A M P was investigated. Decreasing the tumbling frequency of the cyclic AMP derivatives in solvents of high viscosity gave different mobilities of the spin label depending on the position of attachment at the purine nucleus. The results showed that mobility of bulky substituents are most restricted at C-8, due to steric interaction with the ribose, and least at N-6. With the CAMP derivatives buried in the enzyme cleft the situation is quite the contrary. Comparing the tumbling times for the piperidine spin label (I) attached by an NIJ group (corresponds to spacer length of one

2

.

4 6 a Number of atoms

x

Fig.’. L k p c i i r I i v m ( J / tlic rimihliiig riiiie) of X i i i m d w i i i i d (..-1,\.IP clwivutiws upoii the lci1,qh of 117c ,spuwr ( i r i i i . The length of spacer is iiidicatcd as thc number of atoms between Ihc purine nuclcus of adenine and the piperidine or pyi-rohdinc ring containing the nitroxide radical ( c f . Fig.1). ( 0 )N-6, (m) C-2 and (A)C-8 spinlabeled derivatives

atom in Fig. 2), the N-6 derivative is immobilized to the highest and the C-8 derivative to the lowest degree. The great reduction in the mobility of the N-6 derivatives is surprising, as substituents at this position have minimal influence on the binding affinity as outlined above, indicating that the C-6 substituents are not involved in specific interactions between effector and enzyme. These data, however, do not reveal direct information about the enzymic environment at this position. Obviously amino acid side chains are present which reduce the mobility of the spin label but do not take part in interactions which affect binding affinity. Another explanation would assume that contributions which reduce binding affinity (probably steric ones) are nearly equally compensated by those which enhance it (probably hydrophobic ones); the compensation should be independent of how long the substituents are. a situation which is quite unlike those described for the C-2 substituents. The label attachcd to N-6 becomes mobile, however, when the spacer arm reaches a length of 5-8 atoms and is completely unrestricted with spacer arms longer than 9 atoms (Pig.2). Substituents at the C-8 position are obviously not hindered by the enzyme surface. A spacer of only one methylene group is sufficient to obtain the complete mobility of the nitroxide label.

41 5

J . Hoppe, E. Rieke, and K. G. Wagner

T

20 G

Fig. 3 . ESR .spectra of CAMP suhstitutedat A‘-6 by spin label I , hound to lhe prorein kinase in the presence und ubsence qf M g - A T P . The concentration of the enzyme was S p M and that of the spin label derivative 9 pM in 0.15 M potassium phosphate of pH 6.7 containing I S mM mercaptoethanol. The spectra were recorded after 15 min incubation at room temperature. (A) Spectrum in the absence of MgATP; (B) spectrum in the presence of 2 m M ATP and 5 m M magnesium acetate. The kinase was first incubated with ATP and Mg2+ for 15min followed by the addition of the spin label CAMP derivative and a second incubation for lSmin. When the order of addition of Mg-ATP and the cAMP derivative, respectively. was reversed, the same spectrum was obtained

The degree of restriction of spin-labeled substituents at the C-2 position is between those at N-6 and C-8. The dependence of the spacer length indicates a medium depth for the geometry of the receptor site. The correlation times illustrated in Fig.2 are in accord with the affinity values listed in Table2 for the C-2 derivatives. Insertion of only one CH, group between C-2 and the spin label leads to enhancement of the spin label mobility, which is accompanied by an increase of binding affinity. This behavior is obviously a consequence of the release of a great deal of steric hindrance.

The Effect of ATP Two molecules of ATP were reported to bind strongly to the holoenzyme of the rabbit muscle protein kinase (type I) with an apparent K , in the range of 50 - 100 nM [2 - 51. The presence of ATP reduces the binding affinity for CAMP. ESR measurements with spin-labeled cAMP derivatives in the presence of ATP should therefore reveal information about the interrelationship of ATP and cAMP binding. ESR measurements of spin-labeled cAMP derivatives in the presence of the protein kinase and ATP are complicated in that free and enzyme-bound cAMP derivatives are present as a consequence of the reduced binding

