Comp. Biochem. Physiol. Vol. 102B,No. 3, pp. 475--482, 1992 Printed in Great Britain
0305-0491/92 $5.00+ 0.00 © 1992Pergamon Press Ltd
A SOLUBLE CALCIUM-BINDING PROTEIN (SCBP) PRESENT IN DROSOPHILA MELANOGASTER A N D CALLIPHORA ERYTHROCEPHALA MUSCLE CELLS E. KIEHL and J. D'HAESE* Institut fiir Zoologie, Lehrstuhl f/Jr Morphologie und Zellbiologie, Universit~t Diisseldorf, Universitiitsstr. i, D-4000 Diisseldorf 1, Germany
(Received 20 November 1991) Abstract--1. Soluble calcium binding proteins (SCBP) were isolated from homogenates of whole flies, from the thorax and from muscles of Drosophila melanogaster and Calliphora erythrocephala. 2. Crude preparations were obtained by extraction at low ionic strength, acid and heat treatment. The Drosophila protein was purified by gel filtration, hydrophobie interaction and ion exchange chromatography. In contrast to calmodulin the Drosophila SCBP did not bind to phenyl-Sepharose in a Ca2+-dependent way. 3. Both the Drosophila and the Calliphora protein revealed identical properties. 4. The apparent molecular mass of the SCBP is 24 kDa. Separation in urea-PAGE demonstrated the existence of two isoforms. 5. The calcium-binding property was assured by a calcium dependent electrophoretic mobility shift and autoradiography of 45Ca2+-incubated Western blots. 6. The proteins are abundant in the thorax and were even detectable in crude extracts of various muscles (leg muscles and the extracoxal depressor). In contrast, in power muscles and in the thoracic ganglion the proteins could not be observed.
INTRODUCTION Soluble calcium-binding proteins (SCBP) have been found in the muscles of aquatic invertebrate animals, e.g. crayfish (Benzonana et al., 1974), sandworm (Cox and Stein, 1981; Gerday et al., 1981), scallop (Collins et al., 1983) and shrimp (Takagi and Konishi, 1984a, b). Recently, SCBP has been isolated and characterized also from a terrestrial invertebrate, from the body wall muscle of the earthworm Lumbricus termstris (Huch et al., 1988). SCBP are characterized by their solubility in low ionic strength buffers, their acid and heat stability and high affinity calcium-binding. The calcium-binding domain of these proteins, the so-called EF-hand, consists of two c~-helices orientated rectangular to each other, which are connected by a calcium binding loop (Kretsinger, 1976; Heizmann, 1991). The SCBP share all these properties with parvalbumins, found in various---especially fast twitch--vertebrate muscles (Gerday, 1988). The functional significance of parvalbumins is not precisely known, a central role as a soluble relaxation factor is favoured (Piront et al., 1980; Gerday, 1988; Cox, 1989; Gillis et al., 1982; Gillis, 1985) and supported *Author to whom correspondence should be addressed. Abbreviations---CN-PDE, 3':5"-cyclic nucleotide-phosphodiesterase; DEAE, diethylaminoethyl; EGTA, ethylene glycol-bis ffl-aminoethyl ether) N,N,N',N'-tetra-acetic acid; M, mol/l; PAGE, polyacrylamide gel electrophoresis; PMSF, phenylmethylsulfonyl fluoride; SCBP, soluble calcium-binding protein; SDS, sodiumdodecylsulfate; TEMED, N,N,N',N'-tetramethylethylenediamine; Tris, tris(hydroxymethyl)-aminomethane.
by recent experimental results (Rail, 1989; Hou et al., 199 I). Presumably, the SCBP of invertebrate muscles have the same functions as their vertebrate counterpart. With a molecular mass of about 20 kDa they are larger than parvalbumin with an average molecular mass of 12kDa. The amino acid sequences, known so far from SCBP of five species, reveal the presence of four EF-hand domains, but some of them have lost the ability to bind calcium due to point mutations (Gerday, 1988). The homology concerning the general amino acid sequences of SCBP is low. In addition to muscle tissues considerable amounts of parvalbumin have also been found in other organs, like brain, testis and kidney (Heizmann, 1988; Heizmann, 1991). Probably, the same is also true for the SCBP: antibodies raised against Amphioxus SCBP crossreacted with some neurons in the optic lobe of Drosophila (Buchner et al., 1988). Furthermore, a soluble calcium-binding protein was recently isolated from Drosophila and the antibodies raised against it stained ovaries, gut and neural but not muscle tissue (Kelly, 1990). In the present paper the isolation and characterization of a SCBP from Drosophila melanogaster is described, which occurs in two isoforms and is predominantly present in the muscle tissue of the thorax. Similarities to calmodulin, the protein obtained by Kelly (1990), and that we also demonstrate in muscles of Calliphora erythrocephala are discussed. S o m e of the results have already been presented at the XIXth European Conference on Muscle Contraction and Cell Motility (Kiehl and D'Haese, 1991).
475
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E. KIEHL and J. D'HAESE MATERIALS AND METHODS
Materials Drosophila melanogaster and Calliphora erythrocephala were provided by the Institut f/Jr Genetik and by the Institut fiir Zoologie of the Universit/it Diisseldorf, respectively. Except for mutations affecting muscles, various genotypes of Drosophila melanogaster were used for bulk preparations of SCBP from whole fruit flies. Oregon R wildtype flies were used for all other preparations.
SCBP-preparation from whole and dissected flies Fifteen to forty g Drosophila flies were ground to a powder in liquid nitrogen. The powder was extracted with five volumes (v/w) of SCBP-Ex (25 mM Tris-HCl pH 7.5, 1 mM 2-mercaptoethanol, 0.5 mM PMSF, 5/~ M CaC12) and insoluble material was removed by centrifugation (20000g, 30 min, 4°C). The following acid treatment, ammonium sulphate-fractionation, heat-treatment (60°C) and gel filtration chromatography on Ultrogel AcA 54 (PharmaciaLKB) were performed as described by Huch et al. (1988). Fractions of the gel filtration chromatography in the molecular mass range of 20 kDa (mw 20,000-fraction) were pooled and concentrated by ultrafiltration using a YM5 membrane (Amicon, cut-off 5 kDa). Further purification was achieved by ionic exchange (DEAE-cellulose DE 52, Whatman) and hydrophobic interaction chromatography (phenyl-Sepharose, Pharmacia-LKB). Phenyl-Sepharose chromatography was performed in a 2.5 × 13 cm column equilibrated with 50 mM Tris-HC1 pH 7.5, 0.1 mM CaCI2, 1 mM 2-mercaptoethanol, 0.5 mM PMSF. 8.3 mg of the m w 20,000-fraction dialysed against the same buffer were applied. After washing, the column was eluted stepwise with elution-buffer A (50 mM Tris-HC1 pH 7.5, 0.1 mM CaC12, 1 mM 2-mercaptoethanol, 0.5M NaC1, 0.5mM PMSF), elution-buffer B (50 mM Tris-HCl pH 7.5, I mM EGTA, 1 mM 2-mercaptoethanol, 0.5 mM PMSF), water and 8 M urea. For purification on DEAE-cellulose the m w 20,000fraction in DEAE-buffer (15 mM Tris-HC1 pH 7.5, 1 mM 2-mercaptoethanol, 5 # M CaCI2) was applied to the column (1.6 x 6 cm) equilibrated with the same buffer. Proteins were eluted with a linear gradient of 0-300 mM NaC1 in DEAEbuffer (2 x 100 ml). Drosophila and Calliphora flies were dissected with microscissors into head, thorax and abdomen. The parts were collected separately in SCBP-Ex (150-300 #1 for about 200 heads, thoraces or abdomina) and homogenized in a 1 ml glass/glass homogenizer (Pierce). The homogenates were centrifuged in an Eppendorf-type centrifuge (13,000rpm, 20 min), the supernatant (crude extract) was immediately incubated for PAGE or was further fractionated as described above to obtain a 60°C supernatant (crude preparation). Individual muscles and the thoracic ganglion from Drosophila and Calliphora thoraces were prepared in 50% ethanol in H20. Preparation in 50% ethanol was necessary as the tissues were too soft in Ringer solution (184 mM KC1, 46 mM NaCI, 30 mM CaC12, 10 mM Tris-HC1 pH 7.2) to be handled. Ten to twenty Drosophila-organs were collected in l ml SCBP-Ex, homogenized in a 1 ml glass/glass homogenizer (Pierce) and centrifuged (13,000rpm, Eppendorftype centrifuge, 45 min). The supernatant (crude extract) was concentrated to 50/~1 with microconcentrators (Centricon 10, Amicon). In the case of Calliphora 15-20 organs were collected in 700 #1 SCBP-Ex and also processed up to the 60°C supernatant as described above. Concentration was omitted. In addition to the thoracic ganglion the following muscles were collected for a crude preparation: flight control muscles (including basalar muscles, muscles of third axillary, muscles of first axillary), power muscles (dorsal median muscles, tergosternal muscles, tergal remotors of coxa, lateral oblique dorsal muscles), leg muscles (intracoxal
depressor, levator, sternal promotor, sternal remotor of all six legs), extracoxal depressor. For orientation and terminology of Drosophila muscles the detailed description of Miller (1965), and for preparation of Calliphora muscles the description of Heide (1971), was used.
