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Comparative studies on the composition and properties of troponin from fast, slow, and cardiac muscles
ht. 3.
Biochem., x973,&
COMPARATIVE PROPERTIES
[Scientechnica (Publishers) Ltd.]
189-194.
STUDIES
OF TROPONIN
ON THE
COMPOSITION
‘89
AND
FROM FAST, SLOW, AND CARDIAC MUSCLES*
RENATA DABROWSKA,
MARIA DYDYNSIU,
A. SZPACENKO,
AM)
W. DRABIKOWSKI Department of Biochemistryof Nervous System and Muscle, NenekiInstitute of Experimental Biology, 3 Pasteur Street, Warsaw, Poland (&x+d
21 stpumk,
rg72)
ABSTRACT
SDS-polyacrylamide gel electrophoresis reveals differences in the composition between troponin from f&t and those from slow and cardiac muscles. Both latter troponins contain the products of proteolytic splitting of the proper componenta, formed by endogeneous muscle proteinases. 2. The low ability of slow and cardiac muscle troponin to inhibit Mg++-stimulated ATP-ase of actomyosin appears to be the same as that of fast muscle when calculated on the basis of the amount of the component responsible for this property. 3. The weak ability of Sr++ to replace troponin-bound calcium is the same for cardiac aa for skeletal muscle tmponin. I.
THE contraction-relaxation cycle of almost all kinds of muscle under physiological conditions is regulated by changes in the concentrations of Ca++ ions (for review see W&r, 1966, and Ebashi and Endo, 1968). It has already been well documented that the presence of the troponin-tropomyosin complex is essential for this regulation and that troponin is a calcium receptor in the myofib& (Ebashi, Kodama, and Ebashi, 1968; Fuchs and Briggs, x968; Drabikowski, Barylko, Dabrowslq and Nowak, ~$8). Previous studies concerning the role and properties of the troponin-tropomyosin complex were concentrated almost entirely on the mixed rabbit skeletal muscles which are predominantly fast. Some observations performed so far indicated that the basic mechanism of regulation of the contractionrelaxation cycle by Ca ++ in slow skeletal and cardiac muscies is the same as that in the fast ones (Ebashi, Iwakura, Nakajima, Nakaand Ooi, 1966; Dydynska, 1971; m-, * This work was partially supported by a Foreign Research Agreement No. oporg-I of N.I.H. under P.L.480.
Reddy and Honig, rg7r), although the activity of the tropomyosin-troponin complex seems to be in the former case lower (Ebashi and others, 1966; Muir, Weber, and Olson, tgyx; Furukawa and Peter, xgfr). In view of the recent studies showing that troponin is a complex protein (Hartshome, Theincr, and Mueller, 1969; Drabikowski, Dabrowska, and Barylko, I gy I ; Drabikowski, Rafalowska, Dgbrowska, Szpacenko, and Barylko, 1971; Greaser and Gergely, 1gy I ; Wilkinson, Perry, Cole, and Trayer, x972), the content, composition, and activity of troponin isolated from slow, fast, and cardiac muscles were compared in order to elucidate as far as possible the above differences. METHOD Troponin preparations were obtained fi=om: (I) rabbit fast muscles-adductor magnus, gastrocnemiua, and white parts of the vastus (FTP); (2) mbbit slow muscles-so leus, aemitMdinosua, crureua, and intratransvaxalis (STP); (3) bovine heart muscle (HTP). Homogenates of these groups of muscle, after several washings with 50 m&f KCl, were dried by alcohol and ether treatment. The powders were extracted with IM KCI. Fractions salted out between 40-60 per cent ammonium sulphate
D+BROWSKA
‘90
saturation from the extract were collected and used as preparations of the tropomyosin-troponin
AND OTHERS
I
100
200 300 400 TM-TP cofnpt2x ng
Fto. r.-EfIect of cardiac and skeletal tropomyosin-troponin complexes on Mg++-stimulated ATP-ase activity of rabbit synthetic actomyosin. ATP-au activity was measured at 25%. for 5 minutesinomediumcontainin ~omMTtis-HCl, pH7.5, 1omMiCCl,2mMA t-P 2 fl h&G, o-30 mg. synthetic rabbit actom& (reconstituted from actin and myosin mixed in proportion 1 : 3 w/w) and TM-TP complex from cardiac (0, 0) slow (m) or fast (A, L) rabbit skeletal muscle. Solid symbols indicate that 2 mM cthmadiq-ti 8, (ethylamine)-N,N’ tetra-acetate (ECTA) was additionally present.
3. &o&m.
complex (denoted in the text as TM-TP complex). For details of the procedure see Drabikowski, Dabrowska, and Now& (~$3). The yield of preparation (I) (TM-FTP) was about 25 mg. per gramme of alcohol-ether powder, i.e., similar to that obtained from mixed rabbit skeletal muscle (Drabikowski and othas. 1969). and about 18 mn. and 25 mg. for prepara&s-(2) (TM-STP) pn’;l (3) (TM-HTP) , respectively. RESULTS
0
ht.
