A NEW PEPTIDE THROMBIN INHIBITOR FROM STREPTOMYCES GRISEUS

1987 ◽  
Author(s):  
Hua-zhen Liu ◽  
Wei Chen ◽  
Qi-ying Liu ◽  
Xia Zhang ◽  
Li-xiu Wang ◽  
...  
Keyword(s):  
Amino Acid ◽  
High Performance ◽  
Antithrombin Iii ◽  
Streptomyces Griseus ◽  
Exchange Chromatography ◽  
Performic Acid ◽  
Thrombin Inhibitor ◽  
Amino Acid Residues ◽  
Macroporous Resin ◽  
Terminal Residue

A new peptide thrombin inhibitor was found in the Streptomyces griseus strain 254 isolated from a soil sample from Tongan, Fujian province, China, the inhibitor being a secondary metabolic product. The production of the inhibitor reached a maximum after 3 days culture of bacteria at 28°C in a rotary shaker. The inhibitor excreted in the culture filtrate was purified by absorption on macroporous resin, followed by ion exchange chromatography on DEAE-52, CM-32 cellulose, affinity chromatography on the immobilized thrombin and high performance liquid chromatography. The amino acid composition of the inhibitor was determined to be Val(2), Met(l), Ile(l), Leu(2) and Arg (1), similar to that of the amino acid residues around the reactive site of human antithrombin III, the critical plasma inhibitor of thrombin. The NH2-terminal residue of the inhibitor seems to be blocked by the alkyl group due to the negative reaction to ninhydrin, whereas the COO-terminal residue is most likely to be arginal because of that Arg was not found in the amino acid analysis, unless the peptide was oxidized by performic acid before acid hydrolysis. The chromogen substrates Bz-Phe-Val-Arg-PNA and Bz-Gly-Pro-Lys-PNA were used to determine the thrombin and plasmin activities, respectively. Besides thrombin, the purified inhibitor also exhibits a weak inhibitory activities on trypsin and much weak on plasmin, but not on chymotrypsin and other protein-ases.

scholarly journals Isolation and a partial amino acid sequence of insulin from the islet tissue of cod (Gadus callarias)

1968 ◽  
Vol 106 (2) ◽  
pp. 531-541 ◽  
Author(s):  
P T Grant ◽  
K. B. M. Reid
Keyword(s):  
Amino Acid ◽  
Ion Exchange ◽  
Gel Filtration ◽  
Bovine Insulin ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Performic Acid ◽  
Amino Acid Residues ◽  
Islet Tissue ◽  
A Chain

1. Insulin has been isolated by gel filtration and ion-exchange chromatography from extracts of the discrete islet tissue of cod. The final preparation yielded a single band on electrophoresis at two pH values. The biological potency was 11·5 international units/mg. in mouse-convulsion and other assay procedures. 2. Glycine and methionine were shown to be the N-terminal amino acids of the A and B chains respectively. An estimate of the molecular weight together with amino acid analyses indicated that cod insulin, like the bovine hormone, consists of 51 amino acid residues. In contrast, the amino acid composition differs markedly from bovine insulin. 3. Oxidation of insulin with performic acid yielded the A and B peptide chains, which were separated by ion-exchange chromatography. Sequence studies on smaller peptides isolated from enzymic digests or from dilute acetic acid hydrolysates of the two chains have established the sequential order of 14 of the 21 amino acid residues of the A chain and 25 of the 30 amino acid residues of the B chain.

scholarly journals Identification and Characterization of Lactocyclicin Q, a Novel Cyclic Bacteriocin Produced by Lactococcus sp. Strain QU 12

2009 ◽  
Vol 75 (6) ◽  
pp. 1552-1558 ◽  
Author(s):  
Naruhiko Sawa ◽  
Takeshi Zendo ◽  
Junko Kiyofuji ◽  
Koji Fujita ◽  
Kohei Himeno ◽  
...  
Keyword(s):  
Amino Acid ◽  
High Performance ◽  
Hydrophobic Interaction Chromatography ◽  
Heat Stability ◽  
Peptide Bond ◽  
Exchange Chromatography ◽  
Amino Acid Sequences ◽  
Amino Acid Residues ◽  
Gene Encoding ◽  
High Level

