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Isolation and characterization of tropomyosin from fish muscle
Comp. Biochem. Physiol. Vol. 108B, No. 1, pp. 95-106, 1994
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Copyright © 1994 Elsevier Science Ltd Printed in Great Britain. All rights reserved 0305-0491/94 $6.00 + 0.00
Pergamon
Isolation and characterization of tropomyosin from fish muscle D. H. Heeley* and C. Hong Department of Biochemistry, Memorial University, St John's, Newfoundland, Canada, AIB 3X9
A comprehensive survey of tropomyosin from various fish myotomai muscles is reported. The fish tropomyosins were blocked at the N-terminus and, as expected, were found to be of similar amino acid composition, ~-helical content ( > 90% at 10°C) and molecular weight to other vertebrate striated muscle forms. The tropomyosins of salmonids and herring muscle were noticeably heterogeneous when assessed by 2D-PAGE. The distribution of isoforms was tissue-specific: slow muscle contained ~-type tropomyosin while fast muscle contained p-type tropomyosin. In other species (cod, haddock, wolf-fish and sharks) ~-type tropomyosins were present in both kinds of muscle but p-tropomyosin was absent. Key words: Tropomyosin; Fish muscle; Clupea harengus; Salvelinus alpinus; Salrno salar; Gadus morhua; Melanogramrnus aeglefinus; Urophycis; Pollachius virens; Sebastes; Hippoglossus hippoglossus ; Pseudopleuronectes americanus ; Xiphias gladius ; Anarchichas lupus; Isurus oxyrinchus ; Scomber scrobrus, Prionace glauca ; Raja radiata ; Petromyzon marinus.
Cornp. Biochem. Physiol. 108B, 95-106, 1994.
Introduction Fish constitute the largest and most diverse group of vertebrates. They have been recognized as ideal systems for studies in a number of areas including neurobiology, developmental biology and environmental biology (Powers, 1989). The spectacular musculature of fish also offers a number of advantages to the research of muscle contraction. Bony (teleost) fish contain a simple, and long-range, structural order of myofilament lattices which is well suited for X-ray diffraction studies (Harford and Squire, 1990). In addition, while most individual mammalian muscles are composed of an intermingled mixture of fast and slow fibres, these cell types are anatomically segregated in the fish myotome. In round-bodied fish, the slow (dark) muscle, which is used mostly for low-speed cruising, is confined to a seam along the lateral line running from the head to the caudal fin (Johnston et al., 1975; Johnston, 1980; Bone and Marshall, 1982). The more prevalent fast (light) muscle constitutes the remainder of the myotome and
provides the thrust for high-speed motion. Such a discreet separation of muscle types presents a unique opportunity to isolate and characterize fibre-specific forms of the contractile proteins. By and large, however, fish have been underutilized as sources of muscle tissue and muscle protein for biochemical studies. This study was initiated partly to survey some of the properties of contractile proteins from fish and also to investigate the usefulness of the separated muscle types in addressing the biochemical basis for muscle specialization. Here we report the isolation and characterization, from various species, of one of the components of the thin filament complex: tropomyosin.
M a t e r i a l s and M e t h o d s Preparation of tropomyosin Tropomyosin (TM) was purified from the trunk (myotomal) muscles of fish from the following groups: teleost (Arctic char Salvelinus alpinus, Atlantic salmon Salmo salar, Atlantic herring Clupea harengus, Atlantic mackerel Correspondence to: D. H. Heeley,Dept. of Biochemistry, Memorial University, St John's, Newfoundland, Scomber scrobrus, Atlantic cod Gadus morhua, haddock Melanogrammus aeglefinus, hake UroCanada, A1B3X9. Received 20 July 1993; accepted 22 September 1993. phycis, pollack Pollachius virens, ocean perch 95
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D. H. Heeleyand C. Hong
Sebastes, Atlantic halibut Hippoglossus hippoglossus, winter flounder Pseudopleuronectes americanus, swordfish Xiphias gladius, Atlantic wolf-fish Anarchichas lupus), cartilagenous (mako shark Isurus oxyrinchus, blue shark Prionace glauca, thorny skate Raja radiata), and jawless (sea lamprey Petromyzon marinus). Fish were obtained predominantly from commercial outlets and marine research vessels. The freshness of the samples corresponded to storage periods (on ice) varying from a few days up to a week. For species (e.g. salmon, char, herring and mackerel) where the slow (dark) muscle is present in appreciable levels (15-20% of total muscle mass), it was feasible to dissect this lateral line tissue from the more preponderant fast (light) muscle and isolate TM from each category of muscle. The fast and slow fibres of the lamprey are known to be intermingled (Teravainen, 1971; Bone, 1978) so in this instance TM was made on the mixture as it exists in the myotome. For all the other specimens, TM was purified from myotomal sections, composed of fast muscle with either a small amount of associated dark muscle or none at all. The muscle samples (100-1000 g) were initially minced through a medium-size plate and dehydrated with cold ethanol and acetone. TM contained within the high salt extracts from the dried fibres (10-50g) was then isoelectrically precipitated at pH 4.6 and salted out between 40 and 70% saturated (NH4) 2 SO4. After dialysis and lyophilization, the impure protein (100-400 mg) was then applied to a hydroxylapatite column (2.5 × 10-25 cm) equilibrated in 1 M NaC1, 70 mM sodium phosphate, 1 mM dithiothreitol, 0.01% (w/v) sodium azide pH 7.00, at room temperature and washed with at least two column volumes. The TM was then eluted isocratically with two-column volumes of buffer containing 250 mM sodium phosphate. The prepared material (50-300 mg) was shown by SDS-PAGE to be approximately 95% homogeneous.
Electrophoretic methods Electrophoresis was performed on a Bio-Rad mini-Protean II apparatus (Bio-Rad, Richmond, CA). Unless stated otherwise, all electropherograms were 0.75 mm thick and consisted of an acrylamide/N,N-methylene-bis-acylamide (w/v) ratio of 37.5:1. Protein bands were visualized by staining in a shaking bath containing 0.2% (w/v) Coomassie Brilliant Blue R-250 (Kodak) in 50% (v/v) ethanol, 10% (v/v) acetic acid and then destaining in 20% (v/v) ethanol, 10% (v/v) acetic acid. SDS-PAGE was carried out using the method of Laemmli (1970) on 15% (w/v) polyacrylamide slabs at 100-200 V constant voltage. Alkaline urea PAGE on 10%
(w/v) slabs (Perrie and Perry, 1970) was employed to resolve phosphorylated and unphosphorylated forms of TM. The dephosphorylation of fish TMs (2 mg/ml) was achieved with E. coli alkaline phosphatase (Worthington) at an enzyme:substrate mole ratio of 1: 100, for 1-2hr, at 37°C, in 150mM NaC1, 10mM MgC12, 50mM Tris-HC1, pH 8.00. Samples were diluted with an equal volume of 9 M urea, 1 mM dithiothreitol and applied to a pre-run gel. Electrophoresis was for 2-2½ hr at 300 V. The alkaline phosphatase-treated TMs were checked for proteolysis by SDS-PAGE. None was observed for the duration of the incubation. Western transfer from SDS-polyacrylamide gels to polyvinylidene difluoride (Bio-Rad) was done in a Bio-Rad mini-Trans-Blot Electrophoresis Transfer cell. The blotting conditions were: 60 V for 2½hr in 10 mM CAPS, 10% (v/v) methanol, pH 11.00. Membranes were stained briefly (5-10min) in 40% (v/v) methanol, 0.025% (w/v) Coomassie Brilliant Blue R-250 (Kodak) and destained in 50% (v/v) methanol. Twodimensional polyacrylamide gel electrophoresis (2D-PAGE) consisted of an isoelectric focusing step which was done in tubes (diameter --- 0.8 mm, length = l 5 cm). The gel composition was 9 M urea, 2% (w/v) Ampholines (pH 4-6, Pharmacia-LKB, Uppsala, Sweden), 4% (w/v) acrylamide (Bio-Rad), 0.24% (w/v) N,N-methylene-bis-acrylamide (Bio-Rad) and 0.022% (v/v) NP40 (Sigma, St Louis, MO). Muscle extracts were made by homogenizing carefully dissected samples of fast and slow muscle (0.14.2 g) in a 10X (v/w) ratio of saturated urea containing dithiothreitol with a Polytron (Brinkman, Westburg, NY). The extracts were clarified by centrifugation in a microcentrifuge (Brinkman) and loaded (5-15 #1) on to gels which had been briefly pre-run with 0.1 mM sodium thioglycolate in the alkaline electrode solution. Electrophoresis in this dimension was carried out to equilibrium (~4000 Vhr). The gels were then immersed into a solution containing 1.3% (w/v) SDS, 65 mM Tris-HC1, pH 6.8, 13% (v/v) glycerol and a trace of Bromophenol Blue, for 15 rain, before transference to a second-dimension gel (1.00 mm thick) consisting of 15% (w/v) polyacrylamide in the separating phase and 3% (w/v) polyacrylamide in the stacking phase. The running conditions were as described above.
