The species specificity of the contractile protein composition of the bivalve molluscs

THE SPECIES SPECIFITY OF THE CONTRACTILE PROTEIN COMPOSITION OF THE BIVALVE MOLLUSCS Bom A. MAKWLIS’

ANI)

GIOKGI

P. PINAI \’

Lahoratork of Physiological Fcology. Institute 01‘Marine Biologq ol’ thc Far-Fast ol thc Academ- of Sclcnccs of the II.S.S.R.. Vladlvostok 22. (‘.S.S.R. and ‘Lahoratory of Biochemlstry of Cel1 Reproductlon. Institute of Cytologq of thc Acndcm! 01‘Scienccs of thc I1.S.S.R.. LenIngrad 121.1’S.S.R.

Ccntcr

1. Prc,pal-ations of thc contractile proteins isolated from 26 species of hivalve molluscs wcrc atudlcd hq sodium dodecylsulfate (SDS) and Crca- clcctrophorcsls and also hy SDS-disc-clectrophoresls. of molluscs myofihrils which are always present are heavy and 1. It was shoun that componcnts light chains of myosm. paramyosin. actin. and tropomyosin. Tl-opom?osm and light chains 01‘mysln in diffcrent spcc~cs varied in mol. wt and clcctrical charge minor components were detected among the contractile proteins. Their : .4 numhcr unidentified quantitand mohility rcmain constant for a given mollusc genus or famil!. 1. Flcctrophoretic data show that contractile protcm compositlon is speclcs spccific for mollusc~. 5, Possihllity 01‘thr usefulncss 01 these results for the analqsls of qstematic relations in the clahs H~rtrlrru is discuysed

Abstract

I’LTRODI

CTIOU

MATERIALS

Analysis of thc structure and function of the adductor musclcs in different species of molluscs has revealed a great varicty of ultrastructures and contractile activities of these tissues (Scorobovichuk. 1974; Philpott cr trl.. 1960: Morrison $2 Odense. 1974). Comparative biochemicdl study of proteins forming these structures may givc valuable information about the principles of organiration and evolution of contractile apparatus. From thc studying of the protein composition of molJusc adductors we have detected a certain corrclation betwcen the systematic state of mollusc species and pattern of its contractile proteins (Margulis &LPinaev. 1974). To get more precise information we have made an additional sclection of mollusc species which according Scarlato (1974). represent almost al1 the taxonomie groups of the class Bioalria. inhabiting in North-West part of Pacific Ocean. All the known types of contractilc systems always contain thrce proteins: myosin. actin. and tropomyosin. In addition. it was found that thick filaments of molluscs contain also paramyosin (Elliot cr al., 1957). Thcrcfore it was difflcult to expect fluctuations in the composition with regard to these proteins which are considered to constitutc the basis of contractile structure. Rather. we might cxpect to find variation with respect to additional protcins or smal1 modifications of major oncs. If the clectrophorctic methods are used singly. therc is a risk of missing these smal1 variations. Therefore uc‘ have compared data from three methods: SDSclsctrophorcsis. urea-electrophoresis and SDS-discelectrophoresis. which are known to be good for the separation of contractile proteins. In this paper we pose the question: does the composition and propertjes of contratile proteins. determined electrophoretically. serve as a biochemical indicator for taxonomie relations’? 1x9

AND

MFTHODS

We have collected molluscs of 26 species which represent two superorders, seven orders and 16 families (Table 1). Collection was made in Ochotskoe and Japan seas. Immediately after the opening of shells we removed inner or phasic parts of adductors which were either used directly for the extraction of contractile proteins or stored 10 mM Tris-HCI buffer, pH 7.0 at in 50”,, glycerol, -5-10 c.

Myofìbrils or contractlle protein preparations were obtained from fresh or glycerol-treated muscles according to a slightly modified method of Szent-Györgyi and collahorators (Szent-Györgyi ct UI..1973). Muscles were homogenired in 40 mM NaCl. 10 mM Tris HCI, pH 7.0 using Potter or rotator homogenirer. After centrlfugation at XO0.v x 15 min. the upper gel-like part of the pellet was harvested and washed 3~4 times in the same solution. Determination of elcctrophoretic mobilities and partlal Identification of protcins were carried out with the aid of specially isolated tropomyosin and myosin light chalns 01 Mizuhoprctcw y~s.sotv~si.s(Pet). Tropomyosin was prepared from myofihrils dried hy acetone and ether hy thc sedimentation of its I M NaCl extract with 302, acetonr. Myosm light chains and tropomyosin were extracted also from myofibrils of Ruditapes phillipinarum (Rud) as these proteins have electrophoretic mobilities different from analogous proteins of Pet.

