the amino-acid sequence of troponin c from frog skeletal muscle

Eur. J. Biochem. 91, 231 -242 (1978)

The Amino-Acid Sequence of Troponin C from Frog Skeletal Muscle Jean-Paul VAN EERD, Jean-Paul CAPONY, Conception FERRAZ, and Jean-Franqois PECH ERE

Pharmakologisches Institut der Universitat Zurich, and Centre de Recherches de Biochimie Macromoleculaire du Centre National de la Recherche Scientifique, Montpellier

(Received April 19, 1978)

The primary structure of the troponin C from skeletal muscle of the frog Rana esculenta has been determined. The amino acid sequence was deduced from amino acid determinations of peptides obtained after cleavage with cyanogen bromide. Overlapping peptides were isolated from tryptic digests of performic-acid-oxidized troponin C and phthalylated performic-acid-oxidized troponin C. All overlaps have been determined except for the Arg-Ile sequence at position 103- 104, which has been obtained by comparison with homologous troponins C. Frog troponin C consists of one polypeptide chain containing 162 amino acids. The calculated molecular weight is 18299. There is a single cysteine residue at position 101 and a single tyrosine residue at position 112. No histidine or tryptophan residues are present. The amino-terminal amino acid is N-acetylated. The homology of frog troponin C with other skeletal and cardiac troponin C is briefly discussed.

Troponin C is a muscular calcium-binding protein. The binding of calcium ions to troponin C triggers a set of events resulting in muscular contraction [l].

The amino acid sequences of the skeletal troponin C from rabbit [2,3], chicken [4] and man [5] have been determined. Moreover, the primary structure of troponin C from bovine cardiac muscle is known [6,7]. Troponin C shows internal homology and most probably it has evolved by two successive gene du- plications from a small precursor protein [2,8].

Troponin C is homologous to parvalbumins, a group of low-molecular-weight calcium-binding proteins from the muscular sarcoplasm [9]. The exact location of the calcium-binding sites in parvalbumin is known because the three-dimensional structure of a carp parvalbumin has been determined [lo]. Because of the homology between troponin C and parvalbumins, it is possible to locate four tentative calcium- binding sites in skeletal troponin C and three tentative calcium-binding sites in cardiac troponin C.

Contribution No 163 from the Centre de Recherches. Ahbreviutions. EGTA, ethyleneglycol-bis(B-aminoethyl ether)-

N,N,N’,N’-tetraacetic acid; Tos-Phe-CHnC1, ~-1-tosylamide-2- phenylethylchloromethyl ketone; dansyl, 5-dimethylaminonaphtha- lene-l-sulfonyl; CNBr, cyanogen bromide; NMR, nuclear magnetic resonance.

Enzymes. Trypsin (EC 3.4.22.4); carboxypeptidase A (EC 3.4.12.2); carboxypeptidase B (EC 3.4.12.3); carboxypeptidase C (EC 3.4.12.1); post-proline cleaving enzyme (EC 3.4. - .-); pronase (EC 3.4.21.4and EC 3.4.24.4).

We have extended the previous work on the primary structure of muscular troponin C with the amino acid sequence of frog skeletal troponin C. Frog muscle was chosen because it was felt that there is a lack of knowledge on the similarities and dissimilarities of skeletal muscle from frog (often used as an object for studying muscular contraction by physiologists) and rabbit (often used as an object for studying muscular contraction by biochemists). Furthermore, because the frog is a rather primitive vertebrate, it was hoped to find earlier stages of gene duplication, e.g. to find a troponin C consisting of only two domains containing one calcium-binding site each.

In this paper we describe the complete amino acid sequence of frog skeletal troponin C. Preliminary results on the almost complete sequence have been published before [ll].

MATERIALS AND METHODS

Protein Preparation

Troponin C was isolated from the hindlegs of frogs (Ram esculenta) according to Potter and Gergely [12]. The frogs were provided by the Elevages Couitard (St Hilaire de Riez, Vendtte, France). 211 mg pure troponin C was isolated from 680g muscle mince. The purity was checked by disc gel electrophoresis in the presence of sodium dodecyl sulfate [13]. The isolated troponin C showed only one band.

232 Frog Troponin C

Cyanogen Bromide Cleavage

1.9 pmol of native troponin C was cleaved by CNBr in 70% formic acid for 24 h at room tempera-

methionine was added. After cleavage, formic acid and excess CNBr were removed by repeated drying

zymelsubstrate ratio of ljlO0, wiw). The digestion was terminated by adding soybean trypsin inhibitor (inhibitorienzyme ratio, 211, w/w).

The digest was freeze-dried and the peptides were ture [141' About 40 times Of CNBr Over separated on Sephadex (3-75 (two columns, 200 x 1 cm, in series equilibrated with 0.5 % NH4HC03).

by rotary evaporation. Initial separation of the peptide mixture was carried

out on Sephadex G-50 fine (two columns of 200 x 1 cm in series, equilibrated with 10 mM NH4HC03). Puri-

was performed on DEAE-Sephadex A-25 (23 x 0.9 cm) equilibrated with 20 mM Tris-HCI pH 8.5 and eluted with a KCl gradient from 0- 1 .O M, in the same buffer. The peptides were desalted on Sephadex (3-25. Small peptides were first examined by high-voltage paper electrophoresis at pH 6.4 (3 kV for 30 min) and sub-

Fragmentation of the N-Terminal CNBr Peptide

The N-terminal CNBr peptide (CB1) was further fragmented by pronase digestion during 20 h [17],

proline cleaving enzyme [19]. The post-proline cleaving enzyme was kindly provided by Dr R. Walter (Chi- cago).

Amino Acid Analysis

fication Of the large peptides (more than l5 residues) limited acid hydrolysis [18] and digestion with post-

sequently isolated by ion-exchange chromatography on an anionic resin Durrum DA-X2, or a cationic resin, Technicon Chromobeads-P. Peptides were detected by ninhydrin staining before and after alkaline hydrolysis.

Samples were hydrolysed in evacuated sealed tubes with constant boiling HCI for 24 h at 110 "C. Amino acid analysis was performed on a Beckman Multi- chrom amino acid analyser system.

a single

Complete Tryp tic Digestion Sequence Determination

1.2 pmol of performic-acid-oxidized troponin C [15] was treated first with 50 mM EGTA in 6 M guanidine-HC1, thereafter dialyzed against 25 mM EGTA and finally against HzO. The EGTA-treated troponin C was heat denatured in a boiling water bath for 5 inin at pH 5 - 5.5. After adding Tos-Phe-CHzCI-treated trypsin (enzyme/substrate ratio, 3/100, wiw), N-ethyl- morpholine/acetic acid pH 8.5 was added to a final concentration of 0.1 M. The protein concentration was 10 mg/ml. After digestion for 3 h at 38 "C, the digest was lyophilized. The tryptic peptides were first fractionated on Sephadex G-50 fine (2 columns, 200 x 0.9 cm, in 0.5 NH4HC03). Peptides in pool I11 from the Sephadex G-50 column were further purified on DEAE-cellulose DE-32 (15 x 0.9 cm) equilibrated with 5 mM NH4HC03 and eluted with a gradient of NH4HC03. Peptides in pool IV were chromatographed on an anion exchange resin, Durrum DA-X2. Peptides were detected by ninhydrin staining before and after alkaline hydrolysis.

