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A calcium binding protein from Drosophila melanogaster which activates cAMP phosphodiesterase: Comparison of this protein with porcine brain calmodulin
ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 247, No. 1, May 15, pp. 147-154,1986
A Calcium Binding Protein from Drosophila melanogaster Which Activates CAMP Phosphodiesterase: Comparison of This Protein with Porcine Brain Calmodulin’ F. WANDOSELL, Centro
de Biologla
Molecular Received
(CSIC-UAM), July
23,1985,
L. SERRANO,
AND
Autbnoma,
Universidad
and in revised
form
J. AVILA Canto
October
Blanco,
28049
Madrid,
Spain
30, 1985
A calcium binding protein from Drosophila melanogaster has been isolated and characterized. This protein shows several analogies with pig brain calmodulin in its molecular weight, isoelectric point, peptide maps, calcium binding properties, and ability to activate cyclic AMP phosphodiesterase. However, some differences were observed; the most remarkable one is the presence of tryptophan, an amino acid which is absent from all the calmodulins analyzed previously. o 19% Academic PWSS, I~C.
In invertebrates calcium modulator protein, calmodulin, is implicated in different physiological processes in which calcium ions play a regulatory role. Calmodulin was first detected as a protein that stimulated the cyclic nucleotide phosphodiesterase (1, 2). More recently it has been found to activate several other enzymes, such as Ca2+, M$+-ATPase (3,4) or protein kinases (5,6) [for a review see (7)]. In addition, calcium and calmodulin are related to cytoskeleton proteins, because they affect microtubule assembly (8, 9). Calmodulin is present in all species analyzed so far and it has been isolated from different sources [vertebrates and invertebrates (lo), including the lowest eucaryotes (ll)]. Comparison of the structure and function of the calmodulins from different organisms indicates that this molecule is highly conserved, composed of 148 amino acids, containing four structural calcium binding sites that are homologous to each other, especially when the first calcium domain is compared with the third, and the second with the fourth (12). On this basis,
it has been suggested that the calmodulin gene arose by gene duplication of one- or two-domain precursors (13). At present, all calmodulins analyzed contain no tryptophan or cysteine and have only one or two tyrosine residues in calcium loops 3 and 4 (11, 14). The uv absorption spectra reflect a high phenylalanine-to-tyrosine ratio in both vertebrates (8:2) and invertebrates (8:l). An unusual amino acid, trimethyllysine, is also usually present at residue 115 of bovine brain calmodulin. In Drosophila melanogaster, a calmodulin-like protein is involved in the regulation of phosphodiesterase, form II. Mutants affecting this enzyme appears to be defective in associative learning upon a training schedule (15, 16). Therefore, the purification and characterization, in Drosophila, of a protein related to calmodulin could be fundamental to an understanding of the molecular basis of calcium-calmodulin actions in those processes. Partial purification of calmodulin-like proteins has been recently reported (17); however, an extensive study and characterization of such proteins has not yet been performed. In this paper we describe the purification and characterization of a calcium binding
1 This research was supported by grants from Comisi6n Asesora para la Investigacibn Cientifica y T&cnica and Fondo de Investigaciones Sanitarias. 147
0003-9861/86 Copyright All rights
$3.00
0 1986 by Academic Press, Inc. of reproduction in any form reserved.
148
WANDOSELL,
SERRANO,
protein related to calmodulin, from Bwsophila melanogaster, that also shows some important differences from pig brain calmodulin. MATERIALS
AND
METHODS
Putijication of pig brain calmodulin Pig brain calmodulin has been purified essentially as described by Dedman et al. (12) and was tested functionally by its ability to stimulate phosphodiesterase activity. Cyclic AMP phosphodksterase assays. Phosphodiesterase activity was assayed as described by Lin et al (18). Bovine phosphodiesterase (0.15 unit) (from Sigma) was mixed with variable amounts of fractions containing calmodulin. The mixture was incubated at 30°C for 10 min in a buffer containing 40 mM TrisHCl, pH 8, 0.1 mM CaClz, 10 mM MnClz, and 1 mM CAMP. The reaction was stopped by boiling the samples for 3 min and cooling the mixture on ice. Production of AMP was measured by incubation with 5’nucleotidase (0.4 units; Sigma), after which inorganic phosphate was measured as indicated (19). Amino acid analysis. Amino acid analysis was performed on samples of D. m&nogostercaleium binding protein using a Beckman amino acid analyzer. Samples were hydrolyzed for 24 h at 110°C in 6 N HCl. Four different determinations were performed at different protein concentrations. Tryptophan was estimated by the spectophotometric method described by Edelhoch (20). Fluorescence measurements. Fluorescence spectra were obtained using an absolute spectrofluorometer (Schoeffel RSS-1000). Excitation was performed at 280 or 295 nm, at room temperature. Other procedures. Pig brain calmodulin was iodinated by the Bolton-Hunter procedure (21) or alternatively by the chloramine-T reaction (22), when it was used as a molecular weight marker. Protein concentration was determined by the method of Lowry et al. (23). SDS’-polyacrylamide gel electrophoresis was performed according to Laemmli (24) using 5 or 10 to 20% linear gradients. IEF electrophoresis was performed as described by Diez et al. (25) using 2% (w/v) ampholines (pH 3.55). After focusing, the pH was measured on the gel surface with an LKB Multiphor electrode. The gels were fixed and stained with the dye Stains All as has been described (26). Calcium binding activity was determined by equilibrium dialysis using labeled calcium (*CaCl) in 10 mM Tris, pH ‘7, as buffer. Dialyses were
