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Microheterogeneity of desmin in the electric organ and dorsal muscle of the electric eel Electrophorus electricus
Camp. ~iochem.
Pergamon
Physiol. Vol. I I IA, No. 3. pp. 345-350. 1995 Copyright Q 1995 Elsevier Science Ltd
Printed in Great Britain. All rights reserved 0300-9629/95%9.50
+ 0.00
Microheterogeneity of desmin in the electric organ and dorsal muscle of the electric eel Electrophorus electricus M. C. R. Cordeiro,* V. Moura Neto,* M. Benchimol,* M. V. C. Faria_F and C. Chagas* *Institute de Biofisica Carlos Chagas Filho, Centro de Ciencias da Saude, Universidade Federal do Rio de Janeiro, Cidade Universitaria, 21949-900, Rio de Janeiro-RJ, Brazil; and tInstituto de Biologia, Universidade do Estado do Rio de Janeiro-RJ, Brazil Two-dimensional gel electrophoresis of purified desmin from the electric organ of Electrophorus efectricus exhibited five isoforms as opposed to four from the dorsal muscle of the eel. Differences in isoforms may indicate different degrees of desmin phosphorylation in these tissues. The similarities detected in the two less acidic isodesmin from the electric organ and from the dorsal muscle of Electrophorus electricus suggest that the electric organ has conserved some characteristics seen in the dorsal muscle, favoring previous suggestions that considered the electric tissue as a differentiated form of striated muscle. Key words: Intermediate filaments; Electrophorus electricus; Electric organ; Dorsal Microheterogeneity of desmin; Phosphorylation; Isodesmins; IEF; 2D-Gel.
muscle;
Comp. Biochem. Physiol. 1I lA, 345-350, 1995.
Introduction Electrophorus electricus, a freshwater electric eel, has three electric organs: main organ, Hunter and Sachs. These organs are composed of an association of electrocytes or electroplaques. These multinucleated cellular units are highly specialized and polarized, and are disposed along the rostro-caudal axis of the eel. Indeed, the rostra1 non-innervated face of the electrocyte is a Na+ , K+ ATPase enriched membrane, whereas the acetylcholine receptors (ACh-R) are associated with the caudal innervated cellular surface (Keynes and MartinsFerreira, 1953; Olsen et al., 1977). The characteristic polarity of eiectric fish electrocytes has been associated with the physiCorrespondence
lo: Dr Vivaldo Moura Neto, Instituto de Biofisica Carlos Chagas Filho, Centro de Ciincias da Satide, Universidade Federal do Rio de Janeiro, Cidade Universitkia, 21949-900, Rio de Janeiro-RJ, Brazil. Tel. (55 21) 590 3329; Fax (55 21) 280 8193. Received 31 March 1994; revised 5 January 1995; accepted 10 January 1995.
ology of bioelectrogenesis (Chagas and Paes de Carvalho, 1961). The electric discharge is triggered by a synchronized nervous impulse generated at the medulla and transmitted through a nerve network which ends in synapses on only one side of the electrocyte, the caudal surface. There is some evidence suggesting that the electric organs may be considered as a differentiated form of striated muscle. First, numerous invaginations of the electrocyte surface might represent a remnant of the muscle T system, as described by Mathewson et al. (1961) for Electrophorus, and by Srivastava and Baillet-Derbin (1973) for Eigenmania virescens. Second, in the electric tissue of young Electrophorus electricus, myofibrils were observed to be connected by structures that resembled Z-lines (Esquibel et al., 1971), although these structures were absent in the adult fish. Other studies on the fine structure of electrocytes also suggested their striated muscular origin (Machado et al., 1976). 345
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Supporting this proposal, we have shown the presence of two cytoskeletal components in the electric tissue of Electrophorus electricus: a typical striated muscle-alpha-actin (Ayres Sa et al., 1991), and desmin, an intermediate filament (IF) protein (Costa et al., 1986) that is only expressed in skeletal, cardiac, and smooth muscle cells (Bennett et al., 1979). In the present communication, in an attempt to further characterize similarities between the electric organ and striated muscle from electric eel E. electricus, we compare desmin microheterogeneity in these two tissues, using chicken gizzard desmin as a reference.
