FISHERIES SCIENCE 2000; 66: 743–747

INTRODUCTION

Myosin of scallop adductor muscle has two kinds of lightchains, SH-light chain and regulatory light chain (RLC).EDTA treatment of scallop muscle myosin broughtreversible removal of RLC with accompanying loss ofCa-sensitivity, which is characteristic of scallop musclemyosin.1 Unlike skeletal muscle myosin, smooth muscleand scallop muscle myosins are regulated molecules inmuscle contraction. Scallop muscle contraction is regu-lated by direct binding of calcium to myosin RLC,2 andsmooth muscle contraction is triggered by phosphoryla-tion of myosin RLC.

Scallop adductors contain two different types ofmuscle, one striated and the other catch. Catch muscleis unique in that it is capable of maintaining contrac-tive tension for a long time with a very low metabolicturnover (catch contraction). Although the mechanismunderlying catch contraction is not clear, the contribu-tion of paramyosin,3,4 phosphorylation of myosin heavychain (MHC)5 and the RLC6 has been suggested. Theseunique properties of scallop have brought extensivestudies on scallop striated and catch muscle myosins.

The scallop myosins of striated and catch musclesarise from an alternative RNA splicing of a single MHCgene.7 Reverse transcription–polymerase chain reaction(RT-PCR) study using alternative exon specific primersshowed that catch muscle myosin is expressed in mantle,heart and other tissues but not in striated muscle.7

However, there has been no report about the isolationand identification of myosins from tissues other thanmuscle. Many studies about myosin of mollusca such asakazara,8 squid,9 and abalone10 also have been performedin muscle tissues so far.

In the present study, we isolated myosin from mantlepallial cell layer of scallop, the tissue which containsseveral types of non-muscle cells such as mucous cellsand epithelial cells as reported by Tsuji11 and Wada.12 Wereport the structural characteristics and enzymic proper-ties of the pallial cell myosin which are different fromthose of striated and catch muscle myosins.

MATERIALS AND METHODS

Protein preparation

Adductors (striated and catch muscles) and mantle were prepared from scallop, Patinopecten yessoensis. Themantle pallial cell layer was separated from muscle byscraping with a knife. The separated pallial cell layer wasemployed for extraction of myosin.

Original Article

Isolation and characteristics of intrinsic myosin in mantlepallial cell layer of scallop

Takahiro ARAKI AND Yasushi HASEGAWA*

Muroran Institute of Technology, Department of Applied Chemistry, Muroran, Hokkaido 050-0071, Japan

SUMMARY: It has been reported that catch muscle type myosin is expressed in various tissues ofscallop, including gonad, heart, foot and mantle. However, there has been no report about the iso-lation and identification of myosin from tissues other than the adductor muscle. We isolated myosinfrom the mantle pallial cell layer of scallop and compared the structure and the characteristics withthose of striated and catch muscle myosins. Comparison among three kinds of myosins in cleavagepatterns by trypsin and cyanogen bromide revealed that myosin in mantle pallial cell layer (pallial cellmyosin) has a different structure from striated and catch muscle myosins. The Mg2+-ATPase activityof pallial cell myosin is significantly lower compared with those of striated and catch muscle myosins.The present results indicate the existence of the intrinsic myosin in mantle pallial cell layer.

KEY WORDS: ATPase activity, isolation, mantle, myosin, pallial cell layer, peptide map,scallop.

*Corresponding author: Tel: 81-143-46-5750. Fax: 81-143-46-5700.Email: hasegawa@oyna.cc.muroran-it.ac.jp

Received 8 September 1999. Accepted 8 February 2000.

1/100. At an appropriate time, 0.2 mL of the reactionmixture was pipetted up and added to trichloroaceticacid at a final concentration of 3% for termination of the reaction. The mixture was centrifuged at 14 000 gfor 10 min and the resulting precipitate was dissolved in a solution containing 2% SDS, 0.1 M Tris, 0.1% 2-mercaptoethanol, 0.001% bromophenol blue (SDS-buffer) for SDS-PAGE.

Electrophoresis

SDS-PAGE and PAGE in the presence of 8 M urea(urea-PAGE) were carried out according to the methodof Laemmli,13 and Kendrick-Jones et al.14 The gels werestained with Coomassie brilliant blue-G.