All thc protein preparations were treated with 0.1 l.O”,, SDS hq the method of Weher and Oshorn (Weber & Osborn 1969), 8 M urea according Perrie and Perry (Perrie & Perry, 1971) and 2”,, SDS, 50 mM Tris-HCI. pH 6.X (Sheludko, 1975). 25-250mg of protein was applied onto gel. The electrophoresis in polyacrylamide gel was carried out according to the corresponding methods (Weber & Osborn, 1969; Perrie & Perry, 1971: Sheludko, 1975). Coomassie brilliant blue R-250 or G-250 was used for staining of the gels. For the reproducible comparison of protein

1,o-TM MEDTA-LC

90

A.

A

TM

TN-T a

TM

MLC TN-1 TN-C MLC !,O MLC a

b

C

d

Fig. 1. Comparison of three electrophoretic methods for myofibrillar preparation from Mwculus luwyatus. (a) SDS-electrophesis; (b) urea-electrophoresis; (c) SDS-disc-electrophoresis: and (d) SDS-dlscelectrophoregramm for rabbit myofibril. MHC-myosin heavy chains. PM-paramyosin. A-actin. TM-tr-opomyosin. MCL(LC)-myosin light chains and TN-troponin.

Contractile

protein

composition

zones relatike mobilities (Rs) were calculated. This was done by the division of the distance, measured from the top of the gel to the certain zone, to the distance covered by the zone of Per tropomyosin. The latter one was selected as the main marker zone since it was usually situated in the center of the gel in the electrophoretic methods used. The mixtures of myofibrillar preparations and protein marker were applied onto several gels in such a way that the quantity of dinc component in the mixture was increased. whilc that of the other one v.as decreased. This allows US to evaluate not only the mobility and concentration of the main proteins, but that of the minor components too. Molecular weights (mol. wt) of proteins were calculated from the data of SDS-electrophoresis using the plot of the relative mobllities of rabbit myofibrillar proteina versus their mol. WI as standard. Hi:SI LTS

The data obtained by use of the three methods for electrophoresis of myofibrillar preparation from Muscn/r/.~ lu<,riyurrts (Mus/) are shown in Fig. 1. The simpliest mcthod in sense of interpretation of results is SDS-clectrophoresis. vvhich we used for the determination of mol. wt of proteins. Modification of this method (SDS-disc-clectrophoresis) allows US to obtain a high rcsolution capacity and yields maxima1 numher of contractile protein zones (Sheludko. 1975). Finally. using thc urea-electrophoresis we achieved a good rcsolution of actin. tropomyosin and myosin Iight chains. However proteins with very large mol.

of the bivalvc

molluscs

/9I

wt or with smal1 negative charge cannot penetrate deeply into the gel. Electrophoretic data for the 26 species of molluscs are presented in Fig. 2. Relative mobilities recorded are average values of mobilities calculated from 4~ 5 parallel runs. Results of SDS-disc-electrophoresis are not presented in this scheme. hecause a number of faint zones obtamed by this method require additional studies for their identification. Therefore we usc for discussion only that part of these electrophoretic data which is useful for more detailed characterization of protcrn zones. identified in SDS- and urca-elcctrophoresis.

4s it can bc seen from Fig. 2. the main common components of contractile protein preparations are: myosin heavy chains (mol. wt 200,000 dalton), paramyosin (mol. wt 105.000 dalton), actin (mol. wt 45.000 dalton). tropomyosin (mol. wt 30,00@ 39.000 dalton) and myosin light chains (mol. wt about 16.000 dalton). The zones of myosin heavy chains and actin have the highest intensity. while the intensity of the paramyosin zone varies in different species and is minimal in molluscs from the order Prctinidu. These data arc in agreement with those obtained by other authors (Cohen ~‘r al.. 1971). .4fter isolation of tropomyosin and myosin light chains from Pee and Rud we tried to identify the regions corresponding to these proteins on clcctro-

Ftg 2. Scheme 01‘clrctrophoregramms for contractile protems of adductors of 26 mollusc species and I-ahhit muscle. Upper row represents SDS-electrophoregrams. lower ene-- urea-electrophoregramms. Brlcf names of species are in Table 1. 4bbreviations are the same as in Fig. 1. R-myofibrillar preparation from rabhit.

phorcgramms of contractik proteins from othet- spcCICS. Mlosin light chains of molluscs can hc dividcd into two IJ~CS: FDTAund SH-l~ght chalns (S/cn1Ciyörgyi <‘r LI/.. lY73). In our case FDTA-IighI cham\ of PK and Rut/ wcrc idcntifìcd. t’or thc olhcr spwcs WC assigncd JOllCï wit11 R, 1.15 1.;: 2nd R, = 1.60~ 2.00 to /oncs t-DTA- :tnJ SH-liphc chain~ rcspcctiv$;. In addltlon to thc rn+lor- protems to11cs up to X or 30 minor /ones arc ohsel-\cd uith SDS-clcchu phorcsis OI- SDS-disc-clcctrophor~sis. Thc protcins corresponding to these 7011~s are mostl! unidcntiíied. hut thcir molecular wcight have heen dctcrmined.