Gel electrophoresis and Western blotting SDS-polyacrylamide gel electrophoresis (SDS-PAGE) was performed according to Laemmli (1970) on 0.75 mm thick slab gels using the Midget electrophoresis system (Pharmacia LKB). The gels were stained with Coomassie Brilliant Blue R250 by a procedure adopted from Heukeshoven and Dernick (1988) and in some cases poststained with silver according to Blum et al. (1987). Molecular mass standards were phosphorylase B (92.5 kDa), bovine serum albumin (67 kDa), egg albumin (45 kDa), carbonic anhydrase (29kDa), soybean trypsin inhibitor (21 kDa), cytochrome c (12.5 kDa) and bovine lung trypsin inhibitor (6.5 kDa). For electroblotting the gels were equilibrated in 25 mM Tris, 192raM glycine, 20% methanol for 15min and transferred onto 0.2 or 0.45 t~m nitrocellulose membranes (Schleicher and Schuell) with a semi-dry blot system. Protein bands were reversibly visualized by staining with Ponceau S (Sigma). In urea-polyacrylamide gel electrophoresis (urea-PAGE) a calcium dependent change in the electrophoretic mobility of a protein, which can be considered as a qualitative hint for the ability of a protein to bind calcium (Walsh et al., 1984; McDonald and Walsh, 1985), is much more pronounced than in SDS-PAGE. Urea-PAGE was performed essentially as described by Huch et al. (1988). Protein samples were equilibrated in 50 mM carbonate buffer pH 8.0, I mM CaC12 for 20min on ice and prepared for urea-PAGE by adding the same volume of 4 M urea, 33.33% glycerol, 0.006% Bromphenol Blue, 1 mM CaC12. Different amounts of EGTA were added to the samples.
45Ca2+-overlay The 45Ca2+-overlay technique of Maruyama et al. (1984) was used as an identification method for calcium-binding proteins after urea-PAGE. Western blots were washed three times in overlay buffer (60 mM KC1, 5 mM MgC12, 10 mM imidazole-HCl pH 6.9) for 20rain and incubated with 45Ca2+ (7400 Bq/ml in overlay buffer) for 10 min. After a short wash in 50% ethanol (1-5 min) the membranes were dried between two layers of filter paper. Ca2+-binding proteins were identified by exposing the processed nitrocellulose sheets to an X-ray film (Fuji) at -80°C.
Protein assay Protein concentrations were estimated by the method of Bradford (1976) using the Bio-Rad Protein Assay and bovine serum albumin as a standard. RESULTS
Preparation o f S C B P from whole flies The s t a n d a r d isolation procedure is summarized in Fig. 1. Centrifugation of the h o m o g e n a t e results in a n a p p a r e n t loss o f contractile proteins (Fig. 2B, lanes 1 a n d 2; c o m p a r e also Fig. 4, lanes 5-7). Already in the 60°C s u p e r n a t a n t a considerable e n r i c h m e n t of a 24 k D a protein can be observed (Fig. 2B, lane 3). The fractions pooled after gel filtration on A c A 54 are indicated in the elution profile in Fig. 2A. This mw 20,000-fraction already c o n t a i n s the 24 k D a protein as a m a i n c o m p o n e n t (Fig. 2B, lane 4). Phenyl-Sepharose c h r o m a t o g r a p h y of the mw 20,000 fraction did n o t result in a m a r k e d purification. Nearly all proteins did n o t b i n d to the hydro-
477
SCBP from fly muscles
Evidence for Ca2+-binding
Purification-Scheme fraction
~
protein amount
homogenisation in S/BP-Ex
[fly-homogenate I centrifugafion Icrude -extract I
10 0%
pH5,5 treatment [pH5,5 supernatantJ
t~4%
t,.0%(N Ht,)2S0/, _p r ecip it a ti 0n
[l.0*/,tNHd2S0a-supernatanf] I
180O/o(N Ht)2S Or, -petter I l
27*/.
80%1NH/,)2S0/, _p r ecipifafi 0n
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heat-treatment
[60=C-supernatant]
7%
80%(N H/,)2S0/,_pr ecip it at i o n
lrO'C/eO*/o(NHu)zSO,-peltat[
5%
I AcA 5/,-getfittration I mw 20000 -fraction I
1%
Fig. 1. Purification scheme. phobic matrix and eluted with the column buffer (Fig. 2B, lane 5). Purified Drosophila-SCBP was obtained by ionic exchange chromatography on DEAE-cellulose, where two SCBP containing peaks were eluted at about 160 mM NaCI (Fig. 2B, lane 6). About 5 mg of pure SCBP were obtained from 35 g of fruitflies as starting material, which corresponds to a concentration of about 130 mg SCBP per 1 kg flies.
Ca:+-incubated proteins of the mw 20,000fraction supplemented with increasing amounts of the Ca:+-complexing agent EGTA were separated in urea-PAGE. Figure 3A shows that there are two bands in the m, 20,000-fraction--"Cal" and " C a 2 " - - , which reveal a calcium-dependent mobility shift to form a more diffuse band, which can be subdivided into two smaller ones, " E G I " and "EG2". The calcium-binding ability of these proteins was demonstrated by 45Ca2+-overlay after a mobility shift experiment (Fig. 3A, lanes 5-8). Shifting bands showed Ca2÷-binding after autoradiography. The proteins in the mw 20,000-fraction, which showed a mobility shift in urea-PAGE, the bands Cal, Ca2, EG1 and EG2, were cut out of the gel and re-electrophoresed in SDS-PAGE. The four bands of the urea-PAGE comigrate with the mw 24,000 protein of the m w 20,000-fraction (Fig. 3B). These results suggest that the m, 24,000-protein belongs to the SCBP and that it probably occurs in two isoforms.