Tissue homogenates were prepared as follows. Thestriated muscle, catch muscle, and pallial cell layer wereseparately homogenized in a solution containing 0.2 MNaCl, 5 mM MgCl2, 1 mM EGTA, 20 mM Tris-HCl (pH 7.5), 0.1 mM phenylmethylsulfonyl fluoride, 0.1 mMdiisopropyl phosphofluoridate, 0.1% 2-mercaptoethanol,and 5 mM ATP, and then protein concentrations were determined by the biuret method. Samples ofhomogenates were immediately placed in SDS-bufferand boiled.

For urea-PAGE, purified striated muscle, catchmuscle, and pallial cell myosins were precipitated by 3%trichloroacetic acid and the precipitates were dissolvedin 8 M urea, 0.1 M Tris, 0.1% 2-mercaptoethanol, and0.001% bromophenol blue and loaded onto urea-PAGE.

ATPase assay

ATPase activity was measured according to the methodof Lin and Morales.15 Mg2+-ATPase activity was mea-sured at 15°C in a solution containing 0.1 mg/mLmyosin, 50 mM KCl, 20 mM Tris-HCl (pH 7.5), 5 mMMgCl2, 1 mM ATP, and 0.1 mM CaCl2 or 0.5 mM EGTA.

RESULTS

Preparation of pallial cell myosin

The actomyosin solution that was extracted from thewashed tissue homogenate of mantle pallial cell layer wasfractionated at 40–60% ammonium sulfate in the pres-ence of 5 mM MgATP. This led to removal of almost F-actin and contaminated proteins. At the final step of purification procedure, Sepharose CL-4B gel filtrationwas performed to remove minute contaminated proteins.Yield of myosin was usually approximately 1–2 mg from50 g of mantle pallial cell layer. According to the sameprocedure, about 55 mg and 95 mg of myosins could beobtained from 50 g of catch and striated muscle, respec-tively. Fig. 1a shows SDS-PAGE patterns of striated

Preparation of pallial cell myosin

Pallial cell layer was blended in a solution containing 20 mM MgCl2, 20 mM MOPS-NaOH (pH 7.0), and 1 mM EGTA and washed three times with 4 volumes ofthe same solution. The washed tissues were suspended in3 volumes of a solution containing 0.2 M NaCl, 5 mMMgCl2, 1 mM EGTA, 20 mM Tris-HCl (pH 7.5), 0.1 mMphenylmethylsulfonyl fluoride, 0.1 mM diisopropyl phos-phofluoridate, 0.1% 2-mercaptoethanol, and 5 mM ATP.After stirring for 2 h, the suspension was centrifuged at 22 000 ¥g for 20 min. Ammonium sulfate was added to the supernatant to 40% saturation and the resultingprecipitate was discarded, then crude actomyosin was precipitated with 60% saturated ammonium sulfate. The pellet was dissolved in a small volume of solutionconsisting of 0.6 M NaCl, 5% sucrose, 20 mM Tris-HCl(pH 7.5), 1 mM EGTA, 5 mM MgCl2, 0.1 mM phenyl-methylsulfonyl fluoride fluoride, 0.1 mM diisopropylphosphofluoridate, and 0.1% 2-mercaptoethanol (solu-tion A), and then loaded onto Sepharose CL-4B gel fil-tration column (2.6 ¥ 88 cm) equilibrated with solution Aat a flow rate of 24 mL/h. Fractions containing myosinwere pooled and diluted with 12 volumes of 20 mMMgCl2. The precipitated myosin was collected by cen-trifugation at 22 000 ¥g and dissolved in 0.5 M KCl. The purified myosin was stored in 50% glycerin at -20°C.Striated and catch muscle myosins were prepared by thesame method.

Cyanogen bromide digestion

The heavy chains of striated muscle, catch muscle, and pallial cell myosins (50 mg) were separated from the respective light chains by sodium dodecylsulfate–polyacrylamide gel electrophoresis (SDS-PAGE), andthe heavy chain band was cut out after staining. The gelswere soaked in 100 mL distilled water and dried up. Thisstep was repeated three times. The gel slices were incu-bated with 0.15 M CNBr in 70% formic acid for 24 h at room temperature, then dried up. Distilled water (100 mL) was added to the shrunken gels and then driedup to remove absolutely CNBr and formic acid. After 20 mL of 120 mM Tris-HCl (pH 6.8), 2% SDS, and 0.1%2-mercaptoethanol was added, the gel slices were leftstanding for 1 h at room temperature, and then added toan SDS-PAGE well.