Material and Methods Desmin preparation
Either dorsal muscle, the main electric organ of Electrophorus electricus or chicken gizzard was minced and homogenized in 40 mM imidazole-HCl, pH 6.9, 0.6 M KCl, 1 mM EGTA, 1 mM 2-mercaptoethanol and 0.5% Triton X100. The purification steps were performed according to Geisler and Weber (1980) with a slight modification (Costa et al., 1986). The ethanol-precipitated protein fractions were redissolved in urea buffer (6 M urea, 10 mM sodium phosphate, pH 7.5, 5 mM EGTA, 0.1% 2-mercaptoethanol) and analyzed by IEF or 2D electrophoresis. Protein content was measured according to Bradford (1976). Electrophoresis
Two-dimensional (2D) polyacrylamide gel electrophoresis (PAGE) was performed according to O’Farrell (1975). Isoelectric focusing (IEF; first dimension), was run in cylindrical gels (0.25 x 13 cm) in the presence of 9.6 M urea and 2% (w/v) LKB ampholines (mixtures of 5-8 and 3.5-10 in a 4: 1 ratio). Isoelectric focusing was carried out initially at 400 V for 16 hr and then at 800 V for 2 hr. The second dimension was run in a 10% polyacrylamide gel containing sodium dodecyl sulfate (SDS). Desmin analysis by isoelectric focusing was performed as described elsewhere (Moura Neto et al., 1983). Gels were stained with Coomassie Brilliant Blue. Phospholabeling of desmin
The main electric organ of Electrophorus electricus was minced and homogenized in Hepes buffer (20mM Hepes, pH 7.2; 10 mM magnesium acetate; 0.3 mM EGTA). An amount of this homogenate corresponding to 100 pg of protein was then mixed with 5 PCi of (gamma-32P) ATP. After 5 min at room temperature, the phosphorylation reaction was
stopped by adding an equal volume of lysis buffer (O’Farrell, 1975). Labeled samples were immediately run in the 2D electrophoresis. Autoradiograms were obtained by exposing X-ray XKl Kodak films to vacuum-dried gels, for the appropriate time.
Results Isodesmin from the electric organ and dorsal muscle of the eel
Purified desmin preparations from both organs were analyzed by two-dimensional gel electrophoresis. As shown in Fig. la, five desmin isoforms were detected in the electric organ of the eel, as previously described (Costa et al., 1988). However, a parallel run of the dorsal muscle desmin revealed only four isoforms (Fig. 1b). Chicken gizzard desmin (Fig. lc), used by Geisler and Weber (1980) to establish the purification method, was run here as a reference standard and showed, as expected, two isoforms and also a spot of actin contamination. This actin could not be easily seen by Coomassie Blue staining in preparation of eel tissues. However, its migration does not eliminate the real existence of more acidic isodesmin in these tissues. Actin, a 43-kDa protein, migrates faster than desmin in the second dimensional gel electrophoresis (Fig. lc). These results suggest that the characteristic isodesmin patterns of the tissues studied are related to the different isoelectric points of the various isoforms. This could be clearly seen in the comigration experiment where purified desmins were previously mixed and run in the 2D-electrophoresis system. Desmin from the electric organ was mixed with either desmin from the dorsal muscle (Fig. 2a) of the eel or chicken gizzard desmin (Fig. 2b). This last was also mixed with dorsal muscle desmin (Fig. 2~). Fig. 2 (a, b, c) confirms that, when compared with the avian muscle, desmin from the fish muscle and from the electric organ, both show more acidic and more basic variants than the gizzard desmin. Isoelectric focusing (IEF) of purljied desmin
Separation of differently charged isopeptides would be better performed by the IEF technique alone (Costa et al., 1988). When isodesmin from the three tissues was focused in an IEF gel, we could confirm the presence of five desmin isopeptides in the electric organ (Fig. 3Aa), four in the dorsal muscle (Fig. 3Ab) and only two in the avian muscle (Fig. 3Ac). A third peptide spot in this last gel, corresponds to an actin contamination, already visualized in the two dimensional electrophoresis (Fig. lc). Moreover, the five and four spots shown in Fig. 3Aa
Desmin
in Electrophorus electricus
and 3Ab, respectively, seem to be only isoforms of desmin, since, as shown in Fig. 1 (identical samples run in two dimensional gel), five and four desmin spots are clearly shown. If there is some actin contamination in IEF gel, it is not detectable by Coomassie Blue staining. Under
347
these conditions of IEF, differences in isoelectric points and in the relative abundance of isoforms present in these tissues could be clearly demonstrated. For a better visualization, Fig. 3B shows a schematic representation of these electrofocusing data.
Fig. 1. Two-dimensional gel electrophoresis of desmin stained with Coomassie Brilliant Blue. (a) Desmin from the main electric organ of the electric eel Electrophorm electricus; (b) desmin from the striated dorsal muscle of the electric eel; and (c) desmin from chicken gizzard. The small arrowheads indicate five isodesmins in (a), four in (b) and two in (c). The arrow in (c) indicates actin. Insert (a) shows a non-reduced photograph of the electric organ desmin to better visualize the five isodesmins.
