Structure of N-terminal cyanogen bromide fragment of human plasma albumin.

The complete amino acid sequence of 87 residues of cyanogen bromide fragment CB1 (Asp), the N-terminal fragment of human plasma albumine molecule, has been established. The sequence was determined from the characterization of all tryptic peptides and of chymotryptic arginine-containing peptides in the fragment digested. Overlaps were obtained by tryptic and chymotryptic cleavage of the maleylated S-sulfo derivative of fragment CB1(Asp). Residue 34 is the only cysteine residue in the albumin molecule and it was determined in the form of S-carboxymethyl-cysteine. Edman and dansyl-Edman degradation were used for the sequential analysis.     

The primary structure of Lactobacillus casei thymidylate synthetase. II. The complete amino acid sequence of the active site peptide, CNBr 4.

The 102 amino acid residues of CNBr 4, the largest of 5 cyanogen bromide peptides from the Lactobacillus casei thymidylate synthetase were completely sequenced by means of limited tryptic, tryptic, chymotryptic, and staphylococcal protease peptides. CNBr 4 contains both of the cysteines in an enzyme subunit, with the 5-fluorodeoxyuridylate-reactive cysteine at residue 198 and the other at residue 244.     

Thf complete amino acid sequence of C-I (apoLp-Ser), an apolipoprotein from human very low density lipoproteins.

C-I was prepared from very low density lipoproteins of patients with familial type V hyperliporproteinemia. Peptides from tryptic digests of unmodified and succinylated C-I, chymotryptic peptides, and the products of cayanogen bromide cleavage were isolated and characterized. Sequence analysis of tryptic peptides was performed by the dansyl (5-dimethylaminonaphthalene-1-sulfonyl) technique and hydrolytic regeneration of the amino acid residues from the phenylthiocarbamyl derivatives. Alignment of the tryptic fragments within the cyanogen bromide and succinyl-tryptic peptides was confirmed by the overlap chymotryptic peptides. The complete amino acid sequence of C-I, 57 residues in length, does not reveal any obvious basis for its lipophilic properties.     

Primary structure of the B-chain of human plasmin.

The primary structure of the human plasmin B-chain has been determined. It consists of 230 residues divided in three cyanogen bromide fragments: The amino-terminal 24 residues, the carboxy-terminal three residues and the middle 203 residues. Sequence detemination was performed on the tryptic and the chymotryptic peptides obtained from the main cyanogen bromide fragment of this chain. Owing to similarities between some of the overlapping chymotryptic peptides, two different sequences were possible from these results. However, since the homologies with the pancreatic serine proteases and also the B-chains of thrombin and factor XA are pronounced, the arrangement still could be settled. By peptic digestion of partially reduced and S-carboxymethylated B-chain it was shown that there are two interchain disulphide bridges, which connect the A and B-chains of plasmin, involving Cys-5 and Cys-105 from the B-chain. The intrachain disulphides in the B-chain seem to be situated exactly as in chymotrypsin as partly judged from homologies.     

Complete amino acid sequence of a papain-solubilized human histocompatibility antigen HLA-B7. 1. Isolation and amino acid composition of fragments and of tryptic and chymotryptic peptides

As a part of the overall strategy for determining the complete covalent structure of the papain-solubilized portion of the heavy chain of the human histocompatibility antigen HLA-B7, the protein was dissected into various fragments by a combination of partial acid hydrolysis and cyanogen bromide cleavage. After purification by chromatographic procedures, these fragments have been used as a source for tryptic and chymotryptic peptides. Thirty-three major tryptic and twenty-two major chymotryptic peptides were purified in nanomole amounts and their amino acid compositions determined. These peptides account for the whole extent of the polypeptide chain with the exception of the amino-terminal CNBr pentapeptide. They provide the basis for the formal alignment of the acid cleavage and cyanogen bromide fragments of the molecule as well as the source material for the elucidation of the primary structure of the HLA-B7 heavy chain.     

Primary structure of streptococcal proteinase. I Isolation, composition, and amino acid sequences of the tryptic and chymotryptic peptides of cyanogen bromide fragments 1 to 4.

Tryptic and chymotroptic peptides were isolated and characterized from cyanogen bromide fragments 1 to 4 of streptococcal proteinase and subjected to sequence analysis by the Edman degradation, carboxy-peptidase digestion, and hydrolytic regeneration of the amino acid residues from the phenylthiocarbamyl derivatives. The results, together with the sequence data of the cyanogen bromide fragment 5 reported in the accompanying papers, provide the structural formula of streptococcal proteinase.     

Amino Acid Sequence of Cyanogen Bromide Fragment CB I from Ala Chain of Ricin D

The amino acid sequence of the cyanogen bromide (CNBr) fragment CB I from the Ala chain of ricin D, the largest of three CNBr fragments, was established by manual Edman degradation of the peptides obtained by tryptic, chymotryptic or peptic digestion of fragment CB I. The total number of amino acid residues of fragment CB I accounted for 140 (54%) out of 260 residues in the Ala chain of ricin D.     