Amino acid analysis The various TMs (0.5-2 rag) were hydrolysed in 1 ml of 6 M HCI, containing 0.05% (v/v) phenol, in batches of three or four, at the same time as a rabbit ~-TM standard of equivalent purity. Hydrolysates were analysed on a Beckman Model 121 MB Amino Acid Analyser
97
Fish muscletropomyosin using Benson D-X8.25 Cation Xchange Resin, bed size 200 × 2.8 mm. A single-column, threebuffer sodium-citrate elution method was used at a flow rate of 8 ml/hr with buffers and column temperature as per Beckman 118/119 C1 AN001 application notes. Quantitation of the resuits was done using a Hewlett-Packard Computing Integrator Model 3395A. For each individual TM, analyses were done in triplicate at 24, 48 and 72 hr. Values for serine and threonine were extrapolated back to zero time and valine and isoleucine were taken from the 72-hr sample. Tyrosine was taken from the 24-hr sample. Half-cystine was determined as cysteic acid and methionine as methionine sulphone after oxidation in performic acid prior to acid hydrolysis (Moore, 1963). Some of the half-cystine values were confirmed by determination of carboxymethyl cysteine following reaction with iodoacetic acid in 6M guanidine-HC1 (Crestfield et al., 1963) and acid hydrolysis, at 150°C, for 4 hr in the presence of a small amount of dithiothreitol. Under these conditions oxidation of methionine sulphone was minimal and did not interfere with the quantitation of the derivatized cysteine. Tryptophan was measured by hydrolysis in mercaptoethane sulphonic acid (Penke et al., 1974). Correction factors were obtained from the estimated residue numbers of the rabbit standard and used (where necessary) to adjust the corresponding amino acids of the fish TMs. The compositions were then calculated relative to the alanine number which produced a total nearest to 284 residues. Micro sequence analysis Automated Edman degradation was performed on electroblotted samples of TM using an Applied Biosystems pulsed-liquid sequencer, model 475A equipped with an on-line phenylthiohydantoin analyser, model 120A and also an Applied Biosystems pulsed-liquid sequencer, model 473A, equipped with microgradient phenylthiohydantoin analysis. Circular dichroism spectroscopy Far ultraviolet CD spectra (260-190 nm) were measured using a Jasco-J-500A spectropolarimeter and DP 500 N data processor. The instrument was calibrated at 290.5 nm with a 0.06% (w/v) solution of (IS)-( + )-10 camphorsulphonic acid (Sigma), whose concentration had been confirmed by absorbance at 285 nm (extinction coefficient 34.51/mol/cm) (Hennessey and Johnson, 1982). A water-jacketed cell of 0.01 cm pathlength was used, and the temperature (10-20°C) was controlled by circulating water from a thermostatted bath. The mean residue ellipticity, [®] (in deg/cm2/dmol)
at 222 nm was calculated using the equation: Oobs • m [®] - 10. L .~" (1) Proteins were dissolved in 0.1 M KCI, 50 mM sodium phosphate pH 7.00, 0.01% (w/v) sodium azide and 0.5mM DTT. Concentrations, determined by amino acid analysis, were in the range of 0.4-1 mg/ml. The mean residue weight (m) was evaluated as 33,000- 283, since this is typical of all vertebrate skeletal muscle TMs studied to date. Fractional helix values were calculated from the equation: f.-
[®1 -
[®]R[ ® ] . - [o]R
assuming that TM contains no regions of/~street structure. The following parameters were from Chen et al., 1974: [®]r = 1580 at 222 nm [®]n = - 39,500 at 222 nm. (2) Mass spectrometry Electrospray (ES) mass spectra were collected on a VG Quattro (Fisons Instruments, Altrincham, U.K.) spectrometer. Operating voltage of the ES capillary was 3 kV (positive ion mode), cone voltage, 80V and source temperature, 60°C. Horse heart myoglobin (mol. w t = 16951.48) was used as a standard. Tenmicrolitre samples of TM (dissolved in 50% aqueous acetonitrile, 1% formic acid, concn= 1 mg/ml) were infused into the ES chamber at a flow rate of 10/~l/min. The range m/z was 600-1700a.m.u. (scan rate = 8.2-10.2 sec). Results
Electrophoresis offish muscle extracts Two-dimensional gel electrophoresis was performed on urea extracts of small tissue samples taken from the fast (light) and slow (dark) contracting muscles of various mature fish. Teleosts (N = 15) comprised most of the specimens, but cartilaginous (N = 3) and jawless (N = 1) species were also examined. The TMs were identified initially by is®electric point (pH 4.8-5.0) and apparent mol. wt. For some of the samples this was subsequently confirmed by repeating the electrophoresis with the purified protein. The results presented in Fig. l(a) and summarized in Fig. l(b), show all the TMs to be contained within a small window of the electropherogram. Within this cluster there are a number of points of interest. Fast muscles, in the majority of species tested, contained a single form of TM which, in some instances, was partially phosphorylated. This is inferred by the presence of two overlapping spots of similar Mr
98
D. H. Heeley and C. Hong
(see Fig. l a , gel n u m b e r s 15, 2 9 - 3 1 ) a n d d e m o n s t r a t e d for purified cod, s k a t e a n d s h a r k T M s , w h i c h y i e l d e d two b a n d s o n an a l k a l i u r e a gel, one o f w h i c h c o u l d be e l i m i n a t e d b y a l k a l i n e p h o s p h a t a s e t r e a t m e n t (Fig. 2). T h e s t e a d y - s t a t e p h o s p h o r y l a t i o n level in these fish is low
( 1 0 - 1 5 % ) . G e n e r a l l y , it t e n d e d to be m o r e p r o m i n e n t in the u r e a e x t r a c t s t h a n in the p r o t e i n s a m p l e s ( c o m p a r e gel n u m b e r s 31 a n d 32 in Fig. la), p e r h a p s o w i n g to the a c t i o n o f e n d o g e n o u s p h o s p h a t a s e s . A single, s y m m e t r i cal s p o t l a c k i n g a n acidic satellite w a s o b s e r v e d
1
7
13
19
25
31
2
8
14
~
26
32
t
9
15
~+
4
10
§
11
17
~
12
18
~
27
-Q
33
22
30
Fig. l(a). Two-dimensional polyacrylamide gel electrophoresis of urea extracts from a variety of fish muscles. Only the TM-containing window (pH range = 4.8-5.0) of the electropherogram (oriented acid end to the right) is shown, The samples by number are: (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (I 1) (12)
trout (fast) trout (slow) trout (fast+ slow) salmon (fast) salmon (slow) salmon (fast + slow) char (fast) char (slow) herring (fast) herring (slow) herring (fast) + slow) mackerel (fast)
(13) (14) (15) (16) (17) (18) (19) (20) (21) (22) (23) (24)
mackerel (slow) mackerel (fast+ slow) cod (fast, isolated protein) cod (slow) haddock (fast) haddock (slow) wolf-fish (fast) wolf-fish (slow) wolf-fish (fast + slow) flounder pollack sole
(25) (26) (27) (28) (29) (30) (31) (32) (33) (34) (35) (36)
perch swordfish halibut hake thorny skate blue shark mako shark mako shark (isolated protein) lamprey rabbit skeletal muscle rabbit skeletal muscle+ salmon fast (isolated protein) rabbit skeletal muscle+ salmon slow (isolated protein)
In cases where the source of the muscle type is unspecified, the gel is of a fast muscle homogenate. Lamprey is an exception. Here an extract of a mixed tissue sample was used. Isolated protein was loaded on to a few of the gels (numbers 15, 32, 35 and 36). The remaining samples are urea homogenates. Arrows in gels 2, 3, 5, 6, 8, 11, 13 and 14 indicate the presence of fast isoform(s) in the slow muscle extract. Asterisks in gels 15, 30-32 mark a phosphorylated derivative which was also apparent in several other cases. Double arrows in gels 16, 18 and 20 correspond to presumed slow isoforms lying in very close proximity to the fast TM variant. Variations in spot sizes between gels is attributed to unequal sample loadings arising from differences in the homogenization procedure.
Fish muscle tropomyosin
ACIDIC(pH 4.8)
BASIC(pH 5.0)
7/ 9
Fig. l(b) Electrophoretic map of tropomyosins from fast and slow muscles of various fish. The scheme was compiled from the results of numerous mixing experiments. Muscle type is indicated in brackets. Spot resolution has been idealized for the purpose of clarity. Tropomyosinsare coded as follows: (I) (2) (3) (4) (5) (6) (7) (8) (9) (10)
herring (slow) salmon, trout and char (slow, major isoform) cod, haddock and wolf-fish (slow) mackerel(slow) mackerel(fast) herring (fast) skate (fast, unphosphorylated) skate (fast, phosphorylated) shark (fast, unphosphorylated) shark (fast, phosphorylated) = rabbit skeletal muscle ~-TM, and the fast TMs from cod, haddock, wolf-fish, flounder, pollack, sole, perch, swordfish, halibut and hake. * = phosphorylated form of these ~-TMs. fl = rabbit skeletal muscle fl-TM and the fast TMs from salmon, trout and char. The components from lamprey are not included due to the possibility of heterogeneity arising from protein processing as indicated in the text. in trout, salmon and char (Fig. la, gel numbers 1, 4 and 7) raising the question that the modification may not occur in all species. It is possible, however, that the phosphorylation event may be developmentally linked as documented for other animals (Montarass et al., 1981; Heeley et al., 1982) and could be manifest at earlier ages. When the fast T M s were compared to each other and to the ~ and/~ forms from rabbit, two groups were identified. Several teleosts such as
99
cod, haddock, pollack, hake, flounder, sole, halibut, wolf-fish, swordfish and perch constituted one group. All were judged to have identical mobilities and strikingly, only one spot was observed when muscle extracts were spiked with a rabbit ~ - T M standard (data not shown). Thus, these T M s can be classed as ~-like. Using similar criteria, shark and skate TMs appeared to be not quite equivalent to the latter group of fish and perhaps even distinct from each other. Such differences, however, were slight and these TMs were also 0~-like in electrophoretic mobility (summarized Fig. lb). The single spot noted in fast tissues from salmonids (salmon, trout and char) and herring exhibited a higher Mr than ~-TM. Mixing experiments showed the fast TMs to be identical amongst members of the salmonid family (data not shown) and to exactly match the two-dimensional mobility of rabbit fl-TM (Fig. la, gel numbers 34 and 35). Herring T M while also displaying an elevated Mr, had a more basic isoelectric point than fl-TM (Fig. lb). These TMs, at least using the present methods, would appear to be/~-like and therefore distinct from the other fast TMs. Slow muscle biopsies were dissected from the very outside of the lateral line so as to avoid cross-contamination with fast tissue. The results from this set of electropherograms will now be summarized. O f the teleosts that were tested and are understood to contain low levels ( < 5%) of slow muscle in their myotomes (e.g. cod, haddock and wolf-fish), there did not appear to be a dramatically different T M composition to what had been found in fast muscle. Two additional spots were faintly evident above a major spot, which was established by mixing experiments to be identical in mobility to the fast (s-like) isoform (see Fig. l a, gel numbers 16, 18 and 20). This result could be associated with the difficulty in obtaining homogeneous slow muscle completely free of the underlying fast muscle. Also, the two minor components may be smooth muscle T M s originating from blood vessels which infuse the slow muscle. Nevertheless, the slow T M of cod, haddock and
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PTM Fig. 2. Alkaline-urea-polyacrylamide gel electrophoresis of fish tropomyosins before and after dephosphorylation with E. coli alkaline phosphatase. The lanes are: mako shark TM before (1) and after (2) treatment; blue shark TM before (3) and after (4) treatment; thorny skate TM before (5) and after (6) treatment; and cod TM before (7) and after (8) treatment. These TMs were isolated from samples composed predominantly of fast muscle fibres.
