Structural studies on the coat protein of alfalfa mosaic virus. Isolation and characterization of the tryptic peptides and the alignment of the cyanogen-bromide fragments.

The reduced and carboxymethylated coat protein of alfalfa mosaic virus (AMV 425) was fragmented by means of cyanogen-bromide cleavage. The tryptic peptides from the protein and its four cyanogen-bromide fragments were isolated on a preparative scale by combinations of column and paper separation techniques. The tryptic digest of the carboxymethylated protein contained 24 peptides and two free amino acids. All peptides have been characterized by amino acid analyses and end-group determinations. Together the tryptic peptides account for a total chain length of 228 amino acids. The data are in good agreement with previous reports from this laboratory.     

Isolation of Protein Inhibitors of Papain,Trypsin, and α-Amylase in the Grain of Foxtail Millet

The amino acid sequence of Indian peafowl egg-white lysozyme has been identified. The reduced and carboxymethylated lysozyme was digested with trypsin followed by purification of the resulting peptides by reverse-phase HPLC. The tryptic peptides obtained were sequenced using the DABITC/PITC double coupling manual sequencing method. The alignment of the tryptic peptides were deduced by comparison with corresponding peptides of hen egg-white lysozyme. This protein proved to consist of 129 amino acid residues, and a relative molecular mass of 14423 Da was calculated. Amino acid sequence comparison of peafowl lysozyme and other phasianoid bird lysozymes revealed a maximum homology ratio of 98% with turkey lysozyme.     

The primary structure of L-asparaginase from Escherichia coli

The carboxymethylated L-asparaginase from Escherichia coli A-1--3 was fragmented with cyanogen bromide and the resulting peptides were isolated by using gel filtration on Sephadex G-50 and column chromatography on DE-52. The amino acid sequences of the 7 cyanogen bromide peptides thus obtained were established completely or partially by further fragmentation with trypsin, chymotrypsin and pepsin, and the Dansyl Edman method. Based on the above results and the complete sequences of the tryptic peptides from the carboxymethylated L-asparaginase reported in the previous paper, the whole sequence of the enzyme was established. The reported sequence consists of 321 amino acid residues and its calculated molecular weight is 34 080.     

The primary structure of aspartate aminotransferase from pig heart muscle. Partial sequences determined by digestion with thermolysin and elastase

Peptides produced by thermolytic digestion of aminoethylated aspartate aminotransferase and of the oxidized enzyme were isolated and their amino acid sequences determined. Digestion by elastase of the carboxymethylated enzyme gave peptides representing approximately 40% of the primary structure. Fragments from these digests overlapped with previously reported sequences of peptides obtained by peptic and tryptic digestion (Doonan et al., 1972), giving ten composite peptides containing 395 amino acid residues. The amino acid composition of these composite peptides agrees well with that of the intact enzyme. Confirmatory results for some of the present data have been deposited as Supplementary Publication 50018 at the National Lending Library for Science and Technology, Boston Spa, Yorks. LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1973) 131, 5.     

The amino acid sequences of the tryptic, chymotryptic and peptic peptides from the L-2 light chain of rabbit skeletal muscle myosin.

The amino acid sequences of the tryptic peptides from the aminoethylated L-2 light chain of rabbit skeletal muscle myosin were determined by various enzymatic hydrolyses, partial hydrolysis with dilute acetic acid and Edman degradation. The amino acid sequences of the chymotryptic and peptic peptides from the carboxymethylated L-2 light chain were partially analysed in the same manner as the tryptic peptides. The primary structure of the L-2 light chain of rabbit skeletal muscle myosin was deduced from the above results.     

The primary structure of the alpha subunit of protocatechuate 3,4-dioxygenase. I. Isolation and sequence of the tryptic peptides.

The carboxymethylated alpha subunit of protocatechuate 3,4-dioxygenase was digested with trypsin. The 14 tryptic peptides were isolated by ion exchange chromatography on DEAE-Sephadex and by gel filtration chromatography. Automated Edman degradation and carboxypeptidase Y and B digestion were used to establish the sequence of these peptides. Further fragmentation of two tryptic peptides, T3 and T5, by Staphylococcus aureus protease and cyanogen bromide, respectively, was necessary to complete the sequences. The tryptic peptides accounted for a minimum of 199 residues out of a total of 202 residues predicted by amino acid analysis.     