The amino acid sequence of Clostridium pasteurianum iron protein, a component of nitrogenase. II. Cyanogen bromide peptides

A total of 10 cyanogen bromide peptides were isolated from the S-beta-carboxymethyl iron protein of nitrogenase. Purification of these peptides was performed mainly by gel filtration on Sephadex G-50; by ascending paper chromatography using the solvent system of pyridine, isoamyl alcohol, 0.1 M ammonium hydroxide; and also, in some cases, with additional steps such as anion exchange column chromatography on Dowex 1-X2 or ascending paper chromatography in an acidic solvent system or by pyridine precipitation of the cyanogen bromide fragment. Sequenator analyses of three large cyanogen bromide peptides (53 to 72 residues) provided tryptic peptide overlap data for the inner portion of the protein. The cyanogen bromide peptides accounted for all of the 273 amino acid residues which were present in the tryptic peptides isolated from carboxymethyl-iron protein (Tanaka, M., Haniu, M., Yasunobu, K. T., and Mortenson, L. E. (1977) J. Biol. Chem. 252, 7081-7088).     

Dihydrofolate reductase: the amino acid sequence of the enzyme from a methotrexate-resistant mutant of Escherichia coli.

The determination of the amino acid sequence of the enzyme dihydrofolate reductase (5,6,7,8-tetrahydrofolate:NADP+ oxidoreductase, EC 1.5.1.3) from a mutant of Escherichia coli B is described. The 159 residues were positioned by automatic Edman degradation of the whole protein, of the reduced and alkylated cyanogen bromide fragments, and of selected tryptic, chymotryptic, and thermolytic digestion products. An N-bromosuccinimide produced fragment of the largest cyanogen bromide peptide was also used in the sequence determination.     

Rule of antibody structure: the primary structure of a human monoclonal IgA1-immunoglobulin (Myeloma protein Tro). IV. Isolation and characterization of the chymotryptic peptides of the H-chain (author's transl)]

The preceeding papers dealth with the purification and characterization of IgA immunoglobulin Tro and its H- and L-chains, the separation and characterization of the cyanogen bromide fragments and the isolation of the tryptic splitting products of the H-chain. In this paper the chymotryptic peptides of the H-chain are presented. All chymotryptic peptides were isolated and purified parallel to the isolation of tryptic peptides. On the basis of these overlapping splitting products the arrangement of the tryptic peptides in the protein could be determined. These and other splitting products listed in this paper contributed to the complete sequence determination of of the H-chain.     

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

As the initial phase of the determination of the complete covalent structure of a human immunoglobulin A, 52 chymotryptic peptides, ranging in length from 2 to 37 residues, were isolated and characterized from the reduced and carboxymethylated alpha1 heavy chain of the myeloma IgA protein Bur. The peptides were subjected to sequence analysis by the dansylation technique, manual and automatic Edman degradation, and carboxypeptidase digestion. The results, in conjunction with the data on the tryptic and thermolysin peptides and the cyanogen bromide fragments reported in the accompanying papers, established the complete primary structure of a human IgA chain.     

The primary structure of actin from rabbit skeletal muscle. Completion and analysis of the amino acid sequence.

Actin is the principal constituent of the thin filaments of muscle, and in order to provide information basic to understanding the molecular basis of actin function we have studied its amino acid sequence. The isolation, compositions, and sequences of cyanogen bromide peptides, ranging in size from 3 to 44 residues, have previously been reported (ELZINGA, M. (1971) Biochemistry 10, 224-229, and other papers in the present series). The peptides have been aligned by isolation and characterization of tryptic peptides that contain methionine. The isolation of one of the CNBr peptides (CB-14) was complicated by the presence of a Met-Thr bond that was only partially split under standard conditions for cyanogen bromide cleavage in formic acid. In this paper conditions are described for increasing the cleavage at this bond. CB-14 is a tetrapeptide, Thr-Gln-Ile-Hse, and this sequence completes the characterization of the actin cyanogen bromide peptides. Finally, the position of CB-14 in the actin sequence as residues 120 to 123 was established by isolation of a chymotryptic overlap peptide. The complete sequence of the 374 residues of actin is presented.     

Covalent structural analysis of yeast inorganic pyrophosphatase.

The present paper describes the amino acid sequence analysis of the internal and COOH-terminal cyanogen bromide fragments of yeast inorganic pyrophosphatase (Sterner, R., Noyes, C., and Heinrikson, R.L. (1974) Biochemistry 13, 91-99). This information coupled with that derived from earlier structural studies of the enzyme (Sterner, R., AND Heinrikson, R.L. (1975) Arch. Biochem. Biophys. 165, 693-703) provides the complete covalent structure of the pyrophosphatase subunit. The majority of the sequence data was derived from automated Edman degradation of the intact cyanogen bromide fragments and the large tryptic peptides obtained from citraconylated derivates in which cleavages were restricted to arginyl residues. The structural determination was completed by analysis of tryptic and chymotryptic peptides from the decitraconylated fragments. The monomer peptide chain contains 285 amino acid residues and the molecular weight calculated from the sequence analysis is 32,042.     