Limited Tryptic Digestion

0.75 pmol troponin C was oxidized with performic acid and subsequently phthalylated [I61 in order to modify all e-amino groups of the lysine residues rever- sibly. The modified protein could now be digested by trypsin at the arginine residues only. Tryptic digestion of the performic-acid-oxidized and phthalylated protein was carried out at 38 "C during 110 min on 0.1 M N-ethylmorpholinelacetic acid pH 8.5 (en-

Manual Edman Degradation. This was performed as described previously [7]. Phenylthiohydantoin derivatives of amino acids were determined by high performance liquid chromatography according to the method of Frank and Strubert [20]. The phenyltliio- hydantoin derivative of arginine was determined on a Nucleosil C18 column using reversed phase chromatography (mobile phase, 35 % methanol and 4 % acetic acid in water).

Dansyl Edman. Edman degradation followed by dansylation of the new N-terminal amino acid was carried out according to Gray [21]. For peptides T10 and TI 1, methylisothiocyanate was used instead of phenylisothiocyanate for the Edman degradation.

Automatic Edman Degradation. This degradation of peptide T5 (380 nmol) was performed using a sequenator: Sequenceur PS 100 (Socosi, 94 100 Saint- Maur, France). The degradation was carried out in the presence of 2mg hake parvalbumin used as a carrier [22]. Identification of the phenylthiohydantoin derivatives of amino acids was performed by thin- layer chromatography according to Edman and Hen- schen [23] and by gas chromatography (Beckman CG 65 gas chromatograph) according to Pisano and Bronzert [24].

Mass Spectrometry. The amino acid sequence of the blocked CNBr peptide CB1 was elucidated by mass spectrometry. Mass spectra were recorded on an AEI- GEC MS 50 instrument. Sample handling procedures and instrument parameters were carried out according to Morris et al. [25]. About 100 nmol of CBI was

J.-P. van Eerd, J.-P. Capony, C. Ferraz, and J.-F. Pechere 233

treated with deuteroacetic anhydride followed by permethylation [26] before analysis by mass spectrometry.

Curboxypeptidase Digestion. Carboxypeptidase A and B digestion of certain peptides was performed according to Ambler [27]. Carboxypeptidase C digestion was performed according to Zuber [28].

Identification of the Blocked N Terminus

500 nmol CBI in 0.5 ml 2H20 were analysed on a Bruker HX-360 NMR instrument according to Coz- zone and Marchis-Mouren [29]. Analysis of CBl by mass spectrometry also provided information on the nature of the blocking agent of this peptide.

Amide Assignments

Amide assignments of the glutaminyl and aspara- ginyl residues of the peptides sequenced by the dansyl- Edman method was performed by high-voltage paper electrophoresis at pH 6.5 according to Offord [30].

Nomenclature of Peptides

The peptides have been numbered starting from the N terminus of the complete amino acid sequence. CB means CNBr peptide; PhT, tryptic peptide of phthalylated troponin; T, tryptic peptide ; Pr, peptide as a result of digestion with pronase; PP, peptide as a result of cleavage with post-proline cleaving enzyme ; LAH, peptide obtained as a result of limited acid hydrolysis.

RESULTS

The experimental evidence for the determination of the sequence (Fig. 5 - 11 and Tables 3 -7) is presented as a miniprint (reduced format) supplement immediately following the references.

Amino-Acid Composition

The amino acid composition of frog troponin C was determined by amino acid analysis of acid hydro- lysates (Table 1). For isoleucine and valine the values of a 72-h hydrolysate have been used. The single cysteine residue was determined as cysteic acid by amino acid analysis of the performic-acid-oxidized protein. No histidine is present. The presence of around 164 amino acid residues was calculated from the amino acid analysis. The ultraviolet spectrum of frog troponin C (Fig. 1) shows the presence of a small amount of tyrosine (maximum at 278 nm with a shoulder at 286 nm) as well as the characteristic phenylalanine peaks. No tryptophan is present and the phenylalanine/

2i)o 2iO 2i-O 240 280 300 350 Waveiength (nrn)

Fig. t . Ultraviolet spectrum of fiog skeletal troponin C

tyrosine ratio of 8 - 10/1 determined by spectropho- tometry, is in good agreement with the results of the amino acid analysis.

Cyanogen Bromide Peptides

The CNBr peptides of frog troponin C were chromatographed on Sephadex G-50 (Fig. 5, miniprint supplement). The first peak contained uncleaved and partially cleaved troponin C. The next three peaks (designated I, 11 and HI), contained large CNBr peptides, which were further purified on DEAE Sephadex A-25 (Fig. 6, miniprint supplement).

Peak I, which predominantly contained CB9 was still impure at this stage. Peak I1 yielded a pure peptide CB6 and peak IT1 yielded two pure peptides CBIO and CB4.

Starting from tube 170 of the chromatogram de- picted in Fig. 5, aliquots of every fifth tube were analysed by high-voltage paper electrophoresis at pH 6.4. On the basis of that analysis, tubes 170-260 were combined into three pools, designated IV, V and VT. The peptides in pool IV were isolated by ion-exchange chromatography using an anion exchange resin Dur- rum DA-X2. The elution pattern is shown in the upper half of Fig. 7 (miniprint supplement). Three pure peptides (CB1, CB2 and CB3) were isolated. Peptide CB1 was only detected by ninhydrin staining after alkaline hydrolysis. Therefore, CB1 contained a blocked

234 Frog Troponin C

N-terminal amino acid. The peptides in fraction V were isolated by ion-exchange chromatography using a cation-exchange resin, Technicon Chromobeads P. The elution pattern is shown in the lower half of Fig. 7. Two pure peptides (CB8 and CB12) were isolated. Furthermore free homoserine (CB7 and CB11) was isolated, indicating the presence of at least one Met- Met peptide bond in frog troponin C. Fraction VI contained only one peptide CB5, which appeared to be pure.

The amino acid composition of the isolated CNBr peptides is listed in Table 2. The amino acid composition of CB9 is not reported because this peptide was not isolated in a sufficiently pure form. During the sequencing of frog troponin C, it became clear that the polypeptide chain contains two Met-Met sequences (CB7 and CB11). CB12 was the only peptide without homoserine. Therefore CB12 should be the C-terminal CNBr peptide.