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exhibited only a faint Coomassie Blue staining (Fig. 3Aa) is the most phosphorylated isoform, which is explained by its more acidic nature. The most basic desmin (dl) might be considered unphosphorylated, since the very faint labeling seen in this region seems to correspond to the usual background found in similar experiments. Taking into account that the sample run in Fig. 4 was not a purified preparation, but a crude homogenate, other phosphorylated proteins are evidently expected to appear in the autoradiogram.
Discussion
Fig. 2. Two-dimensional gel electrophoresis of pre-mixed samples of purified desmin. Only the desmin regions are shown. (a) Mixed desmins from the electric organ and from the dorsal muscle of the eel. Arrowheads indicate five electric organ isodesmins; (b) mixed desmins from the electric organ and from the chicken gizzard. Arrowheads indicate the first and fifth electric organ isodesmins; (c) mixed desmins from the dorsal muscle and from the chicken gizzard; arrowheads indicate the four dorsal muscle isodesmins. The gel was Coomassie Blue-stained. The lower arrowheads (in b and c) indicate actin contamination.
Isodesmins were named dl-d5 (from the most basic to the most acidic form). The two avian isodesmins are similarly abundant, and run in the same p1 region of d2 and d3 bands as those from fish organs. Furthermore, the isodesmins from the dorsal striated muscle show a more restrictive p1 range as compared with the larger p1 spectrum expressed by isodesmins from the electric tissue. The more acidic fish muscle isoforms are less stained than their counterparts in the electric organ, indicating comparatively lower concentrations than in this organ. Phosphorylation
of desmin
The endogenous kinase systems and cofactors found in the electric organ homogenate were used to produce phosphorylated isodesmin in the presence of (gamma-32P) ATP, after a short incubation at room temperature. An autoradiogram of a gel where radiolabeled homogenate proteins were separated by two dimensional technique is shown in Fig. 4. Desmin (d5) which
In this study we isolated desmin from the epaxial muscle and from the electric organ of Electrophorus electricus by the same procedures used in the purification of this protein from the chicken gizzard (Geisler and Weber, 1980). Desmin from these tissues had the same apparent molecular weight and focused in the same pH region of an IEF gel. However, different isoelectric variants were detected in these three tissues. It is well known that desmin, an intermediate filament protein, exhibits a high degree of tissue specificity (Lazarides, 1980). Its expression is only confined to muscle (Van de Klundert et al., 1993). The regulation of desmin expression is characterized by a developmental pattern. It is one of the first muscular proteins expressed in myotomes (Schaart et al., 1989) and heart muscle (Li et al., 1993). This protein is encoded by a single-copy gene (Capetanaki et al., 1984; Quax et al., 1985; Li et al., 1989) and one single mRNA is produced. However, desmin preparations from chick, duck and quail contain two variants (Lazarides and Balzer, 1978; O’Connor et al., 1979) of identical molecular weights but presenting different isoelectric points. We had already described five isovariants in the electric organ of Electrophorus electricus, which have a molecular weight of 5 1 kDa (Costa et al., 1988). In this report, with two dimensional gel analysis, we detected fine differences among isoelectric points of dorsal muscle and electric organ desmin. In spite of these fine differences, the most basic isoforms (dl and d2) from both muscular and electric tissues, keep an identity relative to the isoelectric point. In particular, dl isoforms from these tissues show identical electrophoretic properties. Although similarities in electrophoretic properties do not imply identical amino acid sequences of proteins, they are strong indicators that these proteins are very similar in their structures. They are, probably, the primary product of the desmin gene. The more basic dl
Desmin
in Electrophorus electricus
349
Fig. 3.(A) Isoelectric focusing gel electrophoresis (IEF) of desmin stained with Coomassie Brilliant Blue. Only the desmin regions are shown. (a) Desmin from the main electric organ of the electric eel Ekcfrophorus efectricus, (b) desmin from the striated dorsal muscle of the electric eel. and (c) desmin from the chicken gizzard. (B) A diagrammatic representation of the IEF pattern observed in Fig. 3A. Desmin variants are labeled dld5. The thickness of each isodesmin band represents the intensity of Coomassie Blue staining observed directly on the gel. (a) desmin of electric organ, (b) dorsal muscle and (c) chicken gizzard. The arrowhead in 3c indicates actin.
isoform from chicken gizzard is, however, more acidic than dl-desmins from the fish tissues, suggesting a different primary peptide structure. It has been proposed that the electric organ of the eel originated from the dorsal muscle of the eel (Mathewson et al., 1961). Our previous data showing the presence of a sarcomeric (striated) alpha-actin in the electric organ (Ayres Sa et al., 1991) the description of Z-line resembling
structures connected to myofibrils in electroplaques of young Electrophorus electricus (Esquibel et al., 1971) and other electron microscopy observations on the fine ultrastructure of the electroplaque (Machado et al., 1976) are in agreement with this postulate.The similarities of isodesmins from dorsal muscle and from the electric organ of E. electricus reported here might provide additional evidence for this origin.