The tryptic peptides of rabbit muscle triose phosphate isomerase

1. The peptides obtained by tryptic digestion of S-[(14)C]carboxymethylated rabbit muscle triose phosphate isomerase have been studied. 2. The first step in the fractionation of the tryptic digest was gel filtration on coupled columns of Sephadex G-25 and G-50. Further fractionation was carried out by paper electrophoresis and paper chromatography. 3. The digest contained 26 peptides and three free amino acids. The sizes of the peptides ranged from two to 29 residues. 4. The sequences of the peptides have been determined. 5. The length of the polypeptide chains is about 250 amino acid residues. 6. The variant sequences encountered were due to partial deamidation; this may be one of the reasons for multiple forms of the enzyme. 7. The chicken and rabbit enzymes are compared. 8. Detailed evidence for the sequences of the tryptic peptides has been deposited as Supplementary Publication SUP 50024 at the British Library, Lending Division (formerly the National Lending Library for Science and Technology), Boston Spa, Yorks. LS23 7BQ, U.K., from whom copies can be obtained on the terms given in Biochem. J. (1973) 131, 5.     

Amino acid sequence of an active fragment of potato proteinase inhibitor IIa.

The complete amino acid sequence of an active fragment of potato proteinase inhibitor IIa has been established by the Edman degradation procedure and the carboxypeptidase technique. Sequence analyses were carried out on the reduced and carboxymethylated active fragment and its tryptic peptides. To aid in the alignment of some tryptic peptides, the partial sequences of two fragments obtained by selective tryptic cleavage of the reactive site peptide bond of inhibitor IIa at acidic pH, with subsequent reduction and carboxymethylation, were also analyzed. The active fragment consisted of 45 amino acid residues including 6 half-cystine residues. Degradation of the intact active fragment by subtilisin [EC 3.4.21.14.] at pH 6.5. yielded 3 cystine-containing peptides. Sequence analyses of these peptides revealed that the 3 disulfide linkages were located between Cys(10) and Cys(24), Cys(14) and Cys(35), and Cys(20) and Cys(43). The reactive site peptide bond of inhibitor IIa, a Lys-Ser bond, was located between positions 32 and 33 of the active fragment. The overall sequence of the active fragment was quite different from those of potato chymotrypsin inhibitor I (subunit A) and potato carboxypeptidase inhibitor.     

Primary structure of a human IgA1 immunoglobulin. III. Isolation, composition, and amino acid sequence of the thermolysin peptides.

As part of the strategy for determination of the complete covalent structure of a human IgA immunoglobulin, 66 peptides were isolated from a thermolysin digest of reduced and carboxymethylated IgA alpha1 chain Bur and were purified. They range in length from 2 to 24 residues. Some of the peptides have been characterized and sequenced in order to supply needed information that was not obtained from the chymotryptic and tryptic peptides. These thermolysin peptides provide much necessary data to produce a rigorous proof for the primary structure of the human alpha1 chain. The remaining peptides from the thermolysin digest whose amino acid composition and NH2-terminal residues were sufficient to identify them unequivocally have also been assigned in the structure. They supply additional information that helps remove ambiguity in the structure, and they provide useful data about the profile of the peptide bonds that are susceptible to thermolysin digestion.     

Evidence for the amino acid sequence of porcine pancreatic elastase

The preparation and purification of tryptic peptides from aminoethylated Dip-elastase and [(14)C]carboxymethylated Dip-elastase, and of peptic peptides from native elastase is described. A summary of the results of chemical studies used to elucidate the amino acid sequence of these peptides is presented. Full details are given in a supplementary paper that has been deposited as Supplementary Publication SUP 50016 at the National Lending Library for Science and Technology, Boston Spa, Yorks. LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1973), 131, 1-20. These results, together with those from previously published papers, are used to establish the complete amino acid sequence of elastase, which is a single polypeptide chain of 240 residues, molecular weight 25900, containing four disulphide bridges.     

The amino acid sequence around four cysteine residues in trout actin

Four unique carboxymethylcysteine-containing peptides were isolated from tryptic and chymotryptic digests of trout muscle actin carboxymethylated with iodo[2-(14)C]acetic acid in 6m-guanidinium chloride. The amino acid sequences of these peptides were determined and showed a high degree of homology with the corresponding sequences from rabbit actin. One of the radioactive peptides was the C-terminal peptide and another sequence probably contained the cysteine residue from the N-terminal region of the protein.     

