Purification of the delta toxin of Staphylococcus aureus

1970 ◽  
Vol 16 (8) ◽  
pp. 703-708 ◽  
Author(s):  
J. D. Caird ◽  
G. M. Wiseman
Keyword(s):  
Amino Acids ◽  
Staphylococcus Aureus ◽  
Amino Acid ◽  
Ammonium Sulphate ◽  
Deae Cellulose ◽  
Aureus Strain ◽  
Terminal Amino Acid ◽  
Terminal Amino ◽  
Two Peaks

An improved procedure for the purification of the delta toxin of Staphylococcus aureus strain E-delta has been devised which relies upon precipitation at pH 4.0 and further treatment with ammonium sulphate. A final step consists of passage twice through a column of DEAE-cellulose. Toxin obtained by this method appeared to be homogeneous on the basis of immunodiffusion and electrophoresis studies. However, two peaks with sedimentation coefficients of 2.8 S and 9.8 S were obtained when toxin was examined in the ultracentrifuge. Proline was identified as the N-terminal amino acid. No other N-terminal amino acids were detected in the purified toxin.

Studies on Nα-acylated proteins: The N-terminal sequences of two muscle enolases

1985 ◽  
Vol 5 (10-11) ◽  
pp. 847-854 ◽  
Author(s):  
Christopher C. Q. Chin ◽  
Finn Wold
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Ion Exchange ◽  
Coho Salmon ◽  
Standard Procedure ◽  
Exchange Chromatography ◽  
Rabbit Muscle ◽  
Terminal Amino Acid ◽  
Terminal Amino ◽  
Acylated Proteins

A standard procedure for the identification of the N-terminal amino acid in Nα-acylated proteins has been developed. After exhaustive proteolysis, the amino acids with blocked α-amino groups are separated from positively charged, free amino acids by ion exchange chromatography and subjected to digestion with acylase I. Amino acid analysis before and after the acylase treatment identifies the blocked N-terminal amino acid. A survey of acylamino acid substrates showed that acytase will liberate all the common amino acids except Asp, Cys or Pro from their N-acetyl- and N-butyryl derivatives, and will also catalyze the hydrolysis of N-formyl-Met and N-myristyl-Val. Thus, the procedure cannot identify acylated Asp, Cys or Pro, nor, because of the ion exchange step, Nα-acyl-derivatives of Arg, Lys or His. Whenever the protease treatment releases free acylamino acids, the remaining amino acids should be detected. When applied to several proteins, the procedure confirmed known N-terminal acylamino acids and identified acyl-Ser in enolases from chum and coho salmon muscle and in pyruvate kinase from rabbit muscle, and acyl-Thr in phosphofructokinase from rabbit muscle. The protease-acylase assay has been used to identify blocked peptides from CNBr- or protease-treated proteins. When such peptides were treated with 1n HCl at 110° for 10 min, sufficient yields of deacylated, mostly intact, peptide were obtained to permit direct automatic sequencing. The N-terminal sequences of rabbit muscle and coho salmon enolase were determined in this way and are compared to each other and to the sequence of yeast enolase.

scholarly journals Determination of Peptide Contact Points in the Human Angiotensin II Type I Receptor (AT1) with Photosensitive Analogs of Angiotensin II

1999 ◽  
Vol 13 (4) ◽  
pp. 578-586 ◽  
Author(s):  
Stéphane A. Laporte ◽  
Antony A. Boucard ◽  
Guy Servant ◽  
Gaétan Guillemette ◽  
Richard Leduc ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Angiotensin Ii ◽  
Transmembrane Domain ◽  
At1 Receptor ◽  
High Yield ◽  
Type I ◽  
Terminal Amino Acid ◽  
Contact Points ◽  
Terminal Amino