Fig. 4. Autoradiogram of a two dimensional gel electrophoresis containing phospholabeled proteins from homogenate of the electric organ of the eel. The arrows indicate labeled isodesmins. Note the strongest labeling in the more acidic isoform (*). The isodesmins dl-d5 are indicated.
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Moreover, the IEF analysis demonstrated that the d3 and d4 isoforms from electric organ were more Coomassie Blue-stained than those from dorsal muscle (Fig. 4), also expressing higher concentrations. Indeed, the isoform d5 was generated only in the electric tissue. The isoelectric variants of desmin can be produced by post-translational modifications as well, as already described in other cytoskeletal proteins. For instance, glutamylation and acetylation generate tubulin isovariants (Edde et al., 1992). However, in the case of desmin, only phosphorylation has been implicated in posttranslational events (Eriksson et al., 1992; LeBoFerreiro et al., 1994). We verified that the eel desmin could be phosphorylated in uitro, as already demonstrated with chick desmin, through the CAMP-dependent desmin kinase (O’Connor et al., 1981). An upgrading phosphorylation of d3, d4 and d5 in the electric tissue would explain the increasing acidity of their isoelectric points. Finally, our data might suggest that the desmin from the electric organ has conserved some characteristics of the desmin from striated muscle, and contribute to previous postulations that consider the electric tissue of the eel as a differentiation of the striated muscle.
alpha-tubulin can enter the tyrosination/detyrosination cycle. Biochemistry 31, 403-410. Eriksson J. E., Opal P. and Goldman R. D. (1992) Intermediate filament dynamics. Curr. Opinion Cell Biol. 4, 99-104.
Esquibel M. A., Alonso I., Meyer H., de Oliveira Castro G. and Chagas C. (1971) Quelques aspects de I’histogtnese et de I’ontogenbse des organes Clectrique chez Electrophorus electricus. L. C.R. Acad. Sci. Paris 283, 196-199. Geisler N. and Weber K. (1980) Purification of desmins from chicken gizzard and hog stomach. Eur. J. Biochem. 111, 425433.
Keynes R. D. and Martins-Ferreira H. (1953) Membrane potentials in the electroplates of the electric eel. J. Physiol. 119,315-351.
Lazarides E. (1980) Intermediate filaments as mechanical integrator of cellular space. Nature 283, 249-256. Lazarides E. and Balzer D. R. (1978) Specificity of desmin to avian and mammalian muscle cells. CeN 14, 429438. LeHo-Ferreira L. R., Moussatcht N. and Moura Neto V. (1994) Rearrangement of intermediate filament network of BHK-21 cells infected with vaccinia virus. Arch. Virol. (in press). Li Z., Lihenbaum A., Butler-Browne G. and Paulin D. (1989) Human desmin coding gene: Complete nucleotide sequence, characterization and regulation of expression during myogenesis and development. Gene 78, 243-254. Li Z.. Marchand P.. Humbert J., Babinet C. and Pauhn D. (1993) Desmin sequence elements regulating skeletal muscle-specific expression in transgenic mice. Development 117, 947-959.
Machado R. D., de Souza W., Cotta Pereira G. and de Ohveira Castro G. (1976) On the fine structure of the electrocyte of Electrophorus electricus L. Cell Tiss. Res. 174, 355-366.
wish to thank Angela Maria Alves Langer for the excellent technical assistance, Celso Fatini for the photographs and Dr M. C. Mello for critical comments. This work was supported by grants from CNPq, FINEP, CAPES, CEPG-UFRJ and FAPERJ. Acknowledgements-We
References Ayres SB L., Moura Neto V., de Oliveira M. M. and Chagas C. (1991) Heterogeneity of purified actin in the electric organ of the electric eel Electrophorus electricus. J. exp. Zool. 257, 43-50.
Bennett G. S., Fellini S., Toyama Y. and Holtzer H. (1979) Redistribution of intermediate filaments subunits during skeletal myogenesis and maturation in vitro. J. CeN Biol. 82, 577-584.
Bradford M. M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anulyt. Biochem. 72, 248-254.
natn. Acad. Sci. U.S.A. 76, 819-823.
