Is troponin the Ca++-receptive protein in the contractile system?

~~nt~iâ~ieat~ri~in~ II'

pp' 1225-i299, 1970 .

Pergamon Press

73 TROPONIB TES Ca~-RECSPTIVS PROTSIA Ig TES COATRACTII.S 3YSTSYl? 11 . Drabikowaki, B . Hary2ko, R. D4browa]~ aad Y .a . Sariats Department o! Bioohemiatry, Nenoki Institute o! Ezperimsntal Biology, lfaraaw, Polaal

(Received 20 August 1970; in final form 16 September 1970)

It has been already well established that the interaction o! myoaia with aotin represents an essential meohaniam of musoular ooatraotion . Thin interaction, manifested is vitro by auperpreoipltation and elevation of the sotivity of Yg-ATP-ass, is a direot lunation of the oonoantration of ionised oaloium in the medium . At a oonoentration of tree Cam lean than 10~7M, aohieved with either musole miorosomea oonaisting of the lragmenta of aarooplasmio retioulum (FBR~,

or oaloium ohslator

ethylene glyool~-(2-aminoethyl)-tetraaoetio aoid ($(ETA), ao interaction between aotin and myosin taken plane . All these atudiea have led to the oarreat ~y aocspted view that oaloium is a phyaiologioal regulator of the oontraotion relazation oyole . Ca m attired in the sarooplasmio retioulum is released into sarooplasma after the atimulua and aotivates oontraotion, whereas relazation is aocompanied by an aooumulation of oaloium in the sarooplaamio retioulum (f or review see 1, 2) . $bashi and gbashi (3) were the first to auoosed is isolating from musole a protein faotor responsible for Ca-sensitivity of the ooatraotila system . Only in itg presenoe does EOTA oauae an inhibition of superpreoipitation and of Supported is part by Foreign Reaearoh Agreement No 05-015-1 of N. LH . under P.L . 480

1225

1226

TROPONIN, Ca m RECEPTIVE PROTEIN

Yol. 9, No . 21

äg-aotivated ATP-see, whereas aotomyosin devoid of this factor (deaeaeiti$ed aotomyosin (4))is no longer sensitive to the ohaagea in the oonoeatratioa of Ca ++

.

This protein factor,

oalled originally native tropomyoaia, was subsequently found by Ebaahi and Hodama (S) to by a oomplez of Hailey"s tropomyoaia and another myoiibrillar protein, named troponin . "eber and ooworkera (6, 7) olearly showed that the interaotion of myosin with aotin is aooomplished by the binding of oaloium to aotomyosin or myof ibrila . The site of the binding remained nakaown however until only reoent7y when it was found that troponin binds ap to about 3 moles of Ca par 10~g with an apparent affinity oonatant around 10

1 (8, 9,

10) . This

observation led to the hypothesis that in musolea oaloium aotivatea oonaentration by binding to troponin moleonlea (8, 9) . Con~equently, during relazation s release of oaloium from its oomp~ez with troponin should ta]a plsoe . Beoent observations have indeed iadioated some oonformational ohangea is troponin dependent on the oonoentration of free oaloium, as refleoted by the ohaages in the diao eleotrophoresia pattern (11,

12 ) .

Preparations of troponin are heterogeaona (13,14) ; is gel eleotrophoresia we have found four olassea of disoa (Fig . 1) . Althoagh this pattern differs from some of those reoently reported (11, 12 ), in our oaae EaTA also oanaed remarhble obangea in diao eleotrophoresia of troponin preparations . Instead of the fastest diao revealing is the preaenoe of Ca++ a nobility egaal to that of the marker and the nezt diao, a new diao appears with an intermediate mobility . It is worthwhile to aentaon that during fractionation of troponin (14) the faeteat diao is always present is that iraotion whioh shows the highest

Vol. 9, No . 21

TROPOHIN, Cam RECEPTIVE PROTEIN

A

1227

6

B!leot o! B(4TA on the Diea $leotrophoresia Pattern o! Tropania Diao eleotrophosesia was perlorssd on 7.S'1~ aor~]amide with Tris-gl,oias bnlier, pH 8 .S in the preaenoe o! 4 lI urea . 100 pg o! tsoponin samples were applied . Staining with Coomaaie blue . Arrow indioatea tha position o! bsoaophenol blne naed as a sarbr .