Identification of the tissue from which the SCBP originates For the identification of the origin of SCBP, fruitflies were separated into head, thorax and abdomen and a crude preparation was carried out separately up to the 60°C-supernatant. SCBP was abundant only in the thorax, in extracts of heads and abdomina no bands in the m, 24,000-range could be detected (Fig. 4, lanes 1, 2 and 5). This was confirmed by urea-PAGE, because only the thorax contained a significant amount of a protein which showed a Ca2+-dependent mobility shift (Fig. 4, lanes 8-13). In the presence of EGTA, the SCBP appears as a diffuse band in urea-PAGE, especially when crude extracts
A
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92500 D-67000 /~5000 29000
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10 20 30 40 50 60 70 Fraction Fig. 2. Elution profile of the AcA 54 gel filtration chromatography (A). Fractions pooled as the rn, 20,000-fraction are indicated by shading. Chromatography was performed in 5 p M CaCI2, l mM 2-mercaptoethanol, 50 mM NH4HGO 3 pH 8.0 on a 1.6 x 85 cm column with a flow rate of 0.25 m]. rain -I. 30 mg of Drosophila crude preparation were applied to the column. SDS-PAGE of various purification steps of SCBP from Drosophila (B). [l] homogenate; [2] crude extract; [3] 60°C supernatant; [4] m, 20,000-fraction; [5] unbound fraction o£ phenyl-sepharose chromatography; [6] purified SCBP after DEAE-chromatography. The wedges mark the positions of the molecular mass markers, the arrow indicates the position of the SCBP-band.
6
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E. KIEHLand J. D'HAESE
PAGE. From the beginning there is only one band with a Ca2+-dependent mobility shift. This argues against a modification that could occur during the preparation procedure and is in favour of the real existence of two isoforms. As the fly thorax consists predominantly of muscles, it was reasonable to assume that muscles are the tissue the SCBP originates from. This was tested laiD-by preparing crude extracts of power muscles, leg Ca2=-.muscles and the extracoxal depressor muscle. In E51,,,-addition crude extracts were prepared from the thoEG2"" racic ganglion, because it might also contain significant amounts of SCBP. SDS-PAGE of these extracts clearly demonstrate that the extracoxal depressor and the leg muscles are the main sources of the SCBP. In extracts of the power muscles and the thoracic ganglion a protein band corresponding to 1 10 50 0 0 10 0 10 the 24 kDa-SCBP is not detectable (Fig. 6). To test the idea, that certain muscles are the source of the SCBP in flies, extracts of Calliphora erythroB M S [al Ca2 E51 E52 cephala were examined for comparison. Only in homogenates and crude extracts of the thorax and the 92500 = extracoxal depressor do two protein bands appear with a calcium-dependent mobility shift in urea67000,.-PAGE, a pronounced one with low and a faint one with higher electrophoretic mobility (Fig. 5, lanes 7 ~5000,.-and 9). In a 45Ca2+-overlay the slower protein bound most of the radioactive calcium (not shown). Re-electrophoresed in SDS-PAGE the proteins comigrate with the mw 24,000-protein of Drosophila. They also 29000 ~-comigrate with a protein band present in extracts of Calliphora thoraces and certain thoracic muscles (Fig. 5). The presence of SCBP in high amounts could additionally be demonstrated for the flight control muscle. On the other hand, it was impossible to 21000 ==" identify this protein in extracts of heads, abdomina, power muscles and thoracic ganglion of Calliphora either with urea-PAGE or with SDS-PAGE. Figure 6 12500==-shows the identical distribution of the mw 24,000-protein in Drosophila and Calliphora. Thus it seems Fig. 3. Evidence for calcium-binding.Urea-PAGE of the mw 20,000-fraction (A): [1-4] m~ 20,000-fractionincubated with permissible to conclude that SCBP in flies is predom1 mM Ca2÷ and electrophoretically separated after addition inantly present in distinct thoracic muscles, at least of EGTA to the indicated final concentration (in raM). in the extracoxal depressor, the leg muscles and the There is a clear difference in the electrophoretic mobility of flight control muscles. Ca2+-loaded (Cal, Ca2) and Ca2+-depleted protein (EG1, EG2). Due to the high EGTA-concentration in the last but DISCUSSION one sample, EGTA diffused into the last lane, thus causing half of the protein to change electrophoretic mobility. [5,6] We have isolated calcium-binding proteins with Electrotransfer of proteins of the mw20,000-fraction after a a molecular mass of 24kDa from Drosophila mobility shift experiment (Ponceau S stained). [7,8] Autoradiography of [5,6] after 45Ca2+-overlay.SDS-PAGE of the melanogaster and Calliphora erythrocephala. The excised bands Cal, Ca2, EG1, and EG2 from the urea- properties of the proteins resemble those of SCBP PAGE (B). M: molecular mass markers; S: mW20,000-frac- from invertebrate muscle tissue. They are abundant tion; the molecular mass markers are indicated by wedges, in muscles and not detectable in abdomina and heads, position of the SCBP-band is marked by the arrow. The two where muscles are less predominant. From Figs 4 and mobility shifting bands in urea-PAGE Cal and Ca2 and 6 it can be inferred that the proteins are present in their Ca2+-free counterparts EG1 and EG2 comigrate with high amounts in the extracoxal depressor, the flight the mw 24,000-protein (SCBP) of the mw 20,000-fraction. control muscles and the leg muscles but not in the power muscles. Neural tissue or the gonads apparently do not contain the proteins. Although Drosophila-calmodulin has an unusual were applied. The ability to bind Ca 2+ is not lost (see high molecular mass of 20 kDa (calculated according Fig. 3A, lanes 6 and 8). In contrast to the extract of whole flies, the thorax to Smith et al., 1987), which could be difficult to extract contains only one mobility shifting band distinguish from the described 24 kDa-proteins in (Fig. 4, lanes 10 and 11). Therefore, every step of the SDS-PAGE, and the CN-PDE activation assay as an preparation procedure up to the 60°C-supernatant identification method for calmodulin has not been was examined for mobility shifting bands in urea- carried out, the present experimental data for the
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SCBP from fly muscles
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479
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92500D'-
67000ZD" 45000D,--
29000==" 21000 =="12500 = . . Fig. 4. Localization of SCBP in Drosophila. Separate preparation of head, thorax and abdomen. According to SDS-PAGE [1-7], only the thorax contains a protein with a molecular mass of 24,000 which shows a calcium dependent mobility shift in urea-PAGE [8-13]. For comparison, Drosophila actomyosin was also electrophoresed. [1] head crude extract; [2] abdomen crude extract; [3] purified SCBP; [4] thorax 60°C-supernatant; [5] thorax crude extract; [6] thorax homogenate; [7] Drosophila actomyosin (MHC myosin heavy chain; A actin; Ltl, Lfl, Lf2, Lf3 isoforms of myosin light chains according to Takano-Ohmura et aL (1983); t: synchronous muscles, f: asynchronous muscles). The wedges mark the positions of the molecular mass markers, the arrow points to the SCBP-band. [8-13] urea-PAGE of [8,9] head crude extract; [10,11] thorax crude extract; [12,13] abdomen crude extract with 1 mM Ca 2+ [8,10,12] or 10mM EGTA [9,11,13].
Drosophila protein suggest that it is different from caimodulin for a n u m b e r of reasons. A typical source of calmodulin is neural tissue: Doyle et al. (1990) succeeded in isolating several calmodulin-clones from a head-specific c D N A - b a n k of Drosophila. This argues for the existence of calmodulin in the Drosophila head, likely the brain. In extracts of the head and the thoracic ganglion we could not observe a SCBP-band. In contrast to calmodulin, the Drosophila protein did not bind to phenyl-Sepharose and the failure to bind to this matrix is regarded as a characteristic feature of SCBP (Gopalakrishna and Anderson, 1982; Cox, 1989). Furthermore, the
uv-spectrum of Drosophila-SCBP indicates the presence of tryptophan (not shown), which is not found in calmodulin. The regulatory light chain of earthworm myosin also contains an EF-hand calcium-binding domain, which is identical with the corresponding myosin light chain LC2 from Drosophila, and binds 45Ca2+ as shown by the 45Ca2+-overlay technique (Carlhoff, 1988). The possibility that myosin light chains contaminated the fractions tested can be excluded, because the main structural muscle proteins present in the homogenate are effectively removed by the first centrifugation (Fig. 4).