O’Connor C. M., Gard D. L. and Lazarides E. (1981) Phosphorylation of intermediate filament protein by AMPc-denendent orotein kinases. Cell 23. 1355143. O’Farrell P: H. (1975) High resolution two-dimensional electrophoresis of proteins. J. biol. Chem. 250,4007402 1. Olsen R. W.. Meunier J. C. and Channeux J. P. (1977) Progress in the purification of the ch;linergic receptor protein from the E. electricus by affinity chromatography. FEBS Lett 28, 96-100.
Capetanaki Y. G., Ngai J. and Lazarides E. (1984) Characterization and regulation in the expression of a gene coding for the intermediate filament protein desmin. Proc. natn. Acad. Sri. U.S.A. 81, 6909-6913. Chagas C. and Paes de Carvalho A. (eds) (1961) Bioelectrogenesis. Elsevier, Amsterdam. Costa M. L., de Oliveira M. M.. Alberti Jr 0.. Moura Neto V. and Chagas C. (1986) Caracterization de la desmine dans I’organe electrique de 1’Electrophorus electricus L. C.R. Seam.
Acad. Sri. 303, 547-550.
Costa M. L., Moura Neto V. and Chagas C. (1988) Desmin heterogeneity in the main electric organ of Eiectrophorus electri&.
Mathewson R., Wachtel A. and Grundfest H. (1961) Fine structure of electroplaques. In Bioelectrogenesis (Edited by Chagas C. and Paes de Carvalho A.), pp. 25-53. Elsevier, Amsterdam. Moura Neto V., Mallat M., Jeantet C. and Prochiantz A. (1983) Microheterogeneity of tubulin proteins in neuronal and alial cells from the mouse brain in culture. EMBO J. 2, 12331248. O’Connor C. M., Balzer Jr D. R. and Lazarides E. (1979) Phosphorylation of subunit proteins of intermediate filaments from chicken muscle and non-muscle cells. Proc.
Biochimie 70, 783-789.
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EddC B., Rossier J., L-eCaer J. P., PromC J. C., Desbruybres E., Gros F. and Denoulet P. (1992) Polyglutamylated
Quax W., Van den Broeck L., Vree Egberts W., Ramaekers F. and Bloemendal H. (1985) Characterization of the hamster desmin gene: expression and formation of desmin filaments in non-muscle cells after gene transfer. Cell 43, 327-338.
Schaart G., Vibahn C., Langmann H. and Ramalker F. (1989) Desmin and titin expression in early postimplantation mouse embryo. Development 107, 585-596. Srivastava C. B. L. and Baillet-Derbin C. (1973): Etude ultrastructurale de I’organe ilectrique d’un Poisson ilectrique a faible decharge: Eigenmania virescens. C.R. Acad. Sci., Paris 217, 2209-2212.
Van de Klundert F. A. J. M., Raats J. M. H. and Bloemendal H. (1993) Intermediate filaments: regulation of gene expression and assembly. Eur. J. Biochem. 214, 351-366.
Pergamon
Physiol. Vol. I I IA, No. 3. pp. 345-350. 1995 Copyright Q 1995 Elsevier Science Ltd
Printed in Great Britain. All rights reserved 0300-9629/95%9.50
+ 0.00
Microheterogeneity of desmin in the electric organ and dorsal muscle of the electric eel Electrophorus electricus M. C. R. Cordeiro,* V. Moura Neto,* M. Benchimol,* M. V. C. Faria_F and C. Chagas* *Institute de Biofisica Carlos Chagas Filho, Centro de Ciencias da Saude, Universidade Federal do Rio de Janeiro, Cidade Universitaria, 21949-900, Rio de Janeiro-RJ, Brazil; and tInstituto de Biologia, Universidade do Estado do Rio de Janeiro-RJ, Brazil Two-dimensional gel electrophoresis of purified desmin from the electric organ of Electrophorus efectricus exhibited five isoforms as opposed to four from the dorsal muscle of the eel. Differences in isoforms may indicate different degrees of desmin phosphorylation in these tissues. The similarities detected in the two less acidic isodesmin from the electric organ and from the dorsal muscle of Electrophorus electricus suggest that the electric organ has conserved some characteristics seen in the dorsal muscle, favoring previous suggestions that considered the electric tissue as a differentiated form of striated muscle. Key words: Intermediate filaments; Electrophorus electricus; Electric organ; Dorsal Microheterogeneity of desmin; Phosphorylation; Isodesmins; IEF; 2D-Gel.
muscle;
Comp. Biochem. Physiol. 1I lA, 345-350, 1995.