AND
AVILA
performed at 4°C for 24 h on an incubation shaker. Concentrations of unlabeled calcium between lo-’ and 10e6 M were used in each experiment, with 0.02 mCi/ml of labeled calcium chloride (“CaCl) (27). Peptide mapping by limited proteolysis of pig calmodulin and D. melanogaster calcium binding proteins, both ‘a’I-labeled on the tyrosine, was performed in the presence of an equal amount of unlabeled pig calmodulin and at an enzyme/protein ration of 1.25% (w/w). The mixture was incubated for 15 min at 30°C and the reaction was stopped by addition of 1% SDS. Proteolytic labeled fragments were analyzed by gel electrophoresis in 20% polyacrylamide gels and autoradiography. Calcium-dependent hydrophobic chromatography of calmodulin using octyl-Sepharose (Pharmacia) was carried out as indicated by Tanaka et al (28). RESULTS
Isolation The calcium binding protein was isolated from D. melanogaster adults. Flies (100 g) were homogenized in an isotonic buffer containing 0.32 M sucrose, 10 InM Na-phosphate buffer, pH ‘7,1 mM EGTA, and 1 InM PMSF, and centrifuged at 100,OOOgfor 35 min at 4°C. The supernatant was collected and dialyzed against 10 mM Tris, pH 8, 1 mM EDTA, 2 mM P-mercaptoethanol, and 1 M NaCl. After dialysis, the extract at 4 mg/ml was boiled for 10 min, cooled on ice, and centrifuged at 10,OOOgfor 30 min at 4°C. The pellet was discarded and the supernatant was dialyzed against 10 mM Tris, pH 8,1 mM EDTA (TE). DEAE-Cellulose Chromatography The dialyzed solution was applied to a DEAE-cellulose column (20 X 5 cm), previously equilibrated in buffer TE, and eluted stepwise with NaCl(O.1 to 0.5 M) in TE. The fractions eluted with 0.3 M NaCl presented the highest specific activity as activator of the CAMP phosphodiesterase in the presence of calcium (data not shown). Gel Filtration
2 Abbreviations used: SDS, sodium dodecyl sulfate; IEF, isoelectric focusing; EGTA, ethylene glycol bis(6-aminoethyl ether) N,N’-tetraacetic acid; PMSF, phenylmethylsulfonyl fluoride; TE buffer, 10 mM Tris, pH 8,1 mM EDTA.
The DEAE-cellulose fractions eluted at 0.3 M NaCl were pooled, concentrated, and purified by filtration on a Sephadex G-75 column (40 X 1 cm), equilibrated in buffer
Ca BINDING
PROTEIN
FROM
TE, using pig brain calmodulin labeled with *%I as an internal marker. Fractions eluted at the same position as pig brain ‘251-calmodulin appear to contain a single peptide with the same molecular weight in gel electrophoresis (Mr 18,000) as porcine brain calmodulin. These fractions were pooled, concentrated, and rechromatographed under the same conditions as above. Protein characterization of this fraction is indicated in Fig. 1. Total purified protein accounted for 1.5 mg.
Drosophila
$30 2
149
wz&nog&er
-
P
0
1
5
Protein
Functional
20
30
Characteristics
Stimulation
of phosphodiesterase
activity. of the purified D.
The biological
activity peptide was examined by its capacity to stimulate phosphodiesterase activity compared to that of pig brain calmodulin. The levels of stimulation produced by both proteins at the same conmelanogaster
‘IO
10 1 pgr I
20
30
40
50
60
70
No Fractions
FIG. 1. Gel filtration on Sephadex G-75. The fractions eluted at 0.3 M NaCl were pooled and chromatographed on Sephadex G-75-20 (40 X 1 cm) in parallel with ‘%I-calmodulin (0) used as an internal marker. The fractions of D. melunogoster protein eluted at the same position (0) as labeled calmodulin were analyzed by electrophoresis on SDS-polyacrylamide gels. The inset shows the electrophoresis of the purified protein. The arrows represent the positions for tubulin (a), lzsI-pig brain calmodulin (b), and cytochrome e (c), used as molecular weight markers.
FIG. 2. Stimulation of cyclic AMP phosphodiesterase. Bovine brain phosphodiesterase (0.15 gg) was mixed with increasing amounts of pig brain calmodulin (0) and purified protein of D. melurwgcz&r (O), as indicated under Materials and Methods, and the stimulation of the phosphodiesterase activity was assayed. The basal activity of phosphodiesterase in the assay results in the hydrolysis of 0.12 pmol of cyclic AMP at 30°C after 5 min of incubation. In the presence of 10 c(g of added protein 2 rmol of cyclic AMP was hydrolyzed under the above conditions.
centration were very similar, indicating an analogous capacity for both proteins as activators CAMP phosphodiesterase (Fig. 2). Calcium binding capacity. The amount of calcium bound to the D. melanogaster peptide was determined by equilibrium dialysis, using 45CaC1 in the presence of different concentrations of unlabeled calcium (10e4 to 10F6 M), and compared to the calcium binding capacity of pig brain calmodulin. The results obtained were represented as a “Scatchard plot.” Figure 3 shows that the purified peptide of D. melanogaster has three high-affinity calcium binding sites (3.2 X lop5 M) and one lower-affinity binding site (0.9 X 10e4 M). Similar results were obtained for pig brain calmodulin (Fig. 3). Limited proteolysis. Calcium binding protein of D. melanogaster and pig brain calmodulin were labeled with ‘%I on tyrosine residues and subjected to limited chymotrypsin digestion as indicated under Materials and Methods. When the peptides obtained were analyzed by SDS-polyacrylamide gel electrophoresis, a labeled fragment of ik?, 8500 was found in both proteins (Fig. 4) after chymotrypsin digestion.
150
WANDOSELL.
SERRANO.
HOLE Cat4 FIG. 3. Calcium-binding capacity. D. mdmogaster calcium-binding protein (0) and pig calmodulin (0) were dialyzed against different concentrations of %‘a (lo4 to 10m6 M). After equilibrium dialysis the amount (moles) of bound calcium was calculated and the results were represented as a Scatchard plot.
