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PurificationandCharacterizationofacAMP-BindingProteinofVolvoxcarterif.nagariensisIyengar

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Eur. J. Biochem. 228, 480-489 (1995) 0 WBS 1995

Purification and characterization of a CAMP-binding protein of Volvox carteri f. nagariensis Iyengar Ortrun FELDWISCH’, Marion LAMMERTZ‘, Eva HARTMAN“, Joachim FELDWISCH*, Klaus PALME2, Bemd JASTORFF3 and Lothar JAENICKE‘

’ Institut fur Biochemie, Universitat zu Koln, Germany Max-Planck-Institut fur Zuchtungsforschung, Koln, Germany Fachbereich Biologie-Chemie, Universitat Bremen, Germany

(Received 25 November 1994) - EJB 94 1813/3

Two CAMP-binding proteins, cbpl and cbp2, were purified from the cytoplasm of the green alga Volvox carteri. Both proteins have a native molecular mass of 90 kDa as determined by gel filtration. cbp2 was purified to apparent electrophoretic homogeneity, having a subunit molecular mass of 42 kDa as determined by SDSPAGE. The cbpl preparation contains a 42-kDa and a 44-kDa band. The CAMP- binding activity is not associated with protein kinase activity. Tryptic peptides of cbp2 were sequenced by automated Edman degradation. Two pairs of peptides differ in one amino acid only, thus pointing to the presence of isoforms of cbp2. Both binding proteins differed from the CAMP-specific phosphodiester- ases of V carteri with respect to charge, molecular mass and binding affinity to N6-CAMP-agarose. Reverse-phase chromatography of the bound ligand revealed that the two binding proteins hydrolyse cAMP to 5’AMP. The binding specificity of purified cbpl and cbp2 was probed by a set of modified cAMP derivatives. Both proteins bind cAMP strictly specifically in the anti conformation; position 1 and 6 of the adenine moiety and at least one of the exocyclic 0 atoms of the ribose cyclic phosphate moiety are essential. 3-Isobutyl-1-methylxanthine is an effective inhibitor of binding but the natural methyl- xyanthines are not. At present it is not clear whether cbpl and cbp2 are individual proteins or isoforms of one another.

Keywords. CAMP-binding protein ; phosphodiesterase ; CAMP-dependant protein kinase ; plant ; peptide sequence.

The dioecious green alga Volvox carteri f. nagariensis repro- duces either vegetatively or sexually [l]. The sexual cycle is induced when a highly specific and active pheromone is sponta- neously formed by a mutational switch in one of lo4 male spher- oids [2]. The pheromone was characterized as a set of glycopro- tein isoinducers containing the same (225kDa) core protein and three N-glycosylated oligosaccharides but differing in the length of the 0-glycosylated carbohydrate side chains [3].

Evidence exists for the involvement of a CAMP-dependent signal chain in the process of induction (for review see [4]). This was the starting point of the present study: the CAMP content of sexual cultures is elevated in comparison to vegetative cul- tures [4, 51. For Chlamydomonas reinhardtii, a closely related chlorophycea, evidence for the involvement of cAMP in sexual differentiation has been given [6].

Signal transduction by cAMP as second messenger is medi- ated by intracellular cAMP receptors called CAMP-binding pro- teins. The only identified CAMP-binding protein of procaryotes is the protein known as CAP or CRP which is able to bind to DNA specifically when it is liganded with cAMP [7]. In eucary- otes the best studied CAMP-binding protein is the regulatory subunit of the CAMP-dependent protein kinase. Binding of

Correspondence to L. Jdenicke, Institut fur Biochemie, Universitat zu Koln, An der Bottmiihle 2, D-50678 Koln, Germany

Abbreviations. cbp, CAMP-binding protein; pk, protein kinase ; pde, phosphodiesterase ; iBuMeXan, 3-isobutyl-I-methylxanthine ; 2’3’cNMP, nucleoside 2’3’-monophosphate; 3’5’cNMP, nucleoside 3’5’-monophos- phate ; CdCaM, Ca2+/calmodulin-dependent.

cAMP to this enzyme results in the activation of the catalytic subunit which possesses the kinase activity (see 181 for review). In contrast to the non-hydrolysing CAMP-binding proteins, cy- clic nucleotide-dependent phosphodiesterases (pde) bind and split cAMP to S’AMP, thus terminating the cAMP signal in transmitting chains. At least five types of different pde isoen- zymes are known in mammalian cells [9] : calmodulin stimulated pde, cGMP-stimulated pde, GMP-specific pde, low-K,,, pde and nonspecific or multifunctional pde.

Evidence for CAMP-binding proteins or phosphodiesterases in photosynthetic organisms exists : CAMP-dependent protein phosphorylation was shown in the algae Cylindrotheca fusi- f o m i s [lo] and Euglena gracilis [ I l l and in the higher plant Lemna paucicostata [12]. In Petunia hybrida [13] a protein ki- nase was identified that phosphorylates Kemptide and is inhib- ited by the regulatory subunit of bovine CAMP-dependent pro- tein kinase. The inhibition can be reversed by addition of CAMP. This enzyme, however, is not stimulated by CAMP. A multifunc- tional pde was partially purified from Lactuca cotyledons [14] and pea roots [15]. Multifunctional pdes hydrolyze both 2‘3‘cNMP (to 2‘- and 3’NMP) and 3‘5’cNMP (to SNMP) in con- trast to the other pde types which hydrolyze only 3‘5’cNMP (to 5’NMP). The nonspecific pdes are not inhibited by methyl- xanthines [16, 171. In addition to the multifunctional pde a sec- ond pde activity was detected in pea roots. This enzyme hydro- lyzes 3’5’cAMP and (preferably) 3’5’cCMP but does not hy- drolyze cGMP at all. It can be inhibited by theophylline and is not stimulated by calmodulin [15]. A pde inhibitable by theo-

Feldwisch et al. (Eur: J. Biochen. 228) 481

phylline and caffeine was partially purified from the roots of Kgna rnungo [18]. In Lernna paucicostata [17] pde activity was partially purified that hydrolyzes both 2'3'cNMP and 3'5'cNMP with a preference for adenosine nucleotides. As this preparation can be stimulated by calmodulin and is inhibited by theophylline it is possible that it contains both a multifunctional and a calmo- dulin-stimulated pde.