Introduction Electrophorus electricus, a freshwater electric eel, has three electric organs: main organ, Hunter and Sachs. These organs are composed of an association of electrocytes or electroplaques. These multinucleated cellular units are highly specialized and polarized, and are disposed along the rostro-caudal axis of the eel. Indeed, the rostra1 non-innervated face of the electrocyte is a Na+ , K+ ATPase enriched membrane, whereas the acetylcholine receptors (ACh-R) are associated with the caudal innervated cellular surface (Keynes and MartinsFerreira, 1953; Olsen et al., 1977). The characteristic polarity of eiectric fish electrocytes has been associated with the physiCorrespondence
lo: Dr Vivaldo Moura Neto, Instituto de Biofisica Carlos Chagas Filho, Centro de Ciincias da Satide, Universidade Federal do Rio de Janeiro, Cidade Universitkia, 21949-900, Rio de Janeiro-RJ, Brazil. Tel. (55 21) 590 3329; Fax (55 21) 280 8193. Received 31 March 1994; revised 5 January 1995; accepted 10 January 1995.
ology of bioelectrogenesis (Chagas and Paes de Carvalho, 1961). The electric discharge is triggered by a synchronized nervous impulse generated at the medulla and transmitted through a nerve network which ends in synapses on only one side of the electrocyte, the caudal surface. There is some evidence suggesting that the electric organs may be considered as a differentiated form of striated muscle. First, numerous invaginations of the electrocyte surface might represent a remnant of the muscle T system, as described by Mathewson et al. (1961) for Electrophorus, and by Srivastava and Baillet-Derbin (1973) for Eigenmania virescens. Second, in the electric tissue of young Electrophorus electricus, myofibrils were observed to be connected by structures that resembled Z-lines (Esquibel et al., 1971), although these structures were absent in the adult fish. Other studies on the fine structure of electrocytes also suggested their striated muscular origin (Machado et al., 1976). 345
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Supporting this proposal, we have shown the presence of two cytoskeletal components in the electric tissue of Electrophorus electricus: a typical striated muscle-alpha-actin (Ayres Sa et al., 1991), and desmin, an intermediate filament (IF) protein (Costa et al., 1986) that is only expressed in skeletal, cardiac, and smooth muscle cells (Bennett et al., 1979). In the present communication, in an attempt to further characterize similarities between the electric organ and striated muscle from electric eel E. electricus, we compare desmin microheterogeneity in these two tissues, using chicken gizzard desmin as a reference.
Material and Methods Desmin preparation
Either dorsal muscle, the main electric organ of Electrophorus electricus or chicken gizzard was minced and homogenized in 40 mM imidazole-HCl, pH 6.9, 0.6 M KCl, 1 mM EGTA, 1 mM 2-mercaptoethanol and 0.5% Triton X100. The purification steps were performed according to Geisler and Weber (1980) with a slight modification (Costa et al., 1986). The ethanol-precipitated protein fractions were redissolved in urea buffer (6 M urea, 10 mM sodium phosphate, pH 7.5, 5 mM EGTA, 0.1% 2-mercaptoethanol) and analyzed by IEF or 2D electrophoresis. Protein content was measured according to Bradford (1976). Electrophoresis
Two-dimensional (2D) polyacrylamide gel electrophoresis (PAGE) was performed according to O’Farrell (1975). Isoelectric focusing (IEF; first dimension), was run in cylindrical gels (0.25 x 13 cm) in the presence of 9.6 M urea and 2% (w/v) LKB ampholines (mixtures of 5-8 and 3.5-10 in a 4: 1 ratio). Isoelectric focusing was carried out initially at 400 V for 16 hr and then at 800 V for 2 hr. The second dimension was run in a 10% polyacrylamide gel containing sodium dodecyl sulfate (SDS). Desmin analysis by isoelectric focusing was performed as described elsewhere (Moura Neto et al., 1983). Gels were stained with Coomassie Brilliant Blue. Phospholabeling of desmin
The main electric organ of Electrophorus electricus was minced and homogenized in Hepes buffer (20mM Hepes, pH 7.2; 10 mM magnesium acetate; 0.3 mM EGTA). An amount of this homogenate corresponding to 100 pg of protein was then mixed with 5 PCi of (gamma-32P) ATP. After 5 min at room temperature, the phosphorylation reaction was
stopped by adding an equal volume of lysis buffer (O’Farrell, 1975). Labeled samples were immediately run in the 2D electrophoresis. Autoradiograms were obtained by exposing X-ray XKl Kodak films to vacuum-dried gels, for the appropriate time.
