James A. Talbot$ and Robert S. Hodges

Transcription

1 THE JOURNAL OF BOLOGCAL CHEMSTRY Val. 256, No. 23. ssue of December O, pp Prnted n U S.A (Receved for publcaton, May 13, 1981) James A. Talbot$ and Robert S. Hodges From the Department of Bochemstry and the Medcal Research Councl Group n Proten Structure and Functon, Unversty of Alberta, Edmonton, Alberta, Canada T6G 2H7 Fve peptde analogs of the actomyosn ATPase nhbtory regon of troponn (Tn-) have been syntheszed by the sold-phase method. One analog was constructed wth a sequence dentcal to that found n speces of Tn- solated from chcken fast, rabbt fast and rabbt slow skeletal muscle. A second peptde was made dentcal to the sequence of the homologous nhbtory regon of Tn- from rabbt cardac muscle. The remanng three analogs were hybrds of the two sequences. We have verfed that rabbt skeletal fast Tn- was a better nhbtor than rabbt cardac Tn- n a rabbt skeletal actomyosn assay system and extended these studes to show that the same results were found n an assay system whch used rabbt cardac actomyosn. Our skeletal fast muscle peptde analog was also a better nhbtor n both assay systems than the cardac Tn- peptde analogs. The hybrd peptdes ndcated that the changng of prolne 11 to a threonne resdue had no effect and suggested that poston 11 was not essental to nhbton. The other amno acd change, the replacement of argnne 113 by a leucne resdue, was shown to be the crucal dfference and resulted n lower actvty. Thus, the dfferences n relatve nhbtory actvty of rabbt skeletal fast and car- dac Tn- can be at least partally and possbly solely explaned by the sngle amno acd substtuton at poston 113. Short actve sequences of protens, lke the Tn- nhbtory regon are potentally of enormous value as probes of structure-functon relatonshps. We have recently establshed that peptdes made by the sold-phase method whch contan the mnmum nhbtory regon (15-114) also nhbt n a tropomyosn-enhanced manner and do so wth hgh actvty (45% on a molar bass) (8, 9). n ths paper, we have constructed fve peptde analogs to examne the mportance of amno acd changes n the nhbtory regon of varous speces of Tn-. The fve analogs were constructed from the sequences determned for Tn- solated from chcken fast, rabbt fast, rabbt slow skeletal muscle and rabbt cardac muscle (7). These peptdes also allow us to examne the contrbuton to the nhbton by the sde chans of amno acds at two nternal postons of the nhbtory regon and then to compare these fndngs to the nhbtory actvty of the parent Tn- from dfferent speces. These fndngs, n conjuncton wth our prevous studes (8, 9), allowed us to desgnate 15 of the 21 resdues n the nhbtory regon as ether mportant or unmportant to nhbton. Ths leaves only sx resdues whose contrbuton s unknown. EXPERMENTAL PROCEDURES Materals-All chemcals and solvents were reagent grade. Dsopropylethylamne, pyrdne, and methylene chlorde were redstlled pror to use. t-butyloxycarbonyl amno acds were purchased from Spnco Dvson, Beckman nstruments, nc., Palo Alto, CA; Proten Research Foundaton, Japan; and Bachem Fne Chemcals, nc., Marna Del Rey, CA. Copoly(styrene, 1% dvny1benzene)chloromethyl resn (.9 mmol of Cl/g of resn) was purchased from Perce Chemcal Co. BoRex 7 (2-4 mesh sodum form) was purchased from Bo-Rad and the Sephadex G-1 (2-4 mesh) from Phar- maca (Canada), Ltd., Dorval, Quebec. ATP was purchased from Terochem Chemcals Ltd., Edmonton, Alberta. Rabbt skeletal 1- Both the troponn complex and tropomyosn actng n contropomyosn was a generous gft from the laboratory of Dr. L. B. Smlle, Department of Bochemstry, Unversty of Alberta, Edmoncert have been demonstrated to be essental for full calcumsenstve control