Except for the multifunctional pde, all of the above-men- tioned CAMP-binding proteins are involved in signaling cas- cades either mediating or terminating the cAMP signal. For our understanding of the influence of cAMP on the sexual differenti- ation of 'I.i carteri, the identification and isolation of a soluble CAMP-binding protein would be an important first step. We re- port now the purification of two soluble CAMP-binding proteins, cbpl and cbp2, the sequence analysis of tryptic peptides of cbp2 and the biochemical characterization.

EXPERIMENTAL PROCEDURES

Materials. DEAE-Sepharose (HR), Sephacryl-200 (HR) and the Mono P HR 5/20 column were obtained from Pharmacia. N6-CAMP- agarose was purchased from Sigma. The Fractogel EMD TMAE 650s and Fractogel TSK Butyl 650s columns were obtained from Merck. [2,8-3H]cAMP (38 Ci/mmol) and [ y-"PIATP (5000 CUmmol) were purchased from Amersham- Buchler. Trypsin (sequencing grade) was obtained from Boeh- ringer, Mannheim.

Growth of organism. Volvox carteri (female strain HKlO from the UT Collection of Algae, kindly provided by Dr. R. C. Starr, Austin, Texas) was grown at 25 "C and 12 klx in Provasoli- Pintner medium as described [19, 201. The cultures were started by a single spheroid in 10 ml medium. After 4 days, the cultures were transferred into 200 ml medium and, after a further 4 days, into 800 ml medium. The algae were collected by filtration, washed several times with medium, and used immediately or stored until use at -20°C.

Preparation of crude extract. The spheroids were gently pressed three times at 345 kPa through a Yeda press. The matrix material was removed by spinning down and washing the cells once or twice on the centrifuge at IOOOOXg for 10 rnin at 4°C. The cell pellet was resuspended in an equal volume of buffer (25 mM Tris/HCl pH 7.5, 5 mM dithioerythritol, 5 mM MgCl,) and sonicated on ice three times for 30 s at 200 W. Cell debris was removed by centrifugation (IOOOOXg, 10 min, 4°C). Mem- branes were sedimented by ultracentrifugation (120000Xg, 45 min, 4°C). The supernatant was the crude extract used in all further experiments.

Purification of CAMP-binding and protein-kinase activ- ity. All purification steps were performed at 4°C. Crude extract (100 ml) prepared from approximately 80 g algae (fresh mass) was applied on top of a DEAE-Sepharose fast flow column (bed volume, 60 ml) fitted on a FPLC chromatography system (Phar- macia). The column was washed with 200 ml equilibration buffer (25 mM Tris/HCl pH 7.5, 1 mM CaCl,, 1 mM MnCI,, 10 mM 2-mercaptoethanol) and eluted with a 200-ml linear gra- dient of 0-500 mM NaCl in equilibration buffer with a flow rate of 2 mumin; 10-ml fractions were collected, and aliquots were assayed immediately for CAMP-binding or protein kinase activity.

Fractions with CAMP-binding or protein kinase activity were pooled, concentrated by ultrafiltration (YM5 membrane) to max- imal 5 ml and loaded onto a S-200 high-resolution column (0.7X100 cm) which was equilibrated and eluted at a flow rate of 0.4 mumin with buffer A (50 mM TrisMCl pH 7.5, 100 mM NaC1, 1 mM MnCl,, 1 mM CaCI,, 10 mM mercaptoethanol);

4-ml fractions were collected and assayed for CAMP-binding or protein kinase activity.

Fractions containing CAMP-binding activity were pooled and stored at -20°C to collect sufficient partially purified CAMP-binding protein (cbp) for the next steps. A total activity of approximately 170 pmo1/36 ml was used for the purification. The cbp was eventually purified by means of a CAMP-agarose column (CAMP attached to cross-linked agarose through the P- amino group with a C, spacer; bed volume, 1 ml) previously equilibrated with buffer A. The column was washed with 7 ml buffer A (low salt wash) followed by 7 ml buffer A containing 750 &I NaCl (high salt wash). Loading and washing of the CAMP-agarose column were performed at a flow rate of 0.2 mV min. The CAMP-binding activity was eluted with buffer A con- taining 10 mM cAMP at a flow rate of 0.05 mumin. The eluate was diluted 1 : 5 in NaC1-free buffer A, concentrated again by ultrafiltration (repeated five times) to a final volume of 500 p1 and assayed for CAMP-binding activity.

Alternatively cbpl and cbp2 were purified by hydrophobic chromatography on Fractogel TSK Butyl 650 S. Prior to hy- drophobic chromatography (NH&SO, solution was added to S- 200 fractions containing CAMP-binding activity to an end con- centration of 50%. After 1 h, precipitated protein was centri- fuged off (SS 34,20OOOXg, 15 min, 4°C). The supernatant con- taining CAMP-binding activity was applied to the column equili- brated with buffer A' [buffer A with 100 mM NaCl replaced by 2.8 M (NH4),S04]. After washing with 50 ml buffer A', the bound proteins were eluted by a linear gradient of buffer B (0 rnin 0 % B, SO min 100 % B, 60 rnin 100 % B; buffer B is buffer A' without (NH,),SO, ; 5-ml fractions were collected and each fraction dialysed against 5 1 buffer against 5 1 buffer B. A flow rate of 1 mumin was maintained throughout. Under these conditions CAMP-binding activity eluted in fraction 13.

Purification of cbpl and cbp2 by ion-exchange ehroma- tography and chromatofocusing. cbpl and cbp2 were sepa- rated by DEAE-Sepharose chromatography. Both cbpl and cbp2 were separately purified further by 5-200 gel filtration. Fractions containing either cbpl or cbp2 activity were desalted by dialysis against equilibration buffer and loaded onto a Fractogel EMD TMAE650 S column (bed volume 14.5 ml) fitted on a FPLC chromatography system (Pharmacia). After washing with 50 ml equilibration buffer, the column was eluted with a 90-ml linear gradient of 0-500 mM NaCl in equilibration buffer with a flow rate of 1 ml/min; 3-ml fractions were collected and assayed for cbp activity. Alternatively to the Fractogel EMD TMAE650 S column, a Mono P HR 5/20 column fitted on a FPLC system (Pharmacia) was used. The Mono P HR 5/20 column was washed with 50 ml equilibration buffer after loading and eluted with 50 ml Polybuffer PB 74TM (diluted 1 : 10 in H,O, pH 4.0) with a flow rate of 1 mVmin; 2-ml fractions were collected. Prior to assay for cbp or protein kinase activity the pH of each fraction was determined and subsequently adjusted to pH 7.5.

