Methionyl Bonds Research Articles

Complete amino acid sequence of the A chain of human complement-classical-pathway enzyme C1r.

The amino acid sequence of human C1r A chain was determined, from sequence analysis performed on fragments obtained from C1r autolytic cleavage, cleavage of methionyl bonds, tryptic cleavages at arginine and lysine residues, and cleavages by staphylococcal proteinase. The polypeptide chain has an N-terminal serine residue and contains 446 amino acid residues (Mr 51,200). The sequence data allow chemical characterization of fragments alpha (positions 1-211), beta (positions 212-279) and gamma (positions 280-446) yielded from C1r autolytic cleavage, and identification of the two major cleavage sites generating these fragments. Position 150 of C1r A chain is occupied by a modified amino acid residue that, upon acid hydrolysis, yields erythro-beta-hydroxyaspartic acid, and that is located in a sequence homologous to the beta-hydroxyaspartic acid-containing regions of Factor IX, Factor X, protein C and protein Z. Sequence comparison reveals internal homology between two segments (positions 10-78 and 186-257). Two carbohydrate moieties are attached to the polypeptide chain, both via asparagine residues at positions 108 and 204. Combined with the previously determined sequence of C1r B chain [Arlaud & Gagnon (1983) Biochemistry 22, 1758-1764], these data give the complete sequence of human C1r.

Biochemical and immunological studies of bovine and porcine neurofilament triplet proteins by peptide mapping after cyanogen bromide cleavage

1. 1. Peptide mapping of the three bovine and porcine neurofilament protein subunits (“L”, “M” and “H”) with apparent mol. wts of 70, 160 and 210 kDa were performed with CNBr, leading to the cleavage of methionyl bonds. 2. 2. We have obtained two characteristic large fragments with molecular weights of 85 kDa for the “M” bovine subunit and 135 kDa for the “H” subunit of bovine neurofilament. 3. 3. A comparison of the electrophoretic patterns of CNBr generated polypeptides of “L” subunit from beef and pig showed that they are highly related structures. 4. 4. The peptide mappings of CNBr peptides of “M” and “H” subunits from beef and pig were significantly different. 5. 5. Antibodies were raised against the 85 kDa and 135 kDa CNBr fragments. Immunoblotting results with anti-85 kDa and anti-135 kDa of beef are in favour of large differences of structure between the “M” subunits from pig and beef. 6. 6. The “H” proteins were very similar and they also showed that the C-terminal part of bovine “H” and “M” proteins share common antigenic determinants.

Complete amino acid sequence of steer liver microsomal NADH-cytochrome b5 reductase.

The complete covalent structure of liver microsomal NADH-cytochrome b5 reductase from steer liver microsomes was determined. Cleavage at methionyl bonds gave 10 peptides accounting for all the residues of the protein. Acid cleavage of the reductase at the Asp-Pro bonds gave three peptides accounting for all the CNBr peptides in the molecule. Subfragmentation of these peptides by chemical and enzymatic cleavage provided overlaps which established all the fragments in an unambiguous sequence of 300 residues, corresponding to Mr 34,110. Limited tryptic digestion cleaved reductase at residues 28 and 119, yielding a preparation having two noncovalently linked peptides having a conformation which binds flavin and retains the structural features essential for NADH-cytochrome b5 activity. A model for the secondary structure of cytochrome b5 reductase is proposed that is based on computer-assisted analysis of the amino acid sequence. In this model the beta-turns are predominant and there is some 25% alpha and 30% beta structure.

Amino acid sequence of the catalytic subunit of bovine type II adenosine cyclic 3',5'-phosphate dependent protein kinase.

The 350-residue amino acid sequence of the catalytic subunit of bovine cardiac muscle adenosine cyclic 3',5'-phosphate dependent protein kinase is described. The protein has a molecular weight of 40 862, which includes an N-tetradecanoyl (myristyl) group blocking the NH2 terminus and phosphate groups at threonine-197 and serine-338. Seven methionyl bonds in the S-carboxymethylated protein were cleaved with cyanogen bromide to yield eight primary peptides. These fragments, and subpeptides generated by cleavage with trypsin, pepsin, chymotrypsin, thermolysin, and Myxobacter AL-1 protease II, were purified and analyzed to yield the majority of the sequence. The primary peptides were aligned by analyses of overlapping peptides, particularly of methione-containing tryptic peptides generated after in vitro [14C]methyl exchange labeling of methionyl residues in the intact protein.

