Characterization of group A streptococcal M-proteins purified by two methods

1976 ◽  
Vol 22 (8) ◽  
pp. 1072-1082
Author(s):  
David C. Straus ◽  
Charles F. Lange
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Molecular Size ◽  
M Protein ◽  
M Proteins ◽  
Group A ◽  
Terminal Amino ◽  
High Concentrations

Ten different group A streptococcal M-protein preparations purified by trichloroacetic acid precipitation and three M-protein preparations purified by cellulose chromatography were examined by SDS and polyacrylamide gel electrophoresis, and analyzed for amino acid composition and N-terminal amino acids. Fingerprinting (both tryptic and chymotryptic) was performed on the cellulose-purified preparations of M1, M12, and M29 proteins which showed these proteins to be structurally related. Trypsin produced maps with 37 to 42 peptides, whereas chymotrypsin digestion resulted in 8 to 12 peptides, depending on the M-type. Sequencing was performed on the M12 protein and tentative identification of nine N-terminal amino acids was made. Molecular weights of the cellulose and TCA-purified M-proteins were determined by SDS gel electrophoresis and chromatography on G-200 Sephadex, with comparable results, indicating molecular size of at least 23 000. The amino acid analyses of the 10 TCA-purified proteins followed the patterns established for M-proteins, with high concentrations of lysine, aspartic acid, glutamic acid, alanine, and leucine. All 10 proteins had L-alanine as their N-terminal amino acid. Evidence for a one way cross-reaction between type 1 and type 29 streptococci was also found.

The Reaction between Prothombin and Staphylocoagulase

1975 ◽  
Author(s):  
H. C. Hemker ◽  
A. D. Muller ◽  
B. M. Bas
Keyword(s):  
Amino Acids ◽  
Molecular Weight ◽  
Amino Acid ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Limited Proteolysis ◽  
Molecular Weights ◽  
Active Reaction ◽  
Stoichiometric Reaction ◽  
Terminal Amino

The reaction between prothrombin and staphylocoagulase is studied.a. Optimal amounts of the active reaction product (coagulase-thrombin) are found when equimolar amounts of prothrombin and staphylocoagulase are added together.b. The molecular weight of coagulase-thrombin equals the sum of the molecular weights of staphylocoagulase and prothrombin, both when estimated by gelfiltration and by SDS-polyacrylamide gel electrophoresis.c. The amino acid composition of coagulase-thrombin is not in contrast with the sum of the amino acid compositions of staphylocoagulase and prothrombin.d. In a preparation of coagulase-thrombin the N-terminal amino acids are those of prothrombin (alanine) and staphylocoagulase (aspartic acid).e. An antibody against coagulase-thrombin precipitates both prothrombin and staphylocoagulase but not thrombin.f. It is concluded that the thrombin acitivity in coagulase-thrombin is a result from a stoichiometric reaction between one molecule of prothrombin and one molecule of staphylocoagulase and that limited proteolysis does not play a role in this mechanism.

scholarly journals Purification and characterization of a labile rat glutathione transferase of the Mu class

1989 ◽  
Vol 260 (3) ◽  
pp. 789-793 ◽  
Author(s):  
A Kispert ◽  
D J Meyer ◽  
E Lalor ◽  
B Coles ◽  
B Ketterer
Keyword(s):  
Amino Acid ◽  
Gel Electrophoresis ◽  
Glutathione Transferase ◽  
Polyacrylamide Gel Electrophoresis ◽  
Amino Acid Residues ◽  
Purification And Characterization ◽  
Terminal Amino Acid ◽  
Terminal Amino ◽  
Cnbr Cleavage

A labile GSH transferase homodimer termed 11-11 was purified from rat testis by GSH-agarose affinity chromatography followed by anion-exchange f.p.l.c. The enzyme is unstable in the absence of thiol(s) and has relatively low affinity for both 1-chloro-2,4-dinitrobenzene (Km 4.4 mM) and GSH (Km(app.) 4.4mM). Its mobility on SDS/polyacrylamide-gel electrophoresis is slightly less than that of subunits 3 and 4 and its pI is 5.2. Subunit 11 has a blocked N-terminal amino acid residue, but after CNBr cleavage fragments accounting for 113 amino acid residues were sequenced and showed 65% homology with corresponding sequences in subunit 4, indicating that it is a member of the Mu family. GSH transferase 11 is a major isoenzyme in testis, epididymis, prostate and brain and present at lower concentrations in other tissues.