Abstract To identify ligand-binding domains of Angiotensin II (AngII) type 1 receptor (AT1), two different radiolabeled photoreactive AngII analogs were prepared by replacing either the first or the last amino acid of the octapeptide by p-benzoyl-l-phenylalanine (Bpa). High yield, specific labeling of the AT1 receptor was obtained with the 125I-[Sar1,Bpa8]AngII analog. Digestion of the covalent 125I-[Sar1,Bpa8]AngII-AT1 complex with V8 protease generated two major fragments of 15.8 kDa and 17.8 kDa, as determined by SDS-PAGE. Treatment of the[ Sar1,Bpa8]AngII-AT1 complex with cyanogen bromide produced a major fragment of 7.5 kDa which, upon further digestion with endoproteinase Lys-C, generated a fragment of 3.6 kDa. Since the 7.5-kDa fragment was sensitive to hydrolysis by 2-nitro-5-thiocyanobenzoic acid, we circumscribed the labeling site of 125I-[Sar1,Bpa8]AngII within amino acids 285 and 295 of the AT1 receptor. When the AT1 receptor was photolabeled with 125I-[Bpa1]AngII, a poor incorporation yield was obtained. Cleavage of the labeled receptor with endoproteinase Lys-C produced a glycopeptide of 31 kDa, which upon deglycosylation showed an apparent molecular mass of 7.5 kDa, delimiting the labeling site of 125I-[Bpa1]AngII within amino acids 147 and 199 of the AT1 receptor. CNBr digestion of the hAT1 I165M mutant receptor narrowed down the labeling site to the fragment 166–199. Taken together, these results indicate that the seventh transmembrane domain of the AT1 receptor interacts strongly with the C-terminal amino acid of[ Sar1, Bpa8]AngII, whereas the N-terminal amino acid of[ Bpa1]AngII interacts with the second extracellular loop of the AT1 receptor.

Nuclear Magnetic Resonance Studies of Phenylthiohydantoin Amino Acids

1975 ◽  
Vol 53 (9) ◽  
pp. 1005-1009 ◽  
Author(s):  
C. S. Tsai ◽  
N. L. Fraser ◽  
H. Avdovich ◽  
J. P. Farant
Keyword(s):  
Amino Acids ◽  
Nuclear Magnetic Resonance ◽  
Amino Acid ◽  
Magnetic Resonance ◽  
Diagnostic Purpose ◽  
Edman Degradation ◽  
Terminal Amino Acid ◽  
Magnetic Resonance Studies ◽  
Terminal Amino ◽  
Derivatives Of

Proton magnetic spectra of 3-phenyl-2-thiohydantoin derivatives of common amino acids in deuterated dimethyl sulfoxide were recorded. Spectral data pertaining to characteristic protons for diagnostic purpose were compiled. Their application to the N-terminal amino acid analysis of peptide by Edman degradation was examined.

scholarly journals Phosphorus (V) porphyrin diaxially substituted with Leu-enkephalin

2004 ◽  
Vol 2 (3) ◽  
pp. 446-455 ◽  
Author(s):  
Mariusz Gajewski ◽  
Leszek Czuchajowski
Keyword(s):  
Amino Acids ◽  
Photodynamic Therapy ◽  
Amino Acid ◽  
Biologically Active ◽  
Solution Phase ◽  
Terminal Amino Acid ◽  
Leucine Enkephalin ◽  
Potential Applications ◽  
Biologically Active Peptide ◽  
Terminal Amino

AbstractSynthesis of the first phosphorus (V) porphyrin-peptide conjugate was successfully accomplished. A biologically active peptide, leucine enkephalin, was constructed on the phosphorus atom of the 5,10,15,20-meso-tetraphenylporphinato dichlorophosphorus (V) chloride. The method involved solution phase peptide synthesis. The first C-terminal amino acid in the sequence of the peptide was axially attached to the porphyrin through a linker, 3-aminopropanol, and the remainder of leucine enkephalin was synthesized by subsequent additions of amino acids. Leucine enkephalin-P(V) porphyrin conjugate represents a new group of compounds, and its synthesis broadens potential applications of P(V) porphyrine, e.g. in photodynamic therapy.

NUCLEOTIDE SEQUENCE OF THE STAPHYLOCOAGULASE GENE FROM STAPHYLOCOCCUS AUREUS STRAIN BB

1987 ◽  
Author(s):  
S Kaida ◽  
T Miyata ◽  
S Kawabata ◽  
T Morita ◽  
Y Yoshizawa ◽  
...  
Keyword(s):  
Amino Acids ◽  
Staphylococcus Aureus ◽  
Amino Acid ◽  
Tandem Repeats ◽  
Molar Ratio ◽  
Clotting Factor ◽  
Aureus Strain ◽  
Activation Process ◽  
Coding Region ◽  
Flanking Region