Isolation and somatomedin activity of bovine growth hormone fragment 87–124

Various bovine growth hormone (GH) fragements were prepared and tested for somatomedin-like activity in vitro. Cyanogen bromide cleavage followed by reduction and alkylation yielded three fragments which were identified as GH (6–124), GH (150–179) and GH (125–149). No consistent effect was found when these preparations were tested for their ability to stimulate in vitro sulfate and thymidine uptake by rat costal cartilage and to compete with [ 125I]iodoinsulin for insulin-binding sites on placenta membranes. A fourth peptide was isolated by cleavage of the tryptophanyl and methionyl bonds of bovine GH using anhydrous heptofluorobutyric acid and cyanogen bromide. In addition to significant amounts of non-specific cleavage products, a peptide having a molecular weight of about 4800 was isolated. The amino terminal residue was leucine and the carboxyl terminal was homoserine. These data, in addition to the amino acid composition, suggested that the peptide corresponded to residues 87–124. Fragment GH (87–124) stimulated sulfate (minimum effective concentration, 5 · 10 −8 M) and thymidine (minimum effective concentration, 10 −8 M) uptake by rat costal cartilage. It also cross-reacted, albeit weakly, with insulin-binding sites on placenta membrane. Maximum displacement was 35% of non-specific binding. These observations demonstrate that somatomedin-like activity can be generated from the growth hormone molecule which is inherently devoid of such activity.

Partial Amino Acid Sequence of an Alkaline Protease Inhibitor, API-2 (b and c)

API-2b and API-2c (alkaline protease inhibitors) were directly subjected to Edman degradation. It was elucidated that API-2C was lacking in Ala-Pro-Ser-Leu-Gly-Ala located in the NH2-terminal region of API-2b, so the two inhibitors were heterogeneous in the NH2-terminal region. Cleavage of the three methionyl bonds in API-2c with cyanogen bromide resulted in three fragments, designated as Peptide 1, Peptide 2 and Peptide 3. It was found that Peptide 1 was located at the NH2-terminal region, Peptide 2 was in the center and Peptide 3 made-up the COOH-terminal region of the API-2 molecule. Partial sequence data showed that most threonine, alanine, glycine and leucine residues were distributed in the NH2-terminal half of the molecule, and the rest of the molecule contained most of the aromatic residues. The amino acid sequence around the reactive site of API-2 (b and c) was very similar to that of S-SI (Streptomyces subtilisin inhibitor) with the exception of the isoleucine residue in place of valine residue in S-SI.

Studies on the structure of aldolase a from chicken muscle

S-Carboxymethylated chicken muscle aldolase was treated with cyanogen bromide to cleave the 4 methionyl bonds per subunit. Five homogeneous fractions were obtained designated fragments I–V. Fragment I was derived from the N-terminus and fragment II from the C-terminus of the enzyme. Reduction of the enzyme with NaB 3H 4 in the presence of dihydroxyacetone phosphate decreases the enzymatic activity by 90%. Fragment III contained the Schiff base-forming lysine residue since more than 83% of the radioactivity introduced by NaB 3H 4 reduction of aldolase-dihydroxyacetone phosphate was found in this fraction. A tryptic peptide of 27 amino acid residues containing the substrate-binding site was isolated. The gross molecular structure of aldolase A from chicken muscle indicates a high degree of homology with mammalian muscle aldolases.

Complete amino acid sequence of the catalytic subunit of bovine cardiac muscle cyclic AMP-dependent protein kinase.

The complete amino acid sequence of the 349-residue catalytic subunit of cyclic AMP-dependent protein kinase from bovine cardiac muscle is presented. The sequence of the subunit (Mr 40,580 including phosphate groups at threonine-196 and serine-337) was derived largely by automated Edman degradation of nine fragments generated from the carboxymethylated protein by cleavage of methionyl bonds with cyanogen bromide. These fragments were aligned along the polypeptide chain by analysis of methionine-containing tryptic peptides isolated from protein radiolabeled in vitro by [14C]methyl exchange at methionyl residues. The molecule contains only two cysteinyl residues, at positions 198 and 342. It is relatively polar, containing clusters of cationic residues toward the amino terminus and anionic residues towards the carboxyl terminus. Predictions of secondary structure suggest the presence of three major domains with approximately half of the residues occurring in alpha-helices and 12% in beta-strands.

