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.     

Rule of antibody structure: the primary structure of a human monoclonal IgA1-immunoglobulin (myeloma protein Tro), V. The arrangement of the tryptic peptides and a discussion of the complete primary structure of the H-chain (author's transl)]

This communication deals with the sequence work done with tryptic and chymotryptic peptides and some cyanogen bromide splitting products. With these peptides, and if necessary with their splitting peptides, the whole primary structure of the alpha1-H-chain of myeloma protein Tro is established. The position of the amides is determined by electrophoresis and digestion with aminopeptidase M. The alpha1-chain Tro comprises 475 amino acid residues. Because of its specific exchanges and deletions the variable part of alpha1-chain Tro belongs to subgroup III of variable parts of H-chains. The switch from the variable to the constant part occurs at position 119/120 and is analogous to other chains which have been sequenced up to now. The large number of cysteine residues in the alpha-chain which may influence the tertiary structure, especially in the hinge and the subsequent CH2-region, is noteworthy. Furthermore, myeloma protein Tro is compared with the other alpha1-chain Bur[5] sequenced in the meantime, and protein But[6], which is an IgA2 molecule of the allotype A2m(2).     

Amino acid sequence of beta-galactosidase. V. Isolation and sequences of chymotryptic peptides.

The amino acid sequence of 72 chymotryptic peptides isolated from 14C-, 3H-labeled carboxymethyl-beta-galactosidase has been determined. A variety of techniques were used in the isolation procedures including separation by solubility, size, and ion exchange and paper chromatography. These peptides contain approximatley 500 amino acids, range in size from 2 to 26 residues, and give overlaps with tryptic peptides of 16 to 55 residues. Peptides from this digest and those reported earlier from tryptic digests account together for the sequence of about 600 of the 1021 residues in the subunit.     

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

The reduced and either aminothylated or carboxymethylated H-chain of the monoclonal IgA1 immunoglobulin Tro was digested with trypsin. The tryptic peptides were isolated by gel and ion-exchange chromatography. Because of different methods of alkylation, the cysteine-containing peptides could be obtained in two forms and showed additional overlaps. Sequence studies performed with these fragments elucidated the primary structure of the protein.     

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 streptococcal proteinase. II. Isolation, composition, and amino acid sequences of the tryptic and chymotryptic peptides of cyanogen bromide fragment 5.

The amino acid sequence of the cyanogen bromide fragment 5 of streptococcal proteinase has been determined. This fragment comprises residues 130 to 253 of the proteinase chain. Six tryptic peptides were isolated from maleylated cyanogen bromide fragment 5, and their alignment was obtained by the overlap of chymotryptic peptides. Sequence analysis of tryptic, chymotryptic, and thermolysin peptides was performed by the 5-deimethylaminoaphthalene-1-sulfonyl technique and carboxypeptidases digestion.     

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.     

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.     

Primary structure of alpha-clostripain light chain

The primary structure of light chain of alpha-clostripain was determined by sequence analysis of peptides derived from tryptic digests purified by reverse-phase high-performance liquid chromatography. The 22 isolated tryptic peptides were aligned by peptides derived from chymotryptic and staphylococcal V8 proteinase digests. The light chain contains 133 amino acids residues and has a relative molecular mass of 15400. The prediction of its secondary structure is given.     

The amino acid sequence of Pseudomonas fluorescens azurin

The amino acid sequence of Pseudomonas fluorescens azurin has been determined. The protein consists of a single peptide chain of 128 residues. There is one intra-chain disulphide bridge. The sequence was determined by isolation of the soluble tryptic peptides, and by exhaustive examination of the products of chymotryptic and peptic digestion. The sequence has been confirmed by the purification and analysis of the seven fragments obtained by cyanogen bromide treatment of the protein.     

Structural destabilization of the recombinant thermophilic TthL11 ribosomal protein by a single amino acid substitution

Thermus thermophilus L11 protein has previously been reported to be resistant against tryptic and chymotryptic proteolysis under native conditions. With a single amino acid substitution, namely Trp101Arg, conformational changes were induced that resulted in the exhibition of specific amino acids that served as targets for tryptic and chymotryptic action and rendered the protein highly unstable even during purification. This unexpected process was evidenced by the isolation with size exclusion gel chromatography of the well-structured chymotryptic N-terminal domain in a high amount and its characterization both by Edman degradation and QTOF-EMS spectroscopy. On the other hand, the substitution of Val38Cys, which did not contribute to structural changes, indicates a very possible implication of this amino acid in the protein methylation process. The data reported in this work illustrate the distinctive amino acid dynamics in a thermophilic protein, which, while serving the function common to its counterparts from mesophilic organisms, has had to adapt to the extreme environmental conditions typical of thermophilic organisms.     