WC have studied thc changcs of contractile protcin composition using mainly the data from SDS- and urca-clectrophoresis, Fig. 2. First of all it is necessar! to nok thc idcntity of protein composition for congeneric or confamilic species. For cxample. fu11 identity of thc composition and mobihtics of minor and mador proteins was found for representati\jss 01 genera .4stui~fc~ (Asttr. 44) and MUSCI~/II.~ (Md. MIWI). famihcs C~~oritlu~ (Cal. Mw. LA. Rut/). (‘urdir-

minor piotcms (pt-otcms uitli mol. ut 4KOOO. 3Y.OIH) and les\ than 35.000 daltonxl and in lhc mohilirlc\ of tropomqosin and PDT.A-light chain. Lattcl- usc of aqatcimaq hc illiistratcd b\ thc smull inobilit\ tropomyosin C‘r~r,s.~~r.~rr ~/i~+r,~ in SDS-ekctrophoresis. Furthermore. according ka thc urea-electrophorests pattern. families differ kom cach othcr IR either lropomyosm or FDTA-light chain mobilit& or bolh. Analogous conclukns ma\ hc di-awn from the changcs of protcin composition in memhers of thc orders -l.\l~o~riJ~~ and I>rwit/tr. It is nc‘cc\har\to ua> that thcrc urc \omc charactcristIcs \vhlcll ul;if! spcclcs hclonging to a particrllar or&-. kar injtancc. \+c‘lound thc prcscncc of two onc Liithin thc range 01 /oncs In\rcad of uwal K I 7

I .i7!

2.0<1 IOt- YIxclcs

01’ (‘i7fotloí1rirla

in

urca-

clectrophoresis. the high Ie~cl of hetcrogcneity of minor protein composition and typical mobilities of myosin light chain in Li~~rrirlrc. and the poor content of minor proteinx m I’c,c,ri~fi~ltr.

Thc data obtained fol- mobilitlcs of the major contractilc proteins. are summarized in the Table 2 from dato (Cl;. SN). Moctridac~ (Mrrc. Spi) and Pcc~titlitltrc~ which rclatlons hetwecn the s)stematic position of (PN,. Swi). However WC have also ohscrvcd thc cases spccics and charactcrlstics of its myofibrillar proteins. of species diffcrences within a family. Roth species can hc \sen. Sincc in ;I grcat number of cases the of MIWI~II.~ diffcrcd from Modioh (Mo4 in thc mohilitk of thcsc proteins arc common for some of numbcr of minor protrins and in thc difkrcnt mohilithc spccics. Mohilitics of tropomyosin and myosin tics of EDTA -~light chain in area clcctrophorcsis. light chains of scallop (PK) arc used as standard The molccular weights of tropomqosins of musclcs IC\& of mohilit). I‘rom which deviations arc countcd. of Pcrnr~itlict and Mrr~omu arc diffcren t (Fig. 2 I. 1t can For c\amplc. mobilitles of FDTA-light chain of L.trr hc seen from electrophorctic pattcrns that thc musclcs atnd :Yur ln ut-ha-elcctroph«rcsis arc cqtul and conof molluscs from thc same genus or fami have a siderably higher than that of Pet. This fact is illusprotcin composition specifìc for this groep of spccics. trated bl 21 sign R, 9 R,““. Symbol C1.r.t reflects the Thc highest Icvcl of dissimilarlty hetwecn gcncra and presencc within the range of Rs = 1.60~2.00 (ureafamilies ma- bc dcmonstrated wlthin thc order C‘Jrtoelectrophoresis) of two zones instead of usual one. dontida. The main diffcrenccs arc in the conknt of S~mbol VUC, mark5 characteristic for a single specie Tablc 2. C‘omparison of mollusc species on the basis of similaritics myosin

EDTA-

and SH-light

m electrophoretic m«bility chams (sec cuplann~ion in te\t)

of tropomyosin

-

* The localiration

of Gly tropomyosin

was nat determined by SDS-dIsc-electrophorehi\

~uncertain)

and

C‘ontractilc

protein

composltlon

with mobilities of zones with Rf > 1. apparently myosin light chains. We have not found any changes in mobilities of myosin heavy chains. paramyosin and actin among the species studied. Therefore only the data for tropomyosin and myosin light chains are presentcd in the tahle. Differenccs in tropomyosin mobilitics wcrc studied by all thrce electrophoretic methods sincc this protcin could hc casllc identificd on electroforegramms. L’sing the data presented in Table I it is possible to group the species according to identity of onr or more fcaturrs. If species are groupcd togethcr on the basis of the completc identity of fcaturcs onc can recogni/e 13 groups: 1. h’l/C 2. MllSl. MUSU 3. Mor/: 4. G/r: 5. .-l,rtr: 6. C,o: 7. PCT. .h: x. Lut. Nm: 9. Astcc. .4srh: 10. C1.c: ll. PUI. CUI. Lim Mu.. Rut/. Mtrc. Spi. Mwo. Mw: 13.