CAMP-binding assay. cAMP binding was measured in a fi- nal volume of 100 p1 containing 50mM Tris/HCl pH7.5, 20 mM NaC1,l mM MnCI,, 1 mM CaCl,, 13 nM ['HICAMP and varying amounts of binding protein solution. Reactions were started by addition of cbp, incubated for 60 rnin at room temper- ature and stopped by addition of 1 ml cooled (-20°C) 96 % ethanol. After incubation of at least 15 rnin at -2O"C, the pro- tein-bound [3H]cAMP was recovered by centrifugation for 5 rnin in a microfuge at 15000 rpm and 4°C. The supernatant was de- canted and drops carefully removed by cotton plugs. The pellet was resuspended in 100 p1 H,O and counted in 1 ml toluene/ POPOP scintillator. Unspecific binding of [3H]cAMP was mea- sured in the presence of l mM unlabeled CAMP. Binding assays

482 Feldwisch et al. (Em J. Biochern. 228)

20000 300 - e

fraction

Fig. 1. Elution profile of CAMP-binding activity from a DEAE-Seph- arose column. Crude extract (approx. 20 mg protein) was applied to a DEAE-Sepharose fast flow column. After washing, the bound proteins were eluted with a 200-ml linear gradient of 0-500 mM NaCl; 10-ml fractions were collected and aliquots assayed for (m) cbp, (0) pk and (A) pde activity; (-) NaC1.

6000 , ~ -~ -- - 2 ' Z 4 O 0 O I

Vo K A HO M C

.cuu 4.c x

53000 ! - E2000 .- I QIOOO 1 I

1 i

0 0 10 20 30 40

fraction

Fig.2. S-200 gel filtration of cbpl and cbp2. After DEAE-Sepharose chromatography, fractions containing cbpl and cbp2 were pooled, con- centrated by ultrafiltration and loaded separately onto a S-200 high-reso- lution column. The column was eluted at a flow rate of 0.4 mumin; 4- ml fractions were collected and aliquots assayed for cbp. The bar marks the fractions used for further purification. For calibration the following proteins were used: K (catalase 250 ma), A (aldolase 159 ma), H (he- moglobin 64.5 m a ) , 0 (ovalbumin 45 m a ) , M (myoglobin 16.9 m a ) , C (cytochrome c 12.5 kDa). (0) cbpl ; (M) cbp2.

were always performed in duplicate; the standard error of the binding value did not exceed 10 %.

Protein kinase assay. Protein kinase activity was measured essentially by the filtration method [21], in a final volume of 100 p1 containing 50 mM Tris/HCl pH 7.0, 5 mM MgCl,, 0.28 nM [y-3zP]ATP, 7 pg histone IIA or dephosphorylated ca- sein and varying amounts of enzyme preparation. After incuba- tion at room temperature for 40 min, the reaction mixture was filtered through a nitrocellulose filter, washed three times with 10 % trichloroacetic acid, stained with 0.01 % Ponceau S and washed with H,O. The filter was air-dried, cut into pieces, and the fixed radioactivity determined by measuring the Cerenkow radiation in a scintillation counter.

Phosphodiesterase assay. Phosphodiesterase activity was determined by incubating 1 pM CAMP with varying amounts of the enzyme in a final volume of 300 pl 50 mM TrisMC1 pH 8.4, 1 mM MnC1, at 40°C. The reaction was stopped after 10- 30 niin by boiling the samples for 2 min. Precipitated protein was removed by centrifugation. The amount of non-hydrolyzed cAMP in the supematant was determined by RIA as described [22] with the following modifications: 40 p1 antibody (4/2C,, 50 pg/ml) in phosphate-buffered saline was mixed with 50 p1 supernatant or CAMP standard (5-50 pmol) and 10 pl

Fig. 3. SDS-gel electrophoresis of (A) cbpl and (B) cbp2 purified by affinity chromatography on N6-CAMP-agarose. Either cbpl or cbp2 purified by DEAE-Sepharose and S-200 gel filtration were applied to a P-CAMP-agarose column. After washing twice under low and high salt conditions the cbp were eluted by addition of 10 mM CAMP. The gel was silver-stained. (A) cbpl: (1) flow through, (2) low salt wash, (3) high salt wash, (4) second low salt wash, ( 5 ) elution with 1OmM CAMP. (B) cbp2: (1) applied material, (2) flow through, ( 3 ) low salt wash, (4) high salt wash, ( 5 ) elution with 10 mM CAMP. Numbers on the side are molecular masses (in kDa) of marker proteins.

['HICAMP (0.3 pmol) and incubated for 60 rnin at room temper- ature. Alternatively a reverse-phase HPLC assay was used: pde solution was incubated with 1 pM cAMP as described above; 200 pl of the supernatant were etheno derivatized [23] and 25 p1 of the resulting etheno-adenine nucleotides separated as de- scribed in the next section. The retention time of etheno-adenine nucleotides prepared as standard was used to identify the reac- tion products of the Volvox pde.

Reverse-phase-chromatography analysis of adenosine nucleotides. CAMP-binding activity was assayed as described above. The pellet containing protein-bound nucleotides was sus- pended in 50 pl H,O, the supernatant was vacuum-dried (Speed- Vac) and suspended in 50 pl H,O. Both fractions were etheno- derivatized as described [23]. The etheno-adenine nucleotides were separated by a non-linear gradient (0-10 rnin 10 % B', 10-18 rnin 30 % B', 18-26 min 40 % B', 27-31 rnin 100 % B', 31-36 min 100 % A"; buffer A" = 10 mM sodium phosphate pH 7.3 ; buffer B' = buffer A" containing 20 % acetonitrile) on a SS 250/6/4 NucleosilR 7 C,, column at a flow rate of 1 ml/ min; 1-ml fractions were collected. Fractions were dried in a Speed-Vac and resuspended in 100 pl H,O; 1 ml scintillation cocktail was added and the radioactivity measured. Due to quenching, this procedure gives only qualitative data. The dif- ferent adenine nucleotides (~'S'CAMP, 2'3'cAMP, S'AMP, 3'AMP, 2'AMP and adenosine) were identified by comparing their retention times with that of unlabeled N' , N6-etheno-adeno- sine nucleotides.

Feldwisch et al. (Euc: J. Biochem. 228) 483

Table 1. Purification of the 1/: curten' CAMP-binding proteins cbpl and cbp2. Binding activity was measured as pmol [3H]cAMP bound under the reaction conditions described in Experimental Procedures. 10 mM cAMP was used to elute cbpl or cbp2 from the N6-CAMP-agarose affinity resin. The amount of binding activity applied to the N6-CAMP-agarose results from three different purifications. The loss of activity (25-48 %) is due to storing at -20°C. Hydrophobic chromatography on Fractogel TSK Butyl 650 S was used instead of affinity chromatography on N6-CAMP- agarose in order to obtain CAMP-binding protein free of CAMP; this preparation (step 4) is not homogeneous.