A mass-spectrometric method for the estimation of the ratio of gamma-carboxyglutamic acid to glutamic acid at specific sites in proteins. Application to the N-terminal region of bovine prothrombin.

The amino acid sequence of the ferredoxin of Brassica napus was determined by using a Beckman 890C sequencer in combination with the characterization of peptides obtained by tryptic and chymotryptic digestion of the protein; some peptides were subdigested with thermolysin. The molecule consists of a single polypeptide chain of 96 amino acid residues and has an unblocked N-terminus. The primary structure shows considerable similarity with other plant-type ferredoxins.     

Primary structure of 20S,22R-cholesterol-hydroxylating cytochrome P-450 from bovine adrenal cortex mitochondria. Study of the structure of peptides obtained by hydrolysis of fragment F1 with proteinase from Staphylococcus aureus

The fragment F1 resulting from the limited tryptic hydrolysis of the native molecule of cytochrome P-450 has been digested with Staphylococcus aureus protease. 24 peptides, covering the whole polypeptide chain of fragment F1, are isolated from the hydrolysate. Analysis of their amino acid sequence in combination with the earlier data on the structure of cytochrome P-450 chymotryptic peptides and fragment F1 tryptic peptides permitted to carry out a reconstruction of large peptide blocks of fragment F1 that comprise 252 amino acid residues.     

The primary structure of human liver manganese superoxide dismutase

The complete amino acid sequence of manganese superoxide dismutase from human liver was determined. The sequence was deduced following characterization of the peptides obtained from tryptic, chymotryptic, and Staphylococcus aureus digests of the apoprotein. Chemical cleavage with dimethyl sulfoxide-hydrobromic acid was also carried out. The amino acid sequence listed below is made up of 196 amino acids and the two subunit polypeptides in the native enzyme appear to be identical. No homology was observed with copper/zinc containing class of superoxide dismutase. Lys-His-Ser-Leu-Pro-Asp-Leu-Pro-Tyr-Asp-Tyr-Gly-Ala-Leu-Glu-Pro-His-Il e -Asn-Ala-Gln-Ile-Met-Gln-Leu-His-His-Ser-Lys-His-His-Ala-Ala-Tyr-Val-Asn -Asn-Leu-Asn-Val-Thr-Gln-Glu-Lys-Tyr-Gln-Glu-Ala-Leu-Ala-Lys-Gly-Asp-Val -Thr-Ala-Gln-Ile-Ala-Leu-Gln-Pro-Ala-Leu-Lys-Phe-Asn-Gly-Gly-Gly-His-Ile -Asn-His-Ser-Ile-Phe-Trp-Thr-Asn-Leu-Ser-Pro-Asn-Gly-Gly-Gly-Gln-Pro-Lys -Gly-Glu-Leu-Leu-Glu-Ala-Ile-Lys-Arg-Asp-Phe-Gly-Ser-Phe-Asp-Lys-Phe-Lys -Gln-Lys-Leu-Thr-Ala-Ala-Ser-Val-Gly-Val-Gln-Gly-Ser-Gly-Trp-Leu-Gly-Phe -Asn-Lys-Gln-Arg-Gly-His-Leu-Gln-Ile-Ala-Ala-Cys-Pro-Asn-Gln-Asp-Pro-Leu -Gln-Gly-Thr-Thr-Gly-Leu-Ile-Pro-Leu-Leu-Gly-Ile-Asp-Val-Trp-Glu-His-Ala -Tyr-Tyr-Leu-Gln-Tyr-Lys-Asn-Val-Arg-Pro-Asp-Tyr-Leu-Lys-Ala-Ile-Trp-Asn -Val-Ile-Asn-Trp-Glu-Asn-Val-Thr-Glu-Arg-Tyr-Met-Ala-Cys-Lys-Lys.     

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.     

The primary structure of the cytotoxin alpha-sarcin

The primary structure of the cytotoxin alpha-sarcin was determined. Eighteen of the 19 tryptic peptides were purified; the other peptide has arginine only. The complete sequence of 17 of the peptides was determined; the sequence of the remaining peptide was determined in part. The sequence of the 39 NH2-terminal residues was obtained by automated Edman degradation. The carboxyl-terminal amino acids were identified after carboxypeptidase treatment. The assignment of the amino acids in the tryptic peptides was confirmed and their alignment established from the sequence of the secondary tryptic peptides obtained after cleavage of citraconylated alpha-sarcin, from the sequence of a 2-(2-nitrophenylsulfenyl)-3-methyl-3'-bromoindolenine peptide, from the sequence of a chymotryptic peptide, and from the sequence of a peptide obtained with Staphylococcus aureus V8 protease. alpha-Sarcin contains 150 amino acid residues; the molecular weight is 16,987. There are disulfide bridges between cysteine residues at positions 6 and 148 and between residues 76 and 132.     