Studies on alkaline protease inhibitor (API-2) produced by Streptomyces griseoincarnatus strain No. KTo-250. Part VII. Partial amino acid sequence of an alkaline protease inhibitor, API-2 (b and c).

API-2b and API-2c (alkaline protease inhibitors) were directly subjected to Edman degradation. It was elucidated that API-2C was lacking in Ala-Pro-Ser-Leu-Gly-Ala located in the NH2-terminal region of API-2b, so the two inhibitors were heterogeneous in the NH2-terminal region. Cleavage of the three methionyl bonds in API-2c with cyanogen bromide resulted in three fragments, designated as Peptide 1, Peptide 2 and Peptide 3. It was found that Peptide 1 was located at the NH2-terminal region, Peptide 2 was in the center and Peptide 3 made-up the COOH-terminal region of the API-2 molecule. Partial sequence data showed that most threonine, alanine, glycine and leucine residues were distributed in the NH2-terminal half of the molecule, and the rest of the molecule contained most of the aromatic residues.The amino acid sequence around the reactive site of API-2 (b and c) was very similar to that of S-SI (Streptomyces subtilisin inhibitor) with the exception of the isoleucine residue in place of valine residue i...

The primary structure of ribosomal protein S7 from E. coli strains K and B

Ribosomal proteins S7 from 30S subunits of Escherichia coli strains K and B differ extensively in their aminoacid compositions. The experimental details which led to the determination of the complete primary structures of proteins S7K and S7B are presented. Protein S7K consists of a single polypeptide chain of 177 aminoacids giving a calculated molecular weight of 19, 732, whereas protein S7B has 153 residues which amount to a molecular weight of 17,131. Aminoacid sequences were determined by a combination of automated Edman degradation of the intact proteins in a modified Beckman sequenator and sequencing of peptides obtained by digestion with trypsin. Staphylococcus aureus protease, thermolysin and pepsin, either by solid-phase Edman degradation or by dansyl-Edman degradation. Additional information about the primary structure was derived from peptides resulting from chemical cleavages of the protein by 2-(2-nitrophenyl-sulphenyl)-3-methyl 3' bromoindolenine at its tryptophanyl bonds and by cyanogen bromide at its methionyl bonds leading to large fragments. The mutational event occurring between S7B and S7K was characterized. Protein S7K contains an additional sequence of 24 aminoacids at its C-terminal end. The aminoacid sequence of both proteins S7K and S7B was compared to the published sequences of the other ribosomal proteins of Escherichia coli and predictions for the secondary structure of these proteins were made.

Sequence of the amino-terminal 349 residues of rabbit muscle glycogen phosphorylase including the sites of covalent and allosteric control.

The sequence of the amino-terminal 349 residues of rabbit muscle glycogen phosphorylase (EC 2.4.1.1) has been determined. Limited proteolysis of native phosphorylase b (841 residues, subunit molecular weight 97 412) by subtilisin BPN', Streptomyces alkaline protease, or elastase yielded two large segments (light and heavy). The light segment isolated from the subtilisin digest was cleaved at methionyl bonds with cyanogen bromide to yield eight major fragments and two minor overlapping fragments. The alignment of the major fragments was obtained by analysis of the two minor fragments, of five tryptic peptides containing methionine and of one large fragment generated by cleavage of an aspartylproline bond. Analysis of two cyanogen bromide fragments (CB14 and CB17) isolated from the intact molecule identified the sites susceptible to limited proteolysis and the overlap between the light and the heavy segments. Serine-14 and tyrosine-155 were identified as the residues involved in the covalent and allosteric controls of the enzyme, respectively. Residues 108 and 142 were identified as the cysteine residues reported to be involved in the aggregation of subunits.

Cleavage of tryptophanyl peptide bonds in cytochrome b5 by cyanogen bromide.