AND DISCUSSION
In the first experiments the activity of all preparations of the tropomyosin-troponin complex to sensitize rabbit skeletal muscle synthetic actomyosin towards the changes of the concentrations of Ca++ was examined. As seen from Fig. 1 the preparations originating fi-om slow and cardiac muscle possess the ability to inhibit ATP-ase activity of actomyosin in the absence of Ca++, although it is somewhat weaker than in the corresponding preparation from fast muscle. All preparations of TM-TP complexes were subsequently fractionated to troponin and tropomyosin at pH 4.5 in I M KCl, as previously described (Drabikowski and others, xg6g). Mercaptoethanol (I mM) was present in all steps of preparation of the tropomyosintroponin complex and troponin. Recent studies from this (Drabikowski and others, r 971; Drabikowski, Rafalowska, and
-.
a
b
Fxo. n.-SDS polyacrylamide-gel electrophoruis of various typu of troponin. Polyacrylamide gel electrophormis in the presence of SDS was performed according to the method of Weba and Osbom (rg6g). 50 pg. of proteins were applied to IO per cent polyacrylamide gels containing 0’1 per cent SDS and 0.01 M sodium phosphate buffer, pH 7.0. Electrophoresis was performed for 4-5 hours at 7 mA per tube. Gels were stained with Coomassie blue. 2, HTP; 6, STP; c, FTP; d, HTP+FTF’ (qua1 parts) ; c, Troponin from mixed rabbit skeletal muscle.
‘97394
TROPONIN
others, 197 I) and other laboratories (Greaser and Gergely, x971; Schaub, Perry, and Hacker, x972) showed that troponin preparations from mixed rabbit skeletal muscle contain four components with the following molecular weights: 3g,ooo, 24,000, 18,000, and 13,000 daltons determined by means of SDS-polyacrylamide-gel electrophoresis. Occasionally an additional band of protein of about 30,000 molecular weight can be seen (Fig. 2, c). The 24,000 component inhibits actomyosin ATP-ase regardless of the concentration of free Ca++ (Greaser and Gergely, 1971; Drab&xv&i, R&slow&a, and othen, 1971; Wilkinson and others, 1g72), whereas the 18,000 component binds Cat+ and abolishes the effect of the 24,000 component in the presence of Ca++ (Greaser and Gergely, 1970; Drabikowski and others, 1971; Schaub and others, 1972 ; Hartshome and Pyun, 197 I). Thus, by recombination of these two proteins the ability of troponin to inhibit actomyoain ATP-ase only in the absence of Ca++canbercstorcd. The electrophontic Pattern of HTP and STP (Fig. 2, a, b), is somewhat di&cnt. Instead of the 3g,ooo component found only in some preparations and in rather small amounts, the 30,000 protein appears in high quantities. The content of 13,000 and lower molecular weight component(s) in STP and HTP is much higher, whereas that of the 24,000 one is rather low. Our previous studies (Drabikowski, R&low&a, and others, 1971) showed that both 3g,ooo and 24,000 proteins are very susceptible to proteolytic digestion and that the 13,000 component seems to originate from the 24,ooo one. Besides proteases of lysosomal origin, active at acid pH, the activity of proteolytic enzymes acting within the neutral pH range and activated by high KC1 concentrations (Noguchi and Kandatsu, 1970) has also to be taken into account as the possible cause of degradation of troponin constituents. It would seem that the activity of the latter enzyme(s) is higher in cardiac and slow muscles than in the fast ones, and that 30,000 daltons protein is the 6rst product of degradation of the 39,000 component. This view seems to be supported by the fact that the
IN MU.9CL.E
*9*
30,000 component is formed under the effect of very low trypsin concentration (Fig. 3). Moreover, in disk electrophoresis of the whole myofibrils from cardiac and slow muscles, besides the 3g,ooo component, the 30,000 one can be seen. Thus, it is very
a.
bed
t
3.-sDs polyurylanlk&+ eleetmFro. phoresis of rabbit skdeml muscle tmponin digested with various concentrations of tqxlirl. sunpled of tTqnmin were d&ted with tlypsin (Sigma) in IO m&f Tris-HCl bu&r, pH 7.5, at 20%. for 20 minutes. Digestion was stopped by addition of soya-bean trypsin inhibitor (Sigma) in the amount of 2 mg. per I mg. trypin. 0, Control undigatcd troponin; 6, c, d, l, Tmponin digested with 0’01,0~03,oo6, and o-10 ug. uypsin Per mg. protein, rqectively. probable that the former protein exists in oiw both in cardiac and slow muscle, but is split during the preparation of troponin. On the other hand, one cannot exclude that 30,000 protein exists in viw. In this respect it is worth mentioning that recently a protein of molecular weight 31,000 has been also found in f%og thin tilaments (Lehman and SzentGyorgyi, t 972) and insect tropanin (Dgbrowska, Bullard, and Winkelman, unpublished). Fig. JA shows the effect of various types of troponin on the Mg++-stimulated ATP-ase activity of rabbit synthetic actomyosin in the presence of rabbit tropomyosin and in the absence of Ca+. As seen, the most active
D+BROWSKA
192
ABD
ht. J. Biochtm.