Step Volume Binding activity Yield Protein Specific activity

ml pmol % mg pmolhg

Crude extract 100 1100 100 20 5s cbpl

1. DEAE-Sepharose 30 270 25 2 135 2. S-200 gel filtration 12 49 4 0.3 163 3. N6-CAMP-agarose eluate 0.75 0.54 0.0.5 0.0001 5400 4. Fractogel TSK

Butyl 6.50 S 5 16 1 0.033 485 cbp2

1. DEAE-Sepharose 60 540 50 5 108 2. S-200 gel filtration 12 108 10 0.7 154 3. N6-CAMP-agarose eluate 0.75 0.76 0.07 0.001 760 4. Fractogel TSK

Butyl 650 S 5 43 4 0.08 540

Peptide separation and amino acid sequencing. Proteins were separated on 12.5 % polyacrylamide gels as described [24]. Proteins were electroblotted onto polyvinylidene difluoride with 50 mM Tris, 50 mM boric acid, pH 8.3 [25] and stained with amido black (0.1 % in 45 % MeOH). The protein band in ques- tion was cut out and incubated in 500 p1 quenching solution (0.2 % polyvinylpyrrolidone in methanol, HPLC grade) for 30 min. The sample was diluted 1 :2 in H20, incubated for 10 min, washed four times with 500 pl H,O and three times with 500 pl 0.1 M Tris/HCl pH 8.0 and resuspended in 100 pl of the same buffer. Digestion with 1 pg trypsin (Boehringer) was for 4 h at 37°C. Peptides were eluted from polyvinylidene difluoride by 80 % formic acid and separated by reverse-phase HPLC using a 250X2.1 mm microbore column (Vydac) and a gradient of buffer A"' (0.06 % trifluoroacetic acid) and buffer B" (0.056 % trifluoroacetic acid, 80 % acetonitrile). The elution program was 0-37 % B" in 63 min, 37-75 % B" in 32 min, 75-100 % B" in 10 min. Peptides were detected by absorption at 214 nm, col- lected by hand and concentrated in a Speed-Vac. Aliquots were used for direct sequence analysis by automated Edman degrada- tion using an Applied Biosystems 477 A pulsed-liquid sequena- tor equipped with an on-line phenylthiohydantoin analyser (120A). Both sequencer and analyser were operated as recom- mended by the manufacturer.

RESULTS

Purification of CAMP-binding protein. A crude extract, pre- pared from a female strain of Volvox carteri, was applied to a DEAE-Sepharose column. After washing, the column was eluted with a linear gradient of 0-500 mM NaCI. The resulting frac- tions were assayed for CAMP-binding activity (Fig. 1). Two peaks eluting at 150 mM and 300 mM NaCl contained CAMP- binding activity and were called cbpl and cbp2, respectively. Rechromatography of either cbpl or cbp2 did not result in fur- ther splitting of the activity. Both peaks eluted again at either 150 mM or 300 mM NaCl.

After concentration by ultrafiltration, cbpl and cbp2 were further purified by S-200 gel filtration separately. The native molecular mass was 90 kDa for both cbpl and cbp2 (Fig. 2). Further purification was achieved by affinity chromatography on N6-CAMP-agarose (70 mg). About 70 % of both cbpl and cbp2

was retained by the affinity resin. The remaining 30 % was not bound either by rechromatography or by using a column con- taining twice as much affinity resin. After washing at low and high salt conditions, the CAMP-binding activity was eluted by adding 10 mM CAMP. As shown in Fig. 3, cbpl was purified almost to electrophoretic homogeneity while cbp2 was purified to apparent electrophoretic homogeneity. cbp2 has a molecular mass of 42 m a . The cbpl preparation contains the 42-kDa band and a 44-kDa band. A summary of the purification scheme for both cbpl and cbp2 is given in Table 1.

Attempts to elute cbp devoid of cAMP or cbp of higher spe- cific activity from the affinity column by varying the pH of the elution buffer or by reducing the cAMP concentration failed, but it was possible to obtain CAMP-free cbpl and cbp2 using ion- exchange, gel filtration and hydrophobic chromatography on Fractogel TSK Butyl 650 S as described in Experimental Pro- cedures. cbpl or cbp2 purified by this procedure represented the fraction of the highest specific activity (500 pmol/mg) contain- ing no free CAMP. It was not, however, brought to homogeneity; further purification, e.g. by hydroxyapatite chromatography, re- sulted only in high losses of activity (up to 50 %).

As cbpl and cbp2 could only be distinguished by their dif- ferent chromatographic behaviour on DEAE-Sepharose, we ana- lysed cbpl and cbp2 purified to step 2 by two additional chro- matography techniques relying on surface charge : Fractogel EMD TMAE650 S and chromatofocusing on Mono P HR 5/20. Chromatographed separately but under identical conditions on Fractogel EMD TMAE650 S, cbpl eluted at 210 mM NaCl while cbp2 eluted in two peaks of activity at 290 mM NaCl and 375 mM NaCl (Fig. 4A). When both cbpl and cbp2 were chromatographed separately but under identical conditions on Mono P HR 5/20, cbpl eluted in two peaks of activity of PI 5.4 and PI 5.2 (Fig. 4B) whereas cbp2 yielded an asymmetric peak with a maximum at PI 4.9 (Fig. 4C). The PI of the activity shoul- der was determined as approximately 5.4.