ABSTRACT Lactococcus sp. strain QU 12, which was isolated from cheese, produced a novel cyclic bacteriocin termed lactocyclicin Q. By using cation-exchange chromatography, hydrophobic interaction chromatography, and reverse-phase high-performance liquid chromatography, lactocyclicin Q was purified from culture supernatant, and its molecular mass was determined to be 6,062.8 Da by mass spectrometry. Lactocyclicin Q has been characterized by its unique antimicrobial spectrum, high level of protease resistance, and heat stability compared to other reported bacteriocins of lactic acid bacteria. The amino acid sequence of lactocyclicin Q was determined chemically, and this compound is composed of 61 amino acid residues that have a cyclic structure with linkage between the N and C termini by a peptide bond. It showed no homology to any other antimicrobial peptide, including cyclic bacteriocins. On the basis of the amino acid sequences obtained, the sequence of the gene encoding the prepeptide lactocyclicin Q was obtained. This is the first report of a cyclic bacteriocin purified from a strain belonging to the genus Lactococcus.

scholarly journals Structural Analysis and Characterization of Lacticin Q, a Novel Bacteriocin Belonging to a New Family of Unmodified Bacteriocins of Gram-Positive Bacteria

2007 ◽  
Vol 73 (9) ◽  
pp. 2871-2877 ◽  
Author(s):  
Koji Fujita ◽  
Shiro Ichimasa ◽  
Takeshi Zendo ◽  
Shoko Koga ◽  
Fuminori Yoneyama ◽  
...  
Keyword(s):  
Amino Acid ◽  
Dna Sequences ◽  
High Performance ◽  
Hypothetical Protein ◽  
Exchange Chromatography ◽  
Amino Acid Residues ◽  
Gram Positive ◽  
Gram Positive Bacteria ◽  
Methionine Residue ◽  
New Family

ABSTRACT Lactococcus lactis QU 5 isolated from corn produces a novel bacteriocin, termed lacticin Q. By acetone precipitation, cation-exchange chromatography, and reverse-phase high-performance liquid chromatography, lacticin Q was purified from the culture supernatant of this organism, and its molecular mass was determined to be 5,926.50 Da by mass spectrometry. Subsequent analyses of amino acid and DNA sequences revealed that lacticin Q comprised 53 amino acid residues and that its N-terminal methionine residue was formylated. In contrast to most bacteriocins produced by gram-positive bacteria, lacticin Q had no N-terminal extensions such as leader or signal sequences. It showed 66% and 48% identity to AucA, a hypothetical protein from Corynebacterium jeikeium plasmid pA501, and aureocin A53, a bacteriocin from Staphylococcus aureus A53, respectively. The characteristics of lacticin Q were determined and compared to those of nisin A. Similar to nisin A, lacticin Q exhibited antibacterial activity against various gram-positive bacteria. Lacticin Q was very stable against heat treatment and changes in pH; in particular, it was stable at alkaline pH values, while nisin A was inactivated. Moreover, lacticin Q induced ATP efflux from a Listeria sp. strain in a shorter time and at a lower concentration than nisin A, indicating that the former affected indicator cells in a different manner from that of the latter. The results described here clarified the fact that lacticin Q belongs to a new family of class II bacteriocins and that it can be employed as an alternative to or in combination with nisin A.

The Enzymic Digestion of Cod Tropomyosin

1962 ◽  
Vol 19 (6) ◽  
pp. 1095-1104
Author(s):  
B. Truscott ◽  
P. L. Hoogland ◽  
P. H. Odense ◽  
A. E. Waddell
Keyword(s):  
Amino Acid ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Performic Acid ◽  
Amino Acid Residues ◽  
Amino Groups ◽  
Terminal Amino Acid ◽  
Enzymic Digestion ◽  
Disulphide Bonds ◽  
Terminal Amino

Tropomyosin from cod muscle can be oxidized with performic acid to cleave disulphide bonds without degradation of other amino acid residues. The ε-amino groups of lysine within the molecule can be substituted readily with carbobenzoxy-groups for protection against digestion by trypsin. The digestions by trypsin of carbobenzoxy-substituted tropomyosin, and by chymotrypsin of oxidized tropomyosin, have been shown to be reproducible, providing peptides suitable for amino acid sequence studies. The peptides so obtained were separated by ion-exchange chromatography using a Beckman/Spinco Amino Acid Analyzer.After treatment with urea, cod tropomyosin does not yield a free N-terminal amino acid as has been reported for rabbit tropomyosin.