OTHERS
It seems that the different activity of troponin was that originating from fast muscle. Inhibition caused by troponin from various types of’ troponin depends mainly on cardiac and slow muscles was neariy the the content of the constituent inhibiting same, but sign&antly lower than that of’ ATP-ase activity, i.e., the 24,000 daltons PTP, although it reaches eventually the same component. Calculation from the densitodepcc of inhibition when STP and HTP grams obtained by scanniq of SDS-poiywe& added in higher amounts. 100
2”” c
0
I 100
50
0
TPug
l50
A
inhibitofy taCtOf J@
B
Ro. q,-Efket of &us types of troponinon Mg++-stimu&d A’TP-iueactivityof rabbit syutbetic in rckrence to total troponin content. II, Percentage actomyosin. A, Percentage of inhibition qxuscd of inhibition apm*rd in refcrencc to tbc content of tbc q,ooo d&onm compoamt cakuked from obtained by sunning of SDS polyacrylamide gels (su Fig. 2). a, HTP; m, STP; A, ~~~~~~~*~~~ tmuxx! of rabbit trqxxnyosin usedin ~~~~0~7~t
oftmponin &ereonditionsasin~~.
I
0
Q25
aso
a75
I.
TP g Fro. 5.--EEect of skeletal muscie (A) and cardiac (a) troponin on the viscosity of rabbit tropomyosin. Viiity measurements were carried out in an Cstwaid viscometer at 21%. in IO mM “Es-HCl buffer, pH 75. Concentration of rabbit tropomyoain ~5 mg,/ml.
acrylamide gels indicated that both STP and HTP contain on the average only I 7-18 per cent of this component, whereas FTP contains about 30 per cent. If the percentage inhibition of the Mg++-stimulated ATP-ase is plotted against the content of the 24,000 component found in various types of troponin, the average percentage of inhibition is almost the same (Fig. 4). (To simplify the calculation, the weak inhibitory activity of the 13,000 component was nq&cted.) Another charact&’ tic property of troponin is its ability to increase the viscoeity of tropomyosin. In general the viscosity of isolated tropomyosin-troponin cornphzt~ @actions salted out between 40-60 per cent ammonium sulphate saturation, sez ulwne) from slow and cardiac muscles was lower than that isolated ikom fast ones. Similarly cardiac and slow muscle troponins have a very weak or no ef&ct on the viscosity of tropomyosin
TROPONIN
‘97334
193
IN MUSCLE
(Fig. 5). This may be attributed to the lack or deficiency of the 39,000 component, since, as our observations indicate, this is the constituent of troponin which interacts with tropomyosin and F-actin (Drabikowski, 1972). Ebashi and others (1968) reported previously that the af%nity of bivalent cations to cardiac troponin is considerably different from that to skeletal muscle troponin, and
Thus, all the results of the present work seem to indicate that there is no substantial difference in the composition of troponin preparations and the properties of its constituents from all muscles investigated, and that the weaker activity of cardiac and slow skeletal muscle troponin is due to higher degradation, probably being the result of higher activity of endogenous proteases.
NOTE (added in proof) Table Z.-ABILITY OF S&l, AND MgCl, TO REPLACE CALCIUMIN SKELETAL Muscle AND Our recent studies (DFROW~KA, R., CAIUXVZTROH>~N BARYLKO, B., NOWAIC,E., and DRABIKO~~KI, %a ADDITION
Bound after Exchange
(c.p.m./mg. protein)
I. None 0’1 mMSrC1, 3. 0.1 mMMgC1, 4. 0.1 mM %aCl, 2.
Samples of tropomyosin-troponin complexes prepared from bovine eardiae and mixed rabbit skeletal muscle (uu tat) mrr meubated with
0.1 mA4 ‘@CL&l, (containin 14,500 c.p.m./mg. protein) &me or in the prcscncc of other catioas, Bs indicratcd. AftcrNbW+!Ilt &WCX 50 PatIXleIIt radioactivity was meanned
postulated that cardiac troponin has a higher affinity to strontium as compared with the skeletal one. Recently Reddy and Honig (1g71), however, showed that the Ca++binding parameters are the same for cardiac and skeletal muscle troponin. Table I shows the results of the exchange of calcium bound to troponin with *sCa++ and the influence of Srf+ and Mg++ ions on this process. As can be seen, incorporation of radioactivity into the cardiac troponin-tropomyosin complex, being the measure of the exchange of calcium, is considerably lower than into the complex from mixed skeletal muscle. The reason of this phenomenon is at present under investigation. The table shows moreover that the weak ability of Sr++ to replace troponinbound calcium found previously for skeletal muscle troponin (Fuchs, 1971; Drabikowski and Barylko, I 97 I) is the same for the cardiac one.
W- (r973), ‘ The origin of 30,000 daltons protein in troponin preparations ‘, FEBS L.&m, 29, 239-242) have clearly indicated that 30,ooo daltons protein originates from 3g,ooo daltons component of troponin as a result of the activity of neutral muscle proteinases. REFERENCES DRABncowsrtt, W. (rg7a), ‘ Composition and properties of tmponin-complex ‘, 4th Int. Biophysics Cvngrwa, Moscow, Abatr. of sympOGal~,p.S8,Shrclio~,iIItheprap. DuuwwsxI, W., aDd BAR-O, B. (197x), ‘Calcium binding Acta biochim. -. by trbDonin’, _
Di.k:~?3”w”iha~o B D~O~BKA R and Now& E: ( rg68), ‘ &&ng of calciu& b; troponin ‘, Bull.