Trypsin digestion

Myosins (0.2 mg/mL) purified from striated muscle, catchmuscle, and mantle pallial cell layer were separately dis-solved in a solution containing 0.6 M KCl, 20 mM Tris-HCl (pH 7.5), 5 mM MgCl2, 1 mM ATP, 1.0 mM CaCl2,and 0.1% 2-mercaptoethanol. Proteolysis was started byadding trypsin at an enzyme-to-myosin weight ratio of

744 FISHERIES SCIENCE T Araki and Y Hasegawa

muscle, catch muscle and pallial cell homogenates.Myosin content, judged by inspection of densitometricscans of SDS-PAGE patterns, was estimated to be about5.9% of the total proteins in pallial cell layer. Themyosin content was about 38.2% in striated muscle and14.0% in catch muscle, suggesting that the myosincontent in mantle pallial cell layer is lower than thosein striated and catch muscles.

The myosin isolated from mantle pallial cell layer iscomposed of 200 kDa heavy chain and 17 kDa lightchain components (Fig. 1b). The light chain component(component A) of pallial cell myosin showed the samemigration with RLC of striated muscle myosin on urea-PAGE (Fig. 1c). Treatment with 10 mM EDTA of puri-fied pallial cell myosin induced the dissociation ofcomponent A (data not shown), suggesting that compo-nent A was RLC. Purified catch muscle myosin containstwo kinds of RLC isoforms (RLC-a and RLC-b),16 whichare not detected in pallial cell myosin (Fig. 1c). Compo-nent B showed the same migration with SH-LC of stri-ated and catch muscle myosins and the density ratio ofcomponent B to component A was nearly 1.0. It is sup-posed that component B is SH-LC.

Comparison of pallial cell myosin with striated andcatch muscle myosins

In order to compare the structure of pallial cell myosinwith those of striated and catch muscle myosins, the

digestions of the heavy chains with CNBr and trypsinwere performed as described in Materials and Methods.Digestions with CNBr gave very similar patterns amongthree kinds of myosins, but the appearance of 60 kDafragments was characteristic in catch muscle MHC (Fig2a), indicating that the primary structure of pallial cellmyosin was different from that of catch muscle myosin.Digestions of purified myosins with trypsin at a 1 : 100(w/w) ratio also resulted in the generation of similar frag-ments from three kinds of myosins, but the degradationrate of pallial cell MHC was clearly slower than those ofthe others (Fig. 2b). The time course of the decay ofMHC band could be followed by plotting log (remain-ing MHC relative intensity) versus digestion time, whichgave pseudo-first-order rate constant (data not shown).The rate constants for striated muscle, catch muscle, and pallial cell MHC were 4.3 ¥ 10-4/s, 0.68 ¥ 10-4/s and0.26 ¥ 10-4/s, respectively. These results show that sus-ceptibility of pallial cell MHC to trypsin is clearly dif-ferent from those of striated and catch muscle myosins.

ATPase activity

In order to compare the enzymic properties of pallial cellmyosin with those of striated and catch muscle myosins,the Mg2+-ATPase activity was investigated (Table 1).The Mg2+-ATPase activity of pallial cell myosin was 0.38± 0.05 mmol Pi/min per mg and 0.14 ± 0.01 mmol Pi/minper mg in the presence and absence of calcium, suggest-

Myosin in mantle pallial cell layer of scallop FISHERIES SCIENCE 745

(a) (b) (c)