Production, Purification, and Characterization of Micrococcin GO5, a Bacteriocin Produced by Micrococcus sp. GO5 Isolated from Kimchi

2005 ◽  
Vol 68 (1) ◽  
pp. 157-163 ◽  
Author(s):  
MI-HEE KIM ◽  
YOON-JUNG KONG ◽  
HONG BAEK ◽  
HYUNG-HWAN HYUN
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Broad Spectrum ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Molecular Size ◽  
High Heat ◽  
Terminal Amino Acid ◽  
Terminal Amino ◽  
Terminal Amino Acid Sequence

Strain GO5, a bacteriocin-producing bacterium, was isolated from green onion kimchi and identified as Micrococcus sp. The bacteriocin, micrococcin GO5, displayed a broad spectrum of inhibitory activity against a variety of pathogenic and nonpathogenic microorganisms, as tested by the spot-on-lawn method; its activity spectrum was almost identical to that of nisin. Micrococcin GO5 was inactivated by trypsin (whereas nisin was not) and was completely stable at 100°C for 30 min and in the pH range of 2.0 to 7.0. Micrococcin GO5 exhibited a typical mode of bactericidal activity against Micrococcus flavus ATCC 10240. It was purified to homogeneity through ammonium sulfate precipitation, ultrafiltration, and CM-Sepharose column chromatography. The molecular mass of micrococcin GO5 was estimated to be about 5.0 kDa by tricine–sodium dodecyl sulfate–polyacrylamide gel electrophoresis and in situ activity assay with the indicator organism. The amino acid sequence of micrococcin GO5 lacks lanthionine and β-methyllanthionine and is rich in hydrophobic amino acids and glycine, providing the basis for the high heat stability of this bacteriocin. The N-terminal amino acid sequence of micrococcin GO5 is Lys-Lys-Ser-Phe-Cys-Gln-Lys, and no homology to bacteriocins reported previously was observed in the amino acid composition or N-terminal amino acid sequence. Based on the physicochemical properties, small molecular size, and inhibition of Listeria monocytogenes, micrococcin GO5 has been placed with the class II bacteriocins, but its broad spectrum of activity differs from that of other bacteriocins in this class.

Radioiodinated Human Fibrinogen. In Vitro Effects of Increasing Iodination

1975 ◽  
Author(s):  
B. E. Ly ◽  
P. Kierulf
Keyword(s):  
Amino Acid ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Clotting Time ◽  
Terminal Amino Acid ◽  
Human Fibrinogen ◽  
Terminal Amino ◽  
In Vitro Effects ◽  
Polypeptide Chains

Fibrinogen preparations with increasing contents of iodine, ranging from 0.2 to 20 atoms of iodine per molecule fibrinogen, were obtained with the ICl method. Aggregation and shortening of the thrombin clotting time occurred when the content of iodine exceeded 3 atoms per molecule.Upon the action of thrombin, the increase in N-terminal glycine, reflecting fibrin formation, was almost identical in native and iodinated fibrinogen. At visible gelation, however, decreased amounts of N-terminal glycine were found in heavily iodinated fibrinogen, thus indicating enhanced fibrin polymerization. N-terminal analysis of heavily iodinated fibrinogen demonstrated a deficiency in N-terminal tyrosine concomitantly with the apparance of a new N-terminal amino acid, identified as mono-iodo-tyrosine.Polyacrylamide gel electrophoresis at pH 8.9 revealed an increase in mobility following extensive iodination, but no shift in the isoelectric point was observed upon isofocusing.Neither clottability nor the behaviour of fibrinogen and its subunit polypeptide chains on SDS-gel electrophoresis was affected by iodination.

scholarly journals Purification and properties of M protein extracted from group A streptococci with pepsin: covalent structure of the amino terminal region of type 24 M antigen

1977 ◽  
Vol 145 (6) ◽  
pp. 1469-1483 ◽  
Author(s):  
EH Beachey ◽  
GH Stollerman ◽  
EY Chiang ◽  
TM Chiang ◽  
JM Seyer ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Terminal Region ◽  
M Protein ◽  
Skin Tests ◽  
Edman Degradation ◽  
Group A Streptococci ◽  
Passive Hemagglutination ◽  
Amino Terminal ◽  
Group A