Acad.
Sk
Pal.
CLII,
16,
Dzt~%. W.. DABROWIMA. R. and BARYLKO, B. I I 97 I ), ’ S&>on and &a&terization of the constituents of troponin ‘, FEBS L&ten, rg x48-151.
.DUIUKO~~IU.W.. DABRO-. R.. and NOWAK, E. (I g6g), ‘I C&&ative s&i& on the corn: wsition and ~xmerties of EGTA-sensitizina &or ‘, Acta b&&n. biophys. Acad. Sci. Hung., 6 I X2-129. DRABII~O~~I~, W., RAFALOWIIKA, U., DWROWSU,
R., SZPACEMO, A., and B--o, B. (x971), ‘The effect of proteolytic enaymu on the
troponin compl&‘, FEB$ Letters; xg, 259-263. DYDY+KA, M. (IgTI), ‘ Studies on the interaction of actomyosin from slow and fast muscles with mmlatorv DI’oteins‘, Abstr. Gmm., 7th FEBS M-et., Vakk, p. 281.. Ewsm, S., and ENW, M. (x988), ‘ Calcium ion and muscle contraction ‘, Prog. Biophys. mol. BioL., 18, 123-183.
H., NAKAMURA,R., and 001, Y. (1g66), ‘ New structural proteins from dog heart and chicken gizzard ‘,
EBASHI, S., IWAKURA, H., N-JIMA,
Biochm. <., m
201-21 I.
I94
D+BROWSKAAND OTHERS
EBASHI,S., KODAMA,A., 2nd EBASHI,F. (IgsS), ‘ Troponin preparation 2nd physiological function ‘, 3. Biochem. (Tab), h 465-477. FUCHII, F. (Ig7I), ‘Properties of the calcium receptor site of troponin ‘, Biochim. biopigvs.d&a, -22 I-229. FUCHI,F., 2nd Bnraas, F. N. (1g68), ‘ The site of c&ium binding in relation to the activation of myofibrillar contraction ‘, 3. ga. Physiol., 31, 655-676. FURUUWA, T., and PETER,J. B. (Ig7I), ‘ Troponin activity of d&rent type3 of muscle fibers ‘, Erpl ~Vwol., 3x, 214-222. GRENER, M. L., 2nd GERQELY, J. (Igto), ‘Calcium binding component of troponin’, Fe& Proc. Fdn Am. Sac. exp. Bioi. (Abstr.), q, 463. GRURR, M. L, and GERO~Y, J. (Ig7I), ‘ Recollstitution of troponin afzivity from three ~3e30mpon~ta ‘, 3. biol. Ch8n1.,2& HARTSFIORNE; D. J., 2nd PYUN, H. Y. (Ig7I), ‘ Calcium binding by the troponin compla 2nd the purification and propertied of troponin A’, Biochim. bio&s. Actu, rag, 689-71 I. I%R~ORNE, D. J., TURNER, M., and bhELLRR, H. (1g6g), ‘ Studia on troponin ‘, Biochim. bio@ys. Ada, 1% 320-330. LZHMAX, W., and Szlwr-GuijR~YI, A. G. (Ig72), ‘Thin f&men~ from dif%xcnt mu2clcs ‘, Abse. Comm.,Anawi Bio&vs. Sot. AU., p. 27ga. Mum, J. R, Wura, A., and c)Ly)Io, R E. (Ig7r), ‘Card&my&~ATP-2u:awm@wn
with that of fast skeletal AM in its native and in an altered conformation ‘, B&him. bio&vs. Acti, ~4, rgg-log. Nooucx~, T., 2nd KANDATSU, M. (Ig7o), ‘ Autolytic brekdown of rat skeletal muscle proteins in the alkaline pH range ‘, Agr. biol. Chmn., 3&s 390-394. REDDY, S.. and HONIO. C. R. (ro71l. ‘ Relaxinn pro&ins ’ of czudiac muscle I; i?& &UC. Fk& Am. Sot. up. Biol. (Abstr.), 30,492. SCHAUB,M. C., PIIRRY, S. V., and HQXER, W. (Ig72), ‘ The regulatory proteins of the myofibril. Characterization 2nd biological activity of the calciumen&izing factor (uoponin A)‘, Bidem. -1.. rob. 2174i.Q~ WRBRR,k-(&8); ‘ Biw+ed CaIcium transport 2nd rclaxiw factor’. Cw. To&s Bioawdss.
we::? ii?
and Oanorw. M. (1060). ‘The reliability -of molecular &eight &e&nations bv dodccvl sul6te nolvacrvlamide pel clcctrophoresis*;3. b&d. &I.; ri, &&2. WILKINSON, J. M., PXRRY,S. V., COLE,H. A, and TRAYER,J. P. (Ig72), ’ The regulatory proteins of the mvofibril. ScIxuation and biohxical activity the com&Gents of inhibitory-f&r preparations ‘, Eiocrcmr, J., xg, 215-228.
oi
Biochem., x973,&
COMPARATIVE PROPERTIES
[Scientechnica (Publishers) Ltd.]