Primary structure of human triosephosphate isomerase

Human placental triosephosphate isomerase was isolated by an improved procedure and recovered with the highest specific activity ever reported. Employing this purification procedure, sufficient amounts of the enzyme were obtained for detailed primary structural studies. For sequences analysis, the enzyme was reduced and carboxymethylated and subjected to tryptic and chymotryptic digestions. The peptide mixtures were separated by high-performance liquid chromatography using octyl or alkylphenyl reverse-phase columns and trifluoroacetic acid/acetonitrile gradient elution systems. Sequence analyses of the intact enzyme, tryptic, chymotryptic, and cyanogen bromide peptides were accomplished using high-sensitivity solid-phase sequencing procedures with either 4-N,N-dimethylaminoazobenzene-4'-isothiocyanate or phenylisothiocyanate. The primary structure of human triosephosphate isomerase is constructed from the alignment of the tryptic peptides with the analysis of the overlapping chymotryptic peptides. The enzyme is a dimeric molecule consisting of two identical polypeptide chains with 248 amino acid residues and a calculated subunit molecular mass of 26,750 daltons. A comparison of the amino acid sequences from the human placental enzyme and from other species such as rabbit, chicken, and coelacanth muscles showed relatively high sequence homology, indicating that the evolution of the enzyme is very conservative. The amino acids of the active-site pocket and the subunit-subunit contact sites exhibit few changes.     

Site of alkylation of the major ribonuclease from Aspergillus saitoi with iodoacetate

A base non-specific and adenylic acid preferential ribonuclease from Aspergillus saitoi (RNase M) was modified by [14C]iodoacetic acid. RNase M was inactivated with concomitant incorporation of about 1 mol equivalent of carboxymethyl group. Carboxymethylated RNase M (CM RNase M) thus obtained was reduced and carboxymethylated (RCM CM RNase M). From tryptic and chymotryptic digests of RCM CM RNase M, two carboxymethylated histidine-containing peptides labeled with radioactivity were isolated. The amino acid sequences of these two peptides were determined to be Thr-Ile-His-Gly-Leu-Trp-Pro-Asp-Asn-Cys-Asp-Gly-Ser-Tyr... and His-Gly-Thr-Cys-Ile-Asn-Thr-Ile-Asp-Pro-Ser-Cys-Tyr-Pro-Asp-Asp-Tyr-Ala. .... The distribution of the radioactivity on the former and latter peptides was 43% and 57%, respectively. The results indicated that two histidine residues are involved in the active site of RNase M, and the modification of either one of the two histidine residues inactivates RNase M. The CD spectrum of carboxymethylated RNase M indicated that some tryptophan residue(s) with a CD band at 287 nm is in the proximity of the active site histidine residues of RNase M.     

The subunit structure of rabbit skeletal-muscle phosphofructokinase and the amino acid sequence of the tryptic peptide containing the highly reactive thiol group.

1. The single highly reactive (class I) thiol group per 80000-mol.wt. subunit of skeletal-muscle phosphofructokinase was specifically carboxymethylated with iodo[2-14C]acetate, and after denaturation the remaining thiol groups were carboxymethylated with bromo[2-3H]acetate. After tryptic digestion and peptide 'mapping' it was found that the 14C radioactivity was in a spot that did not contain significant amounts of 3H radioactivity, so it is concluded that there is not a second, 'buried' cysteine residue within a sequence identical with that of the class-I cysteine peptide. 2. The total number of tryptic peptides as well as the number of those containing cysteine, histidine or tryptophan were inconsistent with the smallest polypeptide chain of phosphofructokinase (mol.wt. about 80000) being composed of two identical amino acid sequences. 3. The amino acid sequence of the tryptic peptide containing the class-I thiol group was shown to be Cys-Lys-Asp-Phe-Arg. This sequence is compared with part of the sequence containing the highly reactive thiol group of phosphorylase.     

Primary structure of a human IgA1 immunoglobulin. II. Isolation, composition, and amino acid sequence of the tryptic peptides of the whole alpha1 chain and its cyanogen bromide fragments.

As part of the strategy for determining the covalent structure of a human IgA1 molecule (Bur), a tryptic digest was prepared of the reduced and carboxymethylated alpha1 heavy chain. In addition to the main experiment, tryptic peptides were prepared from the succinylated aminoethylated alpha1 chain and from fragments obtained by CNBr scission of the alpha1 chain. Complete recovery of the peptides was impeded by the large size of some of the tryptic peptides and of the principal CNBr fragment, and difficulty in separating other glycopeptides. Twenty-eight tryptic peptides of the reduced and carboxymethylated alpha1 chain were purified and sequenced, accounting for more than 300 residues. Additional information was obtained by sequence analysis of trypudies described in this series of papers contributed to the complete sequence analysis of the alpha1 chain.     

The structure and amount of tubulin in cells and tissues.