Quantitative cleavage of peptide bonds adjacent to tryptophanyl and methionyl residues in the polar moiety of cytochrome b5 was effected using cyanogen bromide in the presence of heptafluorobutyric and formic acids. Application of this method to native cytochrome b5 resulted in cleavage at tryptophanyl and methionyl residues in the polar and membranous segments in high yield. Amino acid analysis of peptides isolated from such digests indicated that tyrosine was modified and the derivative eluted in a position preceding lysine; however, the color constant with ninhydrin remained unchanged. Hydriodic acid hydrolysis of the phenylthiohydantoin derivative of the modified tyrosine regenerated the parent amino acid. Peptides containing the altered tyrosine remained susceptible to chymotryptic cleavage at this residue. Cleavage of methionyl bonds could be prevented by methylene blue-sensitized photooxidation prior to cyanogen bromide/anhydrous heptafluorobutyric acid treatment. The conditions employed for photooxidation were selective for methionyl residues, and the yield and specificity of tryptophanyl cleavage by the cyanogen bromide/anhydrous heptafluorobutyric acid method was unaffected.

Purification and properties of rabbit heart muscle aldolase

Fructose diphosphate aldolase ( d-fructose-1,6-biphosphate d-glyceraldehyde3-phosphate lyase, EC 4.1.2.13) from rabbit heart has been purified and obtained in crystalline form. The preparations are homogeneous on the basis of disc gel electrophoresis and ultracentrifugation. The catalytic and the molecular properties indicate that this is aldolase A. A comparison was made between rabbit heart aldolase and the rabbit muscle enzyme. The sedimentation coefficient, energy of activation and Michaelis constant for Fru-1,6- P 2 were found to be identical with the values obtained for the muscle enzyme. As in case of the muscle enzyme, heart aldolase was found to have a broad pH optimum, remarkable stability over a wide pH range, and the ability to form a Schiff base intermediate with dihydroxyacetone phosphate upon reduction with borohydride. Cleavage of the methionyl bonds with CNBr yields the same pattern as obtained with the muscle enzyme.

Allergens of honey bee venom

The allergenic activities of four purified components of honeybee venom were studied by using histamine release from leukocytes of bee sting-allergic patients. The components studied were hyaluronidase, phospholipase A 2, melittin and apamin with molecular weights, respectively, of about 50,000, 15,800, 2840 and 2038 d. In six of the seven patients studied, hyaluronidase and phospholipase were, respectively, on the average about two and eight times more active by weight than the venom. The situation was reversed in one patient in that hyaluronidase and phospholipase A 2 were, respectively, 90 and 0.5 times more active than the venom. With this single exception, hyaluronidase and phospholipase were about equally active on a molar basis as allergens. Melittin was on the average about one-tenth as active as the venom, and apamin was inactive as an allergen. Chemical modifications of phospholipase A 2 were carried out. Succinylation of eight of its eleven amino groups yielded a derivative that retained 4% of the enzymic activity of the native enzyme. Reduction and carboxymethylation of its four disulfide bonds or cyanogen bromide cleavage of its three methionyl bonds yielded enzymatically inactive derivatives. These derivatives showed varying decreases of allergenic activities when compared to the native enzyme. The results indicate that the antigenic determinants of phospholipase depend on the charge, the amino acid sequence and the conformation of the molecule.

On the Primary Structure of Human Plasminogen and Plasmin

Most of the cyanogen bromide fragments obtained from human plasminogen and plasmin have been purified using combinations of gel filtration and ion-exchange chromatography. The purified fragments have been characterized by molecular weight determination (dodecyl sulphate electrophoresis), amino acid analysis, carbohydrate analysis and direct NH2-terminal amino acid sequence determination. Since some of the purified fragments were compounds with uncompletely cleaved methionyl bonds it was possible to clarify the organization of most of the cyanogen bromide fragments in the plasminogen molecule. The fragment containing the arginyl-valyl bond cleaved during the second step of the activation process is further identified. It is also shown that the microheterogeneity that normally exists in human plasminogen probably has its origin in several sites. One such site is situated in the light (B) chain of plasmin, while another is situated in the carboxyterminal part of the heavy (A) chain. Neither of these sites seems to contain sialic acid.

Primary Structure of Human Fibrinogen and Fibrin: II. STRUCTURAL STUDIES ON NH2-TERMINAL PART OF γ CHAIN