189-194.
STUDIES
OF TROPONIN
ON THE
COMPOSITION
‘89
AND
FROM FAST, SLOW, AND CARDIAC MUSCLES*
RENATA DABROWSKA,
MARIA DYDYNSIU,
A. SZPACENKO,
AM)
W. DRABIKOWSKI Department of Biochemistryof Nervous System and Muscle, NenekiInstitute of Experimental Biology, 3 Pasteur Street, Warsaw, Poland (&x+d
21 stpumk,
rg72)
ABSTRACT
SDS-polyacrylamide gel electrophoresis reveals differences in the composition between troponin from f&t and those from slow and cardiac muscles. Both latter troponins contain the products of proteolytic splitting of the proper componenta, formed by endogeneous muscle proteinases. 2. The low ability of slow and cardiac muscle troponin to inhibit Mg++-stimulated ATP-ase of actomyosin appears to be the same as that of fast muscle when calculated on the basis of the amount of the component responsible for this property. 3. The weak ability of Sr++ to replace troponin-bound calcium is the same for cardiac aa for skeletal muscle tmponin. I.
THE contraction-relaxation cycle of almost all kinds of muscle under physiological conditions is regulated by changes in the concentrations of Ca++ ions (for review see W&r, 1966, and Ebashi and Endo, 1968). It has already been well documented that the presence of the troponin-tropomyosin complex is essential for this regulation and that troponin is a calcium receptor in the myofib& (Ebashi, Kodama, and Ebashi, 1968; Fuchs and Briggs, x968; Drabikowski, Barylko, Dabrowslq and Nowak, ~$8). Previous studies concerning the role and properties of the troponin-tropomyosin complex were concentrated almost entirely on the mixed rabbit skeletal muscles which are predominantly fast. Some observations performed so far indicated that the basic mechanism of regulation of the contractionrelaxation cycle by Ca ++ in slow skeletal and cardiac muscies is the same as that in the fast ones (Ebashi, Iwakura, Nakajima, Nakaand Ooi, 1966; Dydynska, 1971; m-, * This work was partially supported by a Foreign Research Agreement No. oporg-I of N.I.H. under P.L.480.
Reddy and Honig, rg7r), although the activity of the tropomyosin-troponin complex seems to be in the former case lower (Ebashi and others, 1966; Muir, Weber, and Olson, tgyx; Furukawa and Peter, xgfr). In view of the recent studies showing that troponin is a complex protein (Hartshome, Theincr, and Mueller, 1969; Drabikowski, Dabrowska, and Barylko, I gy I ; Drabikowski, Rafalowska, Dgbrowska, Szpacenko, and Barylko, 1971; Greaser and Gergely, 1gy I ; Wilkinson, Perry, Cole, and Trayer, x972), the content, composition, and activity of troponin isolated from slow, fast, and cardiac muscles were compared in order to elucidate as far as possible the above differences. METHOD Troponin preparations were obtained fi=om: (I) rabbit fast muscles-adductor magnus, gastrocnemiua, and white parts of the vastus (FTP); (2) mbbit slow muscles-so leus, aemitMdinosua, crureua, and intratransvaxalis (STP); (3) bovine heart muscle (HTP). Homogenates of these groups of muscle, after several washings with 50 m&f KCl, were dried by alcohol and ether treatment. The powders were extracted with IM KCI. Fractions salted out between 40-60 per cent ammonium sulphate
D+BROWSKA
‘90
saturation from the extract were collected and used as preparations of the tropomyosin-troponin
AND OTHERS
I
100
200 300 400 TM-TP cofnpt2x ng
Fto. r.-EfIect of cardiac and skeletal tropomyosin-troponin complexes on Mg++-stimulated ATP-ase activity of rabbit synthetic actomyosin. ATP-au activity was measured at 25%. for 5 minutesinomediumcontainin ~omMTtis-HCl, pH7.5, 1omMiCCl,2mMA t-P 2 fl h&G, o-30 mg. synthetic rabbit actom& (reconstituted from actin and myosin mixed in proportion 1 : 3 w/w) and TM-TP complex from cardiac (0, 0) slow (m) or fast (A, L) rabbit skeletal muscle. Solid symbols indicate that 2 mM cthmadiq-ti 8, (ethylamine)-N,N’ tetra-acetate (ECTA) was additionally present.
3. &o&m.
complex (denoted in the text as TM-TP complex). For details of the procedure see Drabikowski, Dabrowska, and Now& (~$3). The yield of preparation (I) (TM-FTP) was about 25 mg. per gramme of alcohol-ether powder, i.e., similar to that obtained from mixed rabbit skeletal muscle (Drabikowski and othas. 1969). and about 18 mn. and 25 mg. for prepara&s-(2) (TM-STP) pn’;l (3) (TM-HTP) , respectively. RESULTS
0
ht.