92500m,'67000D" 45000D'-
29000
21000
12500 Fig. 5. SCBP in Calliphora. SDS-PAGE of [1] thorax crude extract; [2] extracoxal depressor homogenate; [3] flight control muscle crude extract; [4] purified Drosophila SCBP; [5] upper band and [6] lower band of the two mobility shifting bands from lane 7 reelectrophoresed; urea-PAGE of [7,8] extracoxal depressor homogenate; [9,10] thorax crude extract with 1 mM Ca 2+ (7,9) and I0 mM EGTA (8,10). Mobility shifting proteins are only found in extracts of the thorax and the extracoxal depressor. In SDS-PAGE they comigrate with the Drosophila SCBP and a mw 24,000 protein only found in extracts of muscles of
Calliphora.
480
E. KIEHLand J. D'HArSE
thorax CatIiphora 1
2
extracoxa{ depressor l:alliphora Orosophita
Drosophita 3
1
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92500 ~67000 ~-
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•
92500~" 67000 ~-
45000 ~-
45000"-
29000 ~"
29000
21000 ='-
21000
12500 = "
12500 D.-
thoracic ganglion Cattiphora Drosophita 1
2
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1
power muscles CatIiphora Drosophila 1 2 3 1 2
2
92500~" 67000 ~-
92500~.. 67000~.-
&5000~"
45000"
29000 ~ "
29000 ~"
21000 " -
21000
12500
12500 " "
Fig. 6. Comparison of Calliphora and Drosophila. SDS-PAGE of different organs of Drosophila and Calliphora. [1] homogenate; [2] crude extract; [3] 60°C-supernatant; [*] crude extract of Drosophila leg muscles. In Calliphora as well as Drosophila, SCBP is present in extracts of thorax, extracoxal depressor and leg muscles (only shown for Drosophila), but is lacking in the thoracic ganglion and power muscles. The protein isolated from Drosophila melanogaster can therefore be considered as a calcium-binding protein clearly different from calmodulin and myosin light chains. The same is probably true for the Calliphora-protein, because it exhibits identical properties as tested so far. In different muscles of various organisms a correlation has been found between the amount of parvalbumin or SCBP and the contraction speed of the muscles (Gerday, 1988). Within the thorax the power muscles are the prominent organs. Though these muscles are fast contracting, we were unable to detect SCBP in their extracts. An explanation could be that SCBP as a soluble relaxation factor is not of functional significance, as the contraction-relaxation cycle of these muscles is independent of a fast release and removal of calcium (Riiegg, 1988). In contrast, flight control muscles, leg muscles and the extracoxal depressor muscle with a normal way of activation may depend on the presence of SCBP for fast restoring activability.
In extracts of whole Drosophila-flies always two mobility shifting bands have been observed in ureaPAGE. The interpretation that there are two isoforms of SCBP in Drosophila is complicated by the fact that, in extracts of thoraces, only one shifting band can be found. As the thorax has been proven as the main source of SCBP, it can be excluded that one of the two isoforms originates from head or abdomen. It is therefore assumed that the two isoforms exist in a population of flies, but not necessarily within a single strain. For the preparation of Drosophila-SCBP from whole flies a mixture of flies of different genotypes was used, whereas the SCBP fractions from dissected flies and single organs were prepared from a single Oregon R laboratory strain. Extracts of Calliphora thoraces show a strong and a weak mobility shifting band in urea-PAGE. This can also be explained by the existence of two isoforms, but with a different frequency of the two alleles in the population. This opinion is supported by the recent finding that in five of six crude extracts from thoraces
SCBP from fly muscles of different Drosophila strains two mobility shifting bands and, in one extract, one mobility shifting band were observed. Recently, Kelly (1990) isolated a Ca2+-binding protein from Drosophila with properties very similar to those described in this paper. The main difference between the two proteins is their distribution in the fly: Kelly detected the calcium-binding protein in extracts of various organs of the fly--predominantly in head extracts--but not in the muscles. Furthermore, for preparation Kelly used a phenothiazine chromatography, in which the protein bound to the matrix. As phenothiazine belongs to the hydrophobic matrices to which SCBP do not bind (Cox, 1989; Charbonneau and Cormier, 1979; Jamieson and Vanaman, 1979), this argues for differences between both proteins. It would be interesting, to compare directly the two proteins to decide whether they are really different or not.
REFERENCES
Benzonana G., Cox J., Kohler L. and Stein E. (1974) Characterisation d'une nouvelle mttallo-proteine calcique du myogtne de certains crustacts. C.r. hebd. Sbanc. Acad. Sci. Paris 279, 1491-1493. Blum H., Beier H. and Gross H. (1987) Improved silver staining of plant proteins, RNA and DNA in polyacrylamide gels. Electrophoresis 8, 93-99. Bradford M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analyt. Biochem. 72, 248-254. Buchner E., Bader R., Buchner S., Cox J., Emson P., Florey E., Heizmann C., Hemm C., Hofbauer A. and Oertel W. (1988) Cell specific immuno-probes for the brain of normal and mutant Drosophila melanogaster. I. Wildtype visual system. Cell Tissue Res. 253, 357370. Carlhoff D. (1988) Characterisierung der Proteine aus der schr~iggestreiften Muskulatur des Regenwurms Lumbricus terrestris mit besonderer Beriicksichtigung der Myosingekoppelten Ca2+-Regulation. Doctoral thesis of the Mathematisch-Naturwissenschaftlichen Fakultfit der Universit/it D/isseldorf. Charbonneau H. and Cormier M. J. (1979) Purification of plant calmodulin by fluphenazine-sepharose affinity chromatography. Biochem. biophys. Res. Commun. 90, 1039-1047. Collins J., Johnson J. and Szent-Gytrgyi A. (1983) Purification and characterisation of a scallop sarcoplasmatic calcium binding protein. Biochemistry 22, 341-345. Cox J. A. (1989) Unique calcium binding proteins in invertebrates. In Calcium Binding Proteins in Normal and Transformed Cells (Edited by Pochet R., Lawson E. and Heizmann C.), pp. 67-72. Plenum Publishing Corporation, New York. Cox J. A. and Stein E. A. (1981) Characterization of a new sarcoplasmic calcium-binding protein with magnesiuminduced cooperativity in the binding of calcium. Biochemistry 20, 5430-5436. Doyle K. E., Kovalick G. E., Lee E. and Beckingham K. (1990) Drosophila melanogaster contains a single calmodulin gene. J. molec. Biol. 213, 599~05. Gerday C., Collin S. and Gerardin-Otthiers N. (1981) The soluble calcium-binding protein from muscle of the sandworm. Nereis virens. J. Muscle Res. Cell Motil. 2, 225-238. Gerday C. (1988) Soluble calcium binding proteins in vertebrate and invertebrate muscles. In Calcium and