A . Abort 10S1I Cam preae~ . B . Taopoaia preinonbated at Oo !or 24 honsa with 8 Y area and 1 WI BATA waa need and 1 ~ B8'PA waa added to the bn!les . Cam binding abilltJ. Owing to the poatnlatsd sole a! tropoaia, ?88 or oaloins ohelatora whioh remoTe coat o! the bones Ca lroa golibsila (1S, 1), shonLd also oanae disaooiation a! troponi~oaloinm ooaipUs, and a direot deaoastsation of thin phenomendn was the original aim o! this work . IIaezpeoted],T it waa lonad that

1228

TROPONIN, Ca*+ RECEPTIVE PROTEIN

Vol . 9, No . 21

oaloium bout to troponin is diffioult to remove by fragmented aarooplaamio retioulum, $GTA or ethylenediaminetetraaoetio aoid (SDTA) . Troponin was obtained from low ionio strength eztraot of rabbit aloeletal muaole, ae desoribed before (16), and its bound Ca was ezohanged with

~ sCaC12 .

In one type of ezperiments 0 .1 m1I

~~`CaC12 introduoed for the ezohange remsiaed present in the inonbation mizture during subsequent treatment of troponin with F3H or ohelatora . In this oase the amount of troponin bound 4gCa was measured in the paralled aaaple with the use of Dowez SO ea previoua]y desoribed for aotin (17 ) . In the aeoond type troponin A~Ca was freed of 4S Ca~ with Dowez SO before treatment with FSR or ohelatora . Table 1 shows that nioroaomea, whioh easily remove free Cam, are unable to remove oaloium from its oomplez with troponin . Also when troponin was in the oomplez with tropomyosin its bound oaloium was not removed by FSR . llhen the treatment with miorosomea was performed on troponin treated with Dowez 30 prior to inoubation, a small deorsaae of bound radioaotivity was found . The latter was howeve~e of the acme order as that found after the aeoond Dowez j0 treatment of troponin . The preseaoe of 0 .1 mM EGTA did not osuse any ohange is the amount of radioaotivity bound to troponin . The amount of oaloium released from troponin during inoubation with EDTA or EGTA was determined with the use of a Sephadez G-25 ooluma, on whioh radioaotivity not bound to protein was separated . The results obtained showed that EDTA in a oonoentration twine that of bound oaloium had only a weak disaooiating effect and 1 mM EDTA removed not more than 5096 of

Vol . 9, No . 21

TROPONIN, Ca ++ RECEPTNE PROTEIN

1229

ZABLE 1 Rsmo~el ol 45Ca Bound to Troponin b~ FSR F3R was prepared from rabbit muscles as prewi.onel~ described (18) and purified on continuous encrose gradient . Calcium uptal~e was measured in two ml samples containing 0 .1 1[ âCl, 20 ~ hietidine-HC1, pH ? .2, S m1I Erozalate, ~ mY YgCl 5 ~ A~P 1 .0-1 .5 ng/al of troponin labeled with 5Ca (or ~ .1 dI s(~aCl2 in the control samples) B(iTA as indicated and FSß (50-100 pg protsin/ml) added at 0 ~ims After incubation at room tempsratnre for the time indicated, ~Cs bound b~ F3R was remored by filtration on äillipore filters. A . Troponin sgnlibrated for 20 min with 0 .1 s>< 45CaCl s~de incubation with FSR without re~mowal ot2

u$gC~i2

$zpt .