AND DISCUSSION
In the first experiments the activity of all preparations of the tropomyosin-troponin complex to sensitize rabbit skeletal muscle synthetic actomyosin towards the changes of the concentrations of Ca++ was examined. As seen from Fig. 1 the preparations originating fi-om slow and cardiac muscle possess the ability to inhibit ATP-ase activity of actomyosin in the absence of Ca++, although it is somewhat weaker than in the corresponding preparation from fast muscle. All preparations of TM-TP complexes were subsequently fractionated to troponin and tropomyosin at pH 4.5 in I M KCl, as previously described (Drabikowski and others, xg6g). Mercaptoethanol (I mM) was present in all steps of preparation of the tropomyosintroponin complex and troponin. Recent studies from this (Drabikowski and others, r 971; Drabikowski, Rafalowska, and
-.
a
b
Fxo. n.-SDS polyacrylamide-gel electrophoruis of various typu of troponin. Polyacrylamide gel electrophormis in the presence of SDS was performed according to the method of Weba and Osbom (rg6g). 50 pg. of proteins were applied to IO per cent polyacrylamide gels containing 0’1 per cent SDS and 0.01 M sodium phosphate buffer, pH 7.0. Electrophoresis was performed for 4-5 hours at 7 mA per tube. Gels were stained with Coomassie blue. 2, HTP; 6, STP; c, FTP; d, HTP+FTF’ (qua1 parts) ; c, Troponin from mixed rabbit skeletal muscle.
‘97394
TROPONIN
others, 197 I) and other laboratories (Greaser and Gergely, x971; Schaub, Perry, and Hacker, x972) showed that troponin preparations from mixed rabbit skeletal muscle contain four components with the following molecular weights: 3g,ooo, 24,000, 18,000, and 13,000 daltons determined by means of SDS-polyacrylamide-gel electrophoresis. Occasionally an additional band of protein of about 30,000 molecular weight can be seen (Fig. 2, c). The 24,000 component inhibits actomyosin ATP-ase regardless of the concentration of free Ca++ (Greaser and Gergely, 1971; Drab&xv&i, R&slow&a, and othen, 1971; Wilkinson and others, 1g72), whereas the 18,000 component binds Cat+ and abolishes the effect of the 24,000 component in the presence of Ca++ (Greaser and Gergely, 1970; Drabikowski and others, 1971; Schaub and others, 1972 ; Hartshome and Pyun, 197 I). Thus, by recombination of these two proteins the ability of troponin to inhibit actomyoain ATP-ase only in the absence of Ca++canbercstorcd. The electrophontic Pattern of HTP and STP (Fig. 2, a, b), is somewhat di&cnt. Instead of the 3g,ooo component found only in some preparations and in rather small amounts, the 30,000 protein appears in high quantities. The content of 13,000 and lower molecular weight component(s) in STP and HTP is much higher, whereas that of the 24,000 one is rather low. Our previous studies (Drabikowski, R&low&a, and others, 1971) showed that both 3g,ooo and 24,000 proteins are very susceptible to proteolytic digestion and that the 13,000 component seems to originate from the 24,ooo one. Besides proteases of lysosomal origin, active at acid pH, the activity of proteolytic enzymes acting within the neutral pH range and activated by high KC1 concentrations (Noguchi and Kandatsu, 1970) has also to be taken into account as the possible cause of degradation of troponin constituents. It would seem that the activity of the latter enzyme(s) is higher in cardiac and slow muscles than in the fast ones, and that 30,000 daltons protein is the 6rst product of degradation of the 39,000 component. This view seems to be supported by the fact that the
IN MU.9CL.E
*9*
30,000 component is formed under the effect of very low trypsin concentration (Fig. 3). Moreover, in disk electrophoresis of the whole myofibrils from cardiac and slow muscles, besides the 3g,ooo component, the 30,000 one can be seen. Thus, it is very
a.
bed
t
3.-sDs polyurylanlk&+ eleetmFro. phoresis of rabbit skdeml muscle tmponin digested with various concentrations of tqxlirl. sunpled of tTqnmin were d&ted with tlypsin (Sigma) in IO m&f Tris-HCl bu&r, pH 7.5, at 20%. for 20 minutes. Digestion was stopped by addition of soya-bean trypsin inhibitor (Sigma) in the amount of 2 mg. per I mg. trypin. 0, Control undigatcd troponin; 6, c, d, l, Tmponin digested with 0’01,0~03,oo6, and o-10 ug. uypsin Per mg. protein, rqectively. probable that the former protein exists in oiw both in cardiac and slow muscle, but is split during the preparation of troponin. On the other hand, one cannot exclude that 30,000 protein exists in viw. In this respect it is worth mentioning that recently a protein of molecular weight 31,000 has been also found in f%og thin tilaments (Lehman and SzentGyorgyi, t 972) and insect tropanin (Dgbrowska, Bullard, and Winkelman, unpublished). Fig. JA shows the effect of various types of troponin on the Mg++-stimulated ATP-ase activity of rabbit synthetic actomyosin in the presence of rabbit tropomyosin and in the absence of Ca+. As seen, the most active
D+BROWSKA
192
ABD
ht. J. Biochtm.