Chemical
and Physical
Characteristics
Mokcular weight. The size of the calcium binding protein of Drosophila has been determined by gel filtration. Chromatography of this peptide on Sephadex showed an elution pattern identical to that of ‘%I-calmodulin, as previously shown and both the proteins exhibited the same electrophoretic mobility when analyzed by SDS-polyacrylamide electrophoresis, with a molecular weight of M, 18,000 (Fig. 1). Isoelectric point. The purified peptide was further characterized on isoelectrofocusing gels, showing a single band with an isoelectric point of 4.30 f 0.05. This band was blue in color, when IEF gels were stained with Stains All (Fig. 5). Similar results were obtained when pig brain calmodulin was focused in parallel on IEF gels (data not shown). When both proteins were stained in solution, with Stains All (26), the same absorption spectrum of the dye was observed. Amino acid composition, The amino acid composition of purified D. melunogaster
AND
AVILA
FIG. 4. Limited chymotrypsin cleavage of calmodulin and calcium-binding protein of D. wdmwgmter. Autoradiographic pattern of ‘261-calmodulin (B) and ‘?-calcium binding protein of D. melanogaster (C) obtained by proteolysis with chymotrypsin. (A) Mixture of undigested calmodulin and D. ndmwgaster calcium-binding protein.
calcium binding protein was determined and compared to that of brain calmodulin, plant calmodulin, and calmodulin from a lower eucaryote (Table I). Some differences were found between these proteins, such as a lower relative content of methionine, phenylalanine, and proline but a higher proportion of histidine in the D. melanogaster protein. Minor differences are present in the other amino acids.
migration
(cm)
FIG. 5. Isoelectrofocusing gel electrophoresis. D. melanogoster calcium-binding protein (10 pg) was focused in a pH gradient between 3.5 and 7; the gel was fixed and stained with Stains All. A parallel gel was focused to determine the pH gradient. The arrow indicates the position for D. mdmwgastw calciumbinding protein in the pH gradient.
Ca BINDING
PROTEIN
FROM TABLE
Drosophila melanogaster Asx Thr Ser Glx GUY Ala Val Met Ileu Leu Tyr Phe His LYS Arg Pro ‘b Tml CYS “Reference (28). ‘Reference (29). ’ Spectrophotometric
22.2 12 12 23.2 14.1 14.1 7 2 5.2 8.6 2 3.6 4 10.6 5.7 0 1” 1 ND
Pig brain calmodulin 24 11 5.5 28 12 11 8 8.5 7 9 2 8 1 7 6 2 0 1 0
Drosophila
151
wwlanogaster
I Plant calmodulin”
Dictyostelium calmodulinb
27 9 5 27 11 10 6 7 5 11 1 8 1 8 4 2 0 l-2 1
26.2 9.2 6.2 28.1 11.4 10.4 8 8.7 7.8 10.2 2 7.8 1 8 6.1 1.8 0 0 0
determinations.
Ultraviolet spectrum. The absorbance spectrum of pig brain calmodulin (Fig. 6B) presents an increase in the absorbance in the phenylalanine-tyrosine region (252, 258, 269, and 276 nm) similar to those of other calmodulins. However, the absorbance spectrum of D. melunogabr calcium binding protein (Fig. 6A) shows a different spectrum, the main difference being the presence of tryptophan. A similar result was obtained when the protein was further purified by octyl-Sepharose chromatography (see Materials and Methods). Tryptophan was also observed when the spectrophotometric analysis was performed in the presence of guanidine hydrochloride. Under these conditions a tyrosine/tryptophan ratio of 2/l was calculated by the procedure described by Edelhoch (20). Flucrrescence. Calcium produces several conformational changes in calmodulin after its interaction with the protein; these changes could be monitored by measuring the intrinsic fluorescence of calmodulin in the presence or absence of calcium. Emis-
sion spectra of binding protein shown in Fig. lengths, in which (280 nm) or only
D. melanogaster
calcium and pig calmodulin are 7 at two different wavetyrosine and tryptophan tryptophan (295 nm) could
WhVELENGTH
FIG. 6. Ultraviolet absorption binding protein of D. molonogoster calmodulin (B).
(ml
spectra of calcium(A) and pig brain
152
WANDOSELL,
SERRANO,
100
AND
AVILA
at 280 nm compared by tyrosinate and tryptophan only shows the tryptophan contribution. This is indicated in Fig. 8 which shows once again that the main difference between the spectra of D. melanogaster calcium binding protein and calmodulin is the presence of tryptophan.
I-
-Ca+2
DISCUSSION _e------e__ --W
--
I
290
317.5
345
2% ---___--
372.5
400
(nml 100
WAVELENGTH
(nm 1
FIG. ‘7. Fluorescence spectrum of calcium-binding protein of D. mehwgaster (-) measured at two different wavelengths (230 and 295 nm) compared with the fluorescence of pig brain calmodulin (---), measured at 280 and 235 nm. Upper panel indicates the spectrum in the absence of calcium and the lower panel shows the spectrum in the presence of calcium.