S~!'A Incnba- Free tion, aa min, mY

Ho 1 2

oontrol tro~onin aontrol tro~onin

0 .1 0 .1 0 .1 0,1

5 5 5 5 10 15

0 .1 0:05 0 .1 0.05 0 .05 0 .05

Rsdioaoti~itF c m r sam fe uce no n resoved p fres bound b8 F.98 _ 210 15000 14500 13000 _l2000

10000 20000 20000 20000

18000 18000 18000

B . Troponin alter equilibration with 45Caß1 treated with Dowez 50 prior to inanbatioa with 2F9R Szpt . Incubation lfo

nia .

_ 1

2

5 5

S

5 10

Radioactivity bound to troponin, c sr sam is er ncu s n before with F3R 1680o 14800 14800 14800 11350

y5ooo 11500 12250 112 10150

650 15050 14750 15000

TROPONIN, Cam RECEPTIVE PROTEIN

1230

Vol. 9, No . 21

bound oaloina (Table 2) . The ettaot of B(iTA waa even eoaewhat weaker, is agreement with small ditYsrenoea la the Cam binding oonatanta between these two ohelatora . Yfhen free ~SCa was reaoved before $DT1 treatment, aiailarly to the ezpsriaent a with F3H presented in Table 1B, the etteot of $DT1 waa somewhat more pronounoed . lle were also interested in whether inhibition of aotomyoaia iTP-sae bJ $ßTA is aooompanied bT a release o! bound Ca from tropoain . Prelialaary ezperimeata have shown, however, that when desensitised aotomyoaia was oonbined with a tropogoaia ~SCa-troponin oomple: and subsequentlry iaoubated with 1TP, Yg~ and $1~T1, a liberation o! only a small part of bound Ca took plane . 111 the results of thin work raise the question whether troponin is really a Cam reoeptive protein in myotibxila, iEa affinity to oaloium seems to be too high to to ke part direotl, in the oontraotion-re] .azation oyole . One oan of oourse argue that the affinity of oaloium to tropoain in its isolated form does not retleot the situation in vivo . Our observations seem to bs supported however by the previous findings of weber and ooworkera (6, 7) who have shown that miofibrila attar treatment with mioroso~a or $aT1 still aoataln about 0 .5 - 1.0 pmole of bound Ca per g~ about halt of whioh is ezohaageable . It is very probab],y that thin oaloium oorreaponda to that bound to troponin . The quantity of troponin in musolea oaloulated on the basis of the above value for bound oaloium not removable by EDT1 and on the assumption that troponin oontaina 5 moles of oaloium bound per 105g protein, amounts to about 1 - 2~ of the total miofibrillar proteins i.e . a value oomparable with other rather

Vol. 9, No, 21

TROPONIN, Cam RECEPTIVE PROTEIN

1231

S'aB1+8 2 8tteot ot BDta and 80Ta on the Biading ot Oalaiusl b~ ?roponin a, ? 4roponin (2-3 sg/t1) after equilibration with 0 .1 i 5CaC1p was iaoubated at Oo with EDSa or E~1'a as indioated in the table sad snbssq~t~ passed through Sephadez 0-25 ooham. 8lntion with 4 dI Tris-Hßl, pH 7 .5 . In the protein peat radioaoti~it~ was deteraiaed . 8zpt .

oalainR bound, opa per ~ vrotein

Treatsent

~o

none + 1 i aara~ 1 h

2

9800 4780

none + 1 1 BD~a, 1 h _ _ + 1 i snra, ~ h + 1 i EDTa, 24 h + G Y m~ea, 24 h + 6 H ures and 1 ~i 3Dfd . 24 h

3

4

none + 1 i 80la, 1 h

+ 0 .3 i 8nra, 1 h + 0.3 i ~ ara . 1 h

5

5520 2800 6790 6140 10120 3170 5260 3870 2880 3850

-

8 . ?~

(2-3 sg/wl) after equilibration with 0 .1 i and res~owal o! tree Ca b~ trestssnt with Dowez ~ was iaoubated st Oo for 1 hour with 8~ra at the oonosntratioas indioated and subsequsntl~ ohromatographed on Sephades 0"25 aolwen with 4 aH Tris-~1 pH Z .5 as eluent . In the protein pest radiosot~wit~ was detersinsd .

t.