OTHERS
It seems that the different activity of troponin was that originating from fast muscle. Inhibition caused by troponin from various types of’ troponin depends mainly on cardiac and slow muscles was neariy the the content of the constituent inhibiting same, but sign&antly lower than that of’ ATP-ase activity, i.e., the 24,000 daltons PTP, although it reaches eventually the same component. Calculation from the densitodepcc of inhibition when STP and HTP grams obtained by scanniq of SDS-poiywe& added in higher amounts. 100
2”” c
0
I 100
50
0
TPug
l50
A
inhibitofy taCtOf J@
B
Ro. q,-Efket of &us types of troponinon Mg++-stimu&d A’TP-iueactivityof rabbit syutbetic in rckrence to total troponin content. II, Percentage actomyosin. A, Percentage of inhibition qxuscd of inhibition apm*rd in refcrencc to tbc content of tbc q,ooo d&onm compoamt cakuked from obtained by sunning of SDS polyacrylamide gels (su Fig. 2). a, HTP; m, STP; A, ~~~~~~~*~~~ tmuxx! of rabbit trqxxnyosin usedin ~~~~0~7~t
oftmponin &ereonditionsasin~~.
I
0
Q25
aso
a75
I.
TP g Fro. 5.--EEect of skeletal muscie (A) and cardiac (a) troponin on the viscosity of rabbit tropomyosin. Viiity measurements were carried out in an Cstwaid viscometer at 21%. in IO mM “Es-HCl buffer, pH 75. Concentration of rabbit tropomyoain ~5 mg,/ml.
acrylamide gels indicated that both STP and HTP contain on the average only I 7-18 per cent of this component, whereas FTP contains about 30 per cent. If the percentage inhibition of the Mg++-stimulated ATP-ase is plotted against the content of the 24,000 component found in various types of troponin, the average percentage of inhibition is almost the same (Fig. 4). (To simplify the calculation, the weak inhibitory activity of the 13,000 component was nq&cted.) Another charact&’ tic property of troponin is its ability to increase the viscoeity of tropomyosin. In general the viscosity of isolated tropomyosin-troponin cornphzt~ @actions salted out between 40-60 per cent ammonium sulphate saturation, sez ulwne) from slow and cardiac muscles was lower than that isolated ikom fast ones. Similarly cardiac and slow muscle troponins have a very weak or no ef&ct on the viscosity of tropomyosin
TROPONIN
‘97334
193
IN MUSCLE
(Fig. 5). This may be attributed to the lack or deficiency of the 39,000 component, since, as our observations indicate, this is the constituent of troponin which interacts with tropomyosin and F-actin (Drabikowski, 1972). Ebashi and others (1968) reported previously that the af%nity of bivalent cations to cardiac troponin is considerably different from that to skeletal muscle troponin, and
Thus, all the results of the present work seem to indicate that there is no substantial difference in the composition of troponin preparations and the properties of its constituents from all muscles investigated, and that the weaker activity of cardiac and slow skeletal muscle troponin is due to higher degradation, probably being the result of higher activity of endogenous proteases.
NOTE (added in proof) Table Z.-ABILITY OF S&l, AND MgCl, TO REPLACE CALCIUMIN SKELETAL Muscle AND Our recent studies (DFROW~KA, R., CAIUXVZTROH>~N BARYLKO, B., NOWAIC,E., and DRABIKO~~KI, %a ADDITION
Bound after Exchange
(c.p.m./mg. protein)
I. None 0’1 mMSrC1, 3. 0.1 mMMgC1, 4. 0.1 mM %aCl, 2.
Samples of tropomyosin-troponin complexes prepared from bovine eardiae and mixed rabbit skeletal muscle (uu tat) mrr meubated with
0.1 mA4 ‘@CL&l, (containin 14,500 c.p.m./mg. protein) &me or in the prcscncc of other catioas, Bs indicratcd. AftcrNbW+!Ilt &WCX 50 PatIXleIIt radioactivity was meanned
postulated that cardiac troponin has a higher affinity to strontium as compared with the skeletal one. Recently Reddy and Honig (1g71), however, showed that the Ca++binding parameters are the same for cardiac and skeletal muscle troponin. Table I shows the results of the exchange of calcium bound to troponin with *sCa++ and the influence of Srf+ and Mg++ ions on this process. As can be seen, incorporation of radioactivity into the cardiac troponin-tropomyosin complex, being the measure of the exchange of calcium, is considerably lower than into the complex from mixed skeletal muscle. The reason of this phenomenon is at present under investigation. The table shows moreover that the weak ability of Sr++ to replace troponinbound calcium found previously for skeletal muscle troponin (Fuchs, 1971; Drabikowski and Barylko, I 97 I) is the same for the cardiac one.
W- (r973), ‘ The origin of 30,000 daltons protein in troponin preparations ‘, FEBS L.&m, 29, 239-242) have clearly indicated that 30,ooo daltons protein originates from 3g,ooo daltons component of troponin as a result of the activity of neutral muscle proteinases. REFERENCES DRABncowsrtt, W. (rg7a), ‘ Composition and properties of tmponin-complex ‘, 4th Int. Biophysics Cvngrwa, Moscow, Abatr. of sympOGal~,p.S8,Shrclio~,iIItheprap. DuuwwsxI, W., aDd BAR-O, B. (197x), ‘Calcium binding Acta biochim. -. by trbDonin’, _
Di.k:~?3”w”iha~o B D~O~BKA R and Now& E: ( rg68), ‘ &&ng of calciu& b; troponin ‘, Bull.