Histidine decarboxylase of Lactobacillus 30a. Sequences of the cyanogen bromide peptides from the alpha chain

The alpha chain of histidine decarboxylase contains eight internal methionine residues. After reductive amination to convert the NH2-terminal pyruvoyl residue to an alanyl residue and cyanogen bromide treatment, 13 pure peptides were isolated. Four of these are incomplete cleavage products. The sequence of each of the remaining nine peptides was established by automated and manual degradation of the intact peptides and of smaller peptides obtained from tryptic, chymotryptic, and staphylococcal protease digests of the cyanogen bromide peptides. These results, together with the data on overlapping peptides reported in the accompanying paper (Huynh, Q. K., Recsei, P. A., Vaaler, G. L., and Snell, E. E. (1984) J. Biol. Chem. 259, 2833-2839), establish the complete amino acid sequence of the alpha chain.     

Primary structure of the elongation factor G from Escherichia coli. V. Amino acid sequence of the C-terminal domain

The amino acid sequence of the C-terminal domain of the elongation factor G (EF-G) has been studied. The polypeptide chain of the domain consists of 228 amino acid residues, and contains no tryptophan or cysteine residues. To determine its structure, the peptides obtained as a result of the fragment digestion by staphylococcal glutamic protease, cyanogen bromide cleavage, and tryptic hydrolysis of the fragment modified by maleic anhydride have been analyzed, as well as peptides obtained after hydrolyses of cyanogen bromide fragments with chymotrypsin, thermolysin and trypsin.     

Primary structure of 20S,22R-cholesterol-hydroxylating cytochrome P-450 from bovine adrenal cortex mitochondria. III. Primary structure of peptides produced by hydrolysis of the fragment F2 with proteinase from Staphylococcus aureus

Primary structure of F2 fragment resulting from limited trypsinolysis of the native cytochrome P-450 has been investigated. Hydrolysis of F2 fragment with proteinase from Staphylococcus aureus afforded 18 homogeneous peptides covering the whole polypeptide chain of the fragment. Complete amino acid sequences were established for 16 peptides, two peptides being elucidated partially. The above data in combination with structural study of chymotryptic peptides of cytochrome P-450 and tryptic peptides of F2 fragment led to reconstitution of six peptide blocks of F2 fragment comprising 203 amino acid residues.     

Amino acid sequence of chitinase from Streptomyces erythraeus

The amino acid sequence of chitinase from Streptomyces erythraeus was determined by the conventional method. The amino acid sequences of tryptic peptides of the reduced and S-carboxymethylated protein were determined. The tryptic peptides were aligned by overlapping the amino acid sequences of chymotryptic peptides, lysyl endopeptidase peptides and cyanogen bromide fragments. S. erythraeus chitinase consists of 290 amino acid residues with the molecular weight of 30,400 and has two disulfide bridges at Cys(45)-Cys(89) and Cys(265)-Cys(272). The enzyme has no significant homology with other chitinases, lysozymes, and other proteins.     

Primary structure of a human IgA1 immunoglobulin. IV. Streptococcal IgA1 protease, digestion, Fab and Fc fragments, and the complete amino acid sequence of the alpha 1 heavy chain.

In order to establish the complete amino acid sequence of the human IgA alpha1 chain Bur, IgA1 protease from Streptococcus sanguis was employed to generate Fabalpha and Fcalpha fragments in the final stage of this investigation. Cyanogen bromide cleavage of the Fabalpha fragment followed by reduction and aminoethylation produced the Fd' fragment (residues 84 to 227); this contains part of the variable region (VR), the whole first constant domain (Calpha1), and part of the hinge region of this heavy chain. The tryptic peptides of the Fd' fragment were isolated, characterized, and sequenced. The results together with the data in the preceding papers on chymotryptic, tryptic, and thermolysin peptides permitted the complete amino acid sequence of the human IgA alpha1 chain to be established.     

Mass-spectrometry with ion extraction from solution at atmospheric pressure for peptide mapping

SIE AP mass spectra of tryptic peptides from the cyclo-GMP phosphodiesterase gamma-subunit and of chymotryptic peptides from the cyanogen bromide fragments of the same subunit exhibit MH+ ions for all theoretically possible smaller peptides. These facts show that SIE AP mass spectrometry can be successfully applied to peptide mapping using 1-2 nmoles of the compound.     

Phosphofructokinase: complete amino-acid sequence of the enzyme from Bacillus stearothermophilus

The entire amino acid sequence of the protein subunit of phosphofructokinase from Bacillus stearothermophilus has been established mainly by sequence analysis of cyanogen bromide fragments and of peptides derived from these fragments by further digestion with proteolytic enzymes. Overlaps of the cyanogen bromide fragments as well as peptide sequences necessary to complement and to confirm tentative assignments within the larger peptide fragments were obtained from the sequences of selected peptides isolated from tryptic and chymotryptic digests of the intact S-[14C]-carboxymethylated protein. Sequence information was also provided by automated sequence analysis of the intact protein subunit and of some of the larger peptide fragments. The sequence is as follows: (See Text).