scholarly journals Antigenicity of proteinases from Streptomyces griseus (pronase)

1970 ◽  
Vol 119 (2) ◽  
pp. 339-342 ◽  
Author(s):  
M. Trop ◽  
R. R. Avtalion ◽  
Z. Malik ◽  
A. Pinsky
Keyword(s):  
Amino Acid ◽  
Active Sites ◽  
Streptomyces Griseus ◽  
Ion Exchange Chromatography ◽  
Enzymic Activity ◽  
Exchange Chromatography ◽  
Soya Bean ◽  
Catalytic Site ◽  
Antigenic Sites ◽  
Bovine Trypsin

The five pronase fractions, A1, A2, B, C (trypsin-like), and D (elastolytic), obtained by ion-exchange chromatography, were found to be antigenically distinct. Antibodies to pronase inhibited the enzymic activity of each of the enzyme fractions. Pronase trypsin and bovine trypsin, although resembling each other in enzymic activity and in amino acid sequence around their active sites, did not cross-react antigenically with, nor was their enzymic activity inhibited by, the respective homologous antibodies. Inactivation of pronase trypsin by complexing with soya-bean inhibitor AA, was not associated with a decrease in capacity to precipitate with its antibody. It is assumed that the antigenic sites are located far enough from the catalytic site of the enzyme to allow it to precipitate immunologically even when the catalytic site was blocked.

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.

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.

Quantitative Chromatographic Studies on Urinary Amino Acid Excretion

1968 ◽  
Vol 14 (1) ◽  
pp. 12-21 ◽  
Author(s):  
Richard P Geer ◽  
Richard K Hantman ◽  
Cyrus V Swett
Keyword(s):  
Amino Acid ◽  
Ion Exchange ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Acid Excretion ◽  
Amino Acid Nitrogen ◽  
Urinary Amino

Abstract Amino acid excretions of 82 individuals were quantitatively determined by ion-exchange chromatography. The results are expressed as µmoles amino acid per day, divided by milligrams α-amino acid nitrogen per day. This index is independent of age and provides a more useful method of representation than those presently employed in the literature.

Effect of Deproteinating Agents (Picric Acid and Sulfosalicylic Acid) on Analysis for Free Amino Acids in Swine Blood and Tissue

1969 ◽  
Vol 52 (5) ◽  
pp. 981-984 ◽  
Author(s):  
J E Knipfel ◽  
D A Christensen ◽  
B D Owen
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Ion Exchange ◽  
Free Amino ◽  
Biological Fluids ◽  
Picric Acid ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Sulfosalicylic Acid ◽  
Coefficients Of Variation

Abstract Amino acid analyses were performed on samples of blood, liver tissue, loin muscle, and ham muscle by ion exchange chromatography after deproteination of the samples with picric acid or sulfosalicylic acid (SSA). Resolution of threonine and serine from the ion exchange column was poor when SSA was used as the deproteinating agent. Twelve of sixteen amino acids were higher (P < 0.05) in serum deproteinated with picric acid as compared to concentrations determined after SSA deproteination. Amino acid values for ham muscle tended to be higher after deproteination with picric acid; however, with liver and loin muscle samples, the values were somewhat higher after SSA deproteination. In both serum and tissue analyses, coefficients of variation were lower for niGSt amino acids when picric acid was utilized as the deproteinating agent. The latter observation, in particular, suggests that picric acid is preferable to SSA as a deproteinating agent before amino acid analyses of biological fluids. Standardization of methods of deproteination is needed to allow meaningful comparisons of data.

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.

Isolation of a Crosslinked Peptide from Hitman Fibrin and Development of a Radioimmunoassay

1979 ◽  
Author(s):  
R. Canfield ◽  
B. Lahiri ◽  
R. D’Alisa ◽  
V. Butler ◽  
H. Nossel ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Gel Filtration ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Factor Xiiia ◽  
Cross Linking ◽  
Edman Degradation ◽  
Cnbr Cleavage ◽  
Normal Hemostasis

Factor XIIIa introduces up to six crossllnklng bonds per molecule of fibrin; the bonds between the γ chains on adjacent fibrin molecules form most rapidly. Since cross linking is essential for normal hemostasis and is likely to be important in tests to detect thrombosis, we have attempted to develop a radioimmunoassay that exhibits specificity for the γ chain crosslinks. The immunogen consisted of a 54 amino acid, crosslinked peptide, isolated from purified human γ-γ chains following CNBr cleavage, gel filtration on Sephadex G-50 and ion-exchange chromatography on SP-Sephadex. Amino acid analysis and Edman degradation through step 24 confirmed the sequence of Chen and Doolittle (Biochemistry 10: i486, 1971), and the two degradation steps that failed to liberate the expected PTH-amino acids matched the reported location of the Gin-Lys crosslinks. Antisera were obtained against this immunogen coupled either to bovine thyroglobulin or bovine serum albumin. All antisera elicited bound immunogen that was covalently coupled to ribonuclease radiolabeled with 125I as a tracer. The unlabeled γ-γ, crosslinked peptide effectively inhibited binding (0.03-0.08 picomoles for 50% inhibition), while with some antisera up to 500 times more of the 27 amino acid γ monomer peptide was required for the same degree of inhibition. Fibrinogen and fragment D also were poor Inhibitors. The results Indicate that it is possible by radioimmunoassay to distinguish the COOH-termlnal region of the γ-γ dlmer from that of uncrosslinked molecules.

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.

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