Abstract Chemical cleavage of methionyl bonds in human fibrinogen with cyanogen bromide produces several large fragments. One of these (N-DSK) contains NH2-terminal portions of the three chains of the parent molecule. The NH2-terminal portion of the γ chain of fibrinogen has been isolated. The γ chain fragment has tyrosine as NH2-terminal residue and homoserine as COOH-terminal residue and therefore evidently consists of one single polypeptide chain. It has a molecular weight of 11,500 as determined by ultracentrifugal analysis. molecular weight of 12,700 was found by gel electrophoresis in sodium dodecyl sulfate. The charge heterogeneity of the γ chain fragment observed upon electrophoresis in polyacrylamide gels is probably due to microheterogeneity of its carbohydrate moiety. The NH2-terminal γ chain fragment consists of 78 amino acid residues. The amino acid sequence has been determined. The fragment contains about half of the half-cystines of the whole γ chain. They occur in a cluster, which indicates an uneven distribution of half-cystines within the γ chain of fibrinogen. One of the half-cystine-containing segments shows similarities in sequence with segments in the A α and B β chains. The carbohydrate moiety in the γ chain consists of 2 residues of glucosamine, 6 residues of galactose and mannose, and 1 to 2 residues of sialic acid. The carbohydrate moiety is most likely linked to residue 52. Some differences in specificity of trypsin, plasmin, and thrombin when acting on the γ chain are discussed.

Cleavage of Methionyl Bonds in Taka-amylase A with Cyanogen Bromide

Cleavage of Methionyl Bonds in Taka-amylase A with Cyanogen Bromide

Cleavage of horse immunoglobulin by cyanogen bromide

Treatment of horst IgG with cyanogen bromide in 0·1 M MCl under specified conditions resulted in the cleavage of about two-thirds of its methionyl peptide bonds. A major fragment of about 5 S was isolated from the reaction mixture by gel filtration. The CNBr fragment reacted partially with rabbit anti-(horse IgG), but did not react with rabbit anti-(horse heavy chain). The treatment with CNBr, under the acidic conditions, as well as the treatment with the HCl alone, cause a marked decrease in the biological activity of the immunoglobulins. The cyanogen bromide cleaves the IgG and IgG(T) components of horse serum with equal efficiency, but the digestion products differ in the two cases. Most of the neutralizing anti-Vipera venom activity, retained in the cyanogen bromide-treated immunoglobulin, was found in the fragments derived from IgG(T). It is concluded that, similarly to IgG from other species, the CNBr cleaves preferentially the methionyl bonds in the Fc portion of the horse IgG. While the methionine content of the IgG of various species varies widely, the number and position of cleavage points in all of them is very similar.

Studies on the structure of rabbit muscle aldolase: I. Cleavage with cyanogen bromide: an approach to the determination of the total primary structure

S-Carboxymethylated aldolase was treated with CNBr to cleave the methionyl bonds and the products were separated by chromatography on Sephadex G-75. Five fractions were obtained, designated CnI to CnV according to their order of elution. All fractions except CnI were homogeneous in both N-terminal and C-terminal analyses. CnI was present only in 10% yield and appeared to be an aggregate of peptides. CnII had the N-terminal sequence Pro-His-, and was derived from the N-terminus of aldolase. CnIII, containing C-terminal tyrosine, was the only fragment devoid of homoserine, and was derived from the C-terminus of the enzyme. CnIV contained the Schiff base-forming lysine residue. CnV, with N-terminal sequence Val-Thr-Pro-, represented the overlapping peptide at the COOH-terminal side of this lysine (9). The sequence of the four fragments in the aldolase molecule has thus been deduced as: (NH 2) CnII-CnIV-CnV-CnIII (OH). Since aldolase contains 12 methionine residues, the present results confirm the 4-subunit structure of the enzyme. Fraction CnIII has been shown to contain 2 peptides derived from the C-termini of the α and β chains, and may represent the only region in which the 2 chains differ. The procedure has provided a means to locate in the molecule the functional groups involved in the manifestation of aldolase activity. The significance of this approach to the determination of the total primary structure of the enzyme is discussed.

Subfragments from the Fc fragment of human immunoglobulin G. Isolation and physicochemical charaterization.

A fragment termed fragment Fc' and a related fragment termed fragment pFc' produced by the actions of papain and pepsin respectively on human immunoglobulin G have been isolated and characterized. Amino acid analyses and experiments utilizing cyanogen bromide to cleave the methionyl bonds of the Fc' and pFc' fragments make it possible to locate both fragments within the known chain structure of the immunoglobulin G molecule. The pFc' fragment is probably a non-covalently linked dimer situated at the C-terminal end of the molecule, containing about 232 amino acid residues and having a molecular weight of 26000. The Fc' fragment is a similar dimer of about 182 residues extending from near residue 14 to near residue 105 (numbered from the C-terminal end) of the gamma-chain and has a molecular weight of 21000.