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Calcium Binding Proteins (Edited by Gerday C., Bolls L. and Gilles R,), pp. 23-39. Springer, Berlin. Gillis J. M., Thomason D., Leftvre J. and Kretsinger R. H. (1982) Parvalbumins and muscle relaxation: a computer simulation study. J. Muscle Res. Cell Motil. 3, 377-398. Gillis J. M. (1985) Relaxation of vertebrate skeletal muscle. A synthesis of the biochemical and physiological approaches. Biochim. biophys. Acta 811, 97-145. Gopalakrishna R. and Anderson W. B. (1982) Ca 2+-induced hydrophobic site on calmodulin: application for purification of calmodulin by phenyl-sepharose affinity chromatography. Biochem. biophys. Res. Commun. 104, 830-836. Heide G. (1971) Die Funktion der nicht-flbrill/iren Flugmuskeln yon Calliphora. Teil I, Lage, Insertionsstellen und Innervierungsmuster der Muskeln. Zool, Jb. Physiol. 76, 87-98, Heizmann C. (1988) Parvalbumin in non-muscle cells. In Calcium and Calcium Binding Proteins (Edited by Gerday C., Bolis L. and Gillis R.), pp. 93-101, Springer, Berlin. Heizmann C. (1991) Intracellular calcium-binding proteins: more sites than insights. Trends Biochem. Sci. 16, 98-103. Heukeshoven J. and Dernick R. (1988) Increased sensitivity for Coomassie staining of sodium dodecylsulphate polyacrylamide gels using Phast System Development Unit. Electrophoresis 9, 60-61. Hou T.-t., Rail J. A. and Johnnson J. D. (1991) Role of parvalbumin (PA) in frog skeletal muscle relaxation. J. Muscle Res. Cell Motil. 12, 114. Huch R., D'Haese J. and Gerday C. (1988) A soluble calcium binding protein from the terrestrial annelid Lumbricus terrestris L. J. comp. Physiol. B 158, 325-334. Jamieson G. A. and Vanaman T. C. (1979) Calcium dependent affinity chromatography of calmodulin on an immobilized phenothiazine. Biochem. biophys. Res. Commun. 90, 1048-1056. Kelly L. (1990) Purification and properties of a 23 kDa Ca2+-binding protein from Drosophila melanogaster. Biochem. 3". 271, 661-666. Kiehl E. and D'Haese J. (1991) A soluble calcium-binding protein from Drosophila melanogaster. J. Muscle Res. Cell Motil. 12, 115. Kretsinger R. (1976) Calcium binding proteins. Ann. Rev. Biochem. 45, 239-266. Laemmli U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685. Maruyama K., Mikawa T. and Ebashi S. (1984) Detection of calcium binding proteins by 45Ca autoradiography on nitrocellulose membrane after sodium dodecyl sulphate Gel electrophoresis. J. Biochem. 95, 511-519. McDonald J. R. and Walsh M. P. (1985) Ca2+-binding proteins from bovine brain including a potent inhibitor of protein kinase C. Biochem. J. 232, 559-567. Miller A. (1965) The internal anatomy and histology of the imago of Drosophila melanogaster. In Biology o f Drosophila (Edited by Demerec M.), pp. 420-534. Hafner, New York. Walsh M. P., Valentine V. A., Ngai P. K., Carruthers C. A. and Hollenberg M. D. (1984) Ca2÷-dependent hydrophobic-interaction chromatography. Isolation of a novel CaZ+-binding protein and protein kinase C from bovine brain. Biochem. J. 224, 117-127. Piront A., Lebacq J. and Gillis J. (1980) Physiological properties of calcium binding proteins from crayfish muscle. J. Muscle Res. Cell Motil. 1, 468-469. Rall J. A. (1989) Relationship of isometric unexplained energy production to parvalbumin content in frog skeletal muscle. Prog. Clin. Biol. Res. 315, 117-126. Riiegg J. C. (1988) Calcium in Muscle Activation. Springer, Berlin.
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Smith V. L., Doyle K. E., Maune J. F., Munjaal R. P. and Beckingham K. (1987) Structure and sequence of the Drosophila melanogaster calmodulin gene. J. molec. Biol. 196, 471-485. Takagi T. and Konishi K. (1984a) Amino acid sequence of~ chain of sarcoplasmatic calcium binding protein obtained from shrimp tail muscle. J. Biochem. 95, 1603-1615.
Takagi T. and Konishi K. (1984b) Amino acid sequence of the fl chain of sarcoplasmatic calcium binding protein (SCP) obtained from shrimp tail muscle. J. Biochem. 96, 59-67. Takano-Ohmura H., Hirose G. and Mikawa T. (1983) Separation and identification of Drosophila myosin light chains. J. Biochem. 94, 967-974.
0305-0491/92 $5.00+ 0.00 © 1992Pergamon Press Ltd
A SOLUBLE CALCIUM-BINDING PROTEIN (SCBP) PRESENT IN DROSOPHILA MELANOGASTER A N D CALLIPHORA ERYTHROCEPHALA MUSCLE CELLS E. KIEHL and J. D'HAESE* Institut fiir Zoologie, Lehrstuhl f/Jr Morphologie und Zellbiologie, Universit~t Diisseldorf, Universitiitsstr. i, D-4000 Diisseldorf 1, Germany
(Received 20 November 1991) Abstract--1. Soluble calcium binding proteins (SCBP) were isolated from homogenates of whole flies, from the thorax and from muscles of Drosophila melanogaster and Calliphora erythrocephala. 2. Crude preparations were obtained by extraction at low ionic strength, acid and heat treatment. The Drosophila protein was purified by gel filtration, hydrophobie interaction and ion exchange chromatography. In contrast to calmodulin the Drosophila SCBP did not bind to phenyl-Sepharose in a Ca2+-dependent way. 3. Both the Drosophila and the Calliphora protein revealed identical properties. 4. The apparent molecular mass of the SCBP is 24 kDa. Separation in urea-PAGE demonstrated the existence of two isoforms. 5. The calcium-binding property was assured by a calcium dependent electrophoretic mobility shift and autoradiography of 45Ca2+-incubated Western blots. 6. The proteins are abundant in the thorax and were even detectable in crude extracts of various muscles (leg muscles and the extracoxal depressor). In contrast, in power muscles and in the thoracic ganglion the proteins could not be observed.
INTRODUCTION Soluble calcium-binding proteins (SCBP) have been found in the muscles of aquatic invertebrate animals, e.g. crayfish (Benzonana et al., 1974), sandworm (Cox and Stein, 1981; Gerday et al., 1981), scallop (Collins et al., 1983) and shrimp (Takagi and Konishi, 1984a, b). Recently, SCBP has been isolated and characterized also from a terrestrial invertebrate, from the body wall muscle of the earthworm Lumbricus termstris (Huch et al., 1988). SCBP are characterized by their solubility in low ionic strength buffers, their acid and heat stability and high affinity calcium-binding. The calcium-binding domain of these proteins, the so-called EF-hand, consists of two c~-helices orientated rectangular to each other, which are connected by a calcium binding loop (Kretsinger, 1976; Heizmann, 1991). The SCBP share all these properties with parvalbumins, found in various---especially fast twitch--vertebrate muscles (Gerday, 1988). The functional significance of parvalbumins is not precisely known, a central role as a soluble relaxation factor is favoured (Piront et al., 1980; Gerday, 1988; Cox, 1989; Gillis et al., 1982; Gillis, 1985) and supported *Author to whom correspondence should be addressed. Abbreviations---CN-PDE, 3':5"-cyclic nucleotide-phosphodiesterase; DEAE, diethylaminoethyl; EGTA, ethylene glycol-bis ffl-aminoethyl ether) N,N,N',N'-tetra-acetic acid; M, mol/l; PAGE, polyacrylamide gel electrophoresis; PMSF, phenylmethylsulfonyl fluoride; SCBP, soluble calcium-binding protein; SDS, sodiumdodecylsulfate; TEMED, N,N,N',N'-tetramethylethylenediamine; Tris, tris(hydroxymethyl)-aminomethane.
by recent experimental results (Rail, 1989; Hou et al., 199 I). Presumably, the SCBP of invertebrate muscles have the same functions as their vertebrate counterpart. With a molecular mass of about 20 kDa they are larger than parvalbumin with an average molecular mass of 12kDa. The amino acid sequences, known so far from SCBP of five species, reveal the presence of four EF-hand domains, but some of them have lost the ability to bind calcium due to point mutations (Gerday, 1988). The homology concerning the general amino acid sequences of SCBP is low. In addition to muscle tissues considerable amounts of parvalbumin have also been found in other organs, like brain, testis and kidney (Heizmann, 1988; Heizmann, 1991). Probably, the same is also true for the SCBP: antibodies raised against Amphioxus SCBP crossreacted with some neurons in the optic lobe of Drosophila (Buchner et al., 1988). Furthermore, a soluble calcium-binding protein was recently isolated from Drosophila and the antibodies raised against it stained ovaries, gut and neural but not muscle tissue (Kelly, 1990). In the present paper the isolation and characterization of a SCBP from Drosophila melanogaster is described, which occurs in two isoforms and is predominantly present in the muscle tissue of the thorax. Similarities to calmodulin, the protein obtained by Kelly (1990), and that we also demonstrate in muscles of Calliphora erythrocephala are discussed. S o m e of the results have already been presented at the XIXth European Conference on Muscle Contraction and Cell Motility (Kiehl and D'Haese, 1991).