Trestweat

ov-mß vrot~n

initial

bound attar BeDhad~z

1

pons + 0 , 05 mlI 8D'Pa + 0,1 mr 81rra + o .x rc 81rra

5340 5340 5340 s~4o_-

3040 2800 2000 1580

2

+ 1 i BDTA

8400

1700

123 2

TROPONIN, Ca++ RECEPTIVE PROTEIN

Vol . 9, No. 21

iadireot estimat ions . Iß oaloium direot ~y ooaneoted with the regulation oß musoular aotivity is not bound to troponin, then its site of binding in miofibrils remains to be localized, It is worthwhile to mention that in a recent publioation, which appeared duzing preparation of this paper, Dancker (19) oame to the oonolualon that it is Ca bound to myosin which takes part in the oontraotion-relaxation oycle . I! this is real>~y the oase, the question remains what is the role o! calcium bound to troponin . Rslsrenoea 1 . A. w~ber in "Current Topioa o! Hioenergetioa ", D .R . Sanadi Edr ., Aoademio Presa, Iiew York ead ioadon, 1966, vol . 1, p .203 . 2 . S. Sbaahi and M. 13ado, PrOAr . B1oDh. ldol . Biol . ~~ 123 (1968) . 3 . S . Sbaahi and F . Ebaahi, J. Bioohem. (ropo) ~, 604 (1964) . 4. 3 .Y . Perry, Y. Daviea aad D . Hayter, Bioohem.J . ~, 1C (1966) . 5 . S . Sbaahi aad A. Hodama, J . Bioohem. (Tolpro) ~8, 107 (1965) " 6 . A . Weber and R. Hers, J . Biol . Che~. 2~8, 599 (1963) . 7 . A. Ylebsr, R. Hers and I. Reisa, Hioohemiatr9 ~, 2266 (1969) 8 . S . Sbaahi, A. godama and F . Sbaahi, J . Bioohem. (ropo) ~, 465 (1968) 9 . F . Fuoha and F .N .Hrigga, J . aen .Physiol . ~l, 655 (1968) 10 . W. Drabikowaki, B.Barytko, R.D~browaka and S .Howak, Bull . Aoad . Soi. Poloa. =6, 397 (1968) 11 . T. Wakabayashi aad S . Sbaahi, J . B_ ioohem . (Tolgo) ~, 731

TROPONIN, Ca++ RECEPTIVE PROTEIN

Vol, 9, No. 21 (1968)

12 . Y.C .Sohanb and S .V .Perry, ßioohem. J .

1233

1~, 993 (1969)

13 . D .J .Hartahorne, M.Theiner and H .Mueller, Hioohim . Hioph9a. Aota ~, 320

(1969)

14 . W.Drabikowaki, R .D&brotysl~, B .BaryYko, 1~ .Graeaer aad J .Gergely, III Intern . Hiophyaioa Coagreaa of I.II.P .A .B ., Cambridge, Mass .

1969, Abstraota of Comm ., p . 193 .

15 " A. Websr, R.Here and I.Reiea, J . Gen.Ph9siol .

4~, 679

(1963) 16. VY . Drabikotyaki, R.D4brotyslaa and $ .Notyak, Aota Bioohem. Biophsa . Aoad . Soi . HunB" 4, 112

(1969)

17 " H . Straeleoka-Gotas$ewska and A.Drabikowaki, Aota Hioohim. Polon.

14, 195 (1967)

18 . H.G .Sarsaia and W.Drabikotyaki, Life Soienoes ~, 477 (1969) 19. P.Danoker, Pfl9gera Aroh . 3~, 198 (1970)