Primary Structure of Rabbit Skeletal Muscle Troponin-T SEQUENCE DETERMINATION OF FOUR

The amino acid sequences of four cyanogen bromide (CB) fragments from rabbit skeletal troponin-T have been determined. Fragment CB4 was documented by chymotryptic, thermolytic, tryptic, and 2-(2-nitrophenylsul-fenyl)-3-methyl-3’-bromoindolenine peptides. A combina-tion of automated sequence analysis, tryptic digestion, and penicillocarboxypeptidase-S treatment was used to assemble fragments residues), and the COOH-terminal residues).

From the Medical Research Council of Canada Group on Protein Structure and Function, Department of Biochemistry, University of Alberta, Edmonton, Canada T6G 2H7 The amino acid sequences of four cyanogen bromide (CB) fragments from rabbit skeletal troponin-T have been determined. Fragment CB4 (55 residues) was documented by chymotryptic, thermolytic, tryptic, and 2-(2-nitrophenylsulfenyl)-3-methyl-3'-bromoindolenine peptides. A combination of automated sequence analysis, tryptic digestion, and penicillocarboxypeptidase-S treatment was used to assemble fragments CB5 (24 residues), CB7 (8 residues), and the COOH-terminal CB6 (21 residues).   peptides, each of which was sequenced. The two thermolytic peptides Th8 and Th14 overlapped the regions on either side of tryptophan 205 and tyrosine 227, respectively. BNPS-skatole cleavage at the single tryptophan produced 25-residue peptide B2. This fragment, taken through 14 cycles of the automated Edman procedure, ordered the chymotryptic peptides as being TC5-TCGi-TC6ii and the thermolytic peptides as Th8-Th9-TMii-ThlOi-Thll.
The majority of the acids and amides was determined directly from the mobilities at pH 6.5 of the chymotryptic peptides (Table II). Glutamine 210 was known from peptide Th9 (Table III). Asparagine 188 and aspartic acid 190 were assigned from GLC results (Table I).
CB4, consisting of 55 amino acids, is a basic peptide with a net charge of +4 at neutral pH. Except for 2 clusters of 3 basic residues at the NH, terminus, the acidic and basic moieties are scattered throughout the entire length of the fragment.
Amino Acid Sequence of CB7 -When 90 nmol of the smallest cyanogen bromide fragment was applied to the automated sequence analyzer (Table VII) using Beckman program 090872, 7 out of a total of 8 residues were ordered, leaving only the homoserine at the COOH terminus.
Residue 234 was shown to be alanine from GLC results and the tryptic peptides 'T2 and 'T4 (Table VIII). Two homoserine peptides, 'T3 and 'T4, verified the sequence in the COOH-terminal half of CB7. Asparagine 231 and glutamic acid 237 were determined directly from mobilities at pH 6.5 of peptides 'Tl, 'T3, and 'T4. The complete sequence of CB7 is presented in Fig. 3.
Amino Acid Sequence of CB6 -Since CB6 is the only cyano-gen bromide fragment lacking homoserine, it must occupy the COOH-terminal portion of troponin-T. In two separate runs, samples (250 nmol) of CB6 were taken through 20 cycles on the automated sequence analyzer using Beckman program 090872. The results of one such run are shown in Table VII. The sole remaining amino acid was lysine, indicating that it came after tryptophan 258 and was the last residue of CB6 and therefore of troponin-T.
Penicillocarboxypeptidase-S results (Table IX) confirmed the sequence at the COOH terminus as being Arg-Trp-Lys. This fragment is devoid of glutamyl and aspartyl residues and has an overall charge of +7 at pH 6.5. The complete sequence of CB6 is shown in Fig. 3.
Amino Acid Sequence of CB5-Fragment CB5 (185 nmol) was subjected to automated sequence analysis twice using Beckman program 090872. As shown in Table VII, the automated sequenator went through the entire length of CB5, leaving only the COOH-terminal homoserine residue. Alanine at positions 153, 160,162, and 172 as well as serine at positions 156 and 157 were determined by GLC of the PTH-derivatives.
A tryptic digest of CB5 provided peptides (Table VIII) suitable for assigning the acids and amides. Thus asparagine 154, glutamine 170, and glutamic acid 174 were known directly from the mobilities at pH 6.5 of peptides 'Tl, 'T5, 'T7, and 'T9. Since residue 163 was found to be aspartic acid from the GLC results of Table VII, the single amide moiety of peptide 'T2 was attributed to glutamine 164.
The COOH terminus of CB5 was found to be Glu-Hse by peptide 'T9 (Table VIII). Further confirmation was obtained by penicillocarboxypeptidaseS treatment of the intact cyanogen bromide fragment (Table Xl. Although a mixture of fragments CB6 and CB5 was used, the amino acids derived from CB5 could be distinguished from those of CB6 for the following reasons: (a) the sequence of the COOH terminus of CB6 was already known (Table IX); (b) the enzyme cleaved CB6 at a much slower rate than CB5 so that after 3 min tryptophan and lysine were released from CB6 while alanine, arginine, glutamic acid, and homoserine were removed from CB5. The evidence for the complete sequence of CB5 is summarized in Fig. 3.