Results Isodesmin from the electric organ and dorsal muscle of the eel
Purified desmin preparations from both organs were analyzed by two-dimensional gel electrophoresis. As shown in Fig. la, five desmin isoforms were detected in the electric organ of the eel, as previously described (Costa et al., 1988). However, a parallel run of the dorsal muscle desmin revealed only four isoforms (Fig. 1b). Chicken gizzard desmin (Fig. lc), used by Geisler and Weber (1980) to establish the purification method, was run here as a reference standard and showed, as expected, two isoforms and also a spot of actin contamination. This actin could not be easily seen by Coomassie Blue staining in preparation of eel tissues. However, its migration does not eliminate the real existence of more acidic isodesmin in these tissues. Actin, a 43-kDa protein, migrates faster than desmin in the second dimensional gel electrophoresis (Fig. lc). These results suggest that the characteristic isodesmin patterns of the tissues studied are related to the different isoelectric points of the various isoforms. This could be clearly seen in the comigration experiment where purified desmins were previously mixed and run in the 2D-electrophoresis system. Desmin from the electric organ was mixed with either desmin from the dorsal muscle (Fig. 2a) of the eel or chicken gizzard desmin (Fig. 2b). This last was also mixed with dorsal muscle desmin (Fig. 2~). Fig. 2 (a, b, c) confirms that, when compared with the avian muscle, desmin from the fish muscle and from the electric organ, both show more acidic and more basic variants than the gizzard desmin. Isoelectric focusing (IEF) of purljied desmin
Separation of differently charged isopeptides would be better performed by the IEF technique alone (Costa et al., 1988). When isodesmin from the three tissues was focused in an IEF gel, we could confirm the presence of five desmin isopeptides in the electric organ (Fig. 3Aa), four in the dorsal muscle (Fig. 3Ab) and only two in the avian muscle (Fig. 3Ac). A third peptide spot in this last gel, corresponds to an actin contamination, already visualized in the two dimensional electrophoresis (Fig. lc). Moreover, the five and four spots shown in Fig. 3Aa
Desmin
in Electrophorus electricus
and 3Ab, respectively, seem to be only isoforms of desmin, since, as shown in Fig. 1 (identical samples run in two dimensional gel), five and four desmin spots are clearly shown. If there is some actin contamination in IEF gel, it is not detectable by Coomassie Blue staining. Under
347
these conditions of IEF, differences in isoelectric points and in the relative abundance of isoforms present in these tissues could be clearly demonstrated. For a better visualization, Fig. 3B shows a schematic representation of these electrofocusing data.
Fig. 1. Two-dimensional gel electrophoresis of desmin stained with Coomassie Brilliant Blue. (a) Desmin from the main electric organ of the electric eel Electrophorm electricus; (b) desmin from the striated dorsal muscle of the electric eel; and (c) desmin from chicken gizzard. The small arrowheads indicate five isodesmins in (a), four in (b) and two in (c). The arrow in (c) indicates actin. Insert (a) shows a non-reduced photograph of the electric organ desmin to better visualize the five isodesmins.
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exhibited only a faint Coomassie Blue staining (Fig. 3Aa) is the most phosphorylated isoform, which is explained by its more acidic nature. The most basic desmin (dl) might be considered unphosphorylated, since the very faint labeling seen in this region seems to correspond to the usual background found in similar experiments. Taking into account that the sample run in Fig. 4 was not a purified preparation, but a crude homogenate, other phosphorylated proteins are evidently expected to appear in the autoradiogram.
Discussion
Fig. 2. Two-dimensional gel electrophoresis of pre-mixed samples of purified desmin. Only the desmin regions are shown. (a) Mixed desmins from the electric organ and from the dorsal muscle of the eel. Arrowheads indicate five electric organ isodesmins; (b) mixed desmins from the electric organ and from the chicken gizzard. Arrowheads indicate the first and fifth electric organ isodesmins; (c) mixed desmins from the dorsal muscle and from the chicken gizzard; arrowheads indicate the four dorsal muscle isodesmins. The gel was Coomassie Blue-stained. The lower arrowheads (in b and c) indicate actin contamination.