100
D. H. Heeleyand C. Hong
wolf-fish must be of the at-type since protein is absent from the gel region where fl-TM is located. On the other hand, in fish having a greater supply of dark muscle (salmon, trout, char and herring), there was a clear difference between the fast and slow TMs. When the resolution was particularly high two major spots were resolved in herring (Fig. la, gel number 10) and perhaps salmon as well (Fig. 1a, gel number 5). They were, in both instances, closely related in charge and size. The positions of these spots did not change when the alkaline phosphatase experiment was repeated for the isolated TMs, (data not shown). Therefore, unless substantial dephosphorylation has occurred during the purification, these components are likely to represent two distinct, but very similar, isoforms. As with the fast muscle TMs from these four teleosts, the slow-type components were found to be identical in trout, salmon and char, but these were distinct from herring. In each case, however, the slow TMs had a greater SDS mobility and a more basic isoelectric point than the matching fast isoform, and co-migrated with rabbit ~-TM (Fig. la, gel number 36). Visual inspection of the gels corresponding to trout, salmon and herring (Fig. la, gel numbers 2, 5 and 10) show the slow TMs to be largely exclusive to the slow muscle extracts with less than 5% fast TM present. Furthermore, this was true for muscle pieces taken from several positions along the lateral line of salmon. The cross-contamination of isoforms was greater for Arctic char even though small biopsies were used (Fig. la, gel number 8). In mackerel and lamprey the electrophoretic patterns were more complex in that a greater array of variants was detected. Mackerel slow and fast muscle each contained two isoforms, all having different mobilities (Fig. la, gel numbers 12-14). Lamprey exhibited a cluster of spots (Fig. la, gel number 33). The high number of components in this species is, in part, a reflection of the dispersed mixture of fibres which compose the lamprey's myotome and which make it difficult to obtain homogeneous muscle (Teravainen, 1971; Bone, 1978).
Isolation offish tropomyosins Tropomyosin was prepared from most of the specimens which had been surveyed electrophoretically. For some species (e.g. cod, wolffish and haddock) it was not feasible to dissect sizeable quantities of the slow (dark) muscle. These fish were the ones which showed only minor differences between the TMs from each muscle type. Extraction of TM from slow muscle was, therefore, restricted to fish containing higher levels of this tissue (salmon, char, herring and mackerel).
The TMs were assessed to be approximately 95% homogeneous when examined by SDS-PAGE (Fig. 3). All of the fast muscle isolates yielded one band with the exception of mackerel, where two bands were resolved. For the slow muscle proteins, a significant degree of fast TM contamination (~50% of the total TM) occurred in char (Fig. 3, lane 11) and mackerel (Fig. 3, lane 3) preparations. It was less in the case of herring (~ 20% of the total TM) (Fig. 3, lane 13) and was most satisfactory for salmon (,,~ 5% of the total TM) (Fig. 3, lane 15). The mixing of isoforms during purification could be due to inadequate dissection and, in addition, the presence of intermediate (pink) fibres which lie between the light and dark fibres. Such factors obviously necessitate the employment of chromatographic steps in order to obtain both TMs in homogeneous form. In contrast, salmon appear to be a direct source of fast and slow isoforms since their sharp division, apparent on two-dimensional gels, survives the extraction procedure. All of the slow TMs migrated more rapidly than the fast TMs from the same species. This is well illustrated by inspection of the mixed TM sample from char slow muscle (Fig. 3, lane 11). The inclusion of 5 M urea reduced the mobility of all the fish TMs (Sender, 1971). This is illustrated for a restricted number of samples in Fig. 4 using salmon fast muscle actin as a reference marker. The fish TMs could also be oxidized to disulphide-linked dimers by exposure to atmospheric oxygen, in good correspondence to a rabbit ~-TM standard (Fig. 5) (Johnson and Smillie, 1975; Lehrer, 1975). An exception was the TM isolated from cartilaginous fish where no dimeric species was seen (Fig. 5, lane 3).
Amino acid compositions Compositions were calculated assuming a content of 284 amino acids. This was valid since mass spectrometry of two TMs (cod and mako shark) yielded molecular weights ranging from 32,822 to 32,858. All of the samples were derived from fast muscle preparations which had been shown to contain a single component (Figs la and 3). Mackerel (fast) and lamprey were exceptions. These isolates consisted of more than one form of TM and were analysed as a mixture. The results which are presented in Table 1, show the fish TMs to be much alike and highly similar to rabbit ~-TM. All were devoid of proline and tryptophan, and contained 1 mole of phenylalanine. The major differences are seen for the shark and skate proteins which contain only one residue of glycine and no half-cystine. The lack of this latter residue is consistent with
Fish muscle tropomyosin
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D. H. Heeley and C. Hong
1 2 3 4 5 6 7 8 9 Fig. 4. SDS-polyacrylamide gel electrophoresis of isolated fish tropomyosins in the presence of 5 M urea. A 10% (w/v) gel was used. The lanes are: (1) rabbit ~t-TM; (2) salmon fast muscle actin;(3) cod TM; (4) salmon fast TM; (5) salmon slow TM; (6) thorny skate TM; (7) mako shark TM; (8) lamprey TM; and (9) salmon fast muscle actin. Where the muscle type has not been specified, the TM has been made from a myotomal sample containing predominantly fast muscle. For lamprey, TM was prepared from the entire heterogeneous myotome.
the observations made in the oxidation study (Fig. 5). Finally, cod TM has been analysed before (Odense et al., 1969) and the two compositions are in good agreement. N-terminal analyses The TMs tested were the fast muscle forms from herring, cod, swordfish, perch, blue shark, mako shark and the mixed lamprey sample. All of these were blocked except the lamprey protein. Here, a limited amount of sequence was obtained amounting to 20 residues with strong similarity to residues 12-31 of rabbit ct-TM (Fig. 6). Obviously, since methionine is absent from the sequence, some processing must have occurred, perhaps arising artefactually during post-mortem storage (approximately 1 week) or the isolation procedure. On this point, proteolytic inhibitors were not included in the extraction buffers. Judging from the low yields it is likely that the sequence originates from only a
fraction of the total TM molecules, or one isoform, with the remainder being full length and blocked. Indeed, a partial processing event resulting in an l 1-residue truncation could explain, in part, the multiple mol. wt bands seen on SDS gels (Fig. 3, lane 21) and twodimensional gels (Fig. la, gel number 33). Circular dichroism spectroscopy Fractional values of a-helix (frt) were estimated for a number of TMs, using the magnitude of the CD minima at 222 nm (Fig. 7) and with the assumption that the molecule contains no regions of fl-sheet structure. Values ranging from 0.9 to 1.03 were obtained at 10°C (Table 2), indicating that the fish proteins are similar to other TMs in possessing a high degree of helical structure (Oikawa et al., 1968; Pato and SmiUie, 1981). The or-helical content decreased somewhat when the temperature was raised to 20°C and also when the cod and mako
12345
•, , -
',lllnllB
"
g
,,,,,,
DIMERS
MONOMERS
Fig. 5. Analysis of air-oxidized fish tropomyosins by SDS-polyacrylamide gel electrophoresis. The TMs were dissolved in 0.15M NaCI, 0.2M Tris, 0.01% (w/v) azide pH 8.00, to a concentration of approximately 1 mg/ml and left open to the atmosphere for a period of 2-3 days at room temperature. The subsequent electrophoretic analysis was done on a 10% (w/v) gel. The lanes are as follows: (1) rabbit ~t-TM; (2) salmon fast TM; (3) mako shark TM; (4) herring fast TM; and (5) cod TM. Where the muscle type has not been specified, the TM was made from a myotomal sample composed mainly of fast muscle.
Fish muscle tropomyosin
103
Table 1. Amino acid compositions of fish and rabbit skeletal muscle tropomyosins Rabbit ct Asx Thr Ser Glx Pro Gly Ala Val Met lie Leu Tyr Phe His Lys Arg Trp 1/2-Cys Total
29 8 15 70 0 3 36 9 6 12 33 6 1 2 39 14 0 1 284
Salmon (fast)
31.6(32) 29.4(29) 12.1(12) 9.6(10) 13.8(14) 12.4(12) 67.7(68) 70.3(70) 0 0 4.8 (5) 5.0 (5) 34 33 10.0(10) 8.7 (9) 5.9(6) 6.8(7) 10.8(11) 11.4(11) 33.0(33) 34.0(34) 5.9 (6) 5.4 (5) 1.1 (1) 1.1 (1) 1.2(1) 2.1 (2) 39.9(40) 41.3(41) 13.1(13) 13.4(13) 0 0 1.1 (1) 1.3(1) 287
Halibut Swordfish Asx Thr Ser Glx Pro Gly Ala Val Met lie Leu Tyr Phe His Lys Arg Trp 1/2-Cys
29.4(29) 10.4(10) 12.3(12) 71.3(71) 0 3.0 (3) 33 8.7 (9) 6.3 (6) 10.7(11) 34.6(35) 5.6 (6) 1.2(1) 2.0 (2) 42.4 (42) 13.3(13) 0 1.2(1)
Cod
28.7(29) 9.8 (10) 14.4(14) 70.9(71) 0 3.8 (4) 32 8.8 (9) 7.2 (7) I0.0(10) 34.9(35) 5.9 (6) 1.2(1) 2.0 (2) 41.5 (41) 14.1(14) 0 1.2(1)
Pollack Haddock 29.4(29) 9.9(10) 13.2(13) 69.2(69) 0 4.2 (4) 34 10.1 (10) 6.1(6) 11.8(12) 34.8(35) 5.4 (5) 1.3(1) 2.0(2) 41.2(41) t3.8(14) 0 1.2(1)
283
29.1(29) 10.3(10) 12.6(13) 70.1(70) 0 3.2 (3) 34 8.6(9) 6.1(6) 11.1(11) 33.9(34) 5.9 (6) 1.2(1) 2.1 (2) 40.6(41) 13.6(14) 0 1.4(1)
286
Hake
Flounder
29.0(29) 9.6(10) 12.6(13) 68.3(68) 0 4. i (4) 34 9.2 (9) 6.7(7) 10.7(11) 33.8(34) 5.4 (5) 1.3(1) 2.0(2) 40.0(40) 13.9(14) 0 0.9(1)
29.6(30) 9.7(10) 12.9(13) 72.8(73) 0 3.9 (4) 35 8.2(8) 6.4(6) 10.7(11) 34.3(34) 5.9 (6) 1.1 (1) 1.9(2) 41.1(41) 13.5(13) 0 1.2(1)
284
282
288
Perch
Wolf-fish
Herring
Mackerel
29.2(29) 9.4(9) 12.2(12) 71.8(72) 0 3.4 (3) 33 8.5 (8) 6.6 (7) 10.2(10) 34.7(35) 5.8 (6) 1.1 (1) 1.9 (2) 42.2(42) 12.9(13) 0 1.1 (I)
29.3(29) 9.7(10) 11.3(11) 72.0(72) 0 4.6 (5) 33 7.9 (8) 6.3 (6) 11.2(11) 34.7(35) 5.0 (5) 1.3(1) 1.9 (2) 40.3(40) 12.8(13) 0 1.1 (1)
28.6(29) 13.3(13) 9.7(10) 68.1(68) 0 3.8 (4) 36 9.8 (I0) 6.0 (6) 9.7(10) 32.8(33) 5.9 (6) 1.2(1) 2.9 (3) 37.7(38) 13.3(13) 0 1.0 (1)
30.1(30) 13.0(13) 15.4(15) 68.1(68) 0 4.2 (4) 30 10.6(11) 7.9 (8) 11.0(11) 31.5(31) 6.8 (7) 1.1 (1) 1.7 (2) 40.1 (40) 13.5(13) 0 1.1 (1)
Skate
Blue shark
Mako shark
Lamprey
28.7(29)30.6(31)31.1(31)29.8(30) 10.0(10) 8.0(8) 8.2(8) 10.3(10) 14.8(15) 16.6(17) 16.9(17) 13.4(13) 72.6(73) 67.9(68) 69.3(69) 73.0(73) 0 0 0 0 1.3 (1) 1.5 (1) 1.4 (1) 4.4 (4) 36 37 37 34 8.0 (8) 9.2 (9) 9.3 (9) 10.9(11) 6.9 (7) 5.7 (6) 6.3 (6) 5.2 (5) 10.1(10) 10.9(11) I1.0(11) 10.9(11) 34.7(35) 33.4(33) 33.0(33) 30.6(31) 5.8 (6) 6.2 (6) 6.4 (6) 6.2 (6) 1.1 (1) 1.3(1) 1.2(1) 1.0 (1) 2.0 (2) 2.3 (2) 2.3 (2) 2.0 (2) 39.5(39) 40.3 (40) 39.9 (40) 40.0 (40) 14.0(14) 15.0(15) 15.2(15) 12.6(13) 0 0 0 0 0.2(0) 0.3(0) 0.3(0) 1.0 (1)
Total 284 286 283 282 281 285 286 285 286 285 Analyses were done on TMs isolated from fast muscles, as outlined in Materials and Methods. The number of residues of each amino acid have been calculated relative to alanine to yield a total residue number closest to 284. Some of the half-cystine values were checked by determining carboxymethylcysteine and were in good agreement with those determined by performie acid oxidation. Two determinations were done for salmon, cod, mako and blue shark, herring, mackerel, perch, swordfish and halibut. All other estimates represent a single determination. The composition of rabbit ~-TM is from the published sequence (Stone and Smillie, 1978).