475
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E. KIEHL and J. D'HAESE MATERIALS AND METHODS
Materials Drosophila melanogaster and Calliphora erythrocephala were provided by the Institut f/Jr Genetik and by the Institut fiir Zoologie of the Universit/it Diisseldorf, respectively. Except for mutations affecting muscles, various genotypes of Drosophila melanogaster were used for bulk preparations of SCBP from whole fruit flies. Oregon R wildtype flies were used for all other preparations.
SCBP-preparation from whole and dissected flies Fifteen to forty g Drosophila flies were ground to a powder in liquid nitrogen. The powder was extracted with five volumes (v/w) of SCBP-Ex (25 mM Tris-HCl pH 7.5, 1 mM 2-mercaptoethanol, 0.5 mM PMSF, 5/~ M CaC12) and insoluble material was removed by centrifugation (20000g, 30 min, 4°C). The following acid treatment, ammonium sulphate-fractionation, heat-treatment (60°C) and gel filtration chromatography on Ultrogel AcA 54 (PharmaciaLKB) were performed as described by Huch et al. (1988). Fractions of the gel filtration chromatography in the molecular mass range of 20 kDa (mw 20,000-fraction) were pooled and concentrated by ultrafiltration using a YM5 membrane (Amicon, cut-off 5 kDa). Further purification was achieved by ionic exchange (DEAE-cellulose DE 52, Whatman) and hydrophobic interaction chromatography (phenyl-Sepharose, Pharmacia-LKB). Phenyl-Sepharose chromatography was performed in a 2.5 × 13 cm column equilibrated with 50 mM Tris-HC1 pH 7.5, 0.1 mM CaCI2, 1 mM 2-mercaptoethanol, 0.5 mM PMSF. 8.3 mg of the m w 20,000-fraction dialysed against the same buffer were applied. After washing, the column was eluted stepwise with elution-buffer A (50 mM Tris-HC1 pH 7.5, 0.1 mM CaC12, 1 mM 2-mercaptoethanol, 0.5M NaC1, 0.5mM PMSF), elution-buffer B (50 mM Tris-HCl pH 7.5, I mM EGTA, 1 mM 2-mercaptoethanol, 0.5 mM PMSF), water and 8 M urea. For purification on DEAE-cellulose the m w 20,000fraction in DEAE-buffer (15 mM Tris-HC1 pH 7.5, 1 mM 2-mercaptoethanol, 5 # M CaCI2) was applied to the column (1.6 x 6 cm) equilibrated with the same buffer. Proteins were eluted with a linear gradient of 0-300 mM NaC1 in DEAEbuffer (2 x 100 ml). Drosophila and Calliphora flies were dissected with microscissors into head, thorax and abdomen. The parts were collected separately in SCBP-Ex (150-300 #1 for about 200 heads, thoraces or abdomina) and homogenized in a 1 ml glass/glass homogenizer (Pierce). The homogenates were centrifuged in an Eppendorf-type centrifuge (13,000rpm, 20 min), the supernatant (crude extract) was immediately incubated for PAGE or was further fractionated as described above to obtain a 60°C supernatant (crude preparation). Individual muscles and the thoracic ganglion from Drosophila and Calliphora thoraces were prepared in 50% ethanol in H20. Preparation in 50% ethanol was necessary as the tissues were too soft in Ringer solution (184 mM KC1, 46 mM NaCI, 30 mM CaC12, 10 mM Tris-HC1 pH 7.2) to be handled. Ten to twenty Drosophila-organs were collected in l ml SCBP-Ex, homogenized in a 1 ml glass/glass homogenizer (Pierce) and centrifuged (13,000rpm, Eppendorftype centrifuge, 45 min). The supernatant (crude extract) was concentrated to 50/~1 with microconcentrators (Centricon 10, Amicon). In the case of Calliphora 15-20 organs were collected in 700 #1 SCBP-Ex and also processed up to the 60°C supernatant as described above. Concentration was omitted. In addition to the thoracic ganglion the following muscles were collected for a crude preparation: flight control muscles (including basalar muscles, muscles of third axillary, muscles of first axillary), power muscles (dorsal median muscles, tergosternal muscles, tergal remotors of coxa, lateral oblique dorsal muscles), leg muscles (intracoxal
depressor, levator, sternal promotor, sternal remotor of all six legs), extracoxal depressor. For orientation and terminology of Drosophila muscles the detailed description of Miller (1965), and for preparation of Calliphora muscles the description of Heide (1971), was used.
Gel electrophoresis and Western blotting SDS-polyacrylamide gel electrophoresis (SDS-PAGE) was performed according to Laemmli (1970) on 0.75 mm thick slab gels using the Midget electrophoresis system (Pharmacia LKB). The gels were stained with Coomassie Brilliant Blue R250 by a procedure adopted from Heukeshoven and Dernick (1988) and in some cases poststained with silver according to Blum et al. (1987). Molecular mass standards were phosphorylase B (92.5 kDa), bovine serum albumin (67 kDa), egg albumin (45 kDa), carbonic anhydrase (29kDa), soybean trypsin inhibitor (21 kDa), cytochrome c (12.5 kDa) and bovine lung trypsin inhibitor (6.5 kDa). For electroblotting the gels were equilibrated in 25 mM Tris, 192raM glycine, 20% methanol for 15min and transferred onto 0.2 or 0.45 t~m nitrocellulose membranes (Schleicher and Schuell) with a semi-dry blot system. Protein bands were reversibly visualized by staining with Ponceau S (Sigma). In urea-polyacrylamide gel electrophoresis (urea-PAGE) a calcium dependent change in the electrophoretic mobility of a protein, which can be considered as a qualitative hint for the ability of a protein to bind calcium (Walsh et al., 1984; McDonald and Walsh, 1985), is much more pronounced than in SDS-PAGE. Urea-PAGE was performed essentially as described by Huch et al. (1988). Protein samples were equilibrated in 50 mM carbonate buffer pH 8.0, I mM CaC12 for 20min on ice and prepared for urea-PAGE by adding the same volume of 4 M urea, 33.33% glycerol, 0.006% Bromphenol Blue, 1 mM CaC12. Different amounts of EGTA were added to the samples.
45Ca2+-overlay The 45Ca2+-overlay technique of Maruyama et al. (1984) was used as an identification method for calcium-binding proteins after urea-PAGE. Western blots were washed three times in overlay buffer (60 mM KC1, 5 mM MgC12, 10 mM imidazole-HCl pH 6.9) for 20rain and incubated with 45Ca2+ (7400 Bq/ml in overlay buffer) for 10 min. After a short wash in 50% ethanol (1-5 min) the membranes were dried between two layers of filter paper. Ca2+-binding proteins were identified by exposing the processed nitrocellulose sheets to an X-ray film (Fuji) at -80°C.