The CNBr peptides were sequenced partially by manual Edman degradation and the released phenylthiohydantoin derivatives of amino acids were iden- tified by high performance liquid chromatography. 60- 160 nmol of peptide were used for sequencing. The results of the amino acid sequence determination of the cyanogen bromide peptides are summarized in Table 3.

Tryptic Peptides

Native troponin C is very resistant to tryptic digestion and difficult to denature. Before tryptic digestion, the protein was oxidized by performic acid and further denatured by an EGTA treatment in guanidine-HC1, followed by a heat treatment on a boiling water bath. The tryptic peptides were fractionated first on Sephadex G-50 (see Fig. 8, miniprint supplement). The peaks were analysed by amino acid analysis, dansylation and starting from tube 105 by high-voltage paper electrophoresis at pH 6.5. From this analysis it became evident that pool I consisted of a single pure peptide, T5 containing 37 amino acid residues.

Pool 111, which consisted of a mixture of several peptides was chromatographed on DEAE-cellulose resulting in two peaks (Fig. 9, miniprint supplement). The first peak contained a single pure peptide T3 while the second peak contained a mixture of two peptides. These peptides (T8 and T10) were separated by high-voltage paper electrophoresis at pH 6.5.

Pool IV was chromatographed on an anion-exchange resin Durrum DA-X2 (Fig. 10, miniprint supplement). This allowed the isolation of peptides T1, T2, T4, T6,7, T7, T14 and T15. Peptides T6,7 and T1 were only detected by ninhydrin staining after alkaline hydrolysis indicating that the N-terminal amino acids of these peptides were blocked.

No pure peptides were isolated from pool 11. Pool V contained only salt.

Digestion of 780 nmol of performic-acid-oxidized troponin C with trypsin containing some chymotryptic activity (enzyme/substrate ratio, 31100, w/w, digestion time 16 h), yielded some additional peptides. The digest was chromatographed on Sephadex G-50 under identical conditions as in Fig. 8. Peptide T5 in peak I was shortened due to chymotryptic activity and yielded peptide T5c with C-terminal phenylalanine, as was shown by digestion with carboxypeptidase A. Pep- tide T11 was isolated from the digest from a peak corresponding to pool I1 in Fig.8. Also peptides T12 and T13 were isolated by chromatography of pool IV on Durrum DA-X2 (not shown).

The amino acid composition of the isolated tryptic peptides is listed in Table 4. Peptide T5c is 6 amino acids shorter than peptide T5. No tryptic cleavage occurred at a lysyl residue in peptides T5 and T5c. Peptide T6,7 is a combination peptide of peptides T6 and T7, due to incomplete digestion. Peptide T6 itself was not isolated, neither was peptide T9. T15 does not contain any lysine or arginine residues, so it is the C- terminal tryptic peptide. The number of amide residues of most tryptic peptides was determined from the electrophoretic mobilities at pH 6.5 [30].

Most of the tryptic peptides of frog troponin C were sequenced by the dansyl-Edman procedure (Table 5). The sequence of T5 (37 residues) could be determined by automatic Edman degradation until the penultimate residue. The repetitive yield of the sequencer run was 94 %, as calculated from the amount of the phenylthiohydantoin derivatives of leucine and valine obtained at step 2 and step 36, respectively, and determined by gas chromatography. The C-terminal amino acid (arginine) which was expected because of the specificity of trypsin, combined with the results of amino acid analysis, was confirmed by carboxypeptidase B digestion.

Peptides Obtained after Limited Tryptic Digestion

The peptides resulting from tryptic digestion of performic-acid-oxidized and phthalylated troponin C were chromatographed on Sephadex G-75 (Fig. 1 1, miniprint supplement). All six expected peptides were separated from each other and were isolated in high yields and in a sufficiently pure form. The peak eluting between fractions 100 and 110 consisted mainly of a combination peptide of PhT5 and PhT6, designated PhT5,6, which is the result of incomplete cleavage at arginine residue number 123. Peptide PhT5,6 was isolated in higher yield in a similar experiment from non-oxidized phthalylated troponin C, digested with trypsin for only 30 min instead of 110 min as in the experiment of Fig. 11. The amino acid composition


2,018pprn I A c e t y l

3.00 2.50 200 1.50 100 0.50 Shift, 6 (pprn)

Fig.2. Nuclear magnetic resunance spectrum of cyanugen bromide peptide CBI. The chemical shift of the N-acetyl group is 2.018 pprn downfield from 2,2-dimethyl-2-silapentane-5-sulfonate

of the PhT peptides, including PhT5,6 is listed in Table 6.

Part of the amino acid sequence of PhT4, PhTS and PhT6 has been determined by manual Edman degradation. The identification of the resulting phenylthiohydantoin derivatives of amino acids was performed by thin-layer chromatography. The sequence determination was mainly performed in order to locate the amide positions in the tryptic peptides T6, T7 and TI0 and to establish whether or not T11 contained an amide, because analysis of T11 by high-voltage paper electrophoresis at pH 6.5 did not give a clear answer. Also PhT4 and PhTS contained the tryptic peptides T6 and T9 which had not been isolated after complete tryptic digestion. The N-terminal amino acid glutamine was not cyclised to pyroglutamic acid, which must have been the case with the N-terminal amino acid of T6,7. This is probably because PhT4 was isolated at moderately alkaline pH, while T6,7 was isolated at acidic pH. The results of the amino acid sequence determination of the PhT peptides are summarized in Table 7.

Blocked N-Terminal Cyanogen Bromide Peptide CB1

The blocked N-terminal CNBr peptide CB1 was analysed by nuclear magnetic resonance, because it was expected that the peptide was blocked with an acetyl group. The three protons of the CH3 group of an acetyl group form a singlet at around 2.10 ppm upfield from 2,2-dimethyl-2-silapentane-S-sulfonate [29]. When we look at the NMR spectrum of peptide CBI in 2H20 (Fig.2) we see a singlet at 2.018 ppm. When we compare the area of the singlet with the doublets at 1.242 ppm and 1.356 ppm which can be ascribed to the y-CH3 protons of one threonine residue and the j?-CH3 protons of one alanine residue, respectively [3 11, we may conclude that the singlet

at 2.018 ppm is due to the -CH3 protons of one acetyl group in this peptide.

Prolonged digestion of the octapeptide CB1 with pronase, yielded two tetrapeptides which were separated by high-voltage paper electrophoresis. The C- terminal tetrapeptide, sequenced by the dansyl-Edman method and carboxypeptidase A digestion, yielded the sequence Asp-Gln-Gln-Hse. The N-terminal blocked tetrapeptide had the amino acid composition (Thr, Glx, Pro, Ala). This peptide was subjected to limited acid hydrolysis (1 M HCl, 110 "C for 20 min) and yielded one free N-terminal amino acid residue, viz. alanine. One Edman degradation step revealed glutamic acid or glutamine as new N-terminal amino acid. Carboxypeptidase C digestion of this peptide released only threonine.