Fig. 1 Comparison among striated and catch muscle myosins, and purified pallial cell myosin by polyacrylamide gel electrophore-sis (PAGE). (a) Sodium dodecylsulfate (SDS)-PAGE patterns of pallial cell (lane 1), striated muscle (lane 2), and catch musclehomogenates (lane 3). The homogenates was prepared as described in Materials and Methods, and 15 mg of the homogenates wassubjected to SDS-PAGE. MHC denotes myosin heavy chain. (b) Extract from washed tissue of mantle pallial cell layer (lane 2) andpurified pallial cell myosin (lane 3) were run on 12% SDS-PAGE. Molecular weight markers were run in lane 1. (c) Urea-PAGEfor resolving myosin light chain components. The samples were prepared as described in Materials and Methods. Lane 1, catchmuscle myosin; lane 2, striated muscle myosin; lane 3, pallial cell myosin. A and B show the light chain components of pallial cellmyosin. Regulatory light chain (RLC) a and RLC-b show regulatory light chain isoforms of catch muscle myosin shown by Kondoand Morita.16 The RLC of striated muscle myosin shows the same migration with RLC-b.16

presence of calcium. The pallial cell myosin has a lowersteady-state ATPase activity than myosins of striated and catch muscles, indicating that enzymic properties ofpallial cell myosin are also different from those of stri-ated and catch muscle myosins.

ing that the pallial cell myosin has a calcium-regulatedmechanism similar to striated and catch muscle myosins.In contrast, the Mg2+-ATPase activity of pallial cellmyosin was about 28% that of striated muscle myosinand about 67% that of catch muscle myosin in the

746 FISHERIES SCIENCE T Araki and Y Hasegawa

(b)

(a)

Fig. 2 Cleavage of three kinds of scallop myosins with CNBr (a) and trypsin (b). Each band of scallop MHC was excised aftersodium dodecylsulfate–polyacrylamide gel electrophoresis (SDS-PAGE), and the treatment with CNBr was performed at room tem-perature for 24 h. The digestion fragments were analyzed by 15% SDS-PAGE. (a) Lane 1, striated muscle myosin; lane 2, catchmuscle myosin; lane 3, pallial cell myosin. Molecular weight markers were run in lane M. (b) Striated muscle myosin (1), catchmuscle myosin (2), and pallial cell myosin (3) at 0.2 mg/mL were separately incubated with trypsin at a 100 : 1 (w/w) ratio at 15°Cin the solution consisting 0.6 M KCl, 20 mM Tris-HCl (pH 7.5), 5 mM MgCl2, 1 mM CaCl2, 1 mM ATP, and 0.1% 2-mercap-toethanol. Aliquots were taken after digestion for the times indicated, and 3% trichloroacetic acid was added to terminate the reaction. After centrifugation at 14 000 ¥g for 5 min, the pellet was dissolved in SDS-buffer and analyzed by SDS-PAGE.

The present results suggest that the structural charac-teristics and enzymic properties of pallial cell myosin are different from those of striated and catch musclemyosins. The observed difference is not due to the dif-ference of preparation procedure because we purified stri-ated and catch muscle myosins by the same method asdescribed in Materials and Methods.

DISCUSSION

Digestions of purified striated muscle, catch muscle, andpallial cell myosins with trypsin and CNBr resulted inthe generation of similar fragments from three kinds ofmyosins (Fig. 2). In addition, polyclonal antibody gener-ated against pallial cell myosin recognized striated andcatch muscle MHC, and western blot analysis of frag-ments generated with trypsin treatment of three kinds ofmyosins revealed that the antibody could not distinguishamong the fragments of these three kinds (data notshown). It is supposed that the primary structure ofpallial cell myosin has homology with those of catch andstriated muscle myosins. Nyitray et al. reported that catchmuscle myosin is produced by an alternative splicingfrom the same gene as the striated muscle myosin, andsuggested that another myosin isoform is probablypresent in scallop, which are produced by an alternativesplicing from the same gene.7 Similarity of structuralcharacteristics among the three kinds of myosins maysuggest that pallial cell myosin is also produced by atissue-specific alternative splicing.

The putative amino acid sequences between striatedand catch muscle MHC are well conserved with an iden-tity of 97% although the differences of these myosins aredistinct.7,17 The catch muscle myosin has a considerablylower steady-state ATPase activity than striated musclemyosin,16 and the in vitro motility assay shows that striated muscle myosin moves actin filament faster than catch muscle myosin.17 The two scallop myosins areuseful to determine the correlation between the varia-tions in the heavy chain sequences and the differencesin the ATPase activities.17 The pallial cell myosin has a

lower ATPase activity than striated and catch musclemyosins (Table 1). To determine the full-length sequenceof pallial cell myosin will give more information aboutthe correlation and it will be necessary to determine the characteristics of pallial cell myosin in more detailalthough it could not be determined in the present studybecause of its very low yield.