Partial sequence analysis of cbp2. For amino acid sequence analysis, cbp2 from about 1 kg algae (wet mass) was purified to step 3 (Table 1). After gel-electrophoretic separation, Western blotting and amido black staining, the 42-kDa band (containing approximately 4 pg of cbp2) was cut out from the polyvinyl- idene difluoride membrane and digested with 1 pg trypsin. Pep-

484

100-

Feldwisch et al. ( E m J. Biochem. 228)

............... .. .. ....... .... _.......-I ..... .... g-. ....... '-

'..-..-. .- w - -.-.I

z a 8000 n E 8 6000

.= 4000

- E 2 Q 2000

0

fraction

F E

3

- g 600

300

-100

f q K

&

1: 0 5 10 15 20 25

fraction

l 8 ~

c 8ooo I ~ -- -

I - -

10 15 20 25 30 fraction

Fig. 4. Purification of (A, B) cbpl and (A, C) cbp2 by (A) Fractogel EMD TMAE650 S and (B, C) Mono P HR 5/20 chromatography. (A) cbpl and cbp2 were separated by DEAE-Sepharose chromatography and further purified by S-200 gel filtration separately. They were loaded onto a Fractogel EMD TMAE650 S column and chromatographed under identical conditions in different runs. After washing, the bound proteins were eluted with a linear gradient of 0-500 mM NaCI; 3-m l fractions were collected and assayed for (0) cbpl or (W) cbp2 activity; (-) NaCl. (B) cbpl purified by DEAE-Sepharose chromatography and S-200 gel filtration was applied to a Mono P HR 5/20 column. After washing, the column was eluted with 50 ml Polybuffer PB 74TM; 2-ml fractions were collected and the pH of each fraction determined. Prior to assay for cbpl (0) and pk (A), the pH (-) was adjusted to pH 7.5. (C) cbp2 purified by DEAE-Sepharose chromatography and S-200 gel filtration was ap- plied to a Mono P HR 5/20 column. After washing, the column was eluted with 50ml Polybuffer PB 74TM; 2-nll fractions were collected and the pH of each fraction determined. Prior to assay for cbp2 (W), the pH (-) was adjusted to pH 7.5.

tides were separated on a Vydac C, column, and peptides 9, 15, 28, 49, 50, 53 and 59 (Fig. 5) were sequenced (Table 2). Both peptide pairs 49/50 and 53/59 differ in only one amino acid: 491 50 with GldAla in position 8 ; 53/59 with TyrPhe in position 19. cbpl was not sequenced.

Biochemical characterization of the cbp. For functional analy- sis of the cbps of V carteri, we investigated whether the CAMP-

6 0 i s 8 0

I I 1 53

49 I1 I

30 4 0 50 60 7 0 8 0 90 T i m e ( m t n )

1 53

7 5

30UM 20

Fig. 5. Reverse-phase HPLC separation of tryptic peptides of cbp2 on a Vydac C, column. Elution was performed with a gradient of 0- 100% B" (0.056 % trifluoroacetic acid; 80 % acetonitrile). Fractions were collected by hand. The individual peptides are marked by numbers; those labeled with arrows were sequenced. The elution pattern of a tryp- tic control digest is shown below.

3

Y -7 -6 -5 -4 -3

log ( [ Competitor I/ M )

Fig.6. Competition for CAMP binding of cbpl or Ca/CaM pde by CAMP, cGMP and methylxanthines. cbpl was purified to step 4. cAMP binding was assayed as described in Experimental Procedures. Each sample contained 13 nM ['HICAMP and varying amounts of unla- beled cAMP or competitor (10 nM to 1 mM). The concentration of the added compound was plotted against the binding activity. (A) cbpl from I.: carteri (specific binding activity 500 pmol/mg) competitor: (A) CAMP; (0) iBuMeXan; (W) caffeine. (B) Ca/CaM pde from beef heart (specific binding activity 80 pmol/mg); competitor: (A) CAMP; (0) iBuMeXan; (W) cGMP.

binding activity correlates with protein kinase (pk) or phospho- diesterase (pde) activity in different steps of purification.

As shown in Fig. 1, the main peak of pk activity was eluted at 200 mM NaCl after DEAE-Sepharose chromatography. It

Feldwisch et al. (Eut: J. Biochem. 228) 485

Table 2. Sequence of peptides obtained from cbp2 by tryptic digestion. Peptides 49/50 and 53/59 differ only in positions 8 and 15 (labeled by underlining) respectively.

~ ~ ~~~~~~~~~

Peptide Amino acid sequence

9 Phe-Ala-Tyr-Asp- Ala-Thr-Ly s-Pro-Val-Asn-Glu 15 Ser-Ile-Ser-Val-Thr-Gly -Leu-Lys 28 Phe-Leu-Val-Asp-Gln-Ser-Xaa-Xaa-Xaa-Gly-Tyr-Ala-Ala-Xaa-Glu 49 Ser-Ala-Leu-Glu-Asn-Ser-Val-~-Gly-Asn-Ile-Ala-Gly-Gly-Glu-Ala-~u-Gly-Gln-Phe-Ala-Gln-Val-Gly-Gly-Phe 50 Ser-Ala-Leu-Glu-Asn-Ser-Val-~-Gly-Asn-Ile-Ala-Gly-Gly-Glu-Ala-Leu-Gly-Gln-Phe-Ala-Gln-Val-Gly-Gly-Phe 53 Ala-Asp-Ile-Pro-Ala-Gly-Leu-Val-Thr-Gly-Gly-Gln-Ile-Ala-Ala-Ala-Leu-Pro-~r-Gly-Asn-Thr-Leu-Val-Val-Lys 59 Ala-Asp-Ile-Pro-Ala-Gly-Leu-Val-Thr-Gly-Gly-Gln-Ile-Ala-Ala-Ala-Leu-Pro-~-Gly-Asn-Thr-Leu-Val-V~-Lys

Table 3. Phosphodiesterases of C: carteri. A DEAE-Sepharose column was run as described in Experimental Procedures. The bound protein was eluted with a 400-ml linear gradient of 0-500 mM NaC1; 5-ml fractions were collected and assayed for pde activity by a reverse-phase HPLC assay.

Pde NaCl Activity (product) with substrate Comments

2‘3’cAMP at 3’5’cAMP at

1 PM 30 pM 1 PM 30 pM

pdel 120 2.4 (S’AMP) 2.7 (3’AMP) 2.2 (3’AMP) 1.5 (3’AMP) nonspecific pde pde2 125 - - 4.5 (S’AMP) 9.3 (5’AMP) IowlK,, pde

2.2 (adenosine) 2.4 (adenosine) (K, 1.4 pM;

pde3 175 - - 0.8 (5’AMP) 8.9 (5’AMP) highlK,, pde

V,,, 3.5 pmoYmin)

(K, 31.4 pM; V,,, 6.3 pmollmin)

was, however, not completely separated from cbpl. A complete separation of pk and cbpl was achieved by chromatofocusing (Fig. 4 B). After purification by ion-exchange chromatography and gel filtration, S-200 fractions containing either cbp or pk activity were dialysed and applied to a MonoP HR 5/20 column. cbpl eluted in two peaks at PI 5.4 and 5.2, while pk eluted at PI 5.8 and 5.3 (Fig. 4B).