M protein was extracted from type 24, group A streptococci with pepsin at pH 5.8 and was further purified by ammonium sulfate precipitation, ribonuclease digestion, ion-exchange chromatography, and isoelectric focusing. The purified pepsin extract of M (pep M) protein was shown to be free of nontype-specific immunoreactivity in (a) complement fixation tests with heterologous M antiserum, (b) skin tests in normal adult guinea pigs, and (c) passive hemagglutination tests for the presence of lipoteichoic acid sensitizing or antigenic activity. The pep M24 was highly immunogenic; two of three rabbits developed opsonic antibody titers of 1:256 and the third a titer of 1:32 6 wk after a single injection of 100-pg doses of pep M24 emulsified in complete Freund's adjuvant. The antisera lacked nontype-specific antibodies and produced single precipitin lines in agar gel diffusion tests against crude HC1 extracts of the homologous M protein. Thus, the type-specific antigenic determinant(s) of type 24 M protein appears to be separable from immunotoxic, cross-reactive antigens without loss of immunogenicity in rabbits. The mobility of pep M24 upon electrophoresis in 10 percent sodium dodecyl sulfate pelyacrylamide gel was consistent with an average mol wt of 33,500 daltons. Amino acid analysis demonstrated a predominance of alanine, followed by glutamic acid, lysine, leucine, and aspartic acid. Pep M24 contained an estimated six to seven methionine residues and approximately ten phenylalanine residues per molecule. No other aromatic amino acids were detected. Automatic Edman degradation of pep M24 yielded the sequence of the first 29 amino acids (the amino terminal amino acid being valine) of the amino terminal region of the molecule. The detection of only one new amino acid at each step of Edman degradation confirmed the homogeneity of the purified pep M24.

scholarly journals Purification and properties of tryptophan synthase from baker's yeast (Saccharomyces cerevisiae)

1983 ◽  
Vol 209 (1) ◽  
pp. 151-157 ◽  
Author(s):  
C J Bailey ◽  
P D Turner
Keyword(s):  
Amino Acid ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Baker’S Yeast ◽  
Tryptophan Synthase ◽  
Baker's Yeast ◽  
Terminal Amino Acid ◽  
Yeast Saccharomyces Cerevisiae ◽  
Purification And Properties ◽  
Terminal Amino

Tryptophan synthase was purified from baker's yeast. The purified enzyme exhibited one band on polyacrylamide-gel electrophoresis, had no detectable N-terminal amino acid and C-terminal alanine. The amino acid composition was close to that predicted by recent studies on the DNA sequence of the structural gene for the enzyme. Kinetic parameters for the following three activities were measured: indole-serine condensation, indolylglycerol phosphate lyase and the overall reaction of serine with 1-(indol-3-yl)glycerol 3-phosphate. The Km for indole was much lower than suggested by previous investigations, and the value of 11 microM was measured by a fluorimetric assay.

scholarly journals The structure and mechanism of stem bromelain. Evaluation of the homogeneity of purified stem bromelain, determination of the molecular weight and kinetic analysis of the bromelain-catalysed hydrolysis of N-benzyloxycarbonyl-l-phenylalanyl-l-serine methyl ester

1974 ◽  
Vol 143 (3) ◽  
pp. 575-586 ◽  
Author(s):  
Christopher W. Wharton
Keyword(s):  
Molecular Weight ◽  
Amino Acid ◽  
Methyl Ester ◽  
Gel Electrophoresis ◽  
Ph Dependence ◽  
Polyacrylamide Gel Electrophoresis ◽  
Terminal Amino Acid ◽  
Terminal Amino ◽  
Stem Bromelain ◽  
Hydrolysis Of

1. Purified stem bromelain (EC 3.4.22.4) was eluted from Sephadex G-100 as a single peak. The specific activity across the elution peak was approximately constant towards p-nitrophenyl hippurate but increased with elution volume with N2-benzoyl-l-arginine ethyl ester as substrate. 2. The apparent molecular weight, determined by elution analysis on Sephadex G-100, is 22500±1500, an anomalously low value. 3. Purified stem bromelain was eluted from CM-cellulose CM-32 as a single peak and behaved as a single species during column electrophoresis on Sephadex G-100. 4. Purified stem bromelain migrates as a single band during polyacrylamide-gel electrophoresis under a wide variety of conditions. 5. The molecular weight determined by polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate is 28500±1000. 6. Sedimentation-velocity and equilibrium-ultracentrifugation experiments, under a variety of conditions, indicate that bromelain is an apparently homogeneous single peptide chain of mol.wt. 28400±1400. 7. The N-terminal amino acid composition is 0.64±0.04mol of valine and 0.36±0.04mol of alanine per mol of enzyme of mol.wt. 28500. (The amino acid recovery of the cyanate N-terminal amino acid analysis was standardized by inclusion of carbamoyl-norleucine at the cyclization stage.) 8. The pH-dependence of the Michaelis parameters of the bromelain-catalysed hydrolysis of N-benzyloxycarbonyl-l-phenylalanyl-l-serine methyl ester was determined. 9. The magnitude and pH-dependence of the Michaelis parameters have been interpreted in terms of the mechanism of the enzyme. 10. The enzyme is able to bind N-benzyloxycarbonyl-l-phenylalanyl-l-serine methyl ester relatively strongly but seems unable to make use of the binding energy to promote catalysis.