be detected. Figure 7 indicates that upon the addition of calcium a variation in the fluorescence pattern of D. melanogaster calcium binding protein was found. This involved an increase in the fluorescence of tyrosine and a decrease in the fluorescence of tryptophan. A similar variation in the fluorescence of tyrosine was also observed for pig brain calmodulin (Fig. 7). To calculate the contribution of tyrosine and tryptophan to the fluorescence pattern of the D. melanogaster protein, the experiment described in Fig. ‘7 was repeated in the absence of calcium at a lower pH (pH 2.5). At this pH tyrosine that had a higher pH in salt form (tyrosinate) becames protonated and shifts to the acidic form (tyrosine). As a consequence of the previous shift, the fluorescence spectrum measured
A new calcium binding protein that activates cyclic AMP phosphodiesterase has been identified in D. melanogaster. It was purified following a method which yields homogeneous calmodulin from porcine brain. Characterization of the D. melane gaster protein indicated several analogies but also some differences when it was compared to pig brain calmodulin. Among the analogies we found that the D. melanogaster protein was heat stable and had the same electrophoretic mobility and isoelectric point as calmodulin together with similar calcium binding properties and ability to activate bovine brain cyclic AMP phosphodiesterase. Also, the similarities between chymotryptic patterns of ‘%I-labeled brain calmodulin and ‘%I-labeled calcium binding protein of D. melanogaster suggested that the locations of ‘%I-tyrosines in Drosophila protein may be very similar to those of pig brain calmodulin (loops 3 and 4). The most remarkable difference was the presence in the Drosophila
-. 0 290
-I_
I 317
5
I
I
345 WAVELENGTH
FIG. 8. Fluorescence protein of D. mdanogaster ulin (---) in the presence
372.5 lnml
spectra of calcium-binding (-) and pig brain calmodof 0.1 M HCI.
1 400
Ca BINDING
PROTEIN
FROM
peptide of tryptophan which is absent from all the calmodulins analyzed so far. This feature suggests that the D. melanogaster calcium binding protein may not be a genuine calmodulin since this protein appears to be very conserved among the different sources tested. However, it could be considered a calmodulin-like protein in that it also contains methyllysine, an amino acid which is not usually found in other proteins apart from calmodulins. Different calcium binding proteins with a size and calcium binding properties similar to those of calmodulin have been described (31-33). Examples of such proteins-/3-calcineurin (M, lS,OOO), caligulin (Mr 27,000), CBP-18 (M, l&000), S-100 (M, lO,OOO), or the larger protein CBP-48 (M, 48,000)-have been detected only in vertebrates, in contrast to calmodulin which has been detected in both vertebrates and invertebrates. Moreover, although some of these proteins contain tryptophan such as CBP-18 (33), neither its amino acid composition nor its biological activity as an activator of phosphodiesterase is related to the characteristics found for D. melanogaster calcium binding protein. On the other hand it has been demonstrated that the binding of calcium to calmodulin produces a variation in the fluorescence intensity of tyrosine present at the third and fourth calcium binding loops (9, 34). A similar increase in fluorescence was demonstrated for the tyrosines of Drosophila calcium binding protein. In this case the variation was correlated with a decrease in tryptophan fluorescence. These results suggest that the tyrosines are probably located in calcium binding loops, as in the case of calmodulin, and that tryptophan is probably located outside of these loops. Despite the degree of similarity between the D. melanogaster calcium binding protein and porcine brain calmodulin, a further study involving the analysis of the sequence of D. melanogaster protein is required to determine whether the l?ro sophila protein is a genuine calmodulin. In any case, we suggest that this protein, through its ability to regulate the metabolism of csclic AMP. I mav” be imnortant in
Drosophila
mdnnogaster
some processes related learning (15, 16).
153 to associative
ACKNOWLEDGMENTS We thank Dr. Hargreaves for critical reading and language corrections of the manuscript, A. Valencia and R. Caballero for amino acid determinations, and C. Hermoso for typing the manuscript. REFERENCES 1. CHEUNG, W. J. (1970) B&hem. Biophys. Res. Commun 38,533-538. 2. CHEUNG, W. J. (1980) Science 207,19-27. 3. JARRET, H. W., AND PENNISTON, J. T. (1978) J. BioL Chem 253,4676-4682. 4. HASINAGA, S., AND PRATT, M. M. (1984) Biochemistry 23,3032-3037. 5. COHEN, P., BURCHELL, A., FOULKES, J. G., GOHEN, T., VANAMAN, T. C., AND NAIRN, A. C. (1978) FEBS I&. 92,287-293. 6. BURKE, B. E., AND DE LORENZO, R. J. (1981) Proc. NatL AuuL Sci. USA 78,991-995. 7. KRETSINGER, R. J. (1976) Annu. Rev. B&hem. 45, 239-266. 8. MARCUM, J. M., DEDMAN, J. R., BRINKLEY, B. R., AND MEANS, A. R. (1978) Proc. NatL Acad Sci. USA 75,3771-3775. 9. DEDMAN, J. R., POTTER, J. D., JACKSON, R. L., JOHNSON, J. D., AND MEANS, A. R. (1977) J. BioL Chem. 252,8415-8422. 10. WAISMAN, D., STEVENS, F. C., AND WANG, J. H. (1975) Biochem Biophys. Res. Cwmmun 65,975982. 11. KLEE, C. B., AND VANAMAN, T. C. (1982) A&I. Prrr tein Chem. 35,213-321. 12. DEDMAN, J. R., WELSH, M. J., AND MEANS, A. R. (1978) J. BioL Ch,em. 253,7515-7521. 13. VANAMAN, T. C., SHARIEF, F., AND WATTERSON, D. M. (1977) in Calcium Binding Proteins and Calcium Function (Wasserman et d., eds.), pp. 107-116, Elsevier/North-Holland, New York. 14. WALLACE, R. W., TALLANT, E. A., AND CHEUNG, W. V. (1981) Cold Spring Harbor Symp. &ant. BioL 46, 893-902. 15. SOLTI, M., DEVAY, P., KISS, I., LONDESBOROUGH, Y., AND FRIEDRICH, P. (1983) Biochem. Biophys. Res. Commun 11,652-658. 16. LIVINGSTONE, M., SZIBER, P., AND QUINN, W. (1984) Cell 37, 205-215. 17. WALTER, M., AND KIGER, J. (1984) J. Neurosci. 4, 495-501. 18. LIN, Y. M., LIU, Y. P., AND CHEIJNG, W. Y. (1974) J. BioL Chem. 249,4943-49&l. 19. FISKE, C. H., AND SUBBAROW, Y. (1925) J. Biol Chem+ 66,375-400. 20. EDELHOCH, H. (1976) Biochemistry 6,1948-1954.