s h a r k s a m p l e s were e x p o s e d to a helix-inducing solvent (trifluoroethanol). This o b s e r v a t i o n suggests t h a t all regions o f these T M s c a p a b l e o f f o r m i n g a n ~-helix were a l r e a d y in t h a t c o n f o r m a t i o n with little o r no r a n d o m coil. Small errors in p r o t e i n c o n c e n t r a t i o n ( d e t e r m i n e d by a m i n o acid analysis) c o u l d a c c o u n t for s o m e o f the values being slightly over one.
Discussion T h e results o f this p r e l i m i n a r y investigation indicate t h a t T M s f r o m fish skeletal muscle are highly similar to o t h e r v e r t e b r a t e striated CBP(B) I08/I--H
muscle forms o f the p r o t e i n ( C u m m i n s a n d Perry, 1974; Smillie, 1979). This conclusion is b a s e d collectively on i n f o r m a t i o n derived f r o m e l e c t r o p h o r e t i c separations, a m i n o acid c o m p o sitional analyses, circular d i c h r o i s m spectra, m o l e c u l a r weight d e t e r m i n a t i o n s a n d p a r t i a l sequence d a t a f r o m an ancestral species. O n e o f the aims o f this study was to c a t a l o g u e the p a t t e r n o f T M expression in the simply o r g a n i z e d muscle systems o f the m y o t o m e with the l o n g - t e r m view o f c a r r y i n g o u t struct u r e - f u n c t i o n c o m p a r i s o n s on i s o m o r p h i c forms o f the protein. In this regard, a p a t t e r n consisting o f a small n u m b e r o f exclusively
104
D. H. Heeley and C. Hong
located isoforms (ideally one per cell type) would not only facilitate the isolation of the variant TMs but also the interpretation of functional differences. Many of the species proved to be unsuitable. The drawbacks that were encountered included: insufficient amounts of slow trunk muscle (e.g. cod and haddock), an inability to cleanly separate the fast and slow muscles (e.g. mackerel and char) and an overly complex array of isoforms (e.g. mackerel). Promising results were obtained, however, with salmon and, to a lesser extent, herring. These particular teleosts possess, in addition to the more preponderant fast muscle tissue, a substantial seam of slow muscle just underneath the lateral line. A single isoform was found in the fast tissue of either fish and two closely related forms were detected in the slow. The TMs were, to a large extent, segregated within each of the myotomal muscle compartments and could be prepared from salmon with little mixing. Such a sharp fibre division of fast and slow isoforms is in stark contrast to the TM composition of many mammalian striated muscles where a
A O.
-20-
-40. I
200
2'.20 ~,nrn
i 240
B 0'
g' -20"
-40" I
Rabbit-~ 1
M
2 3 4 5 6 7 8 9 10
D A I K K K M Q M
200
Lamprey
2~02
2 4'0 ~,nm
C 0"
g, - 2 0 Yield (pmol)
11
L
12 13 14 15 16 17
K L D K E N
K A D K E N
27.5 27.0 35.6 31.3 49.5 23.6
18
A
A
29.0
19 20 21 22 23 24 25 26 27 28 29 30 31
L D R A E Q A E A D K K A
I D R A E Q A E T D K K A
22.2 27.9 N.D. 20.6 27.8 16.6 12.9 17.4 6.7 14.6 16.6 21.7 15.7
Fig. 6. Comparison of partial amino acid sequences of lamprey and rabbit ~-tropomyosins. The lamprey and rabbit sequences are aligned for maximum identity. ND = not determined accurately due to integrator malfunction. The residue (ARG) was clearly identifiable at this position by inspecting the chromatogram.
-40-
I
200
I
220
I
240
Fig. 7. Far ultraviolet circular dichroism spectra of fish tropomyosins. The spectra are: A, cod (1.1 mg/ml); B, mako shark (1.1 mg/ml) and C, lamprey (0.4 mg/ml). The buffer was 0.1 M potassium chloride, 50 m M sodium phosphate, 0.01% (w/v) sodium azide, and 1 mM DTT, pH 7.00. Temperature = 10°C.
number of forms coexist within individual fibres (Bronson and Schachat, 1982; Heeley et al., 1983). Traditionally, TMs have been termed either or fl by the criterion of electrophoretic mobility (Cummins and Perry, 1973). On this basis, several fish species (cod, haddock and wolf-fish) would appear to contain ~-like TMs in both fast
Fish muscle tropomyosin Table 2. Comparison of fH values for fish tropomyosins
F. Mako shark (1.1 mg/ml)
10°C 10°C (80% TFE) 20°C
1.03 0.91 0.99
Cod (1.14mg/ml)
10°C 10°C (80% TFE) 20°C
0.95 0.84 0.93
Perch (0.8 mg/ml)
10°C 20°C
0.93 0.86
Mackerel (fast) (0.8 mg/ml)
10°C 20°C
1.01 0.93
Halibut (0.8 mg/ml)
10°C 20°C
0.9 0.87
Blue shark (0.8 mg/ml)
10°C 20°C
0.99 0.94
Lamprey (0.4 mg/ml)
10°C 20°C
1.02 0.85
Concentrations were determined by amino acid analysis. Nor-leucine was used as an internal standard and sample volumes were confirmed by weighing.
and slow muscles and lack fl-TM. This has also been demonstrated for carp (Feller et al., 1990). In other cases, however, the match between muscle type and TM type is quite dissimilar to what has been seen before (Dhoot and Perry, 1979). For salmon and herring, the TM component, having the greater (~-like) electrophoretic mobility, is intriguingly confined to the slow muscle, while the more slowly migrating (fl-like) form is located within the fast muscle. It remains to be seen whether this classification, as tentatively assigned by mobility measurements, will be vindicated by determination of the complete protein sequences. Another question of interest concerns what functional consequences, if any, will arise from the heterogeneity? It should be possible to begin to address this problem by making use of the simple and discreet isoform pattern of the Atlantic salmon. Overall, the findings of this report present a case for employing certain species of fish to conduct biochemical muscle research, particularly as it is feasible to purify, in quantitative amounts, specific isoforms of TM with relative ease and perhaps other thin filament proteins as well. Such components could furnish molecular insights into the connection between protein diversity and muscle performance. Acknowledgements--We thank Doug Hall and Sonya Banfield for the amino acid analyses, Joe Banoub for the mass spectral analyses, John Green for help with species
105
classification, and Jackie Dalton for typing the manuscript. This work was supported by the Medical Research Council of Canada.
References Bone Q. (1978) Locomotor muscle, In Fish Physiology (Edited by Hoar W. S. and Randell D. J.), Vol. 7, pp. 361-424. Academic Press, New York. Bone Q. and Marshall N. B. (1982) In Biology of Fishes. Blackie, London. Bronson D. D. and Schachat F. H. (1982) Heterogeneity of contractile proteins. J. biol. Chem. 257, 3937-3944. Chen Y.-H., Yang J. T. and Chau K. H . (1974) Determination of the helix and fl forms of proteins in aqueous solution by circular dichroism. Biochemistry 13, 3350-3359. Crestfield A. M., Moore S. and Stein W. H. (1963) The preparation and enzymatic hydrolysis of reduced and S-carboxymethylated proteins. J. biol. Chem. 238, 622~527. Cummins P. and Perry S. V. (1973) The subunits and biological activity of polymorphic forms of tropomyosin. Biochem. J. 133, 765-777. Cummins P. and Perry S. V. (1974) Chemical and immunochemical characteristics of tropomyosins from striated and smooth muscles. Biochem. J. 141, 43-49. Dhoot G. K. and Perry S. V. (1979) Distribution of polymorphic forms of troponin components and tropomyosin in skeletal muscle. Nature 278, 714--718. Feller G., D'Haese J. and Gerday Ch. (1990) Tropomyosins from the striated muscles of carp (Cyprinus carpio) and of icefish (Channichthys rhinoceratus). Arch. Int. Physiol. Biochim. 98, 297 306. Harford J. and Squire J. M. (1990) Static and time resolved X-ray diffraction studies of fish muscle. In Molecular Mechanisms in Muscular Contraction (Edited by Squire J. M.), pp. 287-320. Heeley D. H., Moir A. J. G. and Perry S. V. (1982) Phosphorylation of tropomyosin during development in mammalian striated muscle. FEBS Lett. 146, 115-118. Heeley D. H., Dhoot G. K., Frearson N., Perry S. V. and Vrbova G. (1983) The effect of cross-innervation on the tropomyosin composition of rabbit skeletal muscle. FEBS Lett. 152, 282-286. Hennessey J. P.Jr and Johnson C. W. Jr (1982) Experimental errors and their effect on analyzing circular dichroism spectra of proteins. Analyt. Biochem. 125, 177-188. Johnson P. and Smillie L. B. (1975) Rabbit skeletal ~tropomyosin chains are in register. Biochem. biophys. Res. Commun. 64, 1316-1322. Johnston I. A., Ward P. S. and Goldspink G. (1975) Studies on the swimming musculature of the rainbow trout. I. Fibre types. J. Fish Biol. 7, 451-458. Johnston I. A. (1980) Specialisation of fish muscle. In Development and Specialisation of Skeletal Muscle (Edited by Goldspink D. F.), pp. 123-148. Cambridge University Press, London. Laemmli U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680~85. Lehrer S. S. (1975) Intramolecular crosslinking of tropomyosin via disulfide bond formation: evidence for chain register. Proc. natn. Acad. Sci. U.S.A. 72, 3377-3381. Montarras D., Fiszman M. Y. and Gros F. (1981) Characterisation of the tropomyosin present in various chick embryo muscle types and in muscle cells differentiated in vitro. J. biol. Chem. 256, 4081-4086. Moore S. (1963) On the determination of cystine as cysteic acid. J. biol. Chem. 238, 235-237. Odense P. H., Leung T. C., Green W. A. and Dingle J. R.