0

2

4

6

8

Number of atoms

Fig. 4. Dependenci~ofthe tumbling rinzrs of kinuse-hound N-6 spin-label derivatives npon /he length of’lhe spacer arm in the presence of A T P . The length of thc spacer is indicated as the number of atoms between the purine nucleus of adenine and the piperidine or pyrrolidine ring containing the nitroxide radical (cf Fig. 1). For experimental conditions see legend of Fig. 3

affinity. The experimental conditions applied, i.e. with the enzyme concentration (about 5 pM), cannot be improved to overcome this difficulty. For the same reason only the N-6 derivatives with their high binding affinity could be studied in this series. Fig.3 shows a spectrum of cAMP substituted at N-6 by spin label I, (Fig. 1) and protein kinase in the presence and absence of ATP and magnesium ions. The contributions of free and immobilized spin label are evident. The low-field peak of the immobilized fraction is shifted by about 5 G to high-field in the presence of ATP; this can be taken as indicative of increased mobility from a comparison of a series of spectra of the free ligand recorded in solutions of increasing viscosities (cf. [6,16]). The spectra of the immobilized spin label have been graphically simulated and 7 values were assigned by comparison with the reference spectra. Fig.4 shows the results for the N-6 derivatives with different spacer lengths. The z values extracted from these spectra are remarkably lower than those observed in the absence of ATP. The difference is especially evident with cAMP substituted at N-6 by spin label I, (spacer of 1 carbon atom, Fig.2 and 4) with z values of 25 and 11 ns, respectively. Furthermore the dependence of the mobility upon the length of the spacer (Fig. 4) represents a rather shallow transition from immobilized to unrestricted conditions in contrast to the rather steep curvature observed in the absence of ATP (Fig.2). The spectra were independent of whether cAMP was mixed first with the enzyme or ATP, indicating reversible interaction of the two ligands.

416

These effects obviously indicate two different conformations of the CAMPsite either in the absence or in the presence of ATP. The higher mobility of the spin label with ATP should be due to less contact of the base with the enzyme cleft which would correspond to a reduced binding affinity. The different dependence of the mobility upon the spacer length would also indicate a different geometry of the binding site.

CONCLUSIONS The results of the present work can be first used to estimate the depth of the CAMP site (in the absence of ATP), assuming that the spacer arm within the bound spin-labeled ligand prefers an extended conformation. The cleft should be rather deep at the position facing N6 of adenine. a depth of about 1 nm from the C-6 of the purine nucleus to the enzyme surface would correspond to an extended spacer of 7 or 8 atoms. At the C-2 site the nucleotide should be immersed by only about 0.5 nm. The data obtained with the C-8 derivatives, binding to a very shallow region, are complicated by the fact that the adenine base has probably rotated around the glycosidic bond to assume a ,sJ:?'/z conformation. Hence, C-8 spin-label substituents may not be located at the natural C-8 position of the CAMPsite but more likely in the neighhourhood of the C-2 position; obviously they found il very shallow region which does not interact with or immobilize them. A very similar interpretation comes from I-esults on the interaction of the protein kinase with CAMP covalently attached to agarose. Cyclic AMP was coupled by spacers of different lengths and from different positions at the adenine base (c/: [l?] and the following paper [18]). Comparison of the binding affinities (Table 2) and the mobilities of the spin-label derivatives (Fig.2) clearly indicates the qualitative differences between the N-6 and (1-2 series. With the N-6 derivatives there is strong immobilization without significantly affecting the binding affinity, while with the C-2 derivatives both are affected simultaneously. The affinity data of Table3 obtained from a series of diamagnetic C-2 derivatives confirm this supposition. A phenyl substituent. which has about the same volume as the nitroxidc-bearing aliphatic cyclic group, has the same influence on binding affinity when attached directly and by an ethyl spacer, as the respective spin-label derivatives. The increase in binding affinity in the series of the rz-alkane C-2 derivatives up to a length of eight carbon atoms would indicate a rather deep hydrophobic cleft at this position. From the mobility data (Fig.2) the cleft should not be deeper than an alkyl chain of 5 carbon atoms. However, the supposition of extended alkyl chains, which underlie such interpretations. ma! not hold for long chains.