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GERARD,
D.
A Calcium Binding Protein from Drosophila melanogaster Which Activates CAMP Phosphodiesterase: Comparison of This Protein with Porcine Brain Calmodulin’ F. WANDOSELL, Centro
de Biologla
Molecular Received
(CSIC-UAM), July
23,1985,
L. SERRANO,
AND
Autbnoma,
Universidad
and in revised
form
J. AVILA Canto
October
Blanco,
28049
Madrid,
Spain
30, 1985
A calcium binding protein from Drosophila melanogaster has been isolated and characterized. This protein shows several analogies with pig brain calmodulin in its molecular weight, isoelectric point, peptide maps, calcium binding properties, and ability to activate cyclic AMP phosphodiesterase. However, some differences were observed; the most remarkable one is the presence of tryptophan, an amino acid which is absent from all the calmodulins analyzed previously. o 19% Academic PWSS, I~C.
In invertebrates calcium modulator protein, calmodulin, is implicated in different physiological processes in which calcium ions play a regulatory role. Calmodulin was first detected as a protein that stimulated the cyclic nucleotide phosphodiesterase (1, 2). More recently it has been found to activate several other enzymes, such as Ca2+, M$+-ATPase (3,4) or protein kinases (5,6) [for a review see (7)]. In addition, calcium and calmodulin are related to cytoskeleton proteins, because they affect microtubule assembly (8, 9). Calmodulin is present in all species analyzed so far and it has been isolated from different sources [vertebrates and invertebrates (lo), including the lowest eucaryotes (ll)]. Comparison of the structure and function of the calmodulins from different organisms indicates that this molecule is highly conserved, composed of 148 amino acids, containing four structural calcium binding sites that are homologous to each other, especially when the first calcium domain is compared with the third, and the second with the fourth (12). On this basis,
it has been suggested that the calmodulin gene arose by gene duplication of one- or two-domain precursors (13). At present, all calmodulins analyzed contain no tryptophan or cysteine and have only one or two tyrosine residues in calcium loops 3 and 4 (11, 14). The uv absorption spectra reflect a high phenylalanine-to-tyrosine ratio in both vertebrates (8:2) and invertebrates (8:l). An unusual amino acid, trimethyllysine, is also usually present at residue 115 of bovine brain calmodulin. In Drosophila melanogaster, a calmodulin-like protein is involved in the regulation of phosphodiesterase, form II. Mutants affecting this enzyme appears to be defective in associative learning upon a training schedule (15, 16). Therefore, the purification and characterization, in Drosophila, of a protein related to calmodulin could be fundamental to an understanding of the molecular basis of calcium-calmodulin actions in those processes. Partial purification of calmodulin-like proteins has been recently reported (17); however, an extensive study and characterization of such proteins has not yet been performed. In this paper we describe the purification and characterization of a calcium binding
1 This research was supported by grants from Comisi6n Asesora para la Investigacibn Cientifica y T&cnica and Fondo de Investigaciones Sanitarias. 147
0003-9861/86 Copyright All rights
$3.00
0 1986 by Academic Press, Inc. of reproduction in any form reserved.
148
WANDOSELL,
SERRANO,
protein related to calmodulin, from Bwsophila melanogaster, that also shows some important differences from pig brain calmodulin. MATERIALS
AND
METHODS
Putijication of pig brain calmodulin Pig brain calmodulin has been purified essentially as described by Dedman et al. (12) and was tested functionally by its ability to stimulate phosphodiesterase activity. Cyclic AMP phosphodksterase assays. Phosphodiesterase activity was assayed as described by Lin et al (18). Bovine phosphodiesterase (0.15 unit) (from Sigma) was mixed with variable amounts of fractions containing calmodulin. The mixture was incubated at 30°C for 10 min in a buffer containing 40 mM TrisHCl, pH 8, 0.1 mM CaClz, 10 mM MnClz, and 1 mM CAMP. The reaction was stopped by boiling the samples for 3 min and cooling the mixture on ice. Production of AMP was measured by incubation with 5’nucleotidase (0.4 units; Sigma), after which inorganic phosphate was measured as indicated (19). Amino acid analysis. Amino acid analysis was performed on samples of D. m&nogostercaleium binding protein using a Beckman amino acid analyzer. Samples were hydrolyzed for 24 h at 110°C in 6 N HCl. Four different determinations were performed at different protein concentrations. Tryptophan was estimated by the spectophotometric method described by Edelhoch (20). Fluorescence measurements. Fluorescence spectra were obtained using an absolute spectrofluorometer (Schoeffel RSS-1000). Excitation was performed at 280 or 295 nm, at room temperature. Other procedures. Pig brain calmodulin was iodinated by the Bolton-Hunter procedure (21) or alternatively by the chloramine-T reaction (22), when it was used as a molecular weight marker. Protein concentration was determined by the method of Lowry et al. (23). SDS’-polyacrylamide gel electrophoresis was performed according to Laemmli (24) using 5 or 10 to 20% linear gradients. IEF electrophoresis was performed as described by Diez et al. (25) using 2% (w/v) ampholines (pH 3.55). After focusing, the pH was measured on the gel surface with an LKB Multiphor electrode. The gels were fixed and stained with the dye Stains All as has been described (26). Calcium binding activity was determined by equilibrium dialysis using labeled calcium (*CaCl) in 10 mM Tris, pH ‘7, as buffer. Dialyses were