Staphylocoagulase (SC) is a secretary protein produced by several strains of Staphylococcus aureus (S. aureus). This protein forms a molecular complex ("staphylothrombin") with human prothrombin in a molar ratio of 1:1. The complex displays the ability to clot fibrinogen and to hydrolyze the synthetic tripeptide substrates for α-thrombin. The formation of staphylothrombin does not require proteolytic cleavage of the prothrombin molecule, and this mechanism differs markedly from the activation process by either blood-clotting factor Xa or snake venom procoagulant.In the present studies, a pAT153 library containing partial Mbo I-digested DNA prepared from aureus strain BB has been screened with a fibrin gel formation method. The identity of these clones with SC was confirmed by DNA sequence analysis and by comparison of the derived amino acid sequence with that determined for the purified SC protein. One of the positive colonies was isolated and 3.1 Kb of the insert DNA was determined by the dideoxy chain termination method. The results indicated that the insert DNA consists of 148 bp 5' flanking region, protein coding region of 715 amino acids and 746 bp 3' flanking region, and that SC from strain BB is synthesized as a precursor with a signal peptide of 26 amino acids. Thus, the mature form was composed of 689 amino acids with a molecular weight of 77,337- The NH2-terminal sequence (324 amino acids) of SC isolated from S. aureus strain 213 (S. Kawabata et al. (1986): J. Biol. Chem. 261, 527-531) was compared with that of SC derived from strain BB. The sequence homology between them was found to show 57 %. It was also found that SC derived from strain BB was composed of 8 tandem repeats (27 amino acid residues in length) in the COOH-terminal region, although their functions are not known.

scholarly journals o-Quinones formed in plant extracts. Their reactions with amino acids and peptides

1969 ◽  
Vol 112 (5) ◽  
pp. 609-616 ◽  
Author(s):  
W. S. Pierpoint
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Plant Extracts ◽  
Thiol Group ◽  
Amino Group ◽  
Amino Groups ◽  
Terminal Amino Acid ◽  
Secondary Reactions ◽  
Terminal Amino ◽  
Group 5

1. The reactions of amino acids and peptides with the o-quinones produced by the enzymic oxidation of chlorogenic acid and caffeic acid have been studied manometrically and spectrophotometrically. 2. Amino acids, except lysine and cysteine, react primarily through their α-amino groups to give red or brown products. These reactions, which compete with the polymerization of the quinones, are followed by secondary reactions that may absorb oxygen and give products with other colours. 3. The ∈-amino group of lysine reacts with the o-quinones in a similar way. The thiol group of cysteine reacts with the quinones, without absorbing oxygen, giving colourless products. 4. Peptides containing cysteine react with the o-quinones through their thiol group. 5. Other peptides, such as glycyl-leucine and leucylglycine, react primarily through their α-amino group and the overall reaction resembles that of the N-terminal amino acid except that it is quicker. 6. With some peptides, the secondary reactions differ from those that occur between the o-quinones and the N-terminal amino acids. The colours produced from carnosine resemble those produced from histidine rather than those from β-alanine, and the reactions of prolylalanine with o-quinones are more complex than those of proline.

scholarly journals Studies on the high-sulphur proteins of reduced mohair. The isolation and amino acid sequence of protein scmkb-m1.2

1975 ◽  
Vol 145 (3) ◽  
pp. 459-467 ◽  
Author(s):  
D Parris ◽  
L S Swart
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Acid Hydrolysis ◽  
Paper Chromatography ◽  
Deae Cellulose ◽  
Edman Degradation ◽  
Sequence Determination ◽  
Terminal Amino Acid ◽  
Partial Acid Hydrolysis ◽  
Terminal Amino

The complete amino acid sequence of mohair protein, SCMKB-M1.2 (97 residues), was determined. The protein was isolated from reduced and carboxymethylated mohair by chromatography on DEAE-cellulose phosphate. Peptides for sequence determination were obtained by digestion with trypsin, pepsin, chymotrypsin, thermolysin and papain, and were fractionated by DEAE-cellulose chromatography, paper chromatography and electrophoresis. The sequence of the peptides were determined by the Edman degradation method (by use of both the Beckman Sequence and a non-automatic procedure), and by partial acid hydrolysis. The protein is closely homologous to wool protein SCMKB-IIIB2, and also contains acetylated alanine as N-terminal amino acid.

scholarly journals Impact of C-terminal amino acid composition on protein expression in bacteria