Acad.
Sk
Pal.
CLII,
16,
Dzt~%. W.. DABROWIMA. R. and BARYLKO, B. I I 97 I ), ’ S&>on and &a&terization of the constituents of troponin ‘, FEBS L&ten, rg x48-151.
.DUIUKO~~IU.W.. DABRO-. R.. and NOWAK, E. (I g6g), ‘I C&&ative s&i& on the corn: wsition and ~xmerties of EGTA-sensitizina &or ‘, Acta b&&n. biophys. Acad. Sci. Hung., 6 I X2-129. DRABII~O~~I~, W., RAFALOWIIKA, U., DWROWSU,
R., SZPACEMO, A., and B--o, B. (x971), ‘The effect of proteolytic enaymu on the
troponin compl&‘, FEB$ Letters; xg, 259-263. DYDY+KA, M. (IgTI), ‘ Studies on the interaction of actomyosin from slow and fast muscles with mmlatorv DI’oteins‘, Abstr. Gmm., 7th FEBS M-et., Vakk, p. 281.. Ewsm, S., and ENW, M. (x988), ‘ Calcium ion and muscle contraction ‘, Prog. Biophys. mol. BioL., 18, 123-183.
H., NAKAMURA,R., and 001, Y. (1g66), ‘ New structural proteins from dog heart and chicken gizzard ‘,
EBASHI, S., IWAKURA, H., N-JIMA,
Biochm. <., m
201-21 I.
I94
D+BROWSKAAND OTHERS
EBASHI,S., KODAMA,A., 2nd EBASHI,F. (IgsS), ‘ Troponin preparation 2nd physiological function ‘, 3. Biochem. (Tab), h 465-477. FUCHII, F. (Ig7I), ‘Properties of the calcium receptor site of troponin ‘, Biochim. biopigvs.d&a, -22 I-229. FUCHI,F., 2nd Bnraas, F. N. (1g68), ‘ The site of c&ium binding in relation to the activation of myofibrillar contraction ‘, 3. ga. Physiol., 31, 655-676. FURUUWA, T., and PETER,J. B. (Ig7I), ‘ Troponin activity of d&rent type3 of muscle fibers ‘, Erpl ~Vwol., 3x, 214-222. GRENER, M. L., 2nd GERQELY, J. (Igto), ‘Calcium binding component of troponin’, Fe& Proc. Fdn Am. Sac. exp. Bioi. (Abstr.), q, 463. GRURR, M. L, and GERO~Y, J. (Ig7I), ‘ Recollstitution of troponin afzivity from three ~3e30mpon~ta ‘, 3. biol. Ch8n1.,2& HARTSFIORNE; D. J., 2nd PYUN, H. Y. (Ig7I), ‘ Calcium binding by the troponin compla 2nd the purification and propertied of troponin A’, Biochim. bio&s. Actu, rag, 689-71 I. I%R~ORNE, D. J., TURNER, M., and bhELLRR, H. (1g6g), ‘ Studia on troponin ‘, Biochim. bio@ys. Ada, 1% 320-330. LZHMAX, W., and Szlwr-GuijR~YI, A. G. (Ig72), ‘Thin f&men~ from dif%xcnt mu2clcs ‘, Abse. Comm.,Anawi Bio&vs. Sot. AU., p. 27ga. Mum, J. R, Wura, A., and c)Ly)Io, R E. (Ig7r), ‘Card&my&~ATP-2u:awm@wn
with that of fast skeletal AM in its native and in an altered conformation ‘, B&him. bio&vs. Acti, ~4, rgg-log. Nooucx~, T., 2nd KANDATSU, M. (Ig7o), ‘ Autolytic brekdown of rat skeletal muscle proteins in the alkaline pH range ‘, Agr. biol. Chmn., 3&s 390-394. REDDY, S.. and HONIO. C. R. (ro71l. ‘ Relaxinn pro&ins ’ of czudiac muscle I; i?& &UC. Fk& Am. Sot. up. Biol. (Abstr.), 30,492. SCHAUB,M. C., PIIRRY, S. V., and HQXER, W. (Ig72), ‘ The regulatory proteins of the myofibril. Characterization 2nd biological activity of the calciumen&izing factor (uoponin A)‘, Bidem. -1.. rob. 2174i.Q~ WRBRR,k-(&8); ‘ Biw+ed CaIcium transport 2nd rclaxiw factor’. Cw. To&s Bioawdss.
we::? ii?
and Oanorw. M. (1060). ‘The reliability -of molecular &eight &e&nations bv dodccvl sul6te nolvacrvlamide pel clcctrophoresis*;3. b&d. &I.; ri, &&2. WILKINSON, J. M., PXRRY,S. V., COLE,H. A, and TRAYER,J. P. (Ig72), ’ The regulatory proteins of the mvofibril. ScIxuation and biohxical activity the com&Gents of inhibitory-f&r preparations ‘, Eiocrcmr, J., xg, 215-228.
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