AND
AVILA
performed at 4°C for 24 h on an incubation shaker. Concentrations of unlabeled calcium between lo-’ and 10e6 M were used in each experiment, with 0.02 mCi/ml of labeled calcium chloride (“CaCl) (27). Peptide mapping by limited proteolysis of pig calmodulin and D. melanogaster calcium binding proteins, both ‘a’I-labeled on the tyrosine, was performed in the presence of an equal amount of unlabeled pig calmodulin and at an enzyme/protein ration of 1.25% (w/w). The mixture was incubated for 15 min at 30°C and the reaction was stopped by addition of 1% SDS. Proteolytic labeled fragments were analyzed by gel electrophoresis in 20% polyacrylamide gels and autoradiography. Calcium-dependent hydrophobic chromatography of calmodulin using octyl-Sepharose (Pharmacia) was carried out as indicated by Tanaka et al (28). RESULTS
Isolation The calcium binding protein was isolated from D. melanogaster adults. Flies (100 g) were homogenized in an isotonic buffer containing 0.32 M sucrose, 10 InM Na-phosphate buffer, pH ‘7,1 mM EGTA, and 1 InM PMSF, and centrifuged at 100,OOOgfor 35 min at 4°C. The supernatant was collected and dialyzed against 10 mM Tris, pH 8, 1 mM EDTA, 2 mM P-mercaptoethanol, and 1 M NaCl. After dialysis, the extract at 4 mg/ml was boiled for 10 min, cooled on ice, and centrifuged at 10,OOOgfor 30 min at 4°C. The pellet was discarded and the supernatant was dialyzed against 10 mM Tris, pH 8,1 mM EDTA (TE). DEAE-Cellulose Chromatography The dialyzed solution was applied to a DEAE-cellulose column (20 X 5 cm), previously equilibrated in buffer TE, and eluted stepwise with NaCl(O.1 to 0.5 M) in TE. The fractions eluted with 0.3 M NaCl presented the highest specific activity as activator of the CAMP phosphodiesterase in the presence of calcium (data not shown). Gel Filtration
2 Abbreviations used: SDS, sodium dodecyl sulfate; IEF, isoelectric focusing; EGTA, ethylene glycol bis(6-aminoethyl ether) N,N’-tetraacetic acid; PMSF, phenylmethylsulfonyl fluoride; TE buffer, 10 mM Tris, pH 8,1 mM EDTA.
The DEAE-cellulose fractions eluted at 0.3 M NaCl were pooled, concentrated, and purified by filtration on a Sephadex G-75 column (40 X 1 cm), equilibrated in buffer
Ca BINDING
PROTEIN
FROM
TE, using pig brain calmodulin labeled with *%I as an internal marker. Fractions eluted at the same position as pig brain ‘251-calmodulin appear to contain a single peptide with the same molecular weight in gel electrophoresis (Mr 18,000) as porcine brain calmodulin. These fractions were pooled, concentrated, and rechromatographed under the same conditions as above. Protein characterization of this fraction is indicated in Fig. 1. Total purified protein accounted for 1.5 mg.
Drosophila
$30 2
149
wz&nog&er
-
P
0
1
5
Protein
Functional
20
30
Characteristics
Stimulation
of phosphodiesterase
activity. of the purified D.
The biological
activity peptide was examined by its capacity to stimulate phosphodiesterase activity compared to that of pig brain calmodulin. The levels of stimulation produced by both proteins at the same conmelanogaster
‘IO
10 1 pgr I
20
30
40
50
60
70
No Fractions
FIG. 1. Gel filtration on Sephadex G-75. The fractions eluted at 0.3 M NaCl were pooled and chromatographed on Sephadex G-75-20 (40 X 1 cm) in parallel with ‘%I-calmodulin (0) used as an internal marker. The fractions of D. melunogoster protein eluted at the same position (0) as labeled calmodulin were analyzed by electrophoresis on SDS-polyacrylamide gels. The inset shows the electrophoresis of the purified protein. The arrows represent the positions for tubulin (a), lzsI-pig brain calmodulin (b), and cytochrome e (c), used as molecular weight markers.
FIG. 2. Stimulation of cyclic AMP phosphodiesterase. Bovine brain phosphodiesterase (0.15 gg) was mixed with increasing amounts of pig brain calmodulin (0) and purified protein of D. melurwgcz&r (O), as indicated under Materials and Methods, and the stimulation of the phosphodiesterase activity was assayed. The basal activity of phosphodiesterase in the assay results in the hydrolysis of 0.12 pmol of cyclic AMP at 30°C after 5 min of incubation. In the presence of 10 c(g of added protein 2 rmol of cyclic AMP was hydrolyzed under the above conditions.
centration were very similar, indicating an analogous capacity for both proteins as activators CAMP phosphodiesterase (Fig. 2). Calcium binding capacity. The amount of calcium bound to the D. melanogaster peptide was determined by equilibrium dialysis, using 45CaC1 in the presence of different concentrations of unlabeled calcium (10e4 to 10F6 M), and compared to the calcium binding capacity of pig brain calmodulin. The results obtained were represented as a “Scatchard plot.” Figure 3 shows that the purified peptide of D. melanogaster has three high-affinity calcium binding sites (3.2 X lop5 M) and one lower-affinity binding site (0.9 X 10e4 M). Similar results were obtained for pig brain calmodulin (Fig. 3). Limited proteolysis. Calcium binding protein of D. melanogaster and pig brain calmodulin were labeled with ‘%I on tyrosine residues and subjected to limited chymotrypsin digestion as indicated under Materials and Methods. When the peptides obtained were analyzed by SDS-polyacrylamide gel electrophoresis, a labeled fragment of ik?, 8500 was found in both proteins (Fig. 4) after chymotrypsin digestion.
150
WANDOSELL.
SERRANO.
HOLE Cat4 FIG. 3. Calcium-binding capacity. D. mdmogaster calcium-binding protein (0) and pig calmodulin (0) were dialyzed against different concentrations of %‘a (lo4 to 10m6 M). After equilibrium dialysis the amount (moles) of bound calcium was calculated and the results were represented as a Scatchard plot.
