α-Glutamic acid-β-naphthylamidase from Neisseria catarrhalis

1969 ◽  
Vol 15 (11) ◽  
pp. 1293-1300 ◽  
Author(s):  
Sidney T. Cox ◽  
Francis J. Behal
Keyword(s):  
Molecular Weight ◽  
Amino Acid ◽  
Glutamic Acid ◽  
Metal Ion ◽  
Gel Filtration ◽  
Aminopeptidase Activity ◽  
Amino Acid Residues ◽  
Ph Optimum ◽  
Hydrolysis Of ◽  
Derivatives Of

A second bacterial peptidase-like enzyme, arylamidase-II, has been isolated from cell free extracts of Neisseria catarrhalis. Arylamidase-II action is limited primarily to the hydrolysis of α-glutamic acid and α-aspartic acid derivatives of β-naphthylamine and short peptides of glutamic acid. The enzyme was purified 450-fold by gel filtration, ion exchange, and calcium phosphate chromatography. Its pH optimum and molecular weight were 7.7 and 170 000, respectively. Aside from its restricted substrate specificity, arylamidase-II has been found to be closely related in its mechanism of action, molecular weight, pH optimum, and metal ion dependence to arylamidase-I, which catalyzes the hydrolysis of neutral amino acid derivatives of β-naphthylamine. Arylamidase-II exhibits aminopeptidase activity, requiring the amino acid residues in the N-terminal and penultimate position to be of the L-configuration in order for hydrolysis to occur.

Characterization and substrate specificity of fumarylacetoacetate fumarylhydrolase

1977 ◽  
Vol 55 (1) ◽  
pp. 1-8 ◽  
Author(s):  
D. J. Mahuran ◽  
Ronald H. Angus ◽  
Carl V. Braun ◽  
S. S. Sim ◽  
Donald E. Schmidt Jr.
Keyword(s):  
Molecular Weight ◽  
Amino Acid ◽  
Gel Electrophoresis ◽  
Guanidine Hydrochloride ◽  
Gel Filtration ◽  
Sulfhydryl Groups ◽  
Ph Optimum ◽  
Amino Terminal ◽  
Diketo Acids ◽  
Hydrolysis Of

The molecular weight of fumarylacetoacetate fumarylhydrolase (EC 3.7.1.2) is 86 000 ± 10 000, as determined by gel filtration. The enzyme appears to be a dimer with a monomer molecular weight of 38 000 – 43 000, as determined by gel electrophoresis, gel filtration in guanidine–hydrochloride, and ultracentrifugation. The subunits appear to be identical, as only one band is seen in gel electrophoresis, only one protein peak is detected in gel filtration in guanidine–hydrochloride, and only one amino-terminal amino acid (proline) is detected. Three free sulfhydryl groups per denatured monomer are detected by reaction with 5,5′-dithiobis(2-nitrobenzoic acid), while for the active enzyme only two sulfhydryl groups react with this reagent. The extinction coefficients at 260 and 280 nm, the amino acid composition, and the isoelectric point (6.7) of the enzyme are also reported.The enzyme catalyzes the hydrolysis of six 2,4-diketo acids and three 3,5-diketo acids tested. The Km of the substrates is similar but V varies by a factor of 120. The pH optimum is 7.3. The enzyme did not catalyze the hydrolysis of a number of esters tested.

Studies on Chymotrypsin-P. I. Characterization

1971 ◽  
Vol 49 (9) ◽  
pp. 999-1004 ◽  
Author(s):  
M. C. Shaw ◽  
T. Viswanatha
Keyword(s):  
Molecular Weight ◽  
Amino Acid ◽  
Physicochemical Properties ◽  
Gel Filtration ◽  
Amino Acid Residues ◽  
The Difference ◽  
Filtration Technique

The physicochemical properties of chymotrypsin-P obtained by the papain activation of chymotrypsinogen have been investigated. The molecular weight of this enzyme as determined by