of the actomyosn ATPase of both vertebrate ton. Rabbt skeletal and cardac Freeze Bologcals, Rogers, AR. muscle was purchased from Pelskeletal and cardac muscle (1, 2). n contrast, the nhbton Amno Acd Analyss, Peptde, and Proten Quanttaton-The quanttes of peptdes and Tn- used were determned from amno of the actomyosn ATPase, but not the calcum-trggered acd analyses after hydrolyss n 1 ml of 6 N HC n evacuated, sealed release of nhbton, can be medated by Tn- and tropo- tubes for 24 h at 11 C. The mean of the molar ratos of all accurately myosn alone (3-5). t has also been shown that a cyanogen measurable amno acds n the acd hydrolysate was used to calculate bromde generated fragment of Tn- (resdues ) can the concentraton of proten or peptde. Quanttes of the rabbt nhbt the actomyosn ATPase sgnfcantly, n a manner that protens used were determned on a Cary 118C spectrophotometer was greatly enhanced by tropomyosn (6, 7). usng the followng E;;,, and molecular weghts: a-tm, 3.3 (1) and 66, (11); skeletal myosn, 5.88 (12) and 47, (13); cardac myosn, * Ths work was supported by the Medcal Research Councl of Canada. The costs of publcaton of ths artcle were defrayed n part 6.6 (14) and 47, (14); skeletal S-1 (Al), 7.9 (15) and 115, (16); fast Tn-, 6.6 (17) and 21, (18); cardac Tn-, 4.37 (19) and 24, by the payment of page charges. Ths artcle must therefore be hereby (19). marked aduertsement n accordance wth 18 U.S.C. Secton 1734 Peptde Synthess, Cleauage, and Purfcaton-The analogs were solely to ndcate ths fact. syntheszed on a Beckman peptde syntheszer Model 99 usng the $ Recpent of Medcal Research Councl Studentshp. general procedures for sold-phase peptde synthess wth specfc The abbrevatons used are: Tn-, troponn ; Tm, tropomyosn; modfcatons prevously descrbed (8, 9). The peptdes were cleaved S-1 (Al), myosn subfragment 1 alkal lght chan 1; Tn-C, troponn from the resn support n a type HF apparatus wth absorpton C; SDS, sodum dodecyl sulfate; EGTA, ethylene glycol bs(p-am- cylnder from Proten Research Foundaton (8). Purfcaton of the noethyl ether)n,n,n,n -tetraacetc acd; acto-s-1 (Al), actn and peptdes was carred out by on exchange chromatography on BoRex subfragment 1 alkal lght chan 1. 7 usng a pyrdne acetate gradent (8). The effluent was montored 12374

2 Studes on the nhbtory Regon wth a Techncon auto-analyzer wth stream dvson for nnhydrn detecton at 57 nm. Subsequent desaltng was performed on Sephadex G-1 column and the effluent was montored wth a Schoeffel Spectroflow montor and monochromator (SF77 and GM77, respectvely) at 23 nm (8). Preparaton of Synthetc Actomyosn and Acto-S-1(A1) Skeletal actn (8),skeletal myosn (a),and skeletal S-1 (A) (9) were prepared as prevously descrbed. Cardac actn was prepared essentally by the method of Spudch and Watt (2) except that after the fnal dalyss the resultng G-actn was repolymerzed to F-actn and the entre Spudch and Watt procedure was repeated. Ths was necessary because of greater contamnaton manfested as a greensh brown color n the soluton. Any actn not used mmedately was stored n the followng manner. Dthothretol was added to the soluton to make t 1 m~ n fresh dthothretol. The soluton (5-1 mg of G-actn/ml) was added slowly, 1 drop a t a tme, nto a beaker flled wth lqud ntrogen. The actn must be stored n the G form as F-actn denatures. The resultng pellets were stored at -2 OC. When the actn was to be used, t was thawed a t room temperature n the presence of an addtonal amount of fresh dthothretol to make the soluton 1 mm. Actn stored by ths procedure was used after perods of up to four months wthout loss of actvty n actvatng the myosn ATPase. Cardac myosn was purfed by followng very