2019 ◽  
Author(s):  
Marc Weber ◽  
Raul Burgos ◽  
Eva Yus ◽  
Jae-Seong Yang ◽  
Maria Lluch-Senar ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Protein Expression ◽  
Translation Termination ◽  
Terminal Sequence ◽  
Bacterial Taxonomy ◽  
Terminal Amino Acid ◽  
Protein Levels ◽  
Terminal Amino ◽  
The Impact

AbstractThe C-terminal sequence of a protein is involved in processes such as efficiency of translation termination and protein degradation. However, the general relationship between features of this C-terminal sequence and levels of protein expression remains unknown. Here, we identified C-terminal amino acid biases that are ubiquitous across the bacterial taxonomy (1582 genomes). We showed that the frequency is higher for positively charged amino acids (lysine, arginine) while hydrophobic amino acids and threonine are lower. In highly abundant proteins, the C-terminal residue is more conserved. We then studied the impact of C-terminal composition on protein levels in a library of M. pneumoniae mutants, covering all possible combinations of the two last codons. We found that charged and polar residues, in particular lysine, led to higher expression, while hydrophobic and aromatic residues led to lower expression, with a difference in protein levels up to 4-fold. Our results demonstrate that the identity of the last amino acids has a strong influence on protein expression levels and is under selective pressure in highly expressed proteins.

scholarly journals Helix capping propensities in peptides parallel those in proteins

1993 ◽  
Vol 90 (23) ◽  
pp. 11332-11336 ◽  
Author(s):  
A Chakrabartty ◽  
A J Doig ◽  
R L Baldwin
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Rank Order ◽  
Alpha Helix ◽  
Asymmetric Distribution ◽  
Minor Effect ◽  
Terminal Amino Acid ◽  
Helix Stability ◽  
Acetyl Group ◽  
Terminal Amino

Helix content of peptides with various uncharged nonaromatic amino acids at either the N-terminal or C-terminal position has been determined. The choice of N-terminal amino acid has a major effect on helix stability: asparagine is the best, glycine is very good, and glutamine is the worst helix-stabilizing amino acid at this position. The rank order of helix stabilization parallels the frequencies of these amino acids at the N-terminal boundary (N-cap) position of helices in proteins found by Richardson and Richardson [Richardson, J. S. & Richardson, D. C. (1988) Science 240, 1648-1652], and the N-terminal amino acid in a peptide composed of helix-forming amino acids may be considered as the N-cap residue. The choice of C-terminal amino acid has only a minor effect on helix stability. N-capping interactions may be responsible for the asymmetric distribution of helix content within a given peptide found by various workers. An acetyl group on the N-terminal alpha-amino function cancels the N-cap effect and the acetyl group is equivalent to N-terminal asparagine in an unacetylated peptide. Our results demonstrate a close relationship between the mechanisms of alpha-helix formation in peptides and in proteins.

scholarly journals Staphylococcus aureus causing osteomyelitis binds to a nonapeptide sequence in bone sialoprotein

1997 ◽  
Vol 327 (3) ◽  
pp. 825-829 ◽  
Author(s):  
Cecilia RYDÉN ◽  
Hui-Shan TUNG ◽  
Victor NIKOLAEV ◽  
Åke ENGSTRÖM ◽  
Åke OLDBERG
Keyword(s):  
Amino Acids ◽  
Staphylococcus Aureus ◽  
Extracellular Matrix ◽  
Synthetic Peptides ◽  
Core Protein ◽  
Bone Sialoprotein ◽  
Amino Acid Residues ◽  
Terminal Part ◽  
Terminal Amino Acid ◽  
Terminal Amino

Bone sialoprotein is a glycoprotein of the bone and dentine extracellular matrix. This protein consists of 320 amino acids, of which 25% are glutamic and aspartic acid residues. Sialic acid, containing oligosaccharides and tyrosine sulphate residues, supplies additional polyanionic properties. Staphylococcal cells, isolated from patients suffering from infection of bone tissue, bind the bone-derived sialoprotein, an interaction which is specifically inhibited by the recombinant bone sialoprotein core protein. We have previously shown that the 150 N-terminal amino acid residues of bone sialoprotein are responsible for the binding to staphylococcal cells. By using recombinant deleted variants of bone sialoprotein and synthetic peptides, we have now localized the staphylococcal binding site to less than 10 residues within the N-terminal part of the protein.

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