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 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.

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.

Acetylcholinesterase aus dem Gift von Bungarus multicinctus. Reinigung und Eigenschaften/The Acetylcholinesterase of Bungarus multicinctus Venom. Purification and Properties

1979 ◽  
Vol 34 (1-2) ◽  
pp. 27-32 ◽  
Author(s):  
H. Großmann ◽  
M. Weinert ◽  
M. Liefländer
Keyword(s):  
Molecular Weight ◽  
Amino Acid ◽  
Isoelectric Focusing ◽  
Gel Electrophoresis ◽  
Amino Acid Analysis ◽  
Specific Activity ◽  
Terminal Amino Acid ◽  
Purification And Properties ◽  
Gradient Gel Electrophoresis ◽  
Terminal Amino

Abstract Acetylcholinesterase from Banded krait (Bungarus multicinctus) venom has been purified by CM-Sephadex chromatography and affinity chromatography to a specific activity of 4290 U/mg. The purified enzyme is a glycoprotein. It is free of electrophoretically detectable contaminating proteins. A molecular weight of 140 000 ± 5 000 has been determined by gradient gel electrophoresis for the native enzyme. It is split into two equal-sized subunits (Mr 70 000 ± 2 000) by SDS treatment. The N-terminal amino acid analysis gave glycine and serine. The purified acetyl­ cholinesterase can be resolved by disc gel electrophoresis into four and by isoelectric focusing into six isozymes. The pI value of the main isozyme has been found to be 5.98 ± 0.05.

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.

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 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.

Normal Mature Human Enamel Protein

1979 ◽  
Vol 58 (2_suppl) ◽  
pp. 986-987 ◽  
Author(s):  
A. Belcourt
Keyword(s):  
Molecular Weight ◽  
Amino Acid ◽  
Gel Electrophoresis ◽  
Protein Concentration ◽  
Polyacrylamide Gel Electrophoresis ◽  
Organic Matrix ◽  
Low Molecular Weight ◽  
Human Enamel ◽  
Weak Density ◽  
Enamel Protein

Pure enamel was prepared using an original microdissection technic. Protein concentration was 375 μg per gram of enamel. Polyacrylamide gel electrophoresis showed a single fast-migrating zone containing a thin double band. Ultracentrifugation studies suggested that the proteins were of low molecular weight or of weak density. Absorption spectra showed a strong absorbance at 260nm. Amino acid analyses yielded a composition of 25% Gly, 13.5% Glu, 11% Ser, 11% Pro, 2% Cys and 2% Hyp. A glucidic content of 15% was estimated and glucose, galactose, mannose and fucose were identified. The organic matrix of enamel seemed to be constituted of two major glycoproteins probably fibrous but different from keratin.

scholarly journals The purification and properties of the second component of human complement

1978 ◽  
Vol 171 (1) ◽  
pp. 99-107 ◽  
Author(s):  
M A Kerr ◽  
R R Porter
Keyword(s):  
Amino Acid ◽  
Polypeptide Chain ◽  
Alternative Pathway ◽  
Polyacrylamide Gel Electrophoresis ◽  
Complement Factor ◽  
Human Complement ◽  
Terminal Amino Acid ◽  
Factor B ◽  
Antigenic Properties ◽  
Terminal Amino

The second component of human complement (C2) was purified by a combination of euglobulin precipitation, ion-exchange chromatography, (NH4)2SO4 precipitation and affinity chromatography. The final product was homogeneous by the criterion of polyacrylamide-gel electrophoresis and represents a purification of about 4000-fold from serum with 15-20% yield. Component C2 comprises a single carbohydrate-containing polypeptide chain, with an apparent mol.wt. of 102000; alanine is the N-terminal amino acid. The molecule is rapidly cleaved by activated subcomponent C1s with the loss of haemolytic activity to yield two fragments with apparent mol.wts. of 74000 and 34000. These fragments are not linked by disulphide bonds and can be easily separated. A second protein isolated during the purification of component C2 was identified by its haemolytic and antigenic properties as complement Factor B, the protein serving an analogous function to component C2 in the alternative pathway. The protein, which is also a single carbohydrate-containing polypeptide chain, has an apparent mol.wt. of 95000 and threonine as N-terminal amino acid. The amino acid analyses of component C2 and Factor B are compared.

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