closelythe method of Wolodko and Kay (14). The major modfcaton was that after the Guba-Straub soluton had been added to themuscle and the mxture strred, an equal volume of dstlled and doubly deonzed water was added wth strrng. After centrfugaton for 15 mn at 476 X g,the supernatant was dscarded. Ths seemed to rd the mxture of the bulk of the red-brown color presumably mparted by red blood cells and heme-contanng protens. The pellet was re-extracted wth 9 ml of Cuba-Straub soluton contanng 1 mm ATP, as descrbed n the Wolodko and Kay procedure wth the remander of the purfcaton performed exactly as specfed (14). Purfcaton of Tn--Fast skeletal Tn- was prepared by afnty chromatography usng Tn-C coupled to Sepharose 4B (21) and the modfcatons outlned (9). Cardac Tn- was prepared by a smlar method (19) wth the followng modfcatons. Proteolytc actvty was hgh n the cardac muscle extracts whch resulted n a large amount of cleavage of Tn- when Tn- was solated by affnty chromatography from troponn preparatons. For ths reason we used the suggested procedure of solatng the Tn- drectly from a muscle mnce (19) usng a 9 M urea extracton n place of the 8 M urea extracton used for skeletal Tn-. Ths extract was prepared as follows. We blended 3 g of cardac muscle from whchthe fat andblood vessels had been excsed wth a scalpel. The blended muscle was extracted for 15 mn wth 9 ml of Guba-Straub soluton (.3 M KC,.1 M KH2P4,.5 M K2HP,, ph 6.5,4 C) whch was 1 mm n ATP. The mxture was centrfuged at 4,76 X g for 15 mn and the supernatantdscarded to remove myosn. The pellet (1 gm) was added to 5 ml of a soluton that was 9 M urea, 15 mm P-mercaptoethanol, 1 mm CaC2, 5 mm Trs-HC1, ph 8., and strred for 3 mn. Ths was fltered through 1 layer of cheesecloth and centrfuged at 1,825 X g for 15 mn. The supernatant was strred wth the Tn-C-Sepharose 4B conjugate for 3mn (not wth a magnetc bar as ths crushes the resn). The mxture was centrfuged a t 1,5 X g for 15 mn, supernatant dscarded, resn resuspended n 2 ml of 9 M urea affnty buffer and centrfuged agan at 1,5 X g for 15mn. The supernatant was decanted, resn-packed nto a column, and the procedure was contnued dentcal to that explaned n Ref. 19. SDS-polyacrylamde gel electrophoress was done wth 8%acrylamde, 6 M urea gels usng the procedure of Weber and Osborn (22). Assay of nhbtory Actuty-nhbton assays were carred out exactly as prevously descrbed wth one excepton (9). The cardac actomyosn ATPase was allowed to react for 15 mn rather than the 1 mn for the skeletal actomyosn ATPase, to compensate for the lower actvty of cardac actomyosn. of Selected Speces of Tn E N EG?A F r a c t l o n No. FG. 1. Chromatographc purfcaton of Tn- on an affnty column of Tn-C lnked to Sepharose 4B.. For detals of purfcaton, see Expermental Procedures. The column buffer conssted of 8 M urea, 15 m~ P-mercaptoethanol, 1 mm CaC2, 5 mm Trs-HC1 buffer, ph 8. (9 M urea for cardac Tn-). EGTA ndcates the pont at whch the same buffer contanng 1 mm EGTA (ph readusted to 8. wth 5 M KOH) was added. RESULTS To compare the relatve actvtes of cardac and skeletal fast muscle Tn- twas frst necessary to prepare theprotens from ther respectve rabbt muscle sources. Skeletal fast muscle Tn- was prepared by frst makng crude troponn and then solatng Tn- on a Tn-C affnty column (21). Ths procedure proved unsatsfactory for rabbt cardac Tn- because of proteolyss of Tn-. To combat ths we used a 9 M urea buffer extract of the muscle mnce as thesource of Tn- - FG. 2. SDS-urea polyacrylamde gel electrophoress of rabbt cardac Tn-. fast Tn-. and fast Tn-C. The dashed svmbol (-) marks the poston of the proten. a, 4 pg of fast Tn-C; b; 4 pg of fast Tn-; c, 4 pgof cardac Tn-.