The primary structure of iron-superoxide dismutase from Photobacterium leiognathi

The complete amino acid sequence of iron-superoxide dismutase from Photobacterium leiognathi was determined. The sequence was deduced following characterization of the peptides obtained from tryptic, chymotryptic, and Staphylococcus aureus V-8 protease digests of the apoprotein. The amino acid sequence listed below is made up of 193 residues. It is the first complete sequence to be determined for an iron-superoxide dismutase. The iron-superoxide dismutase shows the same order of homology with the manganese-superoxide dismutases as these enzymes show among themselves. No homology was observed with the copper/zinc-containing class of superoxide dismutases. Ala-Phe-Glu-Leu-Pro-Ala-Leu-Pro-Phe-Ala-Met-Asn-Ala-Leu-Glu-Pro-His-Ile- Ser-Gln-Glu-Thr-Leu-Glu-Tyr-His-Tyr-Gly-Lys-His-His-Asn-Thr-Tyr-Val-Val- Lys-Leu-Asn-Gly-Leu-Val-Glu-Gly-Thr-Glu-Leu-Ala-Glu-Lys-Ser-Leu-Glu-Glu- Ile-Ile-Lys-Thr-Ser-Thr-Gly-Gly-Val-Phe-Asn-Asn-Ala-Ala-Gln-Val-Trp-Asn- His-Thr-Phe-Tyr-Trp-Asn-Cys-Leu-Ala-Pro-Asn-Ala-Gly-Gly-Glu-Pro-Thr-Gly- Glu-Val-Ala-Ala-Ala-Ile-Glu-Lys-Ala-Phe-Gly-Ser-Phe-Ala-Glu-Phe-Lys-Ala- Lys-Phe-Thr-Asp-Ser-Ala-Ile-Asn-Asn-Phe-Gly-Ser-Ser-Trp-Thr-Trp-Leu-Val- Lys-Asn-Ala-Asn-Gly-Ser-Leu-Ala-Ile-Val-Asn-Thr-Ser-Asn-Ala-Gly-Cys-Pro- Ile-Thr-Glu-Glu-Gly-Val-Thr-Pro-Leu-Leu-Thr-Val-Asp-Leu-Trp-Glu-His-Ala- Tyr-Tyr-Ile-Asp-Tyr-Arg-Asn-Leu-Arg-Pro-Ser-Tyr-Met-Asp-Gly-Phe-Trp-Ala- Leu-Val-Asn-Trp-Asp-Phe-Val-Ser-Lys-Asn-Leu-Ala-Ala.     

The disulfide structure of mouse lysosome-associated membrane protein 1

The disulfide structure of mouse lysosome-associated membrane protein 1 has been determined by reverse-phase isolation and sequence analysis of the cysteine-containing tryptic fragments of the reduced and non-reduced deglycosylated protein. Half-cystines were distinguished (a) by their localization within tryptic or chymotryptic peptides that formed reverse-phase peaks unique to the reduced digests and (b) by their 3H-carboxymethylation only after reduction of the protein. The disulfide arrangement of the cysteines was assigned after isolation of disulfide-linked peptide pairs. Each pair chromatographed as a peak present in the nonreduced (but not the corresponding reduced) tryptic digest. NH2-terminal sequencing as well as reduction, alkylation, and rechromatography of the disulfide-linked fragments led to the following assignment of disulfide bonds: Cys11 and Cys50, Cys125 and Cys161, Cys198 and Cys235, and Cys303 and Cys340. This structure creates four 36-38-residue loops that are symmetrically placed within the two halves of the protein's intraluminal domain. The loops formed by the Cys11-Cys50 and Cys198-Cys235 bridges are homologous, and the Cys125-Cys161 and Cys303-cys340 loops form a second set of homologous domains. The conservation of cysteine residues among lysosome-associated membrane proteins 1 and 2 suggests that this disulfide arrangement is common to both members of this family of lysosomal membrane glycoproteins.     

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.     

Human pituitary thyrotropin: primary structure of the hormone specific beta subunit

The primary structure of the hormone specific β subunit of human pituitary thyrotropin has been deduced from the composition and complete or partial amino acid sequences of the tryptic and chymotryptic peptides. It consists of 112 amino acid residues with the single oligosaccharide moiety which is assumed to be linked to the asparagine residue at position 23.