The Amino Acid Sequence of Streptomyces griseus Trypsin. II The Tryptic Peptides

1974 ◽  
Vol 52 (5) ◽  
pp. 382-392 ◽  
Author(s):  
L. Jurášek ◽  
L. B. Smillie
Keyword(s):  
Soluble Fraction ◽  
Streptomyces Griseus ◽  
Paper Electrophoresis ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Insoluble Residue ◽  
Limited Extent ◽  
Terminal Sequence ◽  
Tryptic Peptides ◽  
Final Purification

Streptomyces griseus trypsin (S.G.T.) isolated from pronase was reduced, aminoethylated, and digested with trypsin. The soluble peptides were recovered and the insoluble residue redigested with chymotrypsin. Following recovery of the soluble fraction, the insoluble portion was in turn digested with α-lytic protease of Myxobacter 495. The three groups of soluble peptides were separately subjected to ion-exchange chromatography on the Technicon peptide analyzer and to final purification by high-voltage paper electrophoresis. Sequence analysis by the dansyl-Edman procedure provided unique sequences from the soluble tryptic peptides accounting for 65% of the S.G.T. molecule. Peptides obtained from the redigests of the insoluble residues accounted for an additional 20%.Tryptic digestion of dansylated S.G.T. yielded a unique α-NH2 dansylated peptide whose composition showed it to be the same as the NH2-terminal sequence previously postulated for this enzyme. The tryptic peptides isolated in this work have provided overlaps for many of the previously sequenced peptic peptides. Three continuous sequences of 49, 36, and 28 residues have been elucidated.Evidence has also been obtained that the NH2-terminal valine residue was, to a limited extent, aminoethylated during reaction of the reduced protein with ethylenimine.

The Amino Acid Sequence of Streptomyces griseus Trypsin. I. The Peptic Peptides

1973 ◽  
Vol 51 (7) ◽  
pp. 1077-1088 ◽  
Author(s):  
L. Jurášek ◽  
L. B. Smillie
Keyword(s):  
Amino Acid ◽  
Serine Proteases ◽  
System Analysis ◽  
Streptomyces Griseus ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Amino Acid Sequences ◽  
Terminal Sequence ◽  
Unique Amino Acid ◽  
High Voltage Electrophoresis

Streptomyces griseus trypsin (S.G.T.) isolated from pronase was digested with pepsin. The peptic peptides were isolated by high-voltage electrophoresis on paper and ion-exchange chromatography on Chromobead P resin using the Technicon autoanalyzer system. Analysis of the purified peptides provides 28 unique amino acid sequences accounting for approximately 95% of the S.G.T. molecule. A portion of the residues not accounted for can be ascribed to free leucine, phenylalanine, tyrosine, and tryptophan present in the peptic digest. The NH2-terminal sequence of S.G.T. was shown to be Val–Val–Gly–Gly–Thr–Arg–Ala–Ala–Gln–Gly–Glu–Phe and is highly homologous with NH2-terminal sequences of other Asp–Ser–Gly serine proteases.

Zur Totalsynthese des Human-Big-Gastrins I und seines 32-Leucin-Analogons (Vorläufige Mitteilung) / Total Synthesis of Human Big Gastrin I and the 32-Leucine Analogue (Preliminary Communication)

1977 ◽  
Vol 32 (7-8) ◽  
pp. 495-506 ◽  
Author(s):  
E. Wünsch ◽  
G. Wendlberger ◽  
A. Hallett ◽  
E. Jaeger ◽  
S. Knof ◽  
...  
Keyword(s):  
Total Synthesis ◽  
Building Blocks ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Protecting Groups ◽  
Side Chain ◽  
Tertiary Alcohols ◽  
Synthetic Product ◽  
Preliminary Communication ◽  
Final Purification

A new total synthesis of the tetratriacontapeptide amide corresponding to the proposed primary structure of human big gastrin I is described. The synthetic route was based on the preparation of six suitably protected fragments, related to sequence 28 - 34, 23 - 27, 21 - 22, 15-20, 9 - 14, and 1 - 8, to be used as building blocks for the total synthesis. The protecting groups were selected according to the Schwyzer-Wünsch strategy of maximum side chain protection based on tertiary alcohols, also for the imidazol function of histidine. Subsequent assembly of the six fragments by three different pathways using the highly efficient Wünsch-Weygand condensation procedure to ensure minimum racemization, followed by deprotection of the synthetic products via exposure to trifluoroacetic acid and final purification by ion-exchange chromatography on DEAE-Sephadex A-25 and partition chromatography on Sephadex G-25, led to human big gastrin I, homogeneous within the limits of the analytical methods used. The biological activity of the synthetic product proved to be 50 percent higher than that of human little gastrin I. The 32-leucine analogue of human big gastrin I was prepared in the same way.

scholarly journals Confirmation of d-aspartic acid in the novel dipeptide β-aspartylglycine isolated from tissue extract of Aplysia kurodai

1989 ◽  
Vol 263 (2) ◽  
pp. 617-620 ◽  
Author(s):  
M Sato ◽  
T Yamaguchi ◽  
N Kanno ◽  
Y Sato
Keyword(s):  
Aspartic Acid ◽  
Body Wall ◽  
Optical Resolution ◽  
Paper Electrophoresis ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Optical Rotatory Dispersion ◽  
The Novel ◽  
Reactive Compound ◽  
High Concentrations

