Isolation and characterization of the cyanogen bromide fragments of lobster arginine kinase (homarus vulgaris).

Arginine kinase was aminoethylated in order to block the five free thiol groups on the native enzyme, and then submitted to BrCN cleavage. The BrCN resulting peptides were soluble in propionic acid (10 percent) and subsequently submitted to gel-filtration. The large polypeptide subfractions were citraconylated and resubmitted to differnt gelchromatographies, whereas the short peptide subfractions were submitted to preparative paper electrochromatographies. Eight peptides of 2, 11, 17, 25, 61, 82, 86 and 132 amino acid residues were isolated, one of which is the overlapping of two peptides. The amino acid composition and the end group of all the isolated peptides were established. The short peptides (2, 11 and 17 residues) were sequenced. All peptides possess homoserine at C-terminal position because one methionyl residue is situated at the C-terminal position in the native protein. The polypeptide with 132 residues possessed N-acetylated residue at N-terminal position: therefore this polypeptide is located at the N-terminal position in the protein. The sum and account of each amino acid of the seven isolated peptides were compared to those of the intact protein: the sum of the seven peptides is 331 amino acid residues, whereas the whole protein contains 342 residues. The molecular weight of arginine kinase is revised and calculated on the basis of the present results (37, 687).     

Special problems encountered during the cyanogen bromide cleavage of lobster arginine kinase

During structural analysis of Lobster muscle arginine-kinase, we have isolated a CNBr resulting peptide with a blocked N-terminal residue. This peptide was sequenced after unblocking by mild acid treatment (1 N HCl at 100 degrees C for 10 min). The blocked form is not due to the formation of pyroglutamic acid nor is it due to the formation of diketopiperazine. We have applied the experimental conditions used for CNBr cleavage of lobster arginine-kinase to a synthetic peptide the structure of which is similar to the above CNBr peptide. We bring evidence that during CNBr cleavage partial formylation occurs with a possible cyclization of a 7 membered ring of Gly--Asp...     

Amino acid sequence of cyanogen bromide fragments of potato phosphorylase

Amino acid sequence analysis of the cyanogen bromide peptides of potato alpha-glucan phosphorylase was undertaken for comparison with rabbit muscle glycogen phosphorylase and for elucidation of the structural bases for the differences in the catalytic and regulatory properties between the animal and plant enzymes. The potato enzyme was carboxymethylated and cleaved with cyanogen bromide. The 17 distinct fragments produced were isolated by a combination of gel filtration, sulfopropyl ion exchange chromatography, and high performance liquid chromatography. The molecular weights of these fragments are distributed in a range of 300 to 30,000. Fragment CI has a blocked amino terminus, and has the same amino acid sequence as CII, which has been assigned as the amino-terminal fragment of potato phosphorylase. The blocking group was deduced to be an acetyl group from the results of fast atom bombardment mass spectrometry of an amino-terminal pentapeptide. This paper describes the sequence determination of all the cyanogen bromide fragments of potato phosphorylase. The complete structure is presented in the following paper (Nakano, K., and Fukui, T. (1986) J. Biol. Chem. 261, 8230-8236).     

Amino acid sequence studies on the alpha chain of human fibrinogen. Complete sequence of the largest cyanogen bromide fragment

The largest fragment produced by complete cyanogen bromide digestion of the alpha chain of human fibrinogen contains 236 residues and has a calculated molecular weight of 23,949. The complete amino acid sequence of the fragment was determined by the isolation of peptides generated by plasmin, trypsin (including digestion of citraconylated material), staphylococcal protease, and chymotrypsin. In addition, some key subfragmentation was achieved by selective chemical cleavage at tryptophan residues. The fragment has an unusual amino acid composition, more than half of its residues being glycine, serine, threonine, and proline. There are very few nonpolar residues, although 7 of the alpha-chain's 10 tryptophans occur in this fragment. The fragment contains 2 cysteine residues located 30 residues apart which are connected by an intrachain disulfide bond in the native molecule. The tryptophans occur with a definite periodicity that highlights a series of 13-residue homology repeats. The fragment also contains the two principal alpha-chain cross-linking sites.     

Amino acid sequence of a peptide with both molt-inhibiting and hyperglycemic activities in the lobster, Homarus americanus

A hydrophobic peptide of 71 residues was isolated from lobster sinus gland extracts that prolonged intermolt periods and lowered ecdysteroid titers in juvenile lobsters. Removal of the N-terminal pyroglutamyl residue allowed sequencing of 30 of the first 36 residues. Additional data were obtained from HPLC-purified fragments from endoproteinase cleavages (Lys-C, Glu-C, Arg-C, Asp-N), and carboxypeptidase Y digestion. This is the first reported amino acid sequence of a crustacean molt-inhibiting hormone. This peptide also has significant hyperglycemic activity.     