Protein assay Protein concentrations were estimated by the method of Bradford (1976) using the Bio-Rad Protein Assay and bovine serum albumin as a standard. RESULTS
Preparation o f S C B P from whole flies The s t a n d a r d isolation procedure is summarized in Fig. 1. Centrifugation of the h o m o g e n a t e results in a n a p p a r e n t loss o f contractile proteins (Fig. 2B, lanes 1 a n d 2; c o m p a r e also Fig. 4, lanes 5-7). Already in the 60°C s u p e r n a t a n t a considerable e n r i c h m e n t of a 24 k D a protein can be observed (Fig. 2B, lane 3). The fractions pooled after gel filtration on A c A 54 are indicated in the elution profile in Fig. 2A. This mw 20,000-fraction already c o n t a i n s the 24 k D a protein as a m a i n c o m p o n e n t (Fig. 2B, lane 4). Phenyl-Sepharose c h r o m a t o g r a p h y of the mw 20,000 fraction did n o t result in a m a r k e d purification. Nearly all proteins did n o t b i n d to the hydro-
477
SCBP from fly muscles
Evidence for Ca2+-binding
Purification-Scheme fraction
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protein amount
homogenisation in S/BP-Ex
[fly-homogenate I centrifugafion Icrude -extract I
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Fig. 1. Purification scheme. phobic matrix and eluted with the column buffer (Fig. 2B, lane 5). Purified Drosophila-SCBP was obtained by ionic exchange chromatography on DEAE-cellulose, where two SCBP containing peaks were eluted at about 160 mM NaCI (Fig. 2B, lane 6). About 5 mg of pure SCBP were obtained from 35 g of fruitflies as starting material, which corresponds to a concentration of about 130 mg SCBP per 1 kg flies.
Ca:+-incubated proteins of the mw 20,000fraction supplemented with increasing amounts of the Ca:+-complexing agent EGTA were separated in urea-PAGE. Figure 3A shows that there are two bands in the m, 20,000-fraction--"Cal" and " C a 2 " - - , which reveal a calcium-dependent mobility shift to form a more diffuse band, which can be subdivided into two smaller ones, " E G I " and "EG2". The calcium-binding ability of these proteins was demonstrated by 45Ca2+-overlay after a mobility shift experiment (Fig. 3A, lanes 5-8). Shifting bands showed Ca2÷-binding after autoradiography. The proteins in the mw 20,000-fraction, which showed a mobility shift in urea-PAGE, the bands Cal, Ca2, EG1 and EG2, were cut out of the gel and re-electrophoresed in SDS-PAGE. The four bands of the urea-PAGE comigrate with the mw 24,000 protein of the m w 20,000-fraction (Fig. 3B). These results suggest that the m, 24,000-protein belongs to the SCBP and that it probably occurs in two isoforms.
Identification of the tissue from which the SCBP originates For the identification of the origin of SCBP, fruitflies were separated into head, thorax and abdomen and a crude preparation was carried out separately up to the 60°C-supernatant. SCBP was abundant only in the thorax, in extracts of heads and abdomina no bands in the m, 24,000-range could be detected (Fig. 4, lanes 1, 2 and 5). This was confirmed by urea-PAGE, because only the thorax contained a significant amount of a protein which showed a Ca2+-dependent mobility shift (Fig. 4, lanes 8-13). In the presence of EGTA, the SCBP appears as a diffuse band in urea-PAGE, especially when crude extracts
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92500 D-67000 /~5000 29000
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10 20 30 40 50 60 70 Fraction Fig. 2. Elution profile of the AcA 54 gel filtration chromatography (A). Fractions pooled as the rn, 20,000-fraction are indicated by shading. Chromatography was performed in 5 p M CaCI2, l mM 2-mercaptoethanol, 50 mM NH4HGO 3 pH 8.0 on a 1.6 x 85 cm column with a flow rate of 0.25 m]. rain -I. 30 mg of Drosophila crude preparation were applied to the column. SDS-PAGE of various purification steps of SCBP from Drosophila (B). [l] homogenate; [2] crude extract; [3] 60°C supernatant; [4] m, 20,000-fraction; [5] unbound fraction o£ phenyl-sepharose chromatography; [6] purified SCBP after DEAE-chromatography. The wedges mark the positions of the molecular mass markers, the arrow indicates the position of the SCBP-band.
6
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E. KIEHLand J. D'HAESE
PAGE. From the beginning there is only one band with a Ca2+-dependent mobility shift. This argues against a modification that could occur during the preparation procedure and is in favour of the real existence of two isoforms. As the fly thorax consists predominantly of muscles, it was reasonable to assume that muscles are the tissue the SCBP originates from. This was tested laiD-by preparing crude extracts of power muscles, leg Ca2=-.muscles and the extracoxal depressor muscle. In E51,,,-addition crude extracts were prepared from the thoEG2"" racic ganglion, because it might also contain significant amounts of SCBP. SDS-PAGE of these extracts clearly demonstrate that the extracoxal depressor and the leg muscles are the main sources of the SCBP. In extracts of the power muscles and the thoracic ganglion a protein band corresponding to 1 10 50 0 0 10 0 10 the 24 kDa-SCBP is not detectable (Fig. 6). To test the idea, that certain muscles are the source of the SCBP in flies, extracts of Calliphora erythroB M S [al Ca2 E51 E52 cephala were examined for comparison. Only in homogenates and crude extracts of the thorax and the 92500 = extracoxal depressor do two protein bands appear with a calcium-dependent mobility shift in urea67000,.-PAGE, a pronounced one with low and a faint one with higher electrophoretic mobility (Fig. 5, lanes 7 ~5000,.-and 9). In a 45Ca2+-overlay the slower protein bound most of the radioactive calcium (not shown). Re-electrophoresed in SDS-PAGE the proteins comigrate with the mw 24,000-protein of Drosophila. They also 29000 ~-comigrate with a protein band present in extracts of Calliphora thoraces and certain thoracic muscles (Fig. 5). The presence of SCBP in high amounts could additionally be demonstrated for the flight control muscle. On the other hand, it was impossible to 21000 ==" identify this protein in extracts of heads, abdomina, power muscles and thoracic ganglion of Calliphora either with urea-PAGE or with SDS-PAGE. Figure 6 12500==-shows the identical distribution of the mw 24,000-protein in Drosophila and Calliphora. Thus it seems Fig. 3. Evidence for calcium-binding.Urea-PAGE of the mw 20,000-fraction (A): [1-4] m~ 20,000-fractionincubated with permissible to conclude that SCBP in flies is predom1 mM Ca2÷ and electrophoretically separated after addition inantly present in distinct thoracic muscles, at least of EGTA to the indicated final concentration (in raM). in the extracoxal depressor, the leg muscles and the There is a clear difference in the electrophoretic mobility of flight control muscles. Ca2+-loaded (Cal, Ca2) and Ca2+-depleted protein (EG1, EG2). Due to the high EGTA-concentration in the last but DISCUSSION one sample, EGTA diffused into the last lane, thus causing half of the protein to change electrophoretic mobility. [5,6] We have isolated calcium-binding proteins with Electrotransfer of proteins of the mw20,000-fraction after a a molecular mass of 24kDa from Drosophila mobility shift experiment (Ponceau S stained). [7,8] Autoradiography of [5,6] after 45Ca2+-overlay.SDS-PAGE of the melanogaster and Calliphora erythrocephala. The excised bands Cal, Ca2, EG1, and EG2 from the urea- properties of the proteins resemble those of SCBP PAGE (B). M: molecular mass markers; S: mW20,000-frac- from invertebrate muscle tissue. They are abundant tion; the molecular mass markers are indicated by wedges, in muscles and not detectable in abdomina and heads, position of the SCBP-band is marked by the arrow. The two where muscles are less predominant. From Figs 4 and mobility shifting bands in urea-PAGE Cal and Ca2 and 6 it can be inferred that the proteins are present in their Ca2+-free counterparts EG1 and EG2 comigrate with high amounts in the extracoxal depressor, the flight the mw 24,000-protein (SCBP) of the mw 20,000-fraction. control muscles and the leg muscles but not in the power muscles. Neural tissue or the gonads apparently do not contain the proteins. Although Drosophila-calmodulin has an unusual were applied. The ability to bind Ca 2+ is not lost (see high molecular mass of 20 kDa (calculated according Fig. 3A, lanes 6 and 8). In contrast to the extract of whole flies, the thorax to Smith et al., 1987), which could be difficult to extract contains only one mobility shifting band distinguish from the described 24 kDa-proteins in (Fig. 4, lanes 10 and 11). Therefore, every step of the SDS-PAGE, and the CN-PDE activation assay as an preparation procedure up to the 60°C-supernatant identification method for calmodulin has not been was examined for mobility shifting bands in urea- carried out, the present experimental data for the
A
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SCBP from fly muscles
1
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479
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92500D'-
67000ZD" 45000D,--
29000==" 21000 =="12500 = . . Fig. 4. Localization of SCBP in Drosophila. Separate preparation of head, thorax and abdomen. According to SDS-PAGE [1-7], only the thorax contains a protein with a molecular mass of 24,000 which shows a calcium dependent mobility shift in urea-PAGE [8-13]. For comparison, Drosophila actomyosin was also electrophoresed. [1] head crude extract; [2] abdomen crude extract; [3] purified SCBP; [4] thorax 60°C-supernatant; [5] thorax crude extract; [6] thorax homogenate; [7] Drosophila actomyosin (MHC myosin heavy chain; A actin; Ltl, Lfl, Lf2, Lf3 isoforms of myosin light chains according to Takano-Ohmura et aL (1983); t: synchronous muscles, f: asynchronous muscles). The wedges mark the positions of the molecular mass markers, the arrow points to the SCBP-band. [8-13] urea-PAGE of [8,9] head crude extract; [10,11] thorax crude extract; [12,13] abdomen crude extract with 1 mM Ca 2+ [8,10,12] or 10mM EGTA [9,11,13].