3 12376 Studes on the nhbtory Regon of Selected Speces of Tn- rather than the crude troponn. The chromatographc profle of ths one-step purfcaton (Fg. 1) demonstrated ts smplcty whle SDS-polyacrylamde gels (Fg. 2) revealed ts resolvng power. The nature of the contamnatng bands of the cardac Tn- preparaton s unknown. The method of purfcaton would seem to ndcate that they are of hgher molecular weght than cardac Tn- and that they bnd to Tn-C n 8 M urea n a calcum-senstve fashon. The presence of P-mercaptoethanol n the preparaton of the proten sample for electrophoress ndcates that the bands are smply not aggregates of Tn- formed by cystene oxdaton. Scannng the gels after stanng and assumrlg that the same amount of dye s taken up by each of the bands shows that the contamnatng bands represent no more than 8% of the cardac Tn- proten sample. Wth purfed Tn- from both rabbt cardac and skeletal fast muscle, we set out to determne ther relatve nhbtory actvtes n actomyosn ATPase assays. These assays contaned ether cardac or skeletal actn and cardac or skeletal myosn. When the only varable n the actomyosn assays was TABLE nhbtory actvtes of rabbt fast and cardac Tn-Z n actomyosn assays Condtons for assayng the nhbton are found under "Expermental Procedures." m, nanomoles of peptde or proten requred to produce 5% of the total nhbton. 1% nhbton refers to nmol P4/mn/mg of myosn. n, number of nhbton curves constructed from no less than 5 ponts each from whch the 15 was derved. S.D., standard devaton.,,, n S.D. Cardac or skeletal actn: skeletal myosn Tn- fast Tn- cardac Cardac or skeletal actn: cardac myosn Tn- fast Tn- cardac the presence of ether cardac or skeletal actn, no sgnfcant effect on the ATPase actvtes was observed. The actomyosn ATPase results ndcated that skeletal fast Tn- was a better nhbtor than cardac Tn- whether the source of the myosn was cardac or skeletal muscle (Table ). These results were n contrast to another study (23). n that study t was reported that when the source of myosn was rabbt skeletal muscle, rabbt skeletal fast Tn- nhbted better than bovne cardac Tn-; but that wth bovne cardac myosn, bovne cardac Tn- nhbted better than rabbt skeletal fast Tn-. However, t must be kept n mnd that we are workng wth rabbt cardac myosn whch could behave dfferently than the bovne cardac system. Our results (Fg. 3) were smlar n two respects to ths prevous study (23). Frst, the maxmum nhbton of skeletal actomyosn by skeletal Tn- was greater (n the vcnty of 9%) than for the skeletal Tn- nhbton of cardac actomyosn (7%). Second, as llustrated nfg. 3, at least twce as much cardac Tn- s requred for maxmal nhbton of cardac actomyosn compared to the amount of skeletal Tn- requred for maxmal nhbton of skeletal actomyosn. The sequences of rabbt skeletal fast and cardac Tn- (Fg. 4) n the regon correspondng to the rabbt skeletal cyanogen bromde fragment (96-116) dffered by fve amno acds, four substtutons, and one nserton. n the regon that we have recently shown to be the mnmum nhbtory regon (9), there are only two amno acd changes. Therefore, we syntheszed fve analogs that encompassed only the regon and assayed ther nhbtory actvty n a rabbt skeletal acto-s-1 (A) ATPase assay. The results (Table 11) ndcated that the replacement of prolne 11 by a threonne resdue (compare peptdes, 11, and V, or peptdes 111 and V) resulted n no sgnfcant change n actvty. Smlarly, the mere nserton of a resdue between resdues 112 and 113 and any subsequent msalgnment of the sequences (compare peptdes and 11) resulted n no sgnfcant change n actvty. However, the replacement of argnne 113 by a leucne resdue O/O n h b t n a 9 - o a3 ao o a3. ao 1.2 nmol nhlbtor/tubo FG. 3. nhbton by rabbt cardac and fast Tn- of hybrd rabbt actomyosn ATPases. The proten concentratons are 9 pg of actn, 36 pg 1 of myosn and 135pgof tropomyosn n 3 ml of assay mxture/tube. The actomyosn ATPase value n the absence of nhbtor wth skeletal myosn and cardac myosn are 28 and 65 nmol of Pod mn/mg of myosn, respectvely. 