01 thc hlval\u

molluxs

193

identificd. Another way to make the scope of the tablc wider is thc consideration of myosin light chains Ioncs. Identified on SDS-disc-electrophorcsis. These both variants may spread th-e differences of smal1 taxons up to species, but the identification of these zones for alt molluscs studied is a very difficult task. It is obvious that for thc comparative analqsis of all thc protem IOIW. it wilt be usciul to apply methods of thc numericut taxonomy to these data. This GUI giw US much more information about corrclation hctbccn specifity of the contractilc protcin pattcrn and tavonomic positron of mollusc specie. Thc data prescntcd in this paper along with mteresting results obtained by other authors (Logvinenko and Kodolova. 197 I : Cohen <‘t ~rl.. 197 I ) ma! pvc ;III additional information about the fine structurc of taxonomie units of HIIII/IG on thc basis of thc \tud> of homolog! and pt-operties of musclc protcms.

Cli. Scr : I 3. P
It is ohkious that groups arc usually composcd of one or two closcly related species. One large group includes memhcrs of different families ( I cw~rirlw. Mncrridac~etc). Thc larpest differcnces are sccn within the order Crrtodor~titl~ The only representative of superorder Prorohmd~it (NW) stands apart from thc other spccics hecause of the unusual mobilities of its light chains. WC must note cases of divergente between our results and systematic data (Scarlato. 1974). which arc infcrcd to be thc cases of parallelism of species belonging to different orders: Ltrr and NIC. Pm and order 1 c~~c,r,r/tr(sec Table 1 and Table 2).

In the coursc of corrclation of structural and functional features of the contractile apparatus with its molecular organisation a great number of muscle structures have been investigated. Variations in molecular parameters were shown for myosin (Brivio & Florini, 1972), tropomyosin (Woods & Pont, 1971). paramyosin (de Villafranca & Haines. 1974) and others, taken from muscles of different animals. In addition the data on mmor contractile proteins. localiled in thick filamcnts (Offer c’t ul.. 1973) Z-line (Saida & Cillrick. 1974) and M-line (Masaki & Takaiti. 1974) have accumulatcd. We have tricd to define more precisclq thc contractile protein compositlon of different molluscs tissues and used for this purpose three clectrophoretic mcthods. Summariling thc difErcnccs in thc composition of molluscs contractilc protcins of a large number 01 species WC may concli~cl~. 1. The diffcrcnces in thc composition of contractilc protcins of different species of molluscs lic in the variation in thc propertjes of ma.jor proteins and in thc numbcr of minor protelns. 2. Thc composition of contractile protelns of closely relatcd molluscs is identical 3. The changes in composition of myofibrillar protcins are most drastic at the leve1 of families and orders 4. Tablc 7. In which the rclationship between spctics of molluscs and properties of its contractilc proteins is shown. would bc more complete. if we includc in it data on mobilities of similar minor protcins. This wil1 be possihle if the minor protein Tones can lx

RFFERE\CF:S R. P. & FL«RINI J. R. (1972) Myosin: a comparati&e studg. Comp. Bioc/~uni.PIIcs~»/. 41, IB, 99. 104.

BKIVIO

<‘OHI\ C.. S/I ~7-c;tij<;\i1 A. G. ái KI~I>RI~)Kc;\I .\. (;

(I9hO) Filaments organl7atlon 01‘molluscan musclcs. ./ ir/ri.Urri. K<,\. 3. 251 2h9. SAIIN J 11 & I I.I~KIC K W. C. (1973) Purdicatlon and pt-opcrtles of thc iuolated honcyhee Z-disc. .I IIUI/C,C R;cI/. 87. h’l hs.3 Sc AKLA 1-0 0 h. (1474) B~valve molluscs of horth-Ucst part 01 the Pac~tic Occan. Doctoral dlssertation. Lcningrad State I niv. LenIngrad. Sir1 L.I I>hO X S (lY75) Protun composltion of rahlxt mu«lihrils. dctcrmtncd hc thc method of disc-ctectrophorc~ia 111priwncc of sodtum dodccylsulfatc 7;rto/oc/11r (1 YSKI 17, 114x 1154