If one of the cbps of V carteri were the regulatory subunit of a CAMP-dependent protein kinase, it should be possible to measure at least some of the following properties: (a) activation of the pk activity in the presence of 5 pM cAMP since the regu- latory subunit inhibiting pk activity dissociates from the catalytic subunits upon binding of CAMP; (b) a shift in molecular mass of both the cbp and pk activity if 2 mM cAMP is added to the elution buffer of gel filtration (prior to assay CAMP was re- moved by extensive dialysis); (c) inhibition of the pk activity by addition of specific inhibitors such as the purified regulatory subunit from another organism or the Walsh inhibitor (ref. 20 in [261).

Activation of pk activity by addition of cAMP to the assay could not be observed after ion-exchange, gel filtration, affinity or chromatofocusing chromatography, even in the presence of pde inhibitors. S-200 gel filtration of fractions containing cbpl and pk activity in the presence of cAMP did not change the elution pattern, indicating that there is no shift in molecular mass and hence no evidence for dissociation of cbp and pk activities in the presence of CAMP. No change in pk activity was observed after addition of purified R subunit from beef heart (Sigma), Walsh inhibitor from beef heart (Sigma) or after removal of a prospective endogeneous inhibitor by (NH),SO, fractionation as described [26].

A comparison of the elution patterns of cbp and pde activity showed that pde eluted at 200 mM NaCI, intermediate between cbpl and cbp2 (Fig. 1). We determined a molecular mass of 126 kDa for pde by S-200 gel filtration (data not shown) differ- ing from the native molecular mass of 90 kDa for both cbpl and cbp2. Furthermore, pde did not bind to the IP-CAMP-agarose affinity resin used for purification of cbp (100 % pde activity in the flow-through).

The Lineweaver-Burk diagram of pde activity partially puri- fied by DEAE-Sepharose and S-200 chromatography was bipha- sic, indicating the existence of two different pdes with different K, and V,,, values : low-K, pde (K, 1.4 pM, V,,, 3.5 pmol/min) and high-K, pde ( K , 31.3 pM, V,,, 6.3 pmol/min). In order to see whether this was true, we used the following approach. Crude extract prepared from V carteri was applied to a DEAE- Sepharose column as described under Materials and Methods. After washing, the pde activity was eluted with a 400-ml linear gradient of 0-500 mM NaCl and 5-ml fractions were collected. Thus the gradient was flattened in comparison to the conditions used for separation of cbpl and cbp2. As we intended to analyse the specificity towards 2’3‘cAMP and 3’5’cAMP, as well as the products of hydrolysis, we used a reverse-phase HPLC assay as described under Experimental Procedures. Each 5-ml fraction was assayed for pde activity with substrate concentrations (either 2’3’cAMP or 3’5’cAMP) of 1 WM and 30 FM. By this approach it was possible to resolve the pde activity measured by RIA (Fig. 1) into three different activities. The results of the peak fractions are given in Table 3. At 120 mM, NaCl eluted a non- specific pde (pdel) that hydrolyzed both 2’3’cAMP and 3’5‘cAMP to 3’AMP. At 125 mM and 175 mM NaCl two pdes specific for 3‘5’cAMP (pde2 and pde3, respectively), were

486 Feldwisch et al. (Eur: J. Biochem. 228)

8000 SAMP 3’5’cAMP

i

Time (min)

Fig. 7. Reverse-phase HPLC analysis of the adenosine nucleotides after incubation of [3H]cAMP with cbp2 from R curten’. cAMP bind- ing was assayed as described in Experimental Procedures. The pellet containing protein-bound nucleotides and the supernatant containing free nucleotides were separated and etheno-derivatives prepared. The nucleo- tides were separated by a non-linear 36-min gradient of 0-100% B’ (10 mM sodium-phosphate pH 7.3, 20% acetonitrile) on a Nucleosil R 7 C,, column. The radioactivity was measured by scintillation counting. The column was calibrated with unlabeled etheno-derivatized adenine nucleotides. Similar results were obtained for cbpl. Differences are within the scope of the error of measurement. (-) Pellet; (...) superna- tant.

eluted. As the reaction products of pde2 were both 5‘AMP and adenosine, it seems to be associated with a S’nucleotidase, whereas pde3 produces only S’AMP. During kinetic analysis, the low-K,,, enzyme should double its activity when the substrate concentration is increased from 1 to 30 pM CAMP, while the activity of the high-K,,, enzyme should increase tenfold. By this criterion pde2 was identified as the low-K, enzyme and pde3 as the high-K,,, enzyme. The nonspecific enzyme probably had a much higher K,,, as its activity did not change under these sub- strate conditions. After $200 gel filtration, pdel activity was lost while pde2 and pde3 could not be separated by this tech- nique and appeared to have the same molecular mass (126 kDa; data not shown). It has not been possible to purify any pde of K carteri to homogeneity so far.

Phosphodiesterases hydrolysing 3’5‘cAMP to 5’AMP are specifically inhibited by methylxanthines, e.g. 3-isobutyl-l- methylxanthine (iBuMeXan), caffeine, theophylline, theobro- mine. It is known, however, that the regulatory subunit of the CAMP-dependent protein kinase binds iBuMeXan in a dose-de- pendent manner [27], whereas theophylline is no inhibitor of cAMP binding. The effect of CAMP, caffeine and iBuMeXan on the binding activity of cbpl from K carteri is given in Fig. 6A. cAMP reaches the threshold concentration of binding at 100 nM. The threshold concentration is the concentration at which bind- ing is reduced at least by 50 %. iBuMeXan is an effective inhibi- tor of binding. Its threshold concentration (1 pM) is one tenth that of CAMP. In contrast, caffeine and the other methyl- xanthines, theobromine and theophylline (data not shown), are not bound at all (100 % binding at 100 pM concentration). Sim- ilar results were obtained for cbp2 (data not shown). Differences are within the scope of the error in measurement.

For comparison and control, Ca2+/calmodulin-dependent (CdCaM) pde from beef heart (Sigma) was assayed with the cbp assay. Competition experiments with CAMP, cGMP and iBu- MeXan show the following results (Fig. 6B): both iBuMeXan and cGMP are more effective in inhibiting [3H]cAMP binding than CAMP. The threshold concentrations for cAMP and iBu- MeXan were estimated as 100 nM and 1 pM for cbp and 1 mM and 10 pM for CdCaM pde. cGMP has a threshold concentra- tion of 100 pM. Caffeine, theophylline and theobromine are

known inhibitors of CdCaM pde though less effective than iBu- MeXan [28]. These experiments demonstrated that the CafCaM pde can be measured with the cbp assay. The enzyme shows the same binding affinity towards CAMP, cGMP and iBuMeXan as when measured with a pde assay. Both proteins clearly differ in respect to binding of the three natural methylxanthines.