1%., nhbton refers to nmol of P4/mn/ mg of myosn. A, skeletal or cardac actn, skeletal myosn; 6, skeletal or cardac actn, cardac myosn;, cardac Tn-; fast Tn R. F. Asn-61 n-lys-leu-phe-asp-leu-arg-gly-lys-phe-l~s-arg-pr~-pr~-l~u-arg-~r~-v~l -Arg-Met &-@@ C.F. er G n "--Leu +he,met R.S. Pro pro " + FG. 4. Sequence of rabbt cardac (KC.), rabbt fast (RF.), rabbt slow (RS.), and chcken fast (C.F.) n a regon homologous to the nhbtory cyanogen bromde fragment (96-116) of fast Tn-., a deleted resdue when comparng to the rabbt cardac sequence. The numberng scheme s of rabbt skeletal fast Tn-. Nondentcal resdues are crcled.

4 Studes on the nhbtory Regon of Selected Speces of Tn TABLE 1 nhbton by rabbt fast and rabbt cardacpeptde analogs of Tn- n hybrd rabbt actomyosn ATPase assays For a descrpton of assay condtons, see Expermental Procedures. Abbrevatons are the same as Table., resdue deleton. - /Fast and cardac actn: skeletal S-1 1 Fast and cardac actn: cardac (A) myosn Peptde * n S.D. 19, n S.D. Gly -Lys-Phe-Lys-Arg-Pro-Pro-Leu-Arg- Arg-Arg-Val-Arg- Ala Gly Ala Gly V Gly Thr ~ V Gly Peptde 1 s the sequence of rabbt skeletal Tn- and Peptde V s the sequence of rabbt cardac Tn- n the regon (7). (compare peptdes 1 and 111, or peptdes V and V) resulted n a drastc loss n nhbtory actvty of the peptdes. The relatve mportance of these amno acd changes was examned n both skeletal and cardac actn, and acto-s-1 (Al) ATPase assays. No dfferences were found, regardless of the source of the actn. Smlarly, the only mportant amno acd change found was the replacement of argnne 113 by leucne n assays where the myosn was from rabbt cardac muscle and the actn was ether from rabbt skeletal or cardac muscle (Table 11). DSCUSSON The sequences of four speces of Tn- were known when we embarked on ths study (7). These Tn- speces had been solated from rabbt skeletal fast and slow muscle, chcken skeletal fast muscle and rabbt cardac muscle. We have recently shown the mnmum nhbtory regon of rabbt skeletal fast muscle Tn- to be the regon (9). The sequences homologous to ths regon n the other Tn- speces (Fg. 4) were dentcal except for the rabbt cardac Tn- molecule. t has been shown that rabbt skeletal slow muscle Tn- nhbts rabbt skeletal actomyosn ATPase poorly compared to rabbt skeletal fast muscle Tn- (21). Thus, even though the nhbtory regon was dentcal n both protens, the nhbtory actvtes were dfferent. Ths suggests that other portons of the proten must be able to modfy the nhbtory actvty of the regon ether by decreasng or ncreasng actvty. As an example of the former, a peptde generated from rabbt skeletal Tn-, contanng the sequence , exhbted less actvty on a molar bass than the cyanogen bromde fragment (6). As an example of the latter case, other portons of the Tn- molecule may ncrease the nhbtory actvty of Tn- relatve to the nhbtory regon snce our peptdes (8, 9) and the orgnal cyanogen bromde fragment (6) nhbted less well than rabbt skeletal fast Tn-. t seemed farly clear that changes n the proten remote from the nhbtory regon mght result n changed nhbtory actvty, but ths dd not rule out the possblty that dfferences n actvty between speces of Tn- mght be explaned by dfferences wthn the nhbtory regon. Furthermore, rabbt cardac Tn- had been shown to nhbt less well than rabbt skeletal fast Tn- (6) n a rabbt skeletal actomyosn assay system and ths result was duplcated by us n ths paper. To determne whether the dfferences n nhbton could be explaned by the changes n the amno acd sequence n the nhbtory regon, we constructed fve analogs of ths regon usng sold-phase peptde synthess. The fndng that the replacement of argnne 113 by a leucne resdue resulted n a drop n nhbtory actvty allowed us to extend our understandng of the relatonshp between sequence and functon of the nhbtory regon. We concluded that the major effect of ths change was due to the loss of the charged sde chan of argnne, rather than the ntroducton of the bulky sde chan of leucne. We came to ths concluson snce, f anythng, argnne has a bulker sde chan than leucne. Thus, we reasoned that argnne 113 was essental to nhbton. A comparson of all four sequenced Tn- molecules revealed that ths nhbtory regon was one of the most strongly conserved sequences n all of the four protens (7). The concluson drawn from that fndng was that the changes found n rabbt cardac Tn- were unlkely to affect the nhbtory actvty sgnfcantly. To uphold ths pont of vew, t was suggested that the constancy of sequence n the nhbtory regon of Tn-, whch presumably bnds to actn, was not very surprsng n vew of the very conservatve nature of actn tself. We mantan, and our studes have revealed, that changes n ths regon can have drastc effects on the nhbtory actvty. Ths too was consstent wth the conservatve nature of actn. f the majorty of the Tn- nhbtory sequence was strongly conserved as s the sequence to whch t bnds, then any change n the sequence of suffcent magntude, e.g. loss of a postve charge, would qute probably modfy the nhbtory actvty. t has been suggested that a /3 turn mght be formed wth resdues and that ths mght be mportant to the nhbtory actvty. Substtuton of prolne by threonne at ths poston would reduce the /3 turn-formng potental by 65% (24) and as we have shown, resulted n no sgnfcant loss of nhbtory actvty. Whle ths does not elmnate the possble mportance of a /3 turn, ths evdence does weaken ts lkelhood. n addton, snce the replacement of a nonpolar amno acd of pecular geometry (prolne) by a polar amno acd of ordnary geometry (threonne) resulted n no change n nhbtory actvty, we beleve that ths poston (11) was nonessental to the nhbon. A summary of the amno acd resdues found to be essental and nonessental for nhbton by ths and prevous studes (8, 9) are shown n Fg. 5. The essental resdues so far determned n the regon are lysne 15, argnne 113 and valne 114. Snce the mnmum nhbtory regon s very basc (5 of 1 resdues) and two of the basc resdues are essental, we feel that lysne 17, argnne 18, and argnne 112 wll also probably be found to be essental. We are actvely pursung the nvestgaton of the mportance of these resdues and hope to construct an nhbtory peptde constructed of only the essental resdues wth glycnes as spacers. Wth what we already know, we can speculate on the nature of the ste wth whch these peptdes and Tn- nteract. Ths complementary bndng ste n a skeletal muscle system may nclude some knd of hydrophobc pocket to accommodate the valne and at least two acdc amno acd resdues for onc nteracton wth lysne 15 and argnne 113 and more than two f the nteractons wth lysne 17, argnne 18, and argnne 112 prove to be mportant. Examnaton of the actn sequence suggests that there s no lnear sequence on actn complementary to the Tn- nhbtory regon.

5 ~~ ~ ~~ Studes on the nhbtory Regon of Selected Speces of Tn FG. 5. Amno acd sequence of fast skeletal Tn- correspondng to the nhbtory cyanogen bromde fragment (96-116). Amno acds shown by ths and prevous studes (8, 9) to be nonessental (bored) or essental (crcled) for nhbton. TABLE 111 Ponts of smlarty between Tn-Z and the nhbtorv DeDtdes 1. At low onc strength" tropomyosn bnds poorly to F-actn. Addton of Tn- (5, 8) or nhbtory peptde (8) promotes bndng of Tm and the consequent nhbton of the actomyosn ATPase.* At low onc strength" n the absence of tropomyosn, the addton of Tn- (4, 6, 8) or nhbtory peptde (6, 8) results n nhbton. Athghonc strength' tropomyosnbnds to F-actnand the actomyosn ATPase s nhbted up to 6% (5). The addton of Tn- (5) or nhbtory peptde (8) does not sgnfcantly ncrease the extent of tropomyosn bndng but ncreases the nhbton of the actomyosn ATPase to 9%. As actnconcentratons n anactomyosnatpaseassaywere ncreased, keepng the concentraton of Tm constant relatve to actn, the nhbton of Tn- (9) and nhbtory peptdes (9) occurs at lower nhbtor/actn ratos,.e. the nhbton effcency ncreased. n ether rabbt skeletal fast Tn- or ts nhbtorypeptdeanalogsnhbt better than cardac Tn- or ts nhbtory peptde analogs respectvely? Low onc strength 1 m~ Mg2+, 2 mm ATP, 3 mm KC1 (4, 5); 2.5 mm Mg2+, 2.5 mm ATP (6); 2.5 mmmg", 2.5 m~ ATP, 2 mm KC1 (8); Peptdes contanng the nhbtory regon nhbt from 45 to 75% as well as skeletal fast Tn- on a molar bass (6, 8, 9). Hgh onc strength 5 m~ Mgz+, 2 mm ATP, 3 mm KC1 (5), 5 mm Mg", 2.5 m~ ATP, 2 mm KC1 (8). Ths paper. fast or cardac actomyosn ATPase assays, We had reason to suspect that the nhbtory behavor of cardac and skeletal fast Tn- mght be dfferent n a cardac actomyosn system than n a skeletal actomyosn system. Assays have shown bovne cardac Tn- to be a poorer nhbtor than rabbt skeletal fast Tn- n a rabbt skeletal actomyosn system but a better nhbtor n a bovne cardac actomyosn system (23). No comparable experments had been publshed for rabbt cardac Tn-, but f the results were smlar they would make for an exctng test of the peptde analogs. Our results wth the two speces of Tn- ndcate that rabbt cardac and bovne cardac muscles were not analogous n ths respect. n support of ths dfference n the regulaton of contracton between cow and rabbt hearts was the fndng that P-tropomyosn, another regulatory proten, was present to about 2% of the total tropomyosn content n large and slowly beatng hearts (sheep, pg, human) but absent n smaller rapdly beatng hearts (rabbt, gunea pg, dog) (25). Ths dscrepancy between bovne and rabbt cardac Tn- nhbtory behavor may be due to amno acd changes at other than the nhbtory regon. f, however, the dfferences n nhbtory actvty reflected dfferences n the nhbtory regon, we would predct that the bovne cardac Tn- regon does not resemble ether rabbt cardac or rabbt skeletal fast Tn- snce ts behavor was dfferent from ether of these. The amno acd sequence of bovne cardac Tn- has yet to be reported. Though the amno acd sequences of rabbt skeletal fast Tn- and rabbt cardac Tn- (7) are hghly homologous n certan regons and are smlar n sze (cardac Tn-, 215 amno acd resdues; rabbt fast Tn-, 178 resdues) the number of amno acd resdues of dentcal sequence s only 13/178 resdues. Even so, our results suggest that the dfferences n relatve nhbtory actvty of rabbt skeletal and cardac Tn- can be at least partally and possbly solely explaned by the sngle amno acd substtuton at poston 113 (usng the rabbt skeletal fast sequence numberng scheme). 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