Drosophila protein suggest that it is different from caimodulin for a n u m b e r of reasons. A typical source of calmodulin is neural tissue: Doyle et al. (1990) succeeded in isolating several calmodulin-clones from a head-specific c D N A - b a n k of Drosophila. This argues for the existence of calmodulin in the Drosophila head, likely the brain. In extracts of the head and the thoracic ganglion we could not observe a SCBP-band. In contrast to calmodulin, the Drosophila protein did not bind to phenyl-Sepharose and the failure to bind to this matrix is regarded as a characteristic feature of SCBP (Gopalakrishna and Anderson, 1982; Cox, 1989). Furthermore, the
uv-spectrum of Drosophila-SCBP indicates the presence of tryptophan (not shown), which is not found in calmodulin. The regulatory light chain of earthworm myosin also contains an EF-hand calcium-binding domain, which is identical with the corresponding myosin light chain LC2 from Drosophila, and binds 45Ca2+ as shown by the 45Ca2+-overlay technique (Carlhoff, 1988). The possibility that myosin light chains contaminated the fractions tested can be excluded, because the main structural muscle proteins present in the homogenate are effectively removed by the first centrifugation (Fig. 4).
92500m,'67000D" 45000D'-
29000
21000
12500 Fig. 5. SCBP in Calliphora. SDS-PAGE of [1] thorax crude extract; [2] extracoxal depressor homogenate; [3] flight control muscle crude extract; [4] purified Drosophila SCBP; [5] upper band and [6] lower band of the two mobility shifting bands from lane 7 reelectrophoresed; urea-PAGE of [7,8] extracoxal depressor homogenate; [9,10] thorax crude extract with 1 mM Ca 2+ (7,9) and I0 mM EGTA (8,10). Mobility shifting proteins are only found in extracts of the thorax and the extracoxal depressor. In SDS-PAGE they comigrate with the Drosophila SCBP and a mw 24,000 protein only found in extracts of muscles of
Calliphora.
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E. KIEHLand J. D'HArSE
thorax CatIiphora 1
2
extracoxa{ depressor l:alliphora Orosophita
Drosophita 3
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1
3
92500 ~67000 ~-
2
3
1
2
•
92500~" 67000 ~-
45000 ~-
45000"-
29000 ~"
29000
21000 ='-
21000
12500 = "
12500 D.-
thoracic ganglion Cattiphora Drosophita 1
2
3
1
power muscles CatIiphora Drosophila 1 2 3 1 2
2
92500~" 67000 ~-
92500~.. 67000~.-
&5000~"
45000"
29000 ~ "
29000 ~"
21000 " -
21000
12500
12500 " "
Fig. 6. Comparison of Calliphora and Drosophila. SDS-PAGE of different organs of Drosophila and Calliphora. [1] homogenate; [2] crude extract; [3] 60°C-supernatant; [*] crude extract of Drosophila leg muscles. In Calliphora as well as Drosophila, SCBP is present in extracts of thorax, extracoxal depressor and leg muscles (only shown for Drosophila), but is lacking in the thoracic ganglion and power muscles. The protein isolated from Drosophila melanogaster can therefore be considered as a calcium-binding protein clearly different from calmodulin and myosin light chains. The same is probably true for the Calliphora-protein, because it exhibits identical properties as tested so far. In different muscles of various organisms a correlation has been found between the amount of parvalbumin or SCBP and the contraction speed of the muscles (Gerday, 1988). Within the thorax the power muscles are the prominent organs. Though these muscles are fast contracting, we were unable to detect SCBP in their extracts. An explanation could be that SCBP as a soluble relaxation factor is not of functional significance, as the contraction-relaxation cycle of these muscles is independent of a fast release and removal of calcium (Riiegg, 1988). In contrast, flight control muscles, leg muscles and the extracoxal depressor muscle with a normal way of activation may depend on the presence of SCBP for fast restoring activability.
In extracts of whole Drosophila-flies always two mobility shifting bands have been observed in ureaPAGE. The interpretation that there are two isoforms of SCBP in Drosophila is complicated by the fact that, in extracts of thoraces, only one shifting band can be found. As the thorax has been proven as the main source of SCBP, it can be excluded that one of the two isoforms originates from head or abdomen. It is therefore assumed that the two isoforms exist in a population of flies, but not necessarily within a single strain. For the preparation of Drosophila-SCBP from whole flies a mixture of flies of different genotypes was used, whereas the SCBP fractions from dissected flies and single organs were prepared from a single Oregon R laboratory strain. Extracts of Calliphora thoraces show a strong and a weak mobility shifting band in urea-PAGE. This can also be explained by the existence of two isoforms, but with a different frequency of the two alleles in the population. This opinion is supported by the recent finding that in five of six crude extracts from thoraces
SCBP from fly muscles of different Drosophila strains two mobility shifting bands and, in one extract, one mobility shifting band were observed. Recently, Kelly (1990) isolated a Ca2+-binding protein from Drosophila with properties very similar to those described in this paper. The main difference between the two proteins is their distribution in the fly: Kelly detected the calcium-binding protein in extracts of various organs of the fly--predominantly in head extracts--but not in the muscles. Furthermore, for preparation Kelly used a phenothiazine chromatography, in which the protein bound to the matrix. As phenothiazine belongs to the hydrophobic matrices to which SCBP do not bind (Cox, 1989; Charbonneau and Cormier, 1979; Jamieson and Vanaman, 1979), this argues for differences between both proteins. It would be interesting, to compare directly the two proteins to decide whether they are really different or not.
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