Amino acid sequence of the alanyl peptide from cyanogen bromide cleavage of bovine plasma albumin

The amino acid sequence of the alanyl peptide from cyanogen bromide cleavage of bovine plasma albumin has been determined. This peptide has 96 residues and extends the known sequence that begins at the N terminus from 87 to 183 residues.     

Amino acid sequence of the small cyanogen bromide peptide of thermitase, a thermostable serine proteinase from Thermoactinomyces vulgaris. Relation to the subtilisins

The following amino acid sequence of the small cyanogen bromide peptide (mol. wt. 5399) of thermitase from Thermoactinomyces vulgaris has been determined: Ala-Thr-Pro-His-Val-Ala-Gly-Val-Ala-Gly-Leu-Leu-Ala-Ser-Gln-Gly-Arg-Ser-Ala-Ser -Asn-Ile-Arg-Ala-Ala-Ile-Glu-Asn-Thr-Ala-Asp-Lys-Ile-Ser-Gly-Thr-Gly-Thr-Tyr-Trp-Ala-Lys-Gly-Arg-Val-Asn-Ala-Tyr-Lys-Ala-Val-Gln-Tyr. The results obtained support the classification of the enzyme as a serine proteinase of the subtilisin type as proposed in a previous paper (1). This partial sequence extending from the serine residue involved in the active site to the C-terminal amino acid of the enzyme shows a 40% homology with the corresponding part of the subtilisin BPN' or subtilisin Carlsberg molecule but a 56% homology as regards conservative amino acid replacements. The secondary structure of this polypeptide fragment, predicted from the data obtained by the method of Chou & Fasman (2) agrees fairly well, within the limit or error of the method, with the structure of the corresponding part of the subtilisin BPN' molecule. Therefore, as expected, no dramatic changes in the spatial structure appear to account for the higher thermostability of thermitase, at least in this area of the polypeptide chain.     

Amino acid sequence of beta-galactosidase. VII. Isolation of the 24 cyanogen bromide peptides.

All of the 24 cyanogen bromide peptides of beta-galactosidase have been isolated in pure form. Of these 8 ranged in size from 2 to 5 residues and were purified by paper electrophoresis. The 16 large peptides, from 23 to 119 residues, were chromatographed at pH 5.0 on a carboxymethyl-cellulose column in 0.02 M ammonium acetate buffer containing 8 M urea. A number of peptides were obtained in pure from following Sephadex G-50 or G-75 gel filtration. Others were separated on sulfopropyl-Sephadex or diethyl-(2-hydroxylpropylaminoethyl)-Sephadex. There large peptides were obtained in over 50% yield and several others were obtained in more than 25% yield.     

Amino acid sequence studies on cyanogen bromide peptides of chicken caldesmon which bind to calmodulin

Three major calmodulin-binding cyanogen bromide peptides (fragments A, B, and D) were isolated from chicken gizzard muscle caldesmon and their amino acid sequences were determined. The molecular masses of fragments A, B, and D were estimated to 16, 12, and 9 kDa, respectively, by SDS-urea polyacrylamide gel electrophoresis. Fragment A was composed of 102 amino acid residues and contained homoserine at the C terminus. The amino acid sequence from the 37th residue of fragment A corresponds to the N-terminal sequence of the 15 kDa peptide which was obtained by thrombin digestion [Mornet, D., Audemard, E., & Derancourt, J. (1988) Biochem. Biophys. Res. Commun. 154, 564-571]. Thrombin 15 kDa peptide binds to F-actin but does not bind to calmodulin. Thus the N-terminal 36 residues and the C-terminal part from the 37th residue of fragment A are supposed to bind to calmodulin and F-actin, respectively. The sequences of fragments B and D were identical, but fragment D was composed of 64 amino acid residues and ended with tryptophan, whereas fragment B was of 98 or 99 amino acid residues and ended with proline. Both fragments B and D are supposed to be the C-terminal peptides of chicken caldesmon. Fragment B had heterogeneous sequences at the C-terminal region. These results can explain the reported heterogeneity of chicken caldesmon in charge and molecular mass.     

The primary structure of human plasma high density apolipoprotein glutamine I (ApoA-I). I. The amino acid sequence of cyanogen bromide fragment II.

Apolipoprotein glutamine I (apoLP-Gln-I or apoA-I) is one of the major protein constituents of human plasma high density lipoproteins. The protein has 245 amino acid residues, including 3 residues of methionine, and is lacking isoleucine, cystine, and cysteine. Cleavage of apoLP-Gln-I with cyanogen bromide yields four fragments, designated in their order of elution from Bio-Gel P-30 as CNBr I, II, III, and IV. In the present study, we report the complete amino acid sequence of the NH2-terminal fragment, CNBr II, a peptide that contains 90 amino acid residues.