scholarly journals Degradation of Aromatics and Chloroaromatics by Pseudomonas sp. Strain B13: Purification and Characterization of 3-Oxoadipate:Succinyl-Coenzyme A (CoA) Transferase and 3-Oxoadipyl-CoA Thiolase

2002 ◽  
Vol 184 (1) ◽  
pp. 207-215 ◽  
Author(s):  
Stefan R. Kaschabek ◽  
Bernd Kuhn ◽  
Dagmar Müller ◽  
Eberhard Schmidt ◽  
Walter Reineke
Keyword(s):  
Amino Acid ◽  
Molecular Mass ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Polyacrylamide Gel ◽  
Terminal Amino Acid ◽  
Ph Optimum ◽  
Native Molecular Mass ◽  
Coa Transferase ◽  
Terminal Amino

ABSTRACT The degradation of 3-oxoadipate in Pseudomonas sp. strain B13 was investigated and was shown to proceed through 3-oxoadipyl-coenzyme A (CoA) to give acetyl-CoA and succinyl-CoA. 3-Oxoadipate:succinyl-CoA transferase of strain B13 was purified by heat treatment and chromatography on phenyl-Sepharose, Mono-Q, and Superose 6 gels. Estimation of the native molecular mass gave a value of 115,000 ± 5,000 Da with a Superose 12 column. Polyacrylamide gel electrophoresis under denaturing conditions resulted in two distinct bands of equal intensities. The subunit A and B values were 32,900 and 27,000 Da. Therefore it can be assumed that the enzyme is a heterotetramer of the type A2B2 with a molecular mass of 120,000 Da. The N-terminal amino acid sequences of both subunits are as follows: subunit A, AELLTLREAVERFVNDGTVALEGFTHLIPT; subunit B, SAYSTNEMMTVAAARRLKNGAVVFV. The pH optimum was 8.4. K m values were 0.4 and 0.2 mM for 3-oxoadipate and succinyl-CoA, respectively. Reversibility of the reaction with succinate was shown. The transferase of strain B13 failed to convert 2-chloro- and 2-methyl-3-oxoadipate. Some activity was observed with 4-methyl-3-oxoadipate. Even 2-oxoadipate and 3-oxoglutarate were shown to function as poor substrates of the transferase. 3-Oxoadipyl-CoA thiolase was purified by chromatography on DEAE-Sepharose, blue 3GA, and reactive brown-agarose. Estimation of the native molecular mass gave 162,000 ± 5,000 Da with a Superose 6 column. The molecular mass of the subunit of the denatured protein, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was 42 kDa. On the basis of these results, 3-oxoadipyl-CoA thiolase should be a tetramer of the type A4. The N-terminal amino acid sequence of 3-oxoadipyl-CoA thiolase was determined to be SREVYI-DAVRTPIGRFG. The pH optimum was 7.8. K m values were 0.15 and 0.01 mM for 3-oxoadipyl-CoA and CoA, respectively. Sequence analysis of the thiolase terminus revealed high percentages of identity (70 to 85%) with thiolases of different functions. The N termini of the transferase subunits showed about 30 to 35% identical amino acids with the glutaconate-CoA transferase of an anaerobic bacterium but only an identity of 25% with the respective transferases of aromatic compound-degrading organisms was found.

PURIFICATION AND PARTIAL CHEMICAL CHARACTERIZATION OF SHEEP NEUROPHYSIN

1973 ◽  
Vol 74 (2) ◽  
pp. 226-236 ◽  
Author(s):  
Michel Chrétien ◽  
Claude Gilardeau
Keyword(s):  
Amino Acid ◽  
Polyacrylamide Gel Electrophoresis ◽  
Chemical Characterization ◽  
Terminal Amino Acid ◽  
Amino Acid Determination ◽  
Pituitary Glands ◽  
Terminal Amino ◽  
Partial Chemical ◽  
Acid Determination

ABSTRACT A protein isolated from ovine pituitary glands has been purified, and its homogeneity assessed by NH2- and COOH-terminal amino acid determination, ultracentrifugation studies, and polyacrylamide gel electrophoresis after carboxymethylation. Its chemical and immunochemical properties are closely similar to those of beef and pork neurophysins, less similar to those of human neurophysins. It contains no tryptophan (like other neurophysins) or histidine (like all except bovine neurophysin-I and human neurophysins). It has alanine at the NH2-terminus and valine at the COOH-terminus. Its amino acid composition is similar to, but not identical with those of porcine and bovine neurophysins.

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