REFERENCES

1. Chantler PD, Szent-Gyorgyi AG. Regulatory light chains andscallop myosin. Full dissociation, reversibility and co-operativeeffects. J. Mol. Biol. 1980; 138: 473–492.

2. Kendrick-Jones J, Lehman M, Szent-Gyorgyi AG. Regulation inmolluscan muscles. J. Mol. Biol. 1970; 54: 313–326.

3. Achazi RK. Phosphorylation of molluscan paramyosin. PflügersArch. 1979; 379: 197–201.

4. Cooley LB, Johnson WH, Krause S. Phosphorylation of para-myosin and its possible role in the catch mechanism. J. Biol. Chem.1979; 254: 2195–2198.

5. Castellani L, Cohen C. Myosin rod phosphorylation and the catchstate of molluscan muscles. Science 1987; 235: 334–337.

6. Takahashi M, Sohma H, Morita F. The steady state intermediateof scallop smooth muscle myosin ATPase and effect of light chainphosphorylation. A molecular mechanism for catch contraction.J. Biochem. 1988; 104: 102–107.

7. Nyitray L, Jansco A, Ochiai Y, Graf L, Szent-Gyorgyi AG. Scallopstriated and smooth muscle myosin heavy-chain isoforms are pro-duced by alternative RNA splicing from a single gene. Proc. NatlAcad. Sci. USA 1994; 91: 12 686–12 690.

8. Nishita K, Ojima T, Watanabe S. Myosin from striated adductormuscle of Chlamys nipponesis akazara. J. Biochem. 1979; 86:663–673.

9. Yoshitomi B, Konno K. Enzymatic properties of myosin ATPasefrom squid Todarodes pacificus mantle muscle. Nippon SuisanGakkaishi 1982; 48: 581–586.

10. Asakawa T, Azuma N. Enzymatic properties of myosin ATPasefrom abalone Haliotis discus smooth muscle. Nippon SuisanGakkaishi 1987; 53: 1243–1249.

11. Tsuji T. Studies on the mechanism of shell- and pearl-formationin Mollusca. J. Fac. Fish. Pref. Univ. Mie 1960; 5: 1–70.

12. Wada K. Studies on the mineralization of the calcified tissue inmolluscs-VIII. Histological and histochemical studies of mucous-cells on the inner and shell surfaces of mantle of some bivalve andgastropod molluscs. Bull. Natl Pearl Res. Lab. 1966; 11: 1283–1297.

13. Laemmli UK. Cleavage of structural proteins during the assemblyof the head of bacteriophage T4. Nature 1970; 227: 680–685.

14. Kendrick-Jones J, Szentkralyi EM, Szent-Gyorgyi AG. Regulatorylight chains in myosin. J. Mol. Biol. 1976; 104: 747–775.

15. Lin T, Morales MF. Application of a one-step procedure for mea-suring inorganic phosphate in the presence of proteins: The acto-myosin ATPase system. Anal. Biochem. 1977; 77: 10–17.

16. Kondo S, Morita F. Smooth muscle of scallop adductor contains atleast two kinds of myosin. J. Biochem. 1981; 90: 673–681.

17. Kurazawa-Goertz SE, Perreault-Micale CL, Trybus KM, Szent-Gyorgyi AG, Greeves MA. Loop I can modulate ADP affinity,ATPase activity, and motility of different scallop myosins. Tran-sient kinetic analysis of S1 isoforms. Biochemistry 1998; 37:7517–7525.

Myosin in mantle pallial cell layer of scallop FISHERIES SCIENCE 747

Table 1 Mg2+-ATPase activity* of scallop myosins

+Ca2+ (mmol -Ca2+ (mmol Pi/min per mg) Pi/min per mg)

Pallial cell 0.38 ± 0.05 0.14 ± 0.01Striated muscle 1.35 ± 0.20 0.69 ± 0.03Catch muscle 0.56 ± 0.06 0.14 ± 0.03

* The ATPase activity was measured at 15°C using 0.1 mg/mLmyosin in 50 mM KCl, 20 mM Tris-HCl (pH 7.5), 5 mM MgCl2, and1 mM ATP in the presence of 0.5 mM EGTA or 0.1 mM CaCl2. Valuesrepresent the means and the SEM of three to six determinations.