Isodesmins were named dl-d5 (from the most basic to the most acidic form). The two avian isodesmins are similarly abundant, and run in the same p1 region of d2 and d3 bands as those from fish organs. Furthermore, the isodesmins from the dorsal striated muscle show a more restrictive p1 range as compared with the larger p1 spectrum expressed by isodesmins from the electric tissue. The more acidic fish muscle isoforms are less stained than their counterparts in the electric organ, indicating comparatively lower concentrations than in this organ. Phosphorylation
of desmin
The endogenous kinase systems and cofactors found in the electric organ homogenate were used to produce phosphorylated isodesmin in the presence of (gamma-32P) ATP, after a short incubation at room temperature. An autoradiogram of a gel where radiolabeled homogenate proteins were separated by two dimensional technique is shown in Fig. 4. Desmin (d5) which
In this study we isolated desmin from the epaxial muscle and from the electric organ of Electrophorus electricus by the same procedures used in the purification of this protein from the chicken gizzard (Geisler and Weber, 1980). Desmin from these tissues had the same apparent molecular weight and focused in the same pH region of an IEF gel. However, different isoelectric variants were detected in these three tissues. It is well known that desmin, an intermediate filament protein, exhibits a high degree of tissue specificity (Lazarides, 1980). Its expression is only confined to muscle (Van de Klundert et al., 1993). The regulation of desmin expression is characterized by a developmental pattern. It is one of the first muscular proteins expressed in myotomes (Schaart et al., 1989) and heart muscle (Li et al., 1993). This protein is encoded by a single-copy gene (Capetanaki et al., 1984; Quax et al., 1985; Li et al., 1989) and one single mRNA is produced. However, desmin preparations from chick, duck and quail contain two variants (Lazarides and Balzer, 1978; O’Connor et al., 1979) of identical molecular weights but presenting different isoelectric points. We had already described five isovariants in the electric organ of Electrophorus electricus, which have a molecular weight of 5 1 kDa (Costa et al., 1988). In this report, with two dimensional gel analysis, we detected fine differences among isoelectric points of dorsal muscle and electric organ desmin. In spite of these fine differences, the most basic isoforms (dl and d2) from both muscular and electric tissues, keep an identity relative to the isoelectric point. In particular, dl isoforms from these tissues show identical electrophoretic properties. Although similarities in electrophoretic properties do not imply identical amino acid sequences of proteins, they are strong indicators that these proteins are very similar in their structures. They are, probably, the primary product of the desmin gene. The more basic dl
Desmin
in Electrophorus electricus
349
Fig. 3.(A) Isoelectric focusing gel electrophoresis (IEF) of desmin stained with Coomassie Brilliant Blue. Only the desmin regions are shown. (a) Desmin from the main electric organ of the electric eel Ekcfrophorus efectricus, (b) desmin from the striated dorsal muscle of the electric eel. and (c) desmin from the chicken gizzard. (B) A diagrammatic representation of the IEF pattern observed in Fig. 3A. Desmin variants are labeled dld5. The thickness of each isodesmin band represents the intensity of Coomassie Blue staining observed directly on the gel. (a) desmin of electric organ, (b) dorsal muscle and (c) chicken gizzard. The arrowhead in 3c indicates actin.
isoform from chicken gizzard is, however, more acidic than dl-desmins from the fish tissues, suggesting a different primary peptide structure. It has been proposed that the electric organ of the eel originated from the dorsal muscle of the eel (Mathewson et al., 1961). Our previous data showing the presence of a sarcomeric (striated) alpha-actin in the electric organ (Ayres Sa et al., 1991) the description of Z-line resembling
structures connected to myofibrils in electroplaques of young Electrophorus electricus (Esquibel et al., 1971) and other electron microscopy observations on the fine ultrastructure of the electroplaque (Machado et al., 1976) are in agreement with this postulate.The similarities of isodesmins from dorsal muscle and from the electric organ of E. electricus reported here might provide additional evidence for this origin.
Fig. 4. Autoradiogram of a two dimensional gel electrophoresis containing phospholabeled proteins from homogenate of the electric organ of the eel. The arrows indicate labeled isodesmins. Note the strongest labeling in the more acidic isoform (*). The isodesmins dl-d5 are indicated.
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M. C. R. Cordeiro Ed al.
Moreover, the IEF analysis demonstrated that the d3 and d4 isoforms from electric organ were more Coomassie Blue-stained than those from dorsal muscle (Fig. 4), also expressing higher concentrations. Indeed, the isoform d5 was generated only in the electric tissue. The isoelectric variants of desmin can be produced by post-translational modifications as well, as already described in other cytoskeletal proteins. For instance, glutamylation and acetylation generate tubulin isovariants (Edde et al., 1992). However, in the case of desmin, only phosphorylation has been implicated in posttranslational events (Eriksson et al., 1992; LeBoFerreiro et al., 1994). We verified that the eel desmin could be phosphorylated in uitro, as already demonstrated with chick desmin, through the CAMP-dependent desmin kinase (O’Connor et al., 1981). An upgrading phosphorylation of d3, d4 and d5 in the electric tissue would explain the increasing acidity of their isoelectric points. Finally, our data might suggest that the desmin from the electric organ has conserved some characteristics of the desmin from striated muscle, and contribute to previous postulations that consider the electric tissue of the eel as a differentiation of the striated muscle.
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wish to thank Angela Maria Alves Langer for the excellent technical assistance, Celso Fatini for the photographs and Dr M. C. Mello for critical comments. This work was supported by grants from CNPq, FINEP, CAPES, CEPG-UFRJ and FAPERJ. Acknowledgements-We
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