As iBuMeXan is a potent inhibitor of pde we analysed, by reverse-phase-chromatography, whether cbpl and cbp2 are true binding proteins or hydrolyse CAMP. Fig. 7 demonstrates that cAMP is hydrolysed by cbp2 during the assay as only 5’AMP was found in the protein-bound fraction. The supernatant con- tains 3’5‘cAMP and 5‘AMP in the ratio 1 :5 indicating that hy- drolysis is not complete. It was impossible to measure cAMP hydrolysis directly since the detection limit for CAMP is 50 pmol by RIA [22] and 5 pmol by reverse-phase chromatography of fluorescent etheno-CAMP [23].

Specificity of CAMP binding. The CAMP-binding site of the cbps of K carteri was analysed by competition experiments with a set of unlabeled cAMP analogues chosen according to the sys- tematic approach previously developed [29] : each atom or atom group of cAMP presumably interacting with the binding site of the target protein is chemically modified by substituents. Modi- fications in essential positions will prevent binding of the rnodi- fied ligand ; therefore these derivatives are not inhibitors of bind- ing. Modifications in positions not essential for binding, how- ever, will allow binding of the modified ligand; these derivatives are effective competitors of binding.

We used cbpl and cbp2 purified to step 4 (specific activity 500 pmol/mg) for these experiments. The preparations are free of CAMP. The cAMP analogs listed in Fig. 8A were added to the incubation mixture in final concentrations of 1 mM, 100 pM, lOpM, 1 pM, 100nM and 10nM each. Samples containing only [3H]cAMP were used as control (100 % binding). For each cAMP derivative, a dose/response curve was measured. The data presented here result from 2-6 independent measurements with duplicate values. The error in measurement amounted to 5- 20%. The data from the different doselresponse curves were transferred to Fig. 8 B and Fig. 8 C. The threshold concentration of binding, that is the concentration at which binding is reduced by at least 50 % in the doselresponse curve, was determined for each derivative. The binding activity of CAMP and derivatives with a similar biological activity is defined as 6 binding units (bu) on an arbitrary logarithmic scale. Derivatives with a 10- fold higher threshold concentration have a binding activity of 5 bu, analogs with a 100-fold higher threshold concentration have a binding activity of 4 bu, and so on. The threshold concentra- tion of compounds that are not bound is defined a 1 bu. A drop in binding by only 1 or 2 bu is considered to be an unspecific effect of derivatisation, which changes general stereochemical or electronic features. Modifications reducing the biological ac- tivity of the derivative at least 103-fold are assumed to be re- quired for the interaction between cAMP ligand and the CAMP- binding protein [29].

When these criteria are applied to evaluate the binding speci- ficity of cbpl (Fig. 8B) and cbp2 (Fig. 8C) it can be seen that both proteins show a similar general binding specificity, al- though only l-N-oxide-CAMP and 5‘-NH-CAMP have exactly the same biological activity towards cbpl and cbp2. The com- pounds 6-Cl-cAMP, N6-benzoyl-CAMP, N6-butyryl-CAMP, 7- deaza-CAMP, 8-Br-cAMP, dcAMP and (SJ-cAMP[S] differ in binding activity by 1 or 2 bu. As shown for cbpl in Fig. 8B, 7- deaza-CAMP has the same binding activity as cAMP (6 bu), while it was bound 1 bu less than cAMP by cbp2 (Fig. 8 C). The derivatives dcAMP (cbpl, 5 bu; cbp2, 4 bu) and 5’-NH-CAMP (cbpl, 5 bu; cbp2, 5 bu) were bound 1 or 2 bu less than CAMP.

Feldwisch et al. (Eur: J. Biochern. 228) 487

dcAMP (8)

5‘-NH (10) ’ cGMP (12) clMP (13)

3’-NH (9)

(SPWMP[SI (11)

This means that the positions 7, 2’- and 5’- are not essential for CAMP-binding of cbpl and cbp2. The binding activity of 3’- NH-CAMP (cbpl, 5 bu; cbp2, not analysed) is reduced by 1 bu, meaning that position 3’ is not essential for CAMP-binding of cbpl. 8-Br-CAM!? is bound 3 bu less than cAMP by cbpl and 4

.-

A 3 4 5

I

B cAMP (1)

-I

7

bu less by cbp2. Modification of position 8 shifts the syn-anti equilibrium of cAMP (1 : 1 ratio) so that the syn conformation is preferred. The reduced biological activity of 8-Br-CAMP indi- cates that cAMP is presumably bound in the anti conformation by cbpl and cbp2.

The binding activity of derivatives 1-N-oxide-CAMP (cbpl, 3 bu; cbp2, 3 bu) and (SJ-cAMP[S], (cbpl, 1 bu; cbp2, 2 bu) is reduced 103-105-fold, corresponding to a drop of 3-5 bu. From this it follows that position 1 and at least one of the exocy- clic 0 atoms (these are the free 0 atoms of the phosphoryl group) of the cAMP molecule are essential for binding.

The reduced binding activity of 6-C1-CAMP (cbpl, 1 bu; cbp2, 2 bu) and N6-benzoyl-CAMP (cbpl, 3 bu; cbp2, 2 bu) identifies position 6 as an essential binding site for both cbpl and cbp2. The low binding activity of the other cyclic nucleo- tides cGMP (cbpl, 2 bu) and cIMP (cbpl, 2 bu) towards cbpl supports this. The identification of position 6 as an essential binding site is surprising since N6-CAMP-agarose (C, spacer) was successfully used in our purification procedure as an affinity matrix for both cbpl and cbp2. When we analysed N6-butyryl- CAMP, which is similar in structure to the affinity resin, this derivative showed a higher binding activity (cbpl, 5 bu; cbp2, 4 bu).

DISCUSSION

The present study describes the purification of two dimeric CAMP-binding proteins, cbpl and cbp2, from the cytoplasm of Volvox carteri. They are very similar in size, though definitely different in basicity. cbp2 was purified to apparent electropho- retic homogeneity and cbpl almost. Both native binding proteins have a molecular mass of 90 kDa as measured by gel filtration. The estimated subunit mass was 42 kDa for cbp2 and 42 kDa or 44 kDa for cbpl.