The Amino Acid Sequence of Streptomyces griseus Protease A: The Peptic Peptides

1971 ◽  
Vol 49 (10) ◽  
pp. 1083-1097 ◽  
Author(s):  
P. Johnson ◽  
L. B. Smillie
Keyword(s):  
Amino Acid ◽  
Streptomyces Griseus ◽  
Disulfide Bridge ◽  
Exchange Chromatography ◽  
Amino Acid Sequences ◽  
Amino Acid Residues ◽  
Homologous Proteins ◽  
Acidic Peptide ◽  
Dowex 1 ◽  
Peptide Fractions

The peptic peptides of Streptomyces griseus Protease A (excluding the previously characterized disulfide bridge peptic peptides) were fractionated into basic, neutral, and neutral plus acidic peptide fractions by chromatography on Dowex 1 × 2. These three peptide fractions were then fractionated by cation-exchange chromatography on Chromobead P resin using the Technicon autoanalyzer system. Following further purifications on paper, the amino acid compositions and sequences of the peptic peptides were determined. The N-terminal sequence of Protease A has been identified as Ile–Ala–Gly–Gly–Glu–Ala. The numbers of amino acid residues obtained from the amino acid sequences reported are in agreement with those numbers obtained from amino acid analysis of the total protein in the cases of tryptophan, methionine, histidine, proline, phenylalanine, and glutamic acid. Some of the results suggest either the presence of nonidentical but highly homologous proteins in the Protease A preparation or the possibility of repeating sequences in a single molecular species.

scholarly journals Tamm–Horsfall urinary glycoprotein. The chemical composition

1970 ◽  
Vol 120 (2) ◽  
pp. 417-424 ◽  
Author(s):  
A. P. Fletcher ◽  
A. Neuberger ◽  
Wendy A. Ratcliffe
Keyword(s):  
Amino Acid ◽  
Performic Acid ◽  
Acid Oxidation ◽  
Amino Acid Residues ◽  
Carbohydrate Composition ◽  
Thiol Groups ◽  
Terminal Amino Acid ◽  
Cystine Content ◽  
Terminal Amino ◽  
Urinary Glycoprotein

1. A revised amino acid and carbohydrate composition of human Tamm–Horsfall glycoprotein is presented. 2. No significant differences were obtained in the amino acid composition of Tamm–Horsfall glycoprotein isolated from patients with cystic fibrosis. 3. The glycoprotein was shown to possess a high half-cystine content of 1 per 11–12 amino acid residues, which has been confirmed by performic acid oxidation and S-alkylation with iodoacetate and iodoacetamide. No thiol groups were detected in the glycoprotein. 4. Treatment of the glycoprotein with 0.5m-sodium hydroxide at 4°C for 2 days did not release heterosaccharide material, which suggests that the predominant carbohydrate–protein linkages present are not of the O-glycosidic type. 5. No N-terminal amino acid was detected in the glycoprotein.

scholarly journals Isolation and amino-acid sequence of two inhibitors of serine proteinases, members of the squash inhibitor family, from Echinocystis lobata seeds.

1996 ◽  
Vol 43 (3) ◽  
pp. 507-513 ◽  
Author(s):  
D Stachowiak ◽  
A Polanowski ◽  
G Bieniarz ◽  
T Wilusz
Keyword(s):  
Amino Acid ◽  
Proteinase Inhibitors ◽  
Sequence Similarity ◽  
Serine Proteinase ◽  
Exchange Chromatography ◽  
Association Constants ◽  
Cathepsin G ◽  
Amino Acid Residues ◽  
Mature Seeds ◽  
Echinocystis Lobata

Two serine proteinase inhibitors (ELTI I and ELTI II) have been isolated from mature seeds of Echinocystis lobata by ammonium sulfate fractionation, methanol precipitation, ion exchange chromatography, affinity chromatography on immobilized anhydrotrypsin and HPLC. ELTI I and ELTI II consist of 33 and 29 amino-acid residues, respectively. The primary structures of these inhibitors are as follows: ELTI I KEEQRVCPRILMRCKRDSDCLAQCTCQQSGFCG ELTI II RVCPRILMRCKRDSDCLAQCTCQQSGFCG The inhibitors show sequence similarity with the squash inhibitor family. ELTI I differs from ELTI II only by the presence of the NH2-terminal tetrapeptide Lys-Glu-Glu-Gln. The association constants (Ka) of ELTI I and ELTI II with bovine-trypsin were determined to be 6.6 x 10(10) M-1, and 3.1 x 10(11) M-1, whereas the association constants of these inhibitors with cathepsin G were 1.2 x 10(7) M-1, and 1.1 x 10(7) M-1, respectively.