A novel o-phthalaldehyde-reactive compound was found in the h.p.l.c. chromatogram of Aplysia kurodai extract. This compound was isolated by ion-exchange chromatography and preparative high-voltage paper electrophoresis. It was shown by optical-rotatory-dispersion spectrum and optical-resolution h.p.l.c. analysis that this compound consisted of equimolar amounts of D-aspartic acid and glycine. This compound resisted cleavage in the Edman reaction. This peptide was inferred to be beta-D-aspartylglycine, and this was confirmed by synthesis. beta-D-Aspartylglycine was detected in all tissues of Aplysia kurodai, with especially high concentrations in body wall (skin and muscle) and gill.

scholarly journals The specificity of proteinases from Streptomyces griseus (pronase)

1970 ◽  
Vol 116 (1) ◽  
pp. 19-25 ◽  
Author(s):  
Moshe Trop ◽  
Yehudith Birk
Keyword(s):  
Streptomyces Griseus ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Considerable Extent ◽  
Small Peptides ◽  
Diisopropyl Phosphorofluoridate ◽  
Naturally Occurring ◽  
Bovine Trypsin ◽  
Wide Range ◽  
Pancreatic Trypsin Inhibitor

Purification of pronase by ion-exchange chromatography gave four proteolytically active fractions. Fraction A2 contained an endopeptidase that attacks poly l-valine. Fraction B contained an endopeptidase, an aminopeptidase and carboxypeptidases. The activities against hippuryl-L-arginine and hippuryl-L-phenylalanine could be inhibited to a considerable extent by di-isopropyl phosphorofluoridate and by EDTA. Fraction C contained an endopeptidase resembling bovine trypsin. The pure enzyme was completely inactivated by di-isopropyl phosphorofluoridate and pancreatic trypsin inhibitor and to about 90% by other naturally occurring trypsin inhibitors. Fraction D contained an apparently homogeneous endopeptidase, inhibited by diisopropyl phosphorofluoridate, that adsorbed to and hydrolysed elastin. The activity of all these fractions was tested qualitatively against a wide range of small peptides and synthetic substrates.

Thermolytic digest of chicken pepsin

1981 ◽  
Vol 46 (8) ◽  
pp. 1994-2004 ◽  
Author(s):  
Miroslav Baudyš ◽  
Vladimír Kostka ◽  
Helena Keilová
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Linear Structure ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Terminal Amino Acid ◽  
Terminal Amino ◽  
Terminal Amino Acid Sequence ◽  
High Voltage Electrophoresis ◽  
Final Purification

Chicken pepsin prepared by the activation of pepsinogen was digested with thermolysin. The thermolytic digest was fractionated by chromatography on Sephadex G-25 fine. Certain fractions were subsequently subjected to ion exchange chromatography on Dowex 50-X2. The final purification was effected by paper chromatography and high voltage electrophoresis. By these procedures a series of homogeneous peptides was obtained; of the latter 54 nonoverlapping (save for a few exceptions) peptides are described in this paper. These peptides in addition to the thermolytic peptides reported before represent 80% of the linear structure of the whole molecule. The N-terminal amino acid sequence of chicken pepsin is discussed from the viewpoint of the recent data obtained by the analysis of the thermolytic digest.

LOCALIZATION CF FIBRIN POLYMERIZATION SITES BY PHOTQAFFINITY LABELING

1987 ◽  
Author(s):  
C Kuyas ◽  
H Sigrist ◽  
P W Straub
Keyword(s):  
Binding Site ◽  
Polypeptide Chain ◽  
Reversed Phase ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Fibrinopeptide A ◽  
Terminal Sequence ◽  
Selective Labeling ◽  
Fibrin Polymerization ◽  
Polypeptide Chains

Fibrin polymerization is competitively inhibited by the te-trapeptide GlyProArgPro. This peptide corresponds to the N-terminal sequence of the fibrin α-polypeptide chain, which is exposed upon release of fibrinopeptide A by thrombin. A binding site for GlyProArgPro was suggested to be located in the C-terminal end of the 411 amino acids containing γ-chain (Varadi and Scheraga, Biochemistry, 25, 519, 1986). In order to characterize the polymerization domain, GlyProArgProLys-azidoazobenzene, a photoactivable derivative of GlyProArgPro was synthesized. Photoaffinity label was bound to fibrinogen in the dark and photolysis was carried out at 0°C. After reduction and S-carboxymethy-lation of the photoaffinity labeled fibrinogen, the polypeptide chains (Aα, Bβ,γ ) were separated by ion-exchange chromatography. Photolabel binding was monitored imnunologically with anti-azo-benzene antibodies (ELISA, Western blot). Selective labeling of the γ-chain was observed. Labeled γ-chains were further digested with CNBr, and the resulting fragments were separated by reversed phase HPIC, immunologically characterized and identified by Edman degradation. GlyProArgProLys-azidoazobenzene was incorporated in the 18 kD CNBr-fragment (γ95-264). The CNBr-fragments arising from C-terminal end of the γ-chain were not labeled.Our results indicate that the binding site of GlyProArgPro is localized exclusively on γ-chain, within the sequence γ95-264.

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