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(1969) The isolation of cod muscle tropomyosin by heat treatment. Biochim. biophys. Acta 188, 124-131, Oikawa K., Kay C. M. and McCubbin W. D. (1968) The ultraviolet circular dichroism of muscle proteins. Biochim. biophys. Acta 168, 164-167. Pato M. D. and Smillie J. B. 1(1981) Fragments of rabbit striated muscle ~-tropomyosin. J. biol. Chem. 256, 593-601. Penke B., Ferenczi R. and Kovacs K. (1974) A new acid hydrolysis method for determining tryptophan in peptides and proteins. Analyt. Biochem. 60, 45-50. Perrie W. T. and Perry S. V. (1970)An electrophoretic study
of the low-molecular-weight components of myosin. Biochem. J. 119, 31-38. Powers D. A. (1989) Fish as model systems. Science 264, 352-358. Sender P. M. (1971) Muscle fibrils: solubilization and gel electrophoresis. F E B S Lett. 17, 106-110. Smillie L. B. (1979) Structure and functions of tropomyosins from muscle and non-muscle sources. Trends Biol. Sci. 4, 151 155. Stone D. and Smillie L. B. (1978) The amino acid sequence of rabbit skeletal ~-tropomyosin. J. biol. Chem. 253, 1137-1148.
Teravainen H. (1971) Anatomical and physiological studies on muscles of lamprey. J. Neurophysiol. 34, 954-973.
~
Copyright © 1994 Elsevier Science Ltd Printed in Great Britain. All rights reserved 0305-0491/94 $6.00 + 0.00
Pergamon
Isolation and characterization of tropomyosin from fish muscle D. H. Heeley* and C. Hong Department of Biochemistry, Memorial University, St John's, Newfoundland, Canada, AIB 3X9
A comprehensive survey of tropomyosin from various fish myotomai muscles is reported. The fish tropomyosins were blocked at the N-terminus and, as expected, were found to be of similar amino acid composition, ~-helical content ( > 90% at 10°C) and molecular weight to other vertebrate striated muscle forms. The tropomyosins of salmonids and herring muscle were noticeably heterogeneous when assessed by 2D-PAGE. The distribution of isoforms was tissue-specific: slow muscle contained ~-type tropomyosin while fast muscle contained p-type tropomyosin. In other species (cod, haddock, wolf-fish and sharks) ~-type tropomyosins were present in both kinds of muscle but p-tropomyosin was absent. Key words: Tropomyosin; Fish muscle; Clupea harengus; Salvelinus alpinus; Salrno salar; Gadus morhua; Melanogramrnus aeglefinus; Urophycis; Pollachius virens; Sebastes; Hippoglossus hippoglossus ; Pseudopleuronectes americanus ; Xiphias gladius ; Anarchichas lupus; Isurus oxyrinchus ; Scomber scrobrus, Prionace glauca ; Raja radiata ; Petromyzon marinus.
Cornp. Biochem. Physiol. 108B, 95-106, 1994.
Introduction Fish constitute the largest and most diverse group of vertebrates. They have been recognized as ideal systems for studies in a number of areas including neurobiology, developmental biology and environmental biology (Powers, 1989). The spectacular musculature of fish also offers a number of advantages to the research of muscle contraction. Bony (teleost) fish contain a simple, and long-range, structural order of myofilament lattices which is well suited for X-ray diffraction studies (Harford and Squire, 1990). In addition, while most individual mammalian muscles are composed of an intermingled mixture of fast and slow fibres, these cell types are anatomically segregated in the fish myotome. In round-bodied fish, the slow (dark) muscle, which is used mostly for low-speed cruising, is confined to a seam along the lateral line running from the head to the caudal fin (Johnston et al., 1975; Johnston, 1980; Bone and Marshall, 1982). The more prevalent fast (light) muscle constitutes the remainder of the myotome and
provides the thrust for high-speed motion. Such a discreet separation of muscle types presents a unique opportunity to isolate and characterize fibre-specific forms of the contractile proteins. By and large, however, fish have been underutilized as sources of muscle tissue and muscle protein for biochemical studies. This study was initiated partly to survey some of the properties of contractile proteins from fish and also to investigate the usefulness of the separated muscle types in addressing the biochemical basis for muscle specialization. Here we report the isolation and characterization, from various species, of one of the components of the thin filament complex: tropomyosin.
M a t e r i a l s and M e t h o d s Preparation of tropomyosin Tropomyosin (TM) was purified from the trunk (myotomal) muscles of fish from the following groups: teleost (Arctic char Salvelinus alpinus, Atlantic salmon Salmo salar, Atlantic herring Clupea harengus, Atlantic mackerel Correspondence to: D. H. Heeley,Dept. of Biochemistry, Memorial University, St John's, Newfoundland, Scomber scrobrus, Atlantic cod Gadus morhua, haddock Melanogrammus aeglefinus, hake UroCanada, A1B3X9. Received 20 July 1993; accepted 22 September 1993. phycis, pollack Pollachius virens, ocean perch 95
96
D. H. Heeleyand C. Hong
Sebastes, Atlantic halibut Hippoglossus hippoglossus, winter flounder Pseudopleuronectes americanus, swordfish Xiphias gladius, Atlantic wolf-fish Anarchichas lupus), cartilagenous (mako shark Isurus oxyrinchus, blue shark Prionace glauca, thorny skate Raja radiata), and jawless (sea lamprey Petromyzon marinus). Fish were obtained predominantly from commercial outlets and marine research vessels. The freshness of the samples corresponded to storage periods (on ice) varying from a few days up to a week. For species (e.g. salmon, char, herring and mackerel) where the slow (dark) muscle is present in appreciable levels (15-20% of total muscle mass), it was feasible to dissect this lateral line tissue from the more preponderant fast (light) muscle and isolate TM from each category of muscle. The fast and slow fibres of the lamprey are known to be intermingled (Teravainen, 1971; Bone, 1978) so in this instance TM was made on the mixture as it exists in the myotome. For all the other specimens, TM was purified from myotomal sections, composed of fast muscle with either a small amount of associated dark muscle or none at all. The muscle samples (100-1000 g) were initially minced through a medium-size plate and dehydrated with cold ethanol and acetone. TM contained within the high salt extracts from the dried fibres (10-50g) was then isoelectrically precipitated at pH 4.6 and salted out between 40 and 70% saturated (NH4) 2 SO4. After dialysis and lyophilization, the impure protein (100-400 mg) was then applied to a hydroxylapatite column (2.5 × 10-25 cm) equilibrated in 1 M NaC1, 70 mM sodium phosphate, 1 mM dithiothreitol, 0.01% (w/v) sodium azide pH 7.00, at room temperature and washed with at least two column volumes. The TM was then eluted isocratically with two-column volumes of buffer containing 250 mM sodium phosphate. The prepared material (50-300 mg) was shown by SDS-PAGE to be approximately 95% homogeneous.
Electrophoretic methods Electrophoresis was performed on a Bio-Rad mini-Protean II apparatus (Bio-Rad, Richmond, CA). Unless stated otherwise, all electropherograms were 0.75 mm thick and consisted of an acrylamide/N,N-methylene-bis-acylamide (w/v) ratio of 37.5:1. Protein bands were visualized by staining in a shaking bath containing 0.2% (w/v) Coomassie Brilliant Blue R-250 (Kodak) in 50% (v/v) ethanol, 10% (v/v) acetic acid and then destaining in 20% (v/v) ethanol, 10% (v/v) acetic acid. SDS-PAGE was carried out using the method of Laemmli (1970) on 15% (w/v) polyacrylamide slabs at 100-200 V constant voltage. Alkaline urea PAGE on 10%
(w/v) slabs (Perrie and Perry, 1970) was employed to resolve phosphorylated and unphosphorylated forms of TM. The dephosphorylation of fish TMs (2 mg/ml) was achieved with E. coli alkaline phosphatase (Worthington) at an enzyme:substrate mole ratio of 1: 100, for 1-2hr, at 37°C, in 150mM NaC1, 10mM MgC12, 50mM Tris-HC1, pH 8.00. Samples were diluted with an equal volume of 9 M urea, 1 mM dithiothreitol and applied to a pre-run gel. Electrophoresis was for 2-2½ hr at 300 V. The alkaline phosphatase-treated TMs were checked for proteolysis by SDS-PAGE. None was observed for the duration of the incubation. Western transfer from SDS-polyacrylamide gels to polyvinylidene difluoride (Bio-Rad) was done in a Bio-Rad mini-Trans-Blot Electrophoresis Transfer cell. The blotting conditions were: 60 V for 2½hr in 10 mM CAPS, 10% (v/v) methanol, pH 11.00. Membranes were stained briefly (5-10min) in 40% (v/v) methanol, 0.025% (w/v) Coomassie Brilliant Blue R-250 (Kodak) and destained in 50% (v/v) methanol. Twodimensional polyacrylamide gel electrophoresis (2D-PAGE) consisted of an isoelectric focusing step which was done in tubes (diameter --- 0.8 mm, length = l 5 cm). The gel composition was 9 M urea, 2% (w/v) Ampholines (pH 4-6, Pharmacia-LKB, Uppsala, Sweden), 4% (w/v) acrylamide (Bio-Rad), 0.24% (w/v) N,N-methylene-bis-acrylamide (Bio-Rad) and 0.022% (v/v) NP40 (Sigma, St Louis, MO). Muscle extracts were made by homogenizing carefully dissected samples of fast and slow muscle (0.14.2 g) in a 10X (v/w) ratio of saturated urea containing dithiothreitol with a Polytron (Brinkman, Westburg, NY). The extracts were clarified by centrifugation in a microcentrifuge (Brinkman) and loaded (5-15 #1) on to gels which had been briefly pre-run with 0.1 mM sodium thioglycolate in the alkaline electrode solution. Electrophoresis in this dimension was carried out to equilibrium (~4000 Vhr). The gels were then immersed into a solution containing 1.3% (w/v) SDS, 65 mM Tris-HC1, pH 6.8, 13% (v/v) glycerol and a trace of Bromophenol Blue, for 15 rain, before transference to a second-dimension gel (1.00 mm thick) consisting of 15% (w/v) polyacrylamide in the separating phase and 3% (w/v) polyacrylamide in the stacking phase. The running conditions were as described above.