Purification of large peptides using Chemoselective tags

1999 ◽  
Author(s):  
Paolo Mascagni
Keyword(s):  
Amino Acid ◽  
Synthetic Peptides ◽  
Solid Phase ◽  
Solid Phase Peptide Synthesis ◽  
Exchange Chromatography ◽  
Target Sequence ◽  
Coupling Reactions ◽  
Amino Acid Residues ◽  
Quaternary Structures ◽  
Specific Stationary Phase

In solid phase peptide synthesis (SPPS), deletion sequences are generated at each addition of amino acid due to non-quantitative coupling reactions. Their concentration increases exponentially with the length of the peptide chain, and after many cycles not only do they represent a large proportion of the crude preparation, but they can also exhibit physicochemical characteristics similar to the target sequence. Thus, these deletion-sequence contaminants present major problems for removal, or even detection. In general, purification of synthetic peptides by conventional chromatography is based on hydrophobicity differences (using RP-HPLC) and charge differences (using ion-exchange chromatography). For short sequences, the use of one or both techniques is in general sufficient to obtain a product with high purity. However, on increasing the number of amino acid residues, the peptide secondary and progressively tertiary and quaternary structures begin to play an important role and the conformation of the largest peptides can decisively affect their retention behaviour. Furthermore, very closely related impurities such as deletion sequences lacking one or few residues can be chromatographically indistinguishable from the target sequence. Therefore, purification of large synthetic peptides is a complex and time-consuming task that requires the use of several separation techniques with the inevitable dramatic reduction in yields of the final material. Permanent termination (capping) of unreacted chains using a large excess of an acylating agent after each coupling step prevents the formation of deletion sequences and generates N-truncated peptides. However, even under these more favourable conditions, separation of the target sequence from chromatographically similar N-capped polypeptides requires extensive purification. If the target sequence could be specifically and transiently labelled so that the resulting product were selectively recognized by a specific stationary phase, then separation from impurities should be facilitated. This chapter deals with such an approach and in particular with the purification of large polypeptides, assembled by solid phase strategy, using lipophilic and biotin-based 9-fluorenylmethoxycarbonyl (Fmoc) chromatographic probes. Assuming that the formation of deletion sequences is prevented by capping unreacted chains, a reciprocal strategy can be applied that involves functional protection of all polymer-supported peptide chains that are still growing, with a specially chosen affinity reagent or chromatographic probe.

scholarly journals The Primary Structure of Antithrombin-III

1977 ◽  
Author(s):  
T. E. Petersen ◽  
G. Dudek-Wojciechowska ◽  
L. Sottrup-Jensen ◽  
S. Magnusson
Keyword(s):  
Amino Acid ◽  
Antithrombin Iii ◽  
Factor Xa ◽  
Amino Acid Residues ◽  
Disulfide Bridges ◽  
Single Chain ◽  
Terminal Sequence ◽  
Chymotryptic Peptide ◽  
Sequence Region ◽  
Bovine Factor

Human antithrombin-III is a single-chain glycoprotein with three disulfide bridges and four prosthetic glucosamine-based oligosaccharide groups. The disulfide bridges have been established. In four fragments of 208, 168, 3 and 46 amino acid residues, resp. 415 of the appr. 425 residues have been sequenced. The four oligosaccharide groups are attached to four Asn-residues within a sequence region of 95 residues. No extensive sequence homology with the trypsin inhibitors has been observed. One chymotryptic peptide was found to be a substrate for bovine factor Xa, cleaving the arginyl bond in the sequence -Ile-Val-Ala-Glu-Gly-Arg-Asp-. A second peptide is cleaved by thrombin. It is not clear whether these sites are inhibitor sites in the native molecule. Other possible candidates for inhibitor sites are a -Val-Leu-Ile-Leu-Pro-Lys-Pro- sequence (similar to the sequence 40-48 of hirudin, which also includes a -Pro-Lys-Pro- sequence) and also the C-terminal sequence -Gly-Arg-Val-Ala-Asn-Pro-Cys-Val-Lys.

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