Chemical
and Physical
Characteristics
Mokcular weight. The size of the calcium binding protein of Drosophila has been determined by gel filtration. Chromatography of this peptide on Sephadex showed an elution pattern identical to that of ‘%I-calmodulin, as previously shown and both the proteins exhibited the same electrophoretic mobility when analyzed by SDS-polyacrylamide electrophoresis, with a molecular weight of M, 18,000 (Fig. 1). Isoelectric point. The purified peptide was further characterized on isoelectrofocusing gels, showing a single band with an isoelectric point of 4.30 f 0.05. This band was blue in color, when IEF gels were stained with Stains All (Fig. 5). Similar results were obtained when pig brain calmodulin was focused in parallel on IEF gels (data not shown). When both proteins were stained in solution, with Stains All (26), the same absorption spectrum of the dye was observed. Amino acid composition, The amino acid composition of purified D. melunogaster
AND
AVILA
FIG. 4. Limited chymotrypsin cleavage of calmodulin and calcium-binding protein of D. wdmwgmter. Autoradiographic pattern of ‘261-calmodulin (B) and ‘?-calcium binding protein of D. melanogaster (C) obtained by proteolysis with chymotrypsin. (A) Mixture of undigested calmodulin and D. ndmwgaster calcium-binding protein.
calcium binding protein was determined and compared to that of brain calmodulin, plant calmodulin, and calmodulin from a lower eucaryote (Table I). Some differences were found between these proteins, such as a lower relative content of methionine, phenylalanine, and proline but a higher proportion of histidine in the D. melanogaster protein. Minor differences are present in the other amino acids.
migration
(cm)
FIG. 5. Isoelectrofocusing gel electrophoresis. D. melanogoster calcium-binding protein (10 pg) was focused in a pH gradient between 3.5 and 7; the gel was fixed and stained with Stains All. A parallel gel was focused to determine the pH gradient. The arrow indicates the position for D. mdmwgastw calciumbinding protein in the pH gradient.
Ca BINDING
PROTEIN
FROM TABLE
Drosophila melanogaster Asx Thr Ser Glx GUY Ala Val Met Ileu Leu Tyr Phe His LYS Arg Pro ‘b Tml CYS “Reference (28). ‘Reference (29). ’ Spectrophotometric
22.2 12 12 23.2 14.1 14.1 7 2 5.2 8.6 2 3.6 4 10.6 5.7 0 1” 1 ND
Pig brain calmodulin 24 11 5.5 28 12 11 8 8.5 7 9 2 8 1 7 6 2 0 1 0
Drosophila
151
wwlanogaster
I Plant calmodulin”
Dictyostelium calmodulinb
27 9 5 27 11 10 6 7 5 11 1 8 1 8 4 2 0 l-2 1
26.2 9.2 6.2 28.1 11.4 10.4 8 8.7 7.8 10.2 2 7.8 1 8 6.1 1.8 0 0 0
determinations.
Ultraviolet spectrum. The absorbance spectrum of pig brain calmodulin (Fig. 6B) presents an increase in the absorbance in the phenylalanine-tyrosine region (252, 258, 269, and 276 nm) similar to those of other calmodulins. However, the absorbance spectrum of D. melunogabr calcium binding protein (Fig. 6A) shows a different spectrum, the main difference being the presence of tryptophan. A similar result was obtained when the protein was further purified by octyl-Sepharose chromatography (see Materials and Methods). Tryptophan was also observed when the spectrophotometric analysis was performed in the presence of guanidine hydrochloride. Under these conditions a tyrosine/tryptophan ratio of 2/l was calculated by the procedure described by Edelhoch (20). Flucrrescence. Calcium produces several conformational changes in calmodulin after its interaction with the protein; these changes could be monitored by measuring the intrinsic fluorescence of calmodulin in the presence or absence of calcium. Emis-
sion spectra of binding protein shown in Fig. lengths, in which (280 nm) or only
D. melanogaster
calcium and pig calmodulin are 7 at two different wavetyrosine and tryptophan tryptophan (295 nm) could
WhVELENGTH
FIG. 6. Ultraviolet absorption binding protein of D. molonogoster calmodulin (B).
(ml
spectra of calcium(A) and pig brain
152
WANDOSELL,
SERRANO,
100
AND
AVILA
at 280 nm compared by tyrosinate and tryptophan only shows the tryptophan contribution. This is indicated in Fig. 8 which shows once again that the main difference between the spectra of D. melanogaster calcium binding protein and calmodulin is the presence of tryptophan.
I-
-Ca+2
DISCUSSION _e------e__ --W
--
I
290
317.5
345
2% ---___--
372.5
400
(nml 100
WAVELENGTH
(nm 1
FIG. ‘7. Fluorescence spectrum of calcium-binding protein of D. mehwgaster (-) measured at two different wavelengths (230 and 295 nm) compared with the fluorescence of pig brain calmodulin (---), measured at 280 and 235 nm. Upper panel indicates the spectrum in the absence of calcium and the lower panel shows the spectrum in the presence of calcium.
be detected. Figure 7 indicates that upon the addition of calcium a variation in the fluorescence pattern of D. melanogaster calcium binding protein was found. This involved an increase in the fluorescence of tyrosine and a decrease in the fluorescence of tryptophan. A similar variation in the fluorescence of tyrosine was also observed for pig brain calmodulin (Fig. 7). To calculate the contribution of tyrosine and tryptophan to the fluorescence pattern of the D. melanogaster protein, the experiment described in Fig. ‘7 was repeated in the absence of calcium at a lower pH (pH 2.5). At this pH tyrosine that had a higher pH in salt form (tyrosinate) becames protonated and shifts to the acidic form (tyrosine). As a consequence of the previous shift, the fluorescence spectrum measured
A new calcium binding protein that activates cyclic AMP phosphodiesterase has been identified in D. melanogaster. It was purified following a method which yields homogeneous calmodulin from porcine brain. Characterization of the D. melane gaster protein indicated several analogies but also some differences when it was compared to pig brain calmodulin. Among the analogies we found that the D. melanogaster protein was heat stable and had the same electrophoretic mobility and isoelectric point as calmodulin together with similar calcium binding properties and ability to activate bovine brain cyclic AMP phosphodiesterase. Also, the similarities between chymotryptic patterns of ‘%I-labeled brain calmodulin and ‘%I-labeled calcium binding protein of D. melanogaster suggested that the locations of ‘%I-tyrosines in Drosophila protein may be very similar to those of pig brain calmodulin (loops 3 and 4). The most remarkable difference was the presence in the Drosophila
-. 0 290
-I_
I 317
5
I
I
345 WAVELENGTH
FIG. 8. Fluorescence protein of D. mdanogaster ulin (---) in the presence
372.5 lnml
spectra of calcium-binding (-) and pig brain calmodof 0.1 M HCI.