Tryptic peptides of dimeric cbp2 were separated by HPLC and peptides 9, 15, 28, 49, 50, 53 and 59 (Table 2) were se- quenced. The peptide pairs 49/50 and 53/59 differ only in one amino acid: in position 8 (GldAla) and 19 (TyrPhe), respec- tively, pointing to the existence of isoforms of cbp2. This is supported by the separation of cbp2 into two peaks of activity on Fractogel EMD TMAE6.50 S (Fig. 4A) and by the (incomplete) separation on Mono P HR 5/20 (Fig. 4C). At present, an internal repetition of part of the amino acid sequence of cbp2 cannot be excluded.

A comparison of peptide sequences was done with the PIR database release using the programs Wordsearch and Fasta of the Wisgen program parcel [30]. The identification of cbp2 by

-1 7

Fig. 8. Structure of cAMP analogues (A) and schematic representa- tion of their biological activity (B, C). (A) Structure of cAMP ana- logues. (1) Adenosine 3’,5’-monophosphate (CAMP) ; (2) adenosine-N’- oxide 3’,5’-monophosphate (1-N-oxide-CAMP) ; (3) 6-chloropurineribo- side 3’,5’-monophosphate (6-Cl-CAMP) ; (4) N6-benzoyladenosine 3’,5’- monophosphate (N6-benzoyl-CAMP); (5) N6-butyryladenosine 3’,5’- monophosphate (N6-butyry-CAMP); (6) 7-deazaadenosine 3’3’- monophosphate (7-deaza-CAMP); (7) 8-bromoadenosine 3’,5’- monophosphate (8-Br-CAMP); (8) 2’-deoxyadenosine 3’,5’-monophos- phate (dcAMP); (9) 3’-deoxy-3’-aminoadenosine 3’,5’-monophosphate (3’-NH-cAMP) ; (10) 5’-deoxy-5’-aminoadenosine 3’,5’-monophosphate (5’-NH-cAMP) ; (1 1) adenosine 3’,5’-phosphosphorothiate dimethyl- amidate, S, isomer, (S,)-cAMP[S]; (12) cGMP, (13) cIMP. (B, C ) Sche- matic representation of the biological activity of cAMP analogues towards (B) cpbl and (C) cbp2. The binding activity is rated in binding units (bu, see text).

488 Feldwisch et al. (Eul: J. Biochem. 228)

sequence comparison on the protein level was not possible as only weak similarities to known proteins exist.

cbpl and cbp2 differ from one another with respect to sur- face charge when chromatographed on DEAE-Sepharose, Frac- togel EMD TMAE 650 S and Mono P HR 5/20. Their native (90 kDaj and denatured molecular mass (cbpl, 42 or 44 kDa; cbp2, 42 kDa) appears to be the same or at least very similar (Figs 2 and 3). The binding affinity towards cyclic nucleotides and methylxanthines as tested in this study is very similar. It is possible that both cbpl and cbp2 consist of two subunits slightly differing in PI. At present, it cannot be determined whether cbpl or cbp2 are individual proteins or isoforms of one another. The amino acid sequence data obtained for cbp2 make it possible to analyse these further on the DNA level.

Although the purification scheme of cbp is similar to that of CAMP-dependent pk (ref. 3 in [S] j, association of binding activ- ity with pk activity was not found. The known pde activities of I.: carteri are not correlated to the binding activity. The CAMP- binding proteins and the phosphodiesterases differ with respect to surface charge, molecular mass and binding affinity to N6- CAMP - agarose. Analysis by reverse-phase chromatography demonstrates, however, that both cbpl and cbp2 have CAMP- hydrolysing activity, thereby indicating a novel type of pde in GI carteri.

The binding specificity was analysed according to [29] for both cbpl and cbp2: binding was shown to be strictly CAMP- specific, positions 1 and 6 and at least one of the exocyclic 0 atoms are essential binding sites, the cAMP molecule is bound in unti conformation. In contrast, the CAMP-dependent pk binds cAMP in s.yn conformation by hydrogen bonds at position 2’, 3’ and 5’ [29]. The base is bound in a hydrophobic pocket by di- pole-induced dipole interactions [29].

The CAMP-binding specificity of the CAMP-binding proteins from V curteri show similarities to the CdCaM pde from beef heart [31] and the yeast low-K, cAMP phosphodiesterase [32] : none of these CAMP-binding proteins bind the phosphate 3’,5’- diester ring directly. At least one of the exocyclic 0 atoms of the cAMP ligand is essential for these CAMP-binding proteins whereas the 2’-0 position is without influence [31]. Further- more, position 6 of the CAMP ligand is essential for binding to yeast low-K, cAMP phosphodiesterase and the CAMP-binding proteins of I? carteri. Position 7 is, however, only essential for the yeast low-K,-CAMP phosphodiesterase. The latter, in con- trast to cbpl and cbp2, binds cAMP in syn conformation [32]. The CdCaM pde and the CAMP-binding proteins from GI carteri differ with respect to the strict cAMP specificity and to the bind- ing of the methylxanthines. cbpl and cbp2 bind only iBuMeXan while CdCaM pde binds iBuMeXan and the natural methyl- xanthines. iBuMeXan and cGMP have a greater polarizing power than cAMP towards succeptible amino-acid side chains of a binding protein. Phosphodiesterases binding cyclic nucleo- tides by dipole interactions, such as the CdCaM pde from beef heart, have a higher binding affinity to cGMP and iBuMeXan than to cAMP [33]. The strict cAMP specificity of the cbp of V carteri suggests that the positions 1 and 6 are bound by hy- drogen bonds instead of dipole interactions. The binding of iBu- MeXan seems to be as exceptional as the binding of N6-butyryl- CAMP. Perhaps both molecules are recognized by a hydrophobic pocket.

A comparison of the binding patterns of the CAMP-binding proteins of GI carteri and the mammalian cAMP phosphodiester- ase [34] shows that both proteins are strictly CAMP-specific. For both, position 6 seems to be an essential binding site while posi- tion 2‘ is not. Differences in the binding pattern appear to exist in regard to position 7 which is not essential for the CAMP- binding proteins of GI carteri.

The purification and characterization of two CAMP-binding proteins in GI carteri implies the existence of a CAMP-dependent signal chain in this organism. In which way the CAMP cascade is connected with sexual induction of V: carteri will be the sub- ject of further study.

The authors thank Mr. M. Koster for excellent technical assistance. This study was supported by Deutsche Forschungsgemeinscha~ through SFB 74 (molecular biology of the cell) and SFB 243 (molecular analysis of development of cellular systems) and by Fonds der Biologischen Chemie.

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