The NH2-terminals and amino acid compositions of three carboxymethylcysteine-containing peptides produced from carboxymethylated calf brain tubulin by digestion with elastase have been determined. The 3H/14C ratio obtained when these peptides were generated from mixtures of [14C]carboxymethylated calf brain tubulin and the [3H]carboxymethylated proteins of mouse brain or 3T3 cells and subsequently purified indicated that both mouse brain and 3T3 cells contained protein giving rise to peptides homologous to these peptides. This indicates that the tubulins of calf brain, mouse brain, and 3T3 cells are homologous in the regions of amino acid sequence accounting for the peptides examined. From the 3H/14C ratio of the isolated peptides, 13.5% of the protein of mouse brain and 3.5% of the protein of 3T3 cells were estimated to be tubulin.     

Triose phosphate isomerase from the coelacanth. An approach to the rapid determination of an amino acid sequence with small amounts of material

The preparation and purification of cyanogen bromide fragments from [¹⁴C]carboxymethylated coelacanth triose phosphate isomerase is presented. The automated sequencing of these fragments, the lysine-blocked tryptic peptides derived from them, and also of the intact protein, is described. Combination with results from manual sequence analysis has given the 247-residue amino acid sequence of coelacanth triose phosphate isomerase in 4 months, by using 100mg of enzyme. (Two small adjacent peptides were placed by homology with the rabbit enzyme.) Comparison of this sequence with that of the rabbit muscle enzyme shows that 207 (84%) of the residues are identical. This slow rate of evolutionary change (corresponding to two amino acid substitutions per 100 residues per 100 million years) is similar to that found for glyceraldehyde 3-phosphate dehydrogenase. The reliability of sequence information obtained by automated methods is discussed.     

Primary structure of otter (Lutra lutra L.) myoglobin. II. Pepsin peptides of trypsin hydrolysate. Reconstruction of the polypeptide chain of the otter myoglobin globin component

13 peptic peptides have been isolated from the insoluble (at pH 5.0) fraction of the tryptic hydrolysate of main chromatographic component of otter myoglobin and their amino acid composition and N-terminal amino acid sequences have been determined. The isolated peptides contain in total 40 amino acid residues. The results obtained, along with those on tryptic peptides and the comparison with homologous portions of myoglobins of the known primary structure, allowed reconstructing the complete amino acid sequence of otter myoglobin.     

Structure primaire de l'anhydrase carbonique érythrocytaire B humaine: II. Clivage par le bromure de cyanogène et séquence des résidus 1–148

The amino acid sequence of the IICNBr fragment of the human erythrocyte carbonic anhydrase B has been determined. This fragment contains the first 148 of the 260 residues of the N-acetylated single polypeptide chain of the protein. After tryptic hydrolysis of this fragment, eleven peptides have been isolated by gel filtration and chromatography on Dowex 50 W-X₂ or DEAE-Sephadex. Eight of them were identified with already sequenced peptides previously isolated from tryptic hydrolysate of the whole protein. The other three ones were obtained in pure form and sequenced. The combined amino acid content of these eleven peptides only account for 124 of the 148 amino acid residues in the IICNBr fragment. The tryptic attack of the maleylated IICNBr fragment gave three peptides as was expected from the number of arginine residues (2) in this fragment: two arginyl peptides (II₁, II₃) and one homoseryl peptide (II₂). They were purified by gel filtration. The unidentified 24 residue tryptic peptide has been isolated from the demaleylated II₂ tryptic hydrolysate and sequenced. The order of the twelve tryptic peptides of IICNBr fragment has been obtained by study of chymotrypsic peptides isolated from II₁ and IICNBr fragment.     

The amino acid sequence of thiogalactoside transacetylase of Escherichia coli

The amino acid sequence of thiogalactoside transacetylase, a dimer, has been determined. The monomer contains 202 amino acid residues in a single polypeptide chain and has a molecular weight of 22,671. The analysis was carried out by treatment of the carboxymethylated protein with cyanogen bromide and with trypsin. All seven cyanogen bromide peptides were isolated in pure form and were ordered by peptides isolated from tryptic digests. The sequence analysis was aided by determination of the DNA sequence of the lacA gene. The amino terminus of the protein is heterogenous because the initiator methionine is only partially cleaved. Another rather unusual feature of this cytoplasmic protein is a very hydrophobic segment in the center portion of the chain. Comparison of the amino acid sequence of thiogalactoside transacetylase to those of the lac repressor, beta-galactosidase, and lactose permease did not reveal any marked similarities. Therefore, there is no obvious evolutionary relatedness among proteins of the Lactose Operon.