1 400
Ca BINDING
PROTEIN
FROM
peptide of tryptophan which is absent from all the calmodulins analyzed so far. This feature suggests that the D. melanogaster calcium binding protein may not be a genuine calmodulin since this protein appears to be very conserved among the different sources tested. However, it could be considered a calmodulin-like protein in that it also contains methyllysine, an amino acid which is not usually found in other proteins apart from calmodulins. Different calcium binding proteins with a size and calcium binding properties similar to those of calmodulin have been described (31-33). Examples of such proteins-/3-calcineurin (M, lS,OOO), caligulin (Mr 27,000), CBP-18 (M, l&000), S-100 (M, lO,OOO), or the larger protein CBP-48 (M, 48,000)-have been detected only in vertebrates, in contrast to calmodulin which has been detected in both vertebrates and invertebrates. Moreover, although some of these proteins contain tryptophan such as CBP-18 (33), neither its amino acid composition nor its biological activity as an activator of phosphodiesterase is related to the characteristics found for D. melanogaster calcium binding protein. On the other hand it has been demonstrated that the binding of calcium to calmodulin produces a variation in the fluorescence intensity of tyrosine present at the third and fourth calcium binding loops (9, 34). A similar increase in fluorescence was demonstrated for the tyrosines of Drosophila calcium binding protein. In this case the variation was correlated with a decrease in tryptophan fluorescence. These results suggest that the tyrosines are probably located in calcium binding loops, as in the case of calmodulin, and that tryptophan is probably located outside of these loops. Despite the degree of similarity between the D. melanogaster calcium binding protein and porcine brain calmodulin, a further study involving the analysis of the sequence of D. melanogaster protein is required to determine whether the l?ro sophila protein is a genuine calmodulin. In any case, we suggest that this protein, through its ability to regulate the metabolism of csclic AMP. I mav” be imnortant in
Drosophila
mdnnogaster
some processes related learning (15, 16).
153 to associative
ACKNOWLEDGMENTS We thank Dr. Hargreaves for critical reading and language corrections of the manuscript, A. Valencia and R. Caballero for amino acid determinations, and C. Hermoso for typing the manuscript. REFERENCES 1. CHEUNG, W. J. (1970) B&hem. Biophys. Res. Commun 38,533-538. 2. CHEUNG, W. J. (1980) Science 207,19-27. 3. JARRET, H. W., AND PENNISTON, J. T. (1978) J. BioL Chem 253,4676-4682. 4. HASINAGA, S., AND PRATT, M. M. (1984) Biochemistry 23,3032-3037. 5. COHEN, P., BURCHELL, A., FOULKES, J. G., GOHEN, T., VANAMAN, T. C., AND NAIRN, A. C. (1978) FEBS I&. 92,287-293. 6. BURKE, B. E., AND DE LORENZO, R. J. (1981) Proc. NatL AuuL Sci. USA 78,991-995. 7. KRETSINGER, R. J. (1976) Annu. Rev. B&hem. 45, 239-266. 8. MARCUM, J. M., DEDMAN, J. R., BRINKLEY, B. R., AND MEANS, A. R. (1978) Proc. NatL Acad Sci. USA 75,3771-3775. 9. DEDMAN, J. R., POTTER, J. D., JACKSON, R. L., JOHNSON, J. D., AND MEANS, A. R. (1977) J. BioL Chem. 252,8415-8422. 10. WAISMAN, D., STEVENS, F. C., AND WANG, J. H. (1975) Biochem Biophys. Res. Cwmmun 65,975982. 11. KLEE, C. B., AND VANAMAN, T. C. (1982) A&I. Prrr tein Chem. 35,213-321. 12. DEDMAN, J. R., WELSH, M. J., AND MEANS, A. R. (1978) J. BioL Ch,em. 253,7515-7521. 13. VANAMAN, T. C., SHARIEF, F., AND WATTERSON, D. M. (1977) in Calcium Binding Proteins and Calcium Function (Wasserman et d., eds.), pp. 107-116, Elsevier/North-Holland, New York. 14. WALLACE, R. W., TALLANT, E. A., AND CHEUNG, W. V. (1981) Cold Spring Harbor Symp. &ant. BioL 46, 893-902. 15. SOLTI, M., DEVAY, P., KISS, I., LONDESBOROUGH, Y., AND FRIEDRICH, P. (1983) Biochem. Biophys. Res. Commun 11,652-658. 16. LIVINGSTONE, M., SZIBER, P., AND QUINN, W. (1984) Cell 37, 205-215. 17. WALTER, M., AND KIGER, J. (1984) J. Neurosci. 4, 495-501. 18. LIN, Y. M., LIU, Y. P., AND CHEIJNG, W. Y. (1974) J. BioL Chem. 249,4943-49&l. 19. FISKE, C. H., AND SUBBAROW, Y. (1925) J. Biol Chem+ 66,375-400. 20. EDELHOCH, H. (1976) Biochemistry 6,1948-1954.
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