Amino acid analysis The various TMs (0.5-2 rag) were hydrolysed in 1 ml of 6 M HCI, containing 0.05% (v/v) phenol, in batches of three or four, at the same time as a rabbit ~-TM standard of equivalent purity. Hydrolysates were analysed on a Beckman Model 121 MB Amino Acid Analyser
97
Fish muscletropomyosin using Benson D-X8.25 Cation Xchange Resin, bed size 200 × 2.8 mm. A single-column, threebuffer sodium-citrate elution method was used at a flow rate of 8 ml/hr with buffers and column temperature as per Beckman 118/119 C1 AN001 application notes. Quantitation of the resuits was done using a Hewlett-Packard Computing Integrator Model 3395A. For each individual TM, analyses were done in triplicate at 24, 48 and 72 hr. Values for serine and threonine were extrapolated back to zero time and valine and isoleucine were taken from the 72-hr sample. Tyrosine was taken from the 24-hr sample. Half-cystine was determined as cysteic acid and methionine as methionine sulphone after oxidation in performic acid prior to acid hydrolysis (Moore, 1963). Some of the half-cystine values were confirmed by determination of carboxymethyl cysteine following reaction with iodoacetic acid in 6M guanidine-HC1 (Crestfield et al., 1963) and acid hydrolysis, at 150°C, for 4 hr in the presence of a small amount of dithiothreitol. Under these conditions oxidation of methionine sulphone was minimal and did not interfere with the quantitation of the derivatized cysteine. Tryptophan was measured by hydrolysis in mercaptoethane sulphonic acid (Penke et al., 1974). Correction factors were obtained from the estimated residue numbers of the rabbit standard and used (where necessary) to adjust the corresponding amino acids of the fish TMs. The compositions were then calculated relative to the alanine number which produced a total nearest to 284 residues. Micro sequence analysis Automated Edman degradation was performed on electroblotted samples of TM using an Applied Biosystems pulsed-liquid sequencer, model 475A equipped with an on-line phenylthiohydantoin analyser, model 120A and also an Applied Biosystems pulsed-liquid sequencer, model 473A, equipped with microgradient phenylthiohydantoin analysis. Circular dichroism spectroscopy Far ultraviolet CD spectra (260-190 nm) were measured using a Jasco-J-500A spectropolarimeter and DP 500 N data processor. The instrument was calibrated at 290.5 nm with a 0.06% (w/v) solution of (IS)-( + )-10 camphorsulphonic acid (Sigma), whose concentration had been confirmed by absorbance at 285 nm (extinction coefficient 34.51/mol/cm) (Hennessey and Johnson, 1982). A water-jacketed cell of 0.01 cm pathlength was used, and the temperature (10-20°C) was controlled by circulating water from a thermostatted bath. The mean residue ellipticity, [®] (in deg/cm2/dmol)
at 222 nm was calculated using the equation: Oobs • m [®] - 10. L .~" (1) Proteins were dissolved in 0.1 M KCI, 50 mM sodium phosphate pH 7.00, 0.01% (w/v) sodium azide and 0.5mM DTT. Concentrations, determined by amino acid analysis, were in the range of 0.4-1 mg/ml. The mean residue weight (m) was evaluated as 33,000- 283, since this is typical of all vertebrate skeletal muscle TMs studied to date. Fractional helix values were calculated from the equation: f.-
[®1 -
[®]R[ ® ] . - [o]R
assuming that TM contains no regions of/~street structure. The following parameters were from Chen et al., 1974: [®]r = 1580 at 222 nm [®]n = - 39,500 at 222 nm. (2) Mass spectrometry Electrospray (ES) mass spectra were collected on a VG Quattro (Fisons Instruments, Altrincham, U.K.) spectrometer. Operating voltage of the ES capillary was 3 kV (positive ion mode), cone voltage, 80V and source temperature, 60°C. Horse heart myoglobin (mol. w t = 16951.48) was used as a standard. Tenmicrolitre samples of TM (dissolved in 50% aqueous acetonitrile, 1% formic acid, concn= 1 mg/ml) were infused into the ES chamber at a flow rate of 10/~l/min. The range m/z was 600-1700a.m.u. (scan rate = 8.2-10.2 sec). Results
Electrophoresis offish muscle extracts Two-dimensional gel electrophoresis was performed on urea extracts of small tissue samples taken from the fast (light) and slow (dark) contracting muscles of various mature fish. Teleosts (N = 15) comprised most of the specimens, but cartilaginous (N = 3) and jawless (N = 1) species were also examined. The TMs were identified initially by is®electric point (pH 4.8-5.0) and apparent mol. wt. For some of the samples this was subsequently confirmed by repeating the electrophoresis with the purified protein. The results presented in Fig. l(a) and summarized in Fig. l(b), show all the TMs to be contained within a small window of the electropherogram. Within this cluster there are a number of points of interest. Fast muscles, in the majority of species tested, contained a single form of TM which, in some instances, was partially phosphorylated. This is inferred by the presence of two overlapping spots of similar Mr
98
D. H. Heeley and C. Hong
(see Fig. l a , gel n u m b e r s 15, 2 9 - 3 1 ) a n d d e m o n s t r a t e d for purified cod, s k a t e a n d s h a r k T M s , w h i c h y i e l d e d two b a n d s o n an a l k a l i u r e a gel, one o f w h i c h c o u l d be e l i m i n a t e d b y a l k a l i n e p h o s p h a t a s e t r e a t m e n t (Fig. 2). T h e s t e a d y - s t a t e p h o s p h o r y l a t i o n level in these fish is low
( 1 0 - 1 5 % ) . G e n e r a l l y , it t e n d e d to be m o r e p r o m i n e n t in the u r e a e x t r a c t s t h a n in the p r o t e i n s a m p l e s ( c o m p a r e gel n u m b e r s 31 a n d 32 in Fig. la), p e r h a p s o w i n g to the a c t i o n o f e n d o g e n o u s p h o s p h a t a s e s . A single, s y m m e t r i cal s p o t l a c k i n g a n acidic satellite w a s o b s e r v e d
1
7
13
19
25
31
2
8
14
~
26
32
t
9
15
~+
4
10
§
11
17
~
12
18
~
27
-Q
33
22
30
Fig. l(a). Two-dimensional polyacrylamide gel electrophoresis of urea extracts from a variety of fish muscles. Only the TM-containing window (pH range = 4.8-5.0) of the electropherogram (oriented acid end to the right) is shown, The samples by number are: (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (I 1) (12)
trout (fast) trout (slow) trout (fast+ slow) salmon (fast) salmon (slow) salmon (fast + slow) char (fast) char (slow) herring (fast) herring (slow) herring (fast) + slow) mackerel (fast)
(13) (14) (15) (16) (17) (18) (19) (20) (21) (22) (23) (24)
mackerel (slow) mackerel (fast+ slow) cod (fast, isolated protein) cod (slow) haddock (fast) haddock (slow) wolf-fish (fast) wolf-fish (slow) wolf-fish (fast + slow) flounder pollack sole
(25) (26) (27) (28) (29) (30) (31) (32) (33) (34) (35) (36)
perch swordfish halibut hake thorny skate blue shark mako shark mako shark (isolated protein) lamprey rabbit skeletal muscle rabbit skeletal muscle+ salmon fast (isolated protein) rabbit skeletal muscle+ salmon slow (isolated protein)
In cases where the source of the muscle type is unspecified, the gel is of a fast muscle homogenate. Lamprey is an exception. Here an extract of a mixed tissue sample was used. Isolated protein was loaded on to a few of the gels (numbers 15, 32, 35 and 36). The remaining samples are urea homogenates. Arrows in gels 2, 3, 5, 6, 8, 11, 13 and 14 indicate the presence of fast isoform(s) in the slow muscle extract. Asterisks in gels 15, 30-32 mark a phosphorylated derivative which was also apparent in several other cases. Double arrows in gels 16, 18 and 20 correspond to presumed slow isoforms lying in very close proximity to the fast TM variant. Variations in spot sizes between gels is attributed to unequal sample loadings arising from differences in the homogenization procedure.
Fish muscle tropomyosin
ACIDIC(pH 4.8)
BASIC(pH 5.0)
7/ 9
Fig. l(b) Electrophoretic map of tropomyosins from fast and slow muscles of various fish. The scheme was compiled from the results of numerous mixing experiments. Muscle type is indicated in brackets. Spot resolution has been idealized for the purpose of clarity. Tropomyosinsare coded as follows: (I) (2) (3) (4) (5) (6) (7) (8) (9) (10)
herring (slow) salmon, trout and char (slow, major isoform) cod, haddock and wolf-fish (slow) mackerel(slow) mackerel(fast) herring (fast) skate (fast, unphosphorylated) skate (fast, phosphorylated) shark (fast, unphosphorylated) shark (fast, phosphorylated) = rabbit skeletal muscle ~-TM, and the fast TMs from cod, haddock, wolf-fish, flounder, pollack, sole, perch, swordfish, halibut and hake. * = phosphorylated form of these ~-TMs. fl = rabbit skeletal muscle fl-TM and the fast TMs from salmon, trout and char. The components from lamprey are not included due to the possibility of heterogeneity arising from protein processing as indicated in the text. in trout, salmon and char (Fig. la, gel numbers 1, 4 and 7) raising the question that the modification may not occur in all species. It is possible, however, that the phosphorylation event may be developmentally linked as documented for other animals (Montarass et al., 1981; Heeley et al., 1982) and could be manifest at earlier ages. When the fast T M s were compared to each other and to the ~ and/~ forms from rabbit, two groups were identified. Several teleosts such as
99
cod, haddock, pollack, hake, flounder, sole, halibut, wolf-fish, swordfish and perch constituted one group. All were judged to have identical mobilities and strikingly, only one spot was observed when muscle extracts were spiked with a rabbit ~ - T M standard (data not shown). Thus, these T M s can be classed as ~-like. Using similar criteria, shark and skate TMs appeared to be not quite equivalent to the latter group of fish and perhaps even distinct from each other. Such differences, however, were slight and these TMs were also 0~-like in electrophoretic mobility (summarized Fig. lb). The single spot noted in fast tissues from salmonids (salmon, trout and char) and herring exhibited a higher Mr than ~-TM. Mixing experiments showed the fast TMs to be identical amongst members of the salmonid family (data not shown) and to exactly match the two-dimensional mobility of rabbit fl-TM (Fig. la, gel numbers 34 and 35). Herring T M while also displaying an elevated Mr, had a more basic isoelectric point than fl-TM (Fig. lb). These TMs, at least using the present methods, would appear to be/~-like and therefore distinct from the other fast TMs. Slow muscle biopsies were dissected from the very outside of the lateral line so as to avoid cross-contamination with fast tissue. The results from this set of electropherograms will now be summarized. O f the teleosts that were tested and are understood to contain low levels ( < 5%) of slow muscle in their myotomes (e.g. cod, haddock and wolf-fish), there did not appear to be a dramatically different T M composition to what had been found in fast muscle. Two additional spots were faintly evident above a major spot, which was established by mixing experiments to be identical in mobility to the fast (s-like) isoform (see Fig. l a, gel numbers 16, 18 and 20). This result could be associated with the difficulty in obtaining homogeneous slow muscle completely free of the underlying fast muscle. Also, the two minor components may be smooth muscle T M s originating from blood vessels which infuse the slow muscle. Nevertheless, the slow T M of cod, haddock and
12345678
PTM Fig. 2. Alkaline-urea-polyacrylamide gel electrophoresis of fish tropomyosins before and after dephosphorylation with E. coli alkaline phosphatase. The lanes are: mako shark TM before (1) and after (2) treatment; blue shark TM before (3) and after (4) treatment; thorny skate TM before (5) and after (6) treatment; and cod TM before (7) and after (8) treatment. These TMs were isolated from samples composed predominantly of fast muscle fibres.
100
D. H. Heeleyand C. Hong
wolf-fish must be of the at-type since protein is absent from the gel region where fl-TM is located. On the other hand, in fish having a greater supply of dark muscle (salmon, trout, char and herring), there was a clear difference between the fast and slow TMs. When the resolution was particularly high two major spots were resolved in herring (Fig. la, gel number 10) and perhaps salmon as well (Fig. 1a, gel number 5). They were, in both instances, closely related in charge and size. The positions of these spots did not change when the alkaline phosphatase experiment was repeated for the isolated TMs, (data not shown). Therefore, unless substantial dephosphorylation has occurred during the purification, these components are likely to represent two distinct, but very similar, isoforms. As with the fast muscle TMs from these four teleosts, the slow-type components were found to be identical in trout, salmon and char, but these were distinct from herring. In each case, however, the slow TMs had a greater SDS mobility and a more basic isoelectric point than the matching fast isoform, and co-migrated with rabbit ~-TM (Fig. la, gel number 36). Visual inspection of the gels corresponding to trout, salmon and herring (Fig. la, gel numbers 2, 5 and 10) show the slow TMs to be largely exclusive to the slow muscle extracts with less than 5% fast TM present. Furthermore, this was true for muscle pieces taken from several positions along the lateral line of salmon. The cross-contamination of isoforms was greater for Arctic char even though small biopsies were used (Fig. la, gel number 8). In mackerel and lamprey the electrophoretic patterns were more complex in that a greater array of variants was detected. Mackerel slow and fast muscle each contained two isoforms, all having different mobilities (Fig. la, gel numbers 12-14). Lamprey exhibited a cluster of spots (Fig. la, gel number 33). The high number of components in this species is, in part, a reflection of the dispersed mixture of fibres which compose the lamprey's myotome and which make it difficult to obtain homogeneous muscle (Teravainen, 1971; Bone, 1978).
Isolation offish tropomyosins Tropomyosin was prepared from most of the specimens which had been surveyed electrophoretically. For some species (e.g. cod, wolffish and haddock) it was not feasible to dissect sizeable quantities of the slow (dark) muscle. These fish were the ones which showed only minor differences between the TMs from each muscle type. Extraction of TM from slow muscle was, therefore, restricted to fish containing higher levels of this tissue (salmon, char, herring and mackerel).
The TMs were assessed to be approximately 95% homogeneous when examined by SDS-PAGE (Fig. 3). All of the fast muscle isolates yielded one band with the exception of mackerel, where two bands were resolved. For the slow muscle proteins, a significant degree of fast TM contamination (~50% of the total TM) occurred in char (Fig. 3, lane 11) and mackerel (Fig. 3, lane 3) preparations. It was less in the case of herring (~ 20% of the total TM) (Fig. 3, lane 13) and was most satisfactory for salmon (,,~ 5% of the total TM) (Fig. 3, lane 15). The mixing of isoforms during purification could be due to inadequate dissection and, in addition, the presence of intermediate (pink) fibres which lie between the light and dark fibres. Such factors obviously necessitate the employment of chromatographic steps in order to obtain both TMs in homogeneous form. In contrast, salmon appear to be a direct source of fast and slow isoforms since their sharp division, apparent on two-dimensional gels, survives the extraction procedure. All of the slow TMs migrated more rapidly than the fast TMs from the same species. This is well illustrated by inspection of the mixed TM sample from char slow muscle (Fig. 3, lane 11). The inclusion of 5 M urea reduced the mobility of all the fish TMs (Sender, 1971). This is illustrated for a restricted number of samples in Fig. 4 using salmon fast muscle actin as a reference marker. The fish TMs could also be oxidized to disulphide-linked dimers by exposure to atmospheric oxygen, in good correspondence to a rabbit ~-TM standard (Fig. 5) (Johnson and Smillie, 1975; Lehrer, 1975). An exception was the TM isolated from cartilaginous fish where no dimeric species was seen (Fig. 5, lane 3).
Amino acid compositions Compositions were calculated assuming a content of 284 amino acids. This was valid since mass spectrometry of two TMs (cod and mako shark) yielded molecular weights ranging from 32,822 to 32,858. All of the samples were derived from fast muscle preparations which had been shown to contain a single component (Figs la and 3). Mackerel (fast) and lamprey were exceptions. These isolates consisted of more than one form of TM and were analysed as a mixture. The results which are presented in Table 1, show the fish TMs to be much alike and highly similar to rabbit ~-TM. All were devoid of proline and tryptophan, and contained 1 mole of phenylalanine. The major differences are seen for the shark and skate proteins which contain only one residue of glycine and no half-cystine. The lack of this latter residue is consistent with
Fish muscle tropomyosin
lO1
~°
~
~ , ~
t02
D. H. Heeley and C. Hong
1 2 3 4 5 6 7 8 9 Fig. 4. SDS-polyacrylamide gel electrophoresis of isolated fish tropomyosins in the presence of 5 M urea. A 10% (w/v) gel was used. The lanes are: (1) rabbit ~t-TM; (2) salmon fast muscle actin;(3) cod TM; (4) salmon fast TM; (5) salmon slow TM; (6) thorny skate TM; (7) mako shark TM; (8) lamprey TM; and (9) salmon fast muscle actin. Where the muscle type has not been specified, the TM has been made from a myotomal sample containing predominantly fast muscle. For lamprey, TM was prepared from the entire heterogeneous myotome.
the observations made in the oxidation study (Fig. 5). Finally, cod TM has been analysed before (Odense et al., 1969) and the two compositions are in good agreement. N-terminal analyses The TMs tested were the fast muscle forms from herring, cod, swordfish, perch, blue shark, mako shark and the mixed lamprey sample. All of these were blocked except the lamprey protein. Here, a limited amount of sequence was obtained amounting to 20 residues with strong similarity to residues 12-31 of rabbit ct-TM (Fig. 6). Obviously, since methionine is absent from the sequence, some processing must have occurred, perhaps arising artefactually during post-mortem storage (approximately 1 week) or the isolation procedure. On this point, proteolytic inhibitors were not included in the extraction buffers. Judging from the low yields it is likely that the sequence originates from only a
fraction of the total TM molecules, or one isoform, with the remainder being full length and blocked. Indeed, a partial processing event resulting in an l 1-residue truncation could explain, in part, the multiple mol. wt bands seen on SDS gels (Fig. 3, lane 21) and twodimensional gels (Fig. la, gel number 33). Circular dichroism spectroscopy Fractional values of a-helix (frt) were estimated for a number of TMs, using the magnitude of the CD minima at 222 nm (Fig. 7) and with the assumption that the molecule contains no regions of fl-sheet structure. Values ranging from 0.9 to 1.03 were obtained at 10°C (Table 2), indicating that the fish proteins are similar to other TMs in possessing a high degree of helical structure (Oikawa et al., 1968; Pato and SmiUie, 1981). The or-helical content decreased somewhat when the temperature was raised to 20°C and also when the cod and mako
12345
•, , -
',lllnllB
"
g
,,,,,,
DIMERS
MONOMERS
Fig. 5. Analysis of air-oxidized fish tropomyosins by SDS-polyacrylamide gel electrophoresis. The TMs were dissolved in 0.15M NaCI, 0.2M Tris, 0.01% (w/v) azide pH 8.00, to a concentration of approximately 1 mg/ml and left open to the atmosphere for a period of 2-3 days at room temperature. The subsequent electrophoretic analysis was done on a 10% (w/v) gel. The lanes are as follows: (1) rabbit ~t-TM; (2) salmon fast TM; (3) mako shark TM; (4) herring fast TM; and (5) cod TM. Where the muscle type has not been specified, the TM was made from a myotomal sample composed mainly of fast muscle.
Fish muscle tropomyosin
103
Table 1. Amino acid compositions of fish and rabbit skeletal muscle tropomyosins Rabbit ct Asx Thr Ser Glx Pro Gly Ala Val Met lie Leu Tyr Phe His Lys Arg Trp 1/2-Cys Total
29 8 15 70 0 3 36 9 6 12 33 6 1 2 39 14 0 1 284
Salmon (fast)
31.6(32) 29.4(29) 12.1(12) 9.6(10) 13.8(14) 12.4(12) 67.7(68) 70.3(70) 0 0 4.8 (5) 5.0 (5) 34 33 10.0(10) 8.7 (9) 5.9(6) 6.8(7) 10.8(11) 11.4(11) 33.0(33) 34.0(34) 5.9 (6) 5.4 (5) 1.1 (1) 1.1 (1) 1.2(1) 2.1 (2) 39.9(40) 41.3(41) 13.1(13) 13.4(13) 0 0 1.1 (1) 1.3(1) 287
Halibut Swordfish Asx Thr Ser Glx Pro Gly Ala Val Met lie Leu Tyr Phe His Lys Arg Trp 1/2-Cys
29.4(29) 10.4(10) 12.3(12) 71.3(71) 0 3.0 (3) 33 8.7 (9) 6.3 (6) 10.7(11) 34.6(35) 5.6 (6) 1.2(1) 2.0 (2) 42.4 (42) 13.3(13) 0 1.2(1)
Cod
28.7(29) 9.8 (10) 14.4(14) 70.9(71) 0 3.8 (4) 32 8.8 (9) 7.2 (7) I0.0(10) 34.9(35) 5.9 (6) 1.2(1) 2.0 (2) 41.5 (41) 14.1(14) 0 1.2(1)
Pollack Haddock 29.4(29) 9.9(10) 13.2(13) 69.2(69) 0 4.2 (4) 34 10.1 (10) 6.1(6) 11.8(12) 34.8(35) 5.4 (5) 1.3(1) 2.0(2) 41.2(41) t3.8(14) 0 1.2(1)
283
29.1(29) 10.3(10) 12.6(13) 70.1(70) 0 3.2 (3) 34 8.6(9) 6.1(6) 11.1(11) 33.9(34) 5.9 (6) 1.2(1) 2.1 (2) 40.6(41) 13.6(14) 0 1.4(1)
286
Hake
Flounder
29.0(29) 9.6(10) 12.6(13) 68.3(68) 0 4. i (4) 34 9.2 (9) 6.7(7) 10.7(11) 33.8(34) 5.4 (5) 1.3(1) 2.0(2) 40.0(40) 13.9(14) 0 0.9(1)
29.6(30) 9.7(10) 12.9(13) 72.8(73) 0 3.9 (4) 35 8.2(8) 6.4(6) 10.7(11) 34.3(34) 5.9 (6) 1.1 (1) 1.9(2) 41.1(41) 13.5(13) 0 1.2(1)
284
282
288
Perch
Wolf-fish
Herring
Mackerel
29.2(29) 9.4(9) 12.2(12) 71.8(72) 0 3.4 (3) 33 8.5 (8) 6.6 (7) 10.2(10) 34.7(35) 5.8 (6) 1.1 (1) 1.9 (2) 42.2(42) 12.9(13) 0 1.1 (I)
29.3(29) 9.7(10) 11.3(11) 72.0(72) 0 4.6 (5) 33 7.9 (8) 6.3 (6) 11.2(11) 34.7(35) 5.0 (5) 1.3(1) 1.9 (2) 40.3(40) 12.8(13) 0 1.1 (1)
28.6(29) 13.3(13) 9.7(10) 68.1(68) 0 3.8 (4) 36 9.8 (I0) 6.0 (6) 9.7(10) 32.8(33) 5.9 (6) 1.2(1) 2.9 (3) 37.7(38) 13.3(13) 0 1.0 (1)
30.1(30) 13.0(13) 15.4(15) 68.1(68) 0 4.2 (4) 30 10.6(11) 7.9 (8) 11.0(11) 31.5(31) 6.8 (7) 1.1 (1) 1.7 (2) 40.1 (40) 13.5(13) 0 1.1 (1)
Skate
Blue shark
Mako shark
Lamprey
28.7(29)30.6(31)31.1(31)29.8(30) 10.0(10) 8.0(8) 8.2(8) 10.3(10) 14.8(15) 16.6(17) 16.9(17) 13.4(13) 72.6(73) 67.9(68) 69.3(69) 73.0(73) 0 0 0 0 1.3 (1) 1.5 (1) 1.4 (1) 4.4 (4) 36 37 37 34 8.0 (8) 9.2 (9) 9.3 (9) 10.9(11) 6.9 (7) 5.7 (6) 6.3 (6) 5.2 (5) 10.1(10) 10.9(11) I1.0(11) 10.9(11) 34.7(35) 33.4(33) 33.0(33) 30.6(31) 5.8 (6) 6.2 (6) 6.4 (6) 6.2 (6) 1.1 (1) 1.3(1) 1.2(1) 1.0 (1) 2.0 (2) 2.3 (2) 2.3 (2) 2.0 (2) 39.5(39) 40.3 (40) 39.9 (40) 40.0 (40) 14.0(14) 15.0(15) 15.2(15) 12.6(13) 0 0 0 0 0.2(0) 0.3(0) 0.3(0) 1.0 (1)
Total 284 286 283 282 281 285 286 285 286 285 Analyses were done on TMs isolated from fast muscles, as outlined in Materials and Methods. The number of residues of each amino acid have been calculated relative to alanine to yield a total residue number closest to 284. Some of the half-cystine values were checked by determining carboxymethylcysteine and were in good agreement with those determined by performie acid oxidation. Two determinations were done for salmon, cod, mako and blue shark, herring, mackerel, perch, swordfish and halibut. All other estimates represent a single determination. The composition of rabbit ~-TM is from the published sequence (Stone and Smillie, 1978).
s h a r k s a m p l e s were e x p o s e d to a helix-inducing solvent (trifluoroethanol). This o b s e r v a t i o n suggests t h a t all regions o f these T M s c a p a b l e o f f o r m i n g a n ~-helix were a l r e a d y in t h a t c o n f o r m a t i o n with little o r no r a n d o m coil. Small errors in p r o t e i n c o n c e n t r a t i o n ( d e t e r m i n e d by a m i n o acid analysis) c o u l d a c c o u n t for s o m e o f the values being slightly over one.
Discussion T h e results o f this p r e l i m i n a r y investigation indicate t h a t T M s f r o m fish skeletal muscle are highly similar to o t h e r v e r t e b r a t e striated CBP(B) I08/I--H
muscle forms o f the p r o t e i n ( C u m m i n s a n d Perry, 1974; Smillie, 1979). This conclusion is b a s e d collectively on i n f o r m a t i o n derived f r o m e l e c t r o p h o r e t i c separations, a m i n o acid c o m p o sitional analyses, circular d i c h r o i s m spectra, m o l e c u l a r weight d e t e r m i n a t i o n s a n d p a r t i a l sequence d a t a f r o m an ancestral species. O n e o f the aims o f this study was to c a t a l o g u e the p a t t e r n o f T M expression in the simply o r g a n i z e d muscle systems o f the m y o t o m e with the l o n g - t e r m view o f c a r r y i n g o u t struct u r e - f u n c t i o n c o m p a r i s o n s on i s o m o r p h i c forms o f the protein. In this regard, a p a t t e r n consisting o f a small n u m b e r o f exclusively
104
D. H. Heeley and C. Hong
located isoforms (ideally one per cell type) would not only facilitate the isolation of the variant TMs but also the interpretation of functional differences. Many of the species proved to be unsuitable. The drawbacks that were encountered included: insufficient amounts of slow trunk muscle (e.g. cod and haddock), an inability to cleanly separate the fast and slow muscles (e.g. mackerel and char) and an overly complex array of isoforms (e.g. mackerel). Promising results were obtained, however, with salmon and, to a lesser extent, herring. These particular teleosts possess, in addition to the more preponderant fast muscle tissue, a substantial seam of slow muscle just underneath the lateral line. A single isoform was found in the fast tissue of either fish and two closely related forms were detected in the slow. The TMs were, to a large extent, segregated within each of the myotomal muscle compartments and could be prepared from salmon with little mixing. Such a sharp fibre division of fast and slow isoforms is in stark contrast to the TM composition of many mammalian striated muscles where a
A O.
-20-
-40. I
200
2'.20 ~,nrn
i 240
B 0'
g' -20"
-40" I
Rabbit-~ 1
M
2 3 4 5 6 7 8 9 10
D A I K K K M Q M
200
Lamprey
2~02
2 4'0 ~,nm
C 0"
g, - 2 0 Yield (pmol)
11
L
12 13 14 15 16 17
K L D K E N
K A D K E N
27.5 27.0 35.6 31.3 49.5 23.6
18
A
A
29.0
19 20 21 22 23 24 25 26 27 28 29 30 31
L D R A E Q A E A D K K A
I D R A E Q A E T D K K A
22.2 27.9 N.D. 20.6 27.8 16.6 12.9 17.4 6.7 14.6 16.6 21.7 15.7
Fig. 6. Comparison of partial amino acid sequences of lamprey and rabbit ~-tropomyosins. The lamprey and rabbit sequences are aligned for maximum identity. ND = not determined accurately due to integrator malfunction. The residue (ARG) was clearly identifiable at this position by inspecting the chromatogram.
-40-
I
200
I
220
I
240
Fig. 7. Far ultraviolet circular dichroism spectra of fish tropomyosins. The spectra are: A, cod (1.1 mg/ml); B, mako shark (1.1 mg/ml) and C, lamprey (0.4 mg/ml). The buffer was 0.1 M potassium chloride, 50 m M sodium phosphate, 0.01% (w/v) sodium azide, and 1 mM DTT, pH 7.00. Temperature = 10°C.
number of forms coexist within individual fibres (Bronson and Schachat, 1982; Heeley et al., 1983). Traditionally, TMs have been termed either or fl by the criterion of electrophoretic mobility (Cummins and Perry, 1973). On this basis, several fish species (cod, haddock and wolf-fish) would appear to contain ~-like TMs in both fast
Fish muscle tropomyosin Table 2. Comparison of fH values for fish tropomyosins
F. Mako shark (1.1 mg/ml)
10°C 10°C (80% TFE) 20°C
1.03 0.91 0.99
Cod (1.14mg/ml)
10°C 10°C (80% TFE) 20°C
0.95 0.84 0.93
Perch (0.8 mg/ml)
10°C 20°C
0.93 0.86
Mackerel (fast) (0.8 mg/ml)
10°C 20°C
1.01 0.93
Halibut (0.8 mg/ml)
10°C 20°C
0.9 0.87
Blue shark (0.8 mg/ml)
10°C 20°C
0.99 0.94
Lamprey (0.4 mg/ml)
10°C 20°C
1.02 0.85
Concentrations were determined by amino acid analysis. Nor-leucine was used as an internal standard and sample volumes were confirmed by weighing.
and slow muscles and lack fl-TM. This has also been demonstrated for carp (Feller et al., 1990). In other cases, however, the match between muscle type and TM type is quite dissimilar to what has been seen before (Dhoot and Perry, 1979). For salmon and herring, the TM component, having the greater (~-like) electrophoretic mobility, is intriguingly confined to the slow muscle, while the more slowly migrating (fl-like) form is located within the fast muscle. It remains to be seen whether this classification, as tentatively assigned by mobility measurements, will be vindicated by determination of the complete protein sequences. Another question of interest concerns what functional consequences, if any, will arise from the heterogeneity? It should be possible to begin to address this problem by making use of the simple and discreet isoform pattern of the Atlantic salmon. Overall, the findings of this report present a case for employing certain species of fish to conduct biochemical muscle research, particularly as it is feasible to purify, in quantitative amounts, specific isoforms of TM with relative ease and perhaps other thin filament proteins as well. Such components could furnish molecular insights into the connection between protein diversity and muscle performance. Acknowledgements--We thank Doug Hall and Sonya Banfield for the amino acid analyses, Joe Banoub for the mass spectral analyses, John Green for help with species
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classification, and Jackie Dalton for typing the manuscript. This work was supported by the Medical Research Council of Canada.
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