Interaction between Protein Kiiiase and Spin-Labeled CAMP

The differences found for the N-6 derivatives in the presence and absence of ATP needs the following explanation. The enzyme concentration was 5 pM and that of the spin-label CAMPderivatives 9 pM. One can assume that under these conditions and absence of ATP the enzyme has dissociated and the CAMP derivatives are practically all bound to the free regulatory subunit (R,). For this enzyme concentration a K, value (50 'I, activation) of 2 pM (CAMP)can be estimated from the data of Beavo et al. [19]. These authors also reported that the addition of 4 m M ATP leads to an increase in the dissociation constant of CAMP by a factor of 50. Although this factor was determined at a lower enzyme concentration, we can assume that the K, value for activation and dissociation of the enzymc under the conditions of the present experiment (2 mM ATP, 5 pM enzyme) will also increase in the same magnetidue, say at least by a factor of 10, i.r. from 2 p M to at least 20 pM. This would mean that at a concentration of the CAMP derivative of 9 p M most of the enzyme is ~dissociated.'There may be a sinall fraction of free R, : however, as R,-bound spin label obviously is more strongly immobilized than that bound to the holoenzyme, the ESR experiment cannot resolve it. The reduction of the binding affinity of CAMP in the presence of ATP is demonstrated in the appearence of the signal of free spin-labeled CAMP. However, the immobilized fraction of the ligand is obviously- bound in a state different to that observed without ATP, as is seen from the magnitude of the tumbling times and their dependence upon the length of the spacer arms (Fig.2 and 4). This suggests that in the presence of 2 m M ATP a considerable fraction of the CAMP derivative binds to the holoenzyine and that the.,, holoenzyme binding site has a conformation which immobilizes its ligand less strongly and probably with reduced affinity. Hence, the following mechanism of protein kinase activation emerges: the protein kinase holoenzyine is subjected to the known dissociation equilibrium. R,C2 S R, + 2C. This equilibrium is shifted to the left side (especially in the presence of ATP) under physiological enzyme concentrations [19]. Activation by CAMP is equivalent to shifting this equilibrium to the right: this is achieved by the existence of CAMP sites with high affinity at R, and obviously low affinity when K, is associated with the catalytic subunits. In other Lvords, dissociation of the holoenzynie converts the two CAMP sites at R, into deeper clefts with high affinity which strongly immobilize the CAMP ligand.

This work has been supported by the D c L / ~ . s ~ w Fnr.scliungs~emc~ii~rzsc.hUfl (Wa 9 I ) and the Fads t k r C'hotii.sc/ieii Idtlzrstrie. We are grateful ttr Dr V. Wray for linguistic advice and to Mrs R. Jihne for typing the manuscript. We are also very gratefd to Dr J . P. Miller for sending us the C-2 derivatives mentionrd.

417

J. Hoppe, E. Rieke, and K. G. Wagner

REFERENCES I . Krebs, E. G. (1972) Curr. Top. Cell. Re&. 5, 99-133. 2. Beaw, J. A , , Bechtel, P. J. & Krebs, E. G. (1975) A h . Cyclic Nucleotide RL's.5 , 241 -251. 3. Haddox, M. K., Newton, N. E., Hartle, D. K. & Goldberg, N. D. (1972) Biochern. Biophj3s. Rex. Comrnurz. 47, 653-661. 4. Hofmann, F., Beavo, J. A., Bechtel, P. J. & Krebs, E. G. (1975) J . Biol. Chem. 250, 7795 -7801. 5. Hoppe, J., Marutzky, R., Freist, W. & Wagner, K. G. (1977) Eur. J . Biockem. 80, 369 - 372. 6. Hoppe, J. & Wagner, K. G. ( 1 974) Eur. J . Bioclzew7.48, 5 19- 522. 7. Jastorff, B. & Freist, W. (1974) Bioorg. Cheni. 3, 103- 113. 8. Mcycr, R. B., Jr, Uno, H., Robins. R. K., Simon, L. N. & Miller, J. P. (1975) BiocheniistrjJ; 14, 3315-3321. 9. Hoppe, J . (1976) Thesis, Technische Universitat Braunschweig.

10. Rosantsev, E. G. (1970) in Free NitroxylRudicul(Ulrich, H., ed.) pp. 230-231, Plenum Press, New York. 1 I . Ames, B. N . (1966) Methods E~zzymol.8, 1 1 5- 118. 12. Hoppe, J . & Wagner, K. G. (1977) FEES Lett. 74, 95-98. 13. Gill, G. N . & Garren, L. D. (1970) Hiochern. L3ioph.w. Rrs. Comrnun. 39, 335 - 343. 14. Dixon, M. &Webb, E. G. (1964) Enzymes, Logmans, London. 15. Meyer, R. B., Shuman, D. A,, Robins, R . K., Bauer, R. J., Dimmitt, M. K. & Simon, L. N. (1972) Biochenii.~try,/ I , 3704- 2709. 16. Hsia, J. C. & Piette, L. H. (1969) Arch. Biochem. Biopkys. 12Y, 296-307. 17. Rieke, E. (1976) Thesis, Technische Universitiit Braunschweig. 18. Rieke, E., Hoppe. J. &Wagner, K. G. (1978) Eur. J . Biocheni. 83, 4I 9 - 426. 19. Beavo, J . A,, Bechtel, P. J. & Krebs, E. G. (1974) Proc. Null Acucl. Sci. U.S.A. 71, 3580-3583.

J . Hoppe and K. G . Wagner*, Gesellschaft fur Biotechnologische Forschung mbli, Mascheroder Weg 1 , D-3300 Braunschweig-Stockheim, Federal Republic of Germany E. Rieke, E.Merck AG, Postfach 41 19, D-6100 Darmstadt 2, Federal Republic of Germany

* To whom correspondence should be addressed