Digestion of CB1 with post-proline cleaving enzyme [19] followed by dansylation, yielded threonine as free N-terminal amino acid suggesting the presence of a Pro-Thr peptide bond.

Mass spectra of CBI confirmed that this peptide is blocked with an acetyl group. The following sequences were assigned from the spectra : Ac-Ala- Gln-Pro-Thr-Asp-Gln and Asp-Gln-Gln-(Hse/Thr). The second sequence is the result of an N-C cleavage before the aspartic acid residue [32]. No distinction can be made between threonine and homoserine because they have the same mass. Combining both sequences and taking into account the fact that the peptide is cyanogen bromide peptide containing one residue of threonine only, the overall sequence of CB1 is Ac-Ala-Gln-Pro-Thr-Asp-Gln-Gln-Hse.

The Complete Amino Acid Sequence

A summary of the sequence results of the three sets of peptides is presented in Fig. 3. It shows the overlaps of the cyanogen bromide peptides with the tryptic

236 Frog Troponin C

10 20 30 Ac-Ala-Gln-Pro-mr-AspGln-Gln~~-Met-~~~a-Arg-Se~Ph~ku-Ser-Glu-Glu-~t-Ile-~a~lu-Phe-Lys-~a-~a-Phe-~~Met-Ph~~~

CB1 ., cB2 ./ cB3 . . a 4 . -. 7-7777- 77777777 77

‘31-Prl CBl-Pr2 ---- ’ .C CB1-Prl-IW

cB1-PP .L < 2-

4 > T1 ., T2 ., T3

c - 2 4 -4 12 3 4-2 -2-4 A 22

40 50 60 ~ir-AspGly-Gly-Gly-RspIle-Ser-~r-Lys-Glu-Leu-Gly-~r-Val-~~t-Ar~-~~t-leu-Gly-Gln-~r-Pr~~r-Lys-Glu-Glu-~u-~~~a-

cB4 ,,cB5 . , CB6

T3 T4 T5

. 7777777771 777777777777 ./

F L - d A ~ ~ “ - - n n - n - - n - n - n - 7 1 n ~ - x - 7 1 - n - - n <A

T5C

PhT2 PhT3 ’L\

100 110 120 Gln-Gly-Lys-Ser-Glu-Glu-Glu-Leu-Na-Glu-~s-Phe-~g-Ile-Ph~~pLys-~n-~a-~pGly-~r-Ile-AspSer-Glu-Glu-Leu-Gly-Glu-

(cB9 )

PhT4 PhT5 ,- 7 7 7 - - - 7 7 7 7 7 -7 ---I 7 7 7 7 - - - -I- 7 - - - 7 7

PhT5,6 < L A

130 140 150 Ile-leu-Arg-Ser-Ser-Gly-Glu-Se~Il~~r-As~Glu-Glu-Ile-Glu-Glu-Leu-Met-Lys-Asp-Gly-~~Lys-~n-~n-~pGly-LyS-Ile-Asp (CB9) cb10

24 - L l \ ~ 2 ~ ~ ~ \ - - 2 \ - 1 - - _ \ - ) . \ ~ ’ 2-

777-7777-7 T10 ./ T11 (T12,13) T14

PhT5 ., PhT6 ---7 --7 - - --- 7 1 7 7 7 - - 7 7

160 Phe-AspGlu-Phe-ku-Lys-@t-@t-Glu-Gly-Val-GlrDH

>

>

>

>

CBlO >a1 cB12 7777

T14 . / TI5 -2-- l - \ ->----J

PhT6

PhT5,6

Fig, 3. Summary of the determination ofthe amino acid sequence offrog skeletal troponin C. Underneath the amino acid sequence, are shown the peptides isolated. The peptides have been numbered according to their order in the sequence. Peptides whose designation starts with a CB, a PhT or a T represent cyanogen bromide peptides, tryptic peptides after phthalylation and simple tryptic peptides, respectively. Sub- digestions of peptides with pronase, by limited acid hydrolysis, or digestion with post-proline cleaving enzyme arc designated by Pr, LAH and PP respectively. -, Manual Edman step; -. automatic Edman step; -, dansyl-Edman step; -, sequence determined by mass spectrometry; -, carboxypeptidase digestion step. A, carboxypeptidase A ; B, carboxypeptidase B; C , carboxypeptidase C


Frog skeletal troponin C Rabbit skeletal troponin C Human skeletal troponin C Chicken skeletal troponin C Bovine cardiac troponin C

10 A c A Q P T D Q Q M D A R S F L S

Ac d t Q Q a e A R S y L S X (T.D.Q)Q a e A R S y L S

X(A.s.m.T)D Q Q a e A R a F L S A c m d D i y k a A v e q L t

20 30 40 5n ,- E E M I A E F K A A F D M F D - T D G G G D I S T K E L G T V M R M L G Q E E M I A E F K A A F D M F D - a D G G G D I S v K E L G T V M R M L G Q E E M I A E F K A A F D M F D - a D G G G D I S v K E L G T V M R M L G 0 E E M I A E F K A A F D M F D - a D G G G D I S T K E L G T V M R M L G Q E E q k n E F K A A F D i F v Z g a e d G e I S T K E L G k V M R M L G Q

60 70 80 90 T P T K E E L D A I I E E V D E D G S G T I D F E E F L V M M V R Q M K E D A Q T P T K E E L D A I I E E V D E D G S G ~ I D F E E F L ~ M M V R Q M K ~ D A ~ T P T K E E L D A I I E E V D E D G S G T I D F E E F L V M M V R Q M K E D A k n P T K E E L D A I I E E V D E D G S G T I D F E E F L V M M V R Q M K E D A k n P T p E E L q e r n I d E V D E D G S G T v D F d E F L V M M V R c M K d D s k

100 110 120 G K S E E E L A E C F R I F D K N A D G Y I D S E E L G E I L R S S G E G K S E E E L A E C F R I a F D r N A D G Y I D a E E L a E I f R a S G E G K S E E E L A E C F R I F D r N A D G Y I D p E E L a E I f R a S G E G K S E E E L A d C F R I F D K N A D G f I D i E E L G E I L R a t G E G K S E E E L s d l F R m F D K N A D G Y I D ~ E E L k i m L q a t G E

130 140 150 160 S I T D E E I E E L M K D G D K N N D G K I D F D E F L K M M E G V Q h v T D E E I E s L M K D G D K N N D G r I D F D E F L K M b l E G V Q h v T D E E I E s L M K D G D K N N D G r I D F D E F L K M M E G V Q h v T e E d I E d L M K D s D K N N D G r I D F D E F L K M M E G V Q t I T e d d l E E L M K D G D K N N D G r I D ~ D E F L ~ f M k G V e

Fig.4. Alignment of the amino acid sequences of troponin C.fiorn dxferent species determined so .far. The sequences have been segmented so as to make their internal homology more easily perceived. The underlined regions represent the four calcium-binding sites. The amino acid residues are written using the one-letter code. Amino acid residues differing from the corresponding amino acid in frog troponin C sequence are printed in lower-case italics. A, Alanine; C, cysteine; D, asparlic acid; E, glutamic acid; F, phenylalanine; G, glycine; H, histidine; I, isoleitcine; K , lysine; L, leucine; M, methionine; N, asparagine; P, proline; Q, glutamine; R, arginine; S, serine; 7, threonine; V, valinc; Y, tyrosine

peptides. There is no overlapping peptide for the Arg-Ile sequence at position 103-104. In this case the alignment has been deduced from homology of the sequence of frog skeletal troponin C with the sequences of troponin C from rabbit, chicken and human skeletal muscle.

As can be seen in Fig. 10, frog skeletal troponin C consists of a single polypeptide chain of 162 amino acid residues. The calculated molecular weight is 18 299. There is a single cysteine residue at position 101 and a single tyrosine residue at position 112. The N-terminal amino acid of frog troponin C is acetylated. No histidine or tryptophan residues are present.

DISCUSSION

The amino acid sequence of frog skeletal troponin C can be readily aligned with the known amino acid sequences of other troponins C (see Fig.4). Frog troponin C has the same number of amino acid residues as chicken troponin C, which is three amino acid re-

sidues more than rabbit and human skeletal troponin C. These three additional amino acid residues are located at the N-terminal end. There are 20 amino acid substitutions and additions between frog and rabbit troponin C, and 21 between frog and chicken. On the other hand there are 57 amino acid replacements and additions between frog skeletal troponin C and bovine cardiac troponin C. Therefore the differ- ence in amino acid sequence between different muscular tissues is much more pronounced than the dif- ference between species. This had been suggested earlier by van Eerd and Takahashi [6] and has been found for troponin I [33].

There is a possibility that any of the aspartic acid residues at positions 68, 140 and 146 is a deamidated asparagine residue because Asn-Gly sequences are known to be very susceptible to deaniidation, presum- ably via a cyclic imide intermediate [34].

A P-aspartyl-glycyl peptide bond is preferentially formed after ring opening of the cyclic imide.

Such a peptide bond cannot be degraded sequen- tially using the Edman procedure.

238 Frog Troponin C

During the Edman degradation of peptides T2, CB4 and CB10, no sudden decrease in yield was ob- served during the degradation of the aspartic acid residues at positions 68, 140 and 146. Therefore we believe these residues to be true aspartic acid residues.

When comparing the different sequences it is also noticeable that the first two amino acids of rabbit troponin C [3] are Asp-Thr, while the corresponding sequence in frog is Thr-Asp. Furthermore, residues 131 and 133 in chicken [4] are Glu,Asp, while in other skeletal troponins C the sequence is Asp,Glu.

Frog troponin C contains only one tyrosine residue making it an ideal protein for conformation studies using tyrosine fluorescence measurements. It should be noted that in comparison with the preliminary results on the sequence of frog skeletal troponin C [ll], two revisions have been made: residue 88 is glutamic acid, not glutamine; residue 91 is glutamine, not glutamic acid.

Calcium-Binding Sites

Collins et al. [2] assigned four calcium-binding sites to rabbit skeletal troponin C, by homology with the calcium-binding site of parvalbumins. Van Eerd and Takahashi [6] suggested that in bovine cardiac troponin C, the region between residues 30 and 40 has lost its ability to bind calcium because of the insertion of an amino acid between positions 30 and 33, and because of the large number of amino acid replacements in this region.

Compared with rabbit skeletal troponin C, frog skeletal troponin C contains some amino acid replacements in the four calcium-binding regions, but the replacements are conservative so that no drastic changes in those regions can be expected. Therefore, frog skeletal troponin C probably contains four calcium-binding sites like the other skeletal troponins C. The single tyrosine residue of frog skeletal troponin C is located in the third calcium-binding site.

Variable Regions in Troponin C

Most amino acid replacements between the skeletal troponins C are located in two regions (Fig.4). The first region is the N-terminal part of the protein, and the second region is situated between the third and fourth calcium-binding site. If we assume that the troponin C molecule is composed of four homologous domains each consisting of helix - calcium-binding site-helix, we see that the variable region is located in the second helix of the third domain, the loop region between the third and fourth domain, and the first helix of the fourth domain. Particularly note- worthy is position 115. The amino acid at position 11 5 is different for all troponin C’s listed in Fig. 4. It is homologous with the amino acid at position 60

of carp parvalbuniin [2]. When we look at the three- dimensional structure of carp parvalbumin [lo], we see that the amino acid corresponding to position 115 in troponin C is located in a sharp bend. It forms the end of a P-pleated sheet at the beginning of a helix, at a right angle with the pleated sheet structure. Though residue 115 is located in the third calcium- binding site it does not form a ligand for the binding of the calcium ion.

E.xtrapolation of Physiological and Biochemical Data on Muscle

Most physiological experiments on muscle have been performed with frog muscle while most biochemical experiments have been performed on rabbit muscle. It may be asked to what extent the results obtained with frog muscle can be extrapolated to rabbit muscle and vice versa?

There are 17 amino acid replacements and three additions at the N-terminal in frog skeletal troponin C as compared with rabbit skeletal troponin C. However, from what has been mentioned before, there are appa- rently no large differences in structure and calcium- binding properties. Therefore, as far as troponin C is concerned, it seems to be reasonable to extrapolate experimental results from frog muscle to rabbit muscle.

The authors wish to acknowledge gratefully Dr H. Rochat from the Universiti d’Aix-Marseille, for performing a sequencer run on the Socosi sequenator. They thank Dr H. R. Morris from Imperial College, London, for the determination of the amino acid sequence of the blocked N-terminal CNBr peptide by mass spectrometry. They further want to acknowledge Dr R. Walter from the University of Illinois, Chicago, for providing the post-proline cleaving enzyme, Dr J. A. Lenstra from the University of Groningen, The Netherlands, for the NMR analysis of the blocked CNBr peptide and Mrs R. Bertrand and J. Jaureguy-Adell from the Cen- tre National de la Recherche Scientifique in Montpellier for their help with the Technicon peptide analyser. Part of this work was performed during the tenure of a short-term EMBO Fellowship by J.-P. v. E. This study was supported by a grant from the Swiss National Foundation (Grant no 3.2130.73) and by the Dtlegation GenPrale u la Recherche Scientifique et Technique.

REFERENCES 1. Ebashi, S. (1974) Essays Biochem. 10, 1-36. 2. Collins, J. H., Potter, J. D., Horn, M. J., Wilshire, G. & Jack-

3. Collins, J. H., Greaser, M. L., Potter, J. D. & Horn, M. J.

4. Wilkinson, J. M. (1976) FEBS Lett. 70, 254-256. 5. Romero-Herrera, A. E., Castillo, 0. & Lehmann, H. (1976)

6. van Eerd, J. P. & Takahashi, K . (1975) Biochem. Biophys. Res.

7. van Eerd, J. P. & Takahashi, K. (1976) Biochemistry, 15,1171 -

8. Weeds, A. G. & McLachlan, A. D. (1974) Nature (Lnnd.)

9. Pechkre, J. F., Capony, J . P. & Ryden, L. (1971) Eur. J . Bio-

man, N. (1973) FEBSLett. 36, 268-272.

(1977) J . Bid. Chem. 252, 6356-6362.

J . Mol. E d . 8, 251 - 270.

Commun. 64, 122- 121.

1180.

252, 646 - 649.

chem. 23,421 -428.

J.-P. van Eerd, J.-P. Capony, C. Ferraz, and J.-F. Pechkre 239

10. Kretsinger, R. H. & Nockholds, C. E. (1973) J . Bid. Chem.

11. van Eerd, J . P., Capony, J. P. &Pechere, J. F. (1977) in Calcium Binding Proteins and Calcium Function (Wasserman, R. H., Corradino, R. A,, Carafoli, E., Kretsinger, R. H., MacLen- nan, D. H. & Siegel, F. L., eds) pp. 232-238, Elsevier- North Holland Publishing Co., New York.

12. Potter, J. D. & Gergely, J. (1974) Biochemistry, 13,2697-2703. 13. Weber, K. & Osborn, M. (1969) J . Biol. Chem. 244, 4406-

14. Gross, E. & Witkop, B. (1962) J . Biol. Chem. 237, 1856- 1860. 15. Hirs, C. H. W. (1967) Methods Enzymol. 11, 197- 199. 16. Bertrand, R., Pantel, P. & Pechtre, J. F. (1974) Biochimie

17. Smyth, G. D. (1967) MethodsEnzymol. 11, 230-231. 18. Light, A. (1967) Methods Enzymol. I / , 417-420. 19. Koida, M. & Walter, R . (1976) J . Biol. Chem. 251, 7593-

20. Frank, G. & Strubert, W. (1973) Chromatographia, 6,522- 524. 21. Gray, W. R. (1972) Methods Enzymol. 25, 333-344. 22. Rochat, H., Bechis, G., Kopeyan, C., Gregoire, J. & van Riet-

schoten, J. (1976) FEBS Lett. 64, 404-408.

248, 3313-3326.

4412.

(Paris) 56, 5 1 5 - 522.

7599.

23. Edman, P. & Henschen, A. (1975) in Protein Sequence Deter- mination (Needleman, G . B., ed.) 2nd ed., pp. 232-279, Springer-Verlag, Berlin.

24. Pisano, J. J. & Bionzert, J. J., Jr (1969) J . Biol. Chem. 244,

25. Morris, H. R., Williams, D. H. & Ambler, R. P. (1971) Bio-

26. Morris, H. R. (1972) FEBS Left. 22, 257-260. 27. Ambler, R. P. (1972) Methods Enzymol. 25, 143- 154. 28. Zuber, H. (1976) Methods Enzymol. 45, 561 - 568. 29. Cozzone, P. & Marchis-Mouren, G. (1970) FEBS Lett. 9,

30. Offord, R. E. (1977) Methods Enzymol. 47, 51-69. 31. Wuthrich, K. (1976) in N M R in Biological Research: Peptides

and Proteins, chap. 5, North-Holland Publish. Comp., Am- sterdam.

32. Morris, H. R., Williams, 3. H., Midwinter, G. G. & Hartley, B. S. (1974) Biochem. J . 141, 701-713.

33. Wilkinson, J. M. & Grand, R. J. A. (1978) Nature (Lond.)

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145.

J.-P. van Eerd, Biochemisch Laboratorium der Rijksuniversiteit Groningen, Nijenborgh 16, NL-9747 AG Groningen, The Netherlands

J.-P. Capony, Biology Department, Brookhaven National Laboratory, Upton, Long Island, New York, U S A . 11973

C. Ferrai and J.-F. Pechere, Centre de Recherches de Biochimie Macromoleculaire du C.N.R.S., Boite postalc 5051, F-34033 Montpellier-Cedex. France

Supplement to

the amino acid sequence of troponin C f r o m frog skeletal muscle

by Jean-Paul van Eerd, J e a n - P a u l Capony, Conception Ferraz and Jean-Franrjois Pechere

POOL I 1

Fraction mber Fraction number

Figure 5

skeletal troponin C on Sephadex G-50 fine ( 2 columns, 0 . 9 x 2 0 0 cm). ElutlOn buffer 10 mM NH HCO . fractron volume

Fractionation of the cyanogen bromide peptides of frog

Figure 6 Purification of the cyanagen bromide peptides from peaks

I. I1 and I11 of Figure 5 on a column (0.9 x 23 cm) of DEAE- Sephadex A-25 equilibrated with 20 mM T r l s - H C 1 pH 8.5. The PeDtideS were eluted With a KC1 oradient ( 0 - 1.0 M . 75 + 75

1.1 ml, flow Speed 4.4 ml/h. 4 3 . hi in Starting buffer. One fraction contained 1.6 kl. The The fractions were pooled as indicated by the horizontal fractions were pooled as indicated by the horizontal bars. bars. The peptides in the different pools are indicated in The peptides isolated from the different pools are indicated the figure. in the Figure.

240 Frog Troponin C

10 CHROHOBEAOS - P

I 50 100 150

Fraction nmber

Figure 7 Upper half. Purification of the cyanogen bromlde

peptides from pool IV Of Figure 5 by chromatography on a column ( 0 . 9 x 25 c m ) of anion-exchange resin Durrm DA-XZ. Column temperature 45‘C.Elution via a varigrad constan- volume mixing device of 80 ml. Elution sequence: flrSt 6 0 m l of pH 9.2 buffer (1% pyridine, 0.2% dimethylallylamme and 0.004% acetic acid in water), followed by 180 ml of 2.0 M acetic acid. Identification of the peaks was by colour development of a part of the eluate with ninhydrin before alkaline hydrolysis (dashed line) and after alkaline hydrolysis (solid line). The fractions were pooled as indicated by the horizontal bars. The peptides isolated from the different pools are indicated in the Figure.

Lower half. Purification of the cyanogen bromldc peptides from pool V of Figure 5 by chromatography on a column 10.9 x 25 c m ) of cation-exchaye resin, Technicon Chromobeads P. Column temperature 45’C. The peptided were eluted with pyridine-acetic acid buffer with a pH gradlent from pH 3 . 1 to pH 5.0. Identification of the peaks by ninhydrin and pooling of the fractions 8 s indicated r n the legend of the upper half Of this Figure. CB7 and CBll ConsISted of free homoserine.

75 160 125 150

Frochon number

Figure 8 Fractionation of the tryptic peptides of performic acld-

oxidized frog skeletal troponin C on Sephadex G-50 fine (2 columns, 0.9 x 200 c m ) . Elution buffer 0.5% NHa,HCO), fraction volume 1 . 8 3 ml. The fractions were pooles as indicated by the horizontal bars. The peptides isolated from the different pools are indicated in the Figure.

(0

Figure 9

NHhHC03 and eluted with a gradlent of NHrHCOl as indicated in the Figure with the dashed line. Fraction volume 1.5 ml. The fractions were pooled as indicated by the horizontal bars. The peptides isolated from the different pools are indrcated in the Figure.

Fractionation of the peptides of pool TI1 from Figure 8 on DEAE-cellulose DE 32 (0.9 X 15 cm), equilibrated with 5 mM

I

Froction number

Flgure 10

Figure 8 on an anion-exchange r e s m Durrum DA-X2. Condition a s in Figure 7 . upper half. Identification of the peaks by colour develooment of Dart of the eluate with ninhvdrin before alkaline

Purification of the tryptic peptides of pool IV from

hydrolysis (dashed line) and after alkallne’hydrolysls Is01 id line). The fractions were pooled as indicated by the horizontal bars. The peptides isolated from the different pools are indicated ~n the Flgure.

Fraction number

Figure 11 Fractionatron of the tryptzc peptides of performic acid-oxidized and phthalylated frog skeletal troponin C on Sephadex G-75 (2 columns, 1.0 X 200 cm). Elution buffer 0.5% NHIHC03, fractron volume 1.5 ml. The fractions were pooled as indicated by horizontal bars. The peptides isolated from the different pools are indicated in the Figure.

J.-P. van Eerd, J.-P. Capony, C. Ferrdz, and J.-F. Pechkre 24 1

Table I. Amino acid compositlon of frog skeletal troponln C. Residues were determined after 24-h and 72-h hydrolysis and are reported as mol/mol protein.

Amino acid Residues/ Nearest Found in molecule integer sequence

Aspartic acid 23.9

SerlIle 8.9a Threonine 7.9a

Glutamic acid 35.1 Proline 1.9 Glyclne 14.0 Alanine 8.6 Cysteine 0.9b Valine 5.0' Methlonine 10.3 Isoleucine 10.iC Leucine 10.2 Tyrosrne 1.1 Phenylalanine 9.9 Lvslne 10.4

10.3 0 . 0

N H 3 Histidine Arginine 4.9 Tryptophan Od

24 8 9

35 2 14 9 1 5 10 11 10

1 10 10 10 0 5 0

23 8 9

3 3 2 14 9 1 5

1 1 11 10 1

10 10 10

0 5 0 -

Total residues 162

a - The values f o r threonlne and Serine have been corrected assuming 5% and 10% destruction, respectively, after 24-h hydrolysis.

b - Determined as cysteic acid. c - Value after 72-h hydrolysis. d - From ultra-violet spectrum.

Table 2 . The amino acid composition of the cyanogen bromide pept ides of frog skeletal trapanin C. Values in brackets Lndicatc the number of residues of each amino acid found in the sequence Of the peptrdes. The amino acid composition o f CB9 IS not reported because it was not isolated with sufficient purity. The values for threonine and serine have been corrected 5% and 1 0 % for destruction, respectively, after 24-h hydrolysis. Impurities i 0.3 mol/mol peptide have been ignored.

RminO CB1 CB2 CE3 CPd CE5 acid nOl/rnl rnl/rnl rnl/rnl rml/rnl rnl/rnl k w t i c acid 1.0 I11 1.0 (11 1.1 (11 3.0 I31 Thrmnine 0.9 (1) 3.0 (3) *rule 1.6 12) 1.0 11)

Glutmnic acid 2.9 (3) 2.0 (2) 1.3 (11 1.7 (11 noline 0.6 (1) Glycine 4.0 14) Rlanine 0.3 (1) 0.9 (1) 2.9 (31 cysterne V a l U e 0.8 (1)

Kethroninea 0.6 11) 0.6 I11 0.7 (1) 0.8 11) 0.8 (11 ISOleuclne 1.0 (11 1.0 I11 Ieuc- 1.4 I11 1.1 ( I )

Tyrosine

Lysine 1.0 (11 0.1 I11 Arq1"vle- 0.3 (I) 1.0 11)

Phenylalanine 0.7 (11 2.0 (21 0.9 11)

TDbtal 18) (10) 1101 (181 (21 Yield 38% 23% 14% 39% 30%

c86

4.1 I41 2.7 (31 1.2 (1)

8.2 (81 0.6 11) 3.1 (3)

rrOl/nOl

1.1 (1)

1.8 (2) 0.6 (1)

2.2 (3) 2.8 ( 3 )

1.5 (2) 0.8 (1)

(331 47%

a - h -

Determined a s the sum of homoserine and The total yield of free homoserine (CB7

homoserine-lactone. and CB111 is 9%.

Table 3. The a m n o acid sequence of the CNBr peptides of troponin C from frog skeletal muscle. The sign 7 indicates a degradation step by the manual Edman procedure and identification of the resulting phenylthiohydantoln amino acid by high performance liquid chromatography. CB9 w a s not isolated in sufficrently pure state. The amide r n CB8 was determined by the mechod of Offord ( 3 0 ) .

Table 4. The amino acid composition of the tryptic peptides of performic acid-oxidized troponin C from frog skeletal muscle. Values In brackets indicate the number of residues of each amino acid found in the sequence of the peptldes. The tryptic peptides T6, T9, T12 and T13 have not been isolated. Impurities i 0.3 mol/mol peptide have been ignored.

h " 0 TI T2 T3 T4 T5 T5d T6,7 acid nOl/rnl nOl/rnl rnI/rnl rnl/rml rnl/rnl rn l / r rOl rnl/nOl &=tic acid 2.0 12) 4.1 (4) 4.0 (4) 3.7 (41 1.5 (1) Threonlne 1.1 (1) 1.9 12) 1.0 (1) 3.1 (3) 2.8 13) *me 1.1 (2) 1.0 I11 1.3 I11 0.9 11) Glutmc acid 3.1 131 3.1 (3) 1.0 (1) 8.7 18) 8.2 (8) 2.8 (3) Prollne 1.0 11) 1.0 (11 0.9 I l l Glycine 3.1 (3) 1.1 (1) 2.9 (3) 2.9 ( 3 ) 1.1 (1)

Alanme 2.0 ( 2 ) 1.0 (11 2.0 12) 1.1 (11 1.0 11) 0.9 (11 cystelnea Viillne 0.9 (1) 2.7 13) 1.0 (1)

b&t.tnonrneb 1.0 (1) 0.9 (11 0.9 (I) 0.9 Ill 2.2 13) 1.2 I11 1.0 11) 1SO1eucrne 1.0 (1) 0.9 (1) 0.3 11) 2 .0 (3) 3.0 (31 Leuclne 1.1 (1) 2.6 13) 2.2 (21

T p s L n e C

2.1 (21 2.1 ( 2 ) 1.8 (21 2.0 (2) 1.1 (1) 0.9 ( 1 ) 1.0 11) 0.8 (1) 1.7 (2)

Phenylalruime Lyslne Arglnine 1.0 (1) 1.0 (1) 0.9 (1)

%tal (111 I121 I171 17) (371 I311 (91 Armdesd 2-3 0 0 0 n.d. n.d. 2 Yield 61% 40% 69% 19% 548 61% 18%

Iynino T7 T8 T10 TII* T14 T15 acid nOl/rnl rnl/nOl rnl/nul nOl/mSl nOl/rnl rnl/nOl Aspartic acld 1.5 (1) 3.0 13) 1.2 (1) 1 . 8 (2) Thrmnvre 0.9 (1) Ser1ne 0.9 (I) 0.9 (1) 2.1 (3) Glut-c acid 1.9 (2) 4.1 (4) 3.2 13) 5.0 (5) 1.2 I11 2.0 (2) Pmllne Glycrne 1.0 (1) 1.9 (2) 1.2 (I) 1.1 (1)

Nan- 0.8 (1) 1.1 (1) 1.0 Ill cysteinea 0.8 11) Vallne 1.0 11)

Methionineb 1.8 12)

1ao1eucine 2.0 12) 2.2 (2) 1.0 (1) Lemme 1.0 (1) 1.7 (21 1.2 (11 1.0 (11

Phenylalanine 1.0 I11 2.1 I21

Arginlne 1.0 (1) 1.1 (11

Tyrosin2 0 (1)

Lysine 0.8 (1) 0.8 0.9 (1)

Total (6) (101 (16) (151 (8) I 6 1

Mmdesd 1 0 1 0-1 0 1

Yield 8% 41% 30% 47% 32% 41%

a - Determined as cysteic acid b - Determined as methionine sulfone c - Destroyed during performic acid-oxidation d - Determined by the method of Offord (301 % - Isolated from another digest with trypsin containing

chymotryptic activLty

242 J.-P. van Eerd, J.-P. Capony, C. Ferraz, and J.-F. Pechere: Frog Troponin C

Table 5. The imino acid sequence of the tryptic peptides Of performic acid-oxidized troponin C from frog skeletal muscle . The Sign 2 indicates a degradation step by the dansyl-Edman procedure. The sign -indicates a degradation step by the automatic seauencer.5; . a n d 5 mean determined bv

Table 7 . The amino acid sequence of the tryptic peptides of performic acid-oxidized and phthalylated traponin C from frog skeletal muscle. The slgn 7 indicates a degradation Step by the manual Edman procedure. The Sign indicates determined by dansylation.

carboxypeptidase A , resp. B digestion. Peptldei-Tb, T9, T12 and T13 have not been isolated. PhTl

T1

T2

T3

T4

T5

T5cx

T6.7

T7

T8

T10=

2

Tll=

TI4

TI5

(no armdes)

(1 mdel

X - Other digestion With trypsin containing chymotryptic

** - Edman degradation u s i n g methylisothiacyanate instead activity

of phenylisothiocyanate.

Table 6. The amino acid composition of the tryptic peptides of performic acid-oxidized and phthalylated tioponin C from frog skeletal muscle. Values in brackets indicate the number of residues of each amino acid found in the sequence of the peptides. Impurities 1 0 . 3 mol/mol peptide have been ignored.

Rmum PhTl PhT2 PhT3 PhT4 PhT5 PhT6 PhT5.8 acid rnl/rnl rnl/rnl rnl/rnl rnl/rnl nol/rnl nOl/rnl rnl/nDl

Avartic acid 1.9 (21 4.1 (4) 4.2 (4 ) 1.2 (1) 3.5 (41 7.5 (8) 9.4 (12)

Tnreonine 1.1 (1) 2.7 ( 3 ) 2.7 13) 1.1 (1) 1.2 I 1) Ser1ne 2.6 (31 1.1 I11 0.9 (11 0.9 ( 1 ) 2.6 (3) 3.0 ( 4) Glutamic acid 3.2 13) 4.7 (4) 8.2 18) 6.2 ( 7 ) 3.3 13) 8.4 (81 11.2 111)

Pml- 0.8 11) 0.9 (1)

Rlanlne 1.9 12) 2.9 13) 1.2 ( I ) 1.8 (2) 1.1 (1) 1.1 ( 11 Glycine 3.8 (4) 3.1 13) 1 . 2 (1) 2.0 (21 4.1 (4) 5.4 ( 61

1.0 (11 0.9 (1) 2 . 3 13) 1.1 (11 1 . 1

1.2 I11 2.9 13) 2.8 13) 0.9 11) 2.8 13) 3.0 2.0 (2) 2.3 ( 3 ) 2.9 (3) 2.9 (3) 4.9 2.1 12) 2.9 131 1.0 11) 2.0 ( 2 ) 2.0 (2) 3.2

0 (1) 0 4.0 I41 2.2 12) 1.0 11) 1.1 (1) 1.9 (2) 3.1 2.3 I21 1.5 (1) 2.0 (2) 1.1 11) 4.6 (41 4.4

1.0 I11 1.0 11) 0.9 I11 1.0 11) 1.1 (1) 1.0

1 )

3) 6)

4 )

11 3 ) 5) 1)

lbta1 (11) (36) (37) (19) (201 (39) (59) Yield 72% 70% 84% 67% 68% 858 18%

a - Determined a5 CySteiC acid b - Determined as methionine s u l f o n e c - Destroyed during performic acid-oxidation X - Isolated f rom non-oxidized phthalylated troponin C

and digested With trypsin for a shorter period of time (30 min Instead of 110 min)

PhTZ

PhT3

PhT4

PhT5

PhT6

PhT5,6'

* Isolated from "on-oxidirdd phthalylated troponin C digested with trypsin for a Shorter period of time 130 min instead of 110 min).

the amino-acid sequence of troponin c from frog skeletal muscle

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