Biochemical and structural heterogeneity of fast and slow muscles of the blue crab Portunus trituberculatus

Comp. Biochem. PhysioL Vol. 90B, No. 2, pp. 355-360, 1988 Printed in Great Britain

0305-0491/88 $3.00+0.00 © 1988 Pergamon Press pie

BIOCHEMICAL A N D STRUCTURAL HETEROGENEITY OF FAST A N D SLOW MUSCLES OF THE BLUE CRAB PORTUNUS TRITUBERCULATUS Y. OCHIAI, Y. KARIYA and K. HASHIMOTO Laboratory of Marine Biochemistry, Faculty of Agriculture, University of Tokyo, Bunkyo-ku, Tokyo 113, Japan (Received 23 July 1987)

Abstract--1. Two types of muscle (white and yellowish) in the swimming leg of the blue crab were compared in respect to protein composition, electrophoretic patterns of protein fractions, myofibrillar ATPase activity and ultrastructure. 2. Proportions of water-soluble and stroma protein fractions were larger in the yellowish than in the white muscle. Myofibrillar Ca2+-ATPase activity of white muscle was less thermostable than in the yellowish muscle counterpart. 3. Electron microscopy showed that yellowish muscle was abundant in mitochondria, though the arrangement of thick and thin filaments hardly differ between the two types of muscles. 4. The results obtained suggested that white and yellowish muscles of the blue crab correspond to fast and slow muscles, respectively.

INTRODUCTION

Skeletal muscles are differentiated into many types, according to their roles or modes of movement: e.g. shortening velocity, enzyme activities such as myofibrillar ATPase activity and amounts of mitochondria and respiratory pigment. In particular, myosin ATPase activity is roughly parallel to the contraction speed of the muscle (B/tr~iny, 1967). Higher vertebrates are generally endowed with two types of muscle, fast (white) and slow (red) muscles. The former is characterized by a high shortening velocity and thus suitable for burst locomotion, whereas the latter has a low shortening velocity and a high content of myoglobin, making it suitable for ordinary movement or maintenance of posture. A similar specialization is also true for fish muscle, which can be classified into ordinary (white) and dark (red) muscles, corresponding to fast and slow types, respectively (Watabe and Hashimoto, 1980; Tsukamoto, 1984). Dark muscle is usually localized along the lateral line of fish. Blue crabs and some other crabs swim actively. Their fifth pereiopods (swimming legs) are shaped as an oar, suitable for swimming. The muscle of this leg contains a yellowish part, in addition to the ordinary white part (Ochiai and Hashimoto, 1985), as is the case with the musculature of the root part of this leg. Yellowish muscle, just like dark muscle of fish, seemed to be involved in the swimming of the crab. Morphological and histological descriptions of the yellowish or analogous muscles have been made (Ogata and Mori, 1964; Hoyle and MacNeil, 1968; Morin and McLaulin, 1973; Ogonowski and Lang, 1979; Silverman and Charlton, 1980). We reported previously that the yellowish color was exclusively due to pteridines (Ochiai and Hashimoto, 1985). However, the chemical composition and function of yellowish muscle for the most part remains to be elucidated. 355

Under these circumstances, we examined and compared both blue crab muscles with respect to protein composition, electrophoretic patterns of protein fractions, myofibrillar ATPase activity, along with ultrastructure. The results obtained showed that the yellowish muscle is a highly oxidative tissue, enabling this crab to swim actively.

MATERIALS AND METHODS

Materials Live specimens of the blue crab Portunus trituberculatus (average body weight, about 400 g) were purchased at the Tokyo Central Wholesale Market. Swimming legs and their roots were excised and yellowish and white muscles were macroscopicaUy separated from each other on ice. Protein composition Protein composition was examined according to the method of Hashimoto et al. (1979). Briefly, the muscle was fractionated into water-soluble (sarcoplasmic), salt-soluble (myofibrillar), alkali-soluble and stroma protein fractions, and those four fractions were determined for nitrogen content by a miero-Kjeldahl method. Preparation of myofibril and myosin Myofibrils were prepared according to Lehman (1977) and Perry and Grey (1956) with some modifications. Minced muscle was washed successively with 30vol of 10raM sodium phosphate buffer (pH 7. I) containing 0.3 M sucrose, 1 mM MgCI2, 1 mM ethylene glycol bis(]Laminoethylether)-N,N,N',N'-tetraacetic acid (EGTA) and 0.1 M NaCI and with 5 vol of 39 mM borate buffer (pH 6.9) containing 0.1 M KC1. This procedure was repeated four more times. Finally, the myofibrils obtained were suspended in 50 mM Tris-maleate buffer (pH 7.5) and used for analyses. Myosin was prepared from each muscle according to Stafford et al. (1979). Light chain subunit fraction was separated by urea treatment of myosin according to Lowey and Holt (1972).

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Measurement of Ca2+-ATPase activity Myofibrillar Ca2+-ATPase activity was measured in 50mM Tris-maleate or glycine-KOH buffer containing 1 mM CaC12, 50 mM KCI, 1 mM ATP at a protein concentration of 0.1-0.2 mg/ml. To the mixture was added 0.1 mM sodium azide as a mitochondrial Ca2+-ATPase inhibitor (Ogonowski and Lang, 1979). The pH-activity relationship was examined in 50 mM Tris-maleate buffer (pH 5-8.5) and 50 mM glycine-KOH buffer (pH 9-12) at 25°C. Temperature--aetivity relationship was studied in 50mM Trismaleate (pH 8.0) between 20-45°C. The reaction was stopped by addition of 5% trichloroacetic acid and the Pi liberated was determined according to Fiske and SubbaRow (1925). Electrophoresis Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed according to Laemmli (1970) in 15% slab gels. Two dimensional PAGE was performed according to Mikawa et al. (1981). For the first dimension, 3% gels in a capillary tube (0.8 mm diameter, 6 cm length) were used. The concentration of Ampholine (LKB) was 2%, with a volume ratio of Ampholine (pH 3.5-10)/Ampholine (pH 4--6) of 0.25. For the second dimension, mini slab gels (15%, 0.8 mm thickness) were used. After the run, the gels were stained as usual with Coomassie brilliant blue R-250. Electron microscopy Electron microscopy was performed as reported previously (Ochiai et al., 1985). The muscle was prefixed in 4% glutaraldehyde, and stained with 1% uranyl acetate. After dehydration, it was embedded in Poly/Bed 812 (Polysciences Inc.). Micrographs were taken with a JEM 100-S type transmission electron microscope (JEOL Co.) at magnifications of 5000-30,000. Protein concentration Protein concentration was determined by the biuret method (Gornall et al., 1949).

Table 1. Protein compositionof white and yellowishmusclesfrom blue crab (rag N/g) Protein White Yellowish fraction muscle muscle Water-soluble 9.6 (41.0)* 10.7 (48.7) Salt-soluble 7.5 (32.1) 6.4 (29.1) Alkali-soluble 5.8 (24.8) 4.0 (18.2) Stroma 0.5 (2.1) 0.9 (4.1) Total 23.4 (100.0) 22.0 (100.1) Non-protein nitrogen 9.8 8.6 *Numbers in parenthesis represent the percentage distribution of protein nitrogen. fraction was 9.6 and 10.7 mg N/g muscle, or 41 and 49% of total protein nitrogen for white and yellowish muscles, respectively. The proportion of this fraction was clearly higher in yellowish muscle, reminding us of fish dark muscle, which also has a higher watersoluble protein content. On the other hand, saltsoluble protein fraction of white muscle showed a higher nitrogen content (7.5 mg N/g) than that of yellowish muscle (6.4 mg N/g). These nitrogen contents accounted for 32 and 29%, respectively, of total protein nitrogen. Alkali-soluble protein fraction, consisting mostly of denatured myofibrillar proteins, was 5.8 and 4.0 mg N/g for white and yellowish muscles, respectively. Nitrogen levels of this fraction were much higher than those for both muscles of some fishes (0.4-1.8rag N/g) (Hashimoto et al., 1979), suggesting that blue crab myofibrillar proteins generally are more labile than the corresponding proteins of fish. The content of stroma protein was 0.5 and 0.9 mg N/g, or 2.1 and 4.1% in total protein nitrogen for white and yellowish muscles, respectively. This again reminded us of similar differences between ordinary and dark muscles of mackerel and sardine (Hashimoto et al., 1979). Electrophoresis

RESULTS AND DISCUSSION

Protein composition

The protein composition of white and yellowish muscles is shown in Table 1. Water-soluble protein

W

Y

W

SDS-PAGE patterns of water-soluble and saltsoluble protein fraction from white and yellowish muscles are shown in Fig. 1. Clear differences were observed in the pattern of water-soluble protein fraction (Fig. I a). The fraction from yellowish muscle

W

Y

Y

"~ MHC PM A

MLC

a

b

c

Fig. 1. SDS-polyacrylamide gel elctrophoresis of water-soluble (a) salt-soluble (b) protein fractions, and myosins (c) from white (W) and yellowish (Y) muscles of blue crab. 15% gel. The bars in (a) represent protein bands specific to yellowish muscle. Abbreviations: MHC, myosin heavy chain; PM, paramyosin; A, actin; MLC, myosin light chains. The arrows in (c) indicate myosin light chains.

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WATER-SOLUGLE

SALT-SOLUBLE

WHITE

YELLOWISH +

OH-

H÷

Fig. 2. Two dimensional polyacrylamide gel electrophoresis of water-soluble and salt-soluble protein fractions from white and yellowish muscle of blue crab. First dimension: isoelectric focusing on 3% polyacrylamide gels; 2nd dimension: SDS-polyacrylamide gel electrophoresis on 15% gels. Asterisks indicate the spots corresponding to actin. Arrowheads indicate the spots of actin.

exhibited more bands than that from white muscle, suggesting the presence of more enzymes. In this connection, the water-soluble fraction of dark muscle of sardine and mackerel showed more protein bands than the ordinary muscle (Hashimoto et al., 1979). On the other hand, the patterns of salt-soluble protein fraction hardly differed between the two types of muscles, i.e. the fractions from both muscles were mainly composed of mytYsin, paramyosin and actin (Fig. lb). Then the two protein fractions were applied to two dimensional P A G E (Fig. 2). The water-soluble fraction of either muscle gave rise to a dense spot (indicated by the asterisk in the left half of Fig. 2). These spots were considered to be actin based on the mol. wt and isoelectric point. In the case of yellowish muscle, two additional dense spots appeared in the lower t o o l wt range. In addition, some minor differences were recognized between both patterns.

The salt-soluble fractions of both muscles resembled each other, as far as the higher tool. wt substances (myosin heavy chain, paramyosin, actin, etc.) are concerned (Fig. 2, right half). However, large differences were recognized between both muscles in the lower mol. wt range, suggesting the occurrence of isoforms of myosin light chains or troponin subunits. Further experiments, however, are necessary for their identification. When analyzed by SDS-PAGE (Fig. lc), each myosin gave rise to two light chains (indicated by arrows), in addition to a heavy chain. The two light chains were in almost equimolar amounts with each myosin, as examined by densitometry of Coomassie Brilliant Blue-stained gels (data not shown). Their mol. wts were 18,000 and 22,000, irrespective of the type of myosin. In this connection, each myosin showed also two light chains in urea-PAGE (Perrie and Perry, 1970) (data not shown).

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5

6

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10

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Fig. 3, pH-activity curves of myofibrillar Ca2+-ATPase from white and yellowish muscles of blue crab. The activity was measured in 50 mM Tris-maleate buffer (pH 5-8.5) or 50 mM glycine-KOH buffer (pH 9-12) at 25°C. The assay solution contained 1 mM CaC12, 50 mM KC1, 1 mM ATP and 0.1-0.2mg/ml of myofibril. O, White muscle, O, yellowish muscle.

Fig. 4. Temperature-activity curves of myofibrillar Ca2+ATPase from white and yellowish muscles of blue crab. The activity was measured in 50 mM Tris-maleate buffer (pH 8.0) containing 1 mM CaC12, 50 mM KC1, I mM ATP and 0.1-0.2 mg]ml of myofibril. ©, White muscle; O, yellowish muscle.

Myofibrillar Ca2 +-A TPase activity The pH-activity curves of both myofibrillar Ca2+-ATPases are shown in Fig. 3. White muscle myofibrils showed a maximum at around pH 6.5, the activity being 0.78#mol Pi/min.mg. On the other hand, yellowish muscle myofibrils showed a lower activity as a whole, with a maximum at around pH 6. The myofibrillar ATPase activity of each muscle tended to decrease with increasing pH. In the range of pH 10-11, the activity of yellowish muscle myofibrils somewhat exceeded that of white muscle counterpart. This suggests that yellowish muscle myofibrils are generally more stable than those of white muscle at alkaline pH. In the present experiments, the assay system contained 0.1 mM sodium azide as the inhibitor against mitochondrial Ca2+-ATPase activity which might be contaminated. However, the myofibriUar ATPase activity in the presence of sodium azide was more than 90% of that in its absence over the pH range examined (data not shown). It follows that the present preparations were almost free from mitochondria. Temperature-activity relationship was examined (Fig. 4). Activity maxima appeared at around 30 and 32.5°C for white and yellowish muscle myofibrils, respectively. The activity values were 0.55 and 0.45#mol Pi/min.mg, respectively. The activity of yellowish muscle myofibrils was rather lower than that of white muscle myofibrils in the range from 20-37.5°C. In connection to this, watabe et al. (1983) reported that mackerel ordinary muscle myofibrils exhibited maximum ATPase activity at 32.5°C, whereas dark muscle counterpart showed maximum activity at around 35°C. Kariya et al. (1986) performed similar experiments on the arm and mantle muscle myofibrils of octopus and found that the optimum temperature of mantle myofibril was 42.5°C, being somewhat higher than that of arm muscle counterpart (40°C). They also found much

more mitochondria in mantle muscle and claimed that this muscle resembles fish dark muscle rather than the ordinary muscle. At around 40°C, the activity of yellowish muscle myofibrils exceeded that of white muscle myofibrils, indicating that the former is more thermo-resistant. However, both myofibrils were completely inactivated at 45°C. Also, Watabe et al. (1983) reported that mackerel dark muscle myofibrils still maintained some activity even at 50°C, the temperature at which ordinary muscle myofibrils lose activity completely. Electron microscopy Electron micrographs of the cross section of white and yellowish muscles are shown in Fig. 5. Thick filaments of about 20 nm diameter were surrounded by 12 thin filaments, though the arrangement of thin filaments was partly disordered (refer to the inserted figures in Fig. 5). As far as the disposition of thin filaments is concerned, blue crab muscles seem to differ from vertebrate muscle in which six thin filaments surround each thick filament. In addition, all thick filaments from both crab muscles possessed a hollow in the core. Maeda (1978) also observed a hollow in the core of thick filaments of blue crab white muscle. The cross section of muscle cell was generally wider in the white than yellowish muscle. This also seems to support'the view that blue crab white and yellowish muscles correspond to fish ordinary and dark muscles, respectively. Microscopy of longitudinal sections showed that both muscles of blue crab were cross-striated. An example of the longitudinal section of yellowish muscle is shown in Fig. 6. It shows the presence of abundant mitochondria, which was reminiscent of highly oxidative muscles such as the mantle muscle of octopus (Kariya et al., 1986). As reported previously, the color of blue crab yellowish muscle is due

Fast and slow muscles of the blue crab

WHITE MUSCLE

YELLOWISHMUSCLE

Fig. 5. Electron microscopy of the cross sections of white and yellowish muscles of blue crab. The bar represents 1 pm. The inserted figures at the right top are the enlarged ( x 4) micrographs of the indicated regions.

Fig. 6. Electron microscopy of the longitudinal section of yellowish muscle of blue crab, showing the array of mitochondria. The bar represents 1/zm.

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to the presence of pteridines (Ochiai et al., 1985), though their possible role in this highly oxidative tissue remains to be solved. It was suggested from these results that white and yellowish muscles of blue crab correspond to fast and slow muscles, respectively, and that yellowish muscle enables this crab to swim actively. Acknowledgements--This study was supported in part by a Grant-in-Aid from the Ministry of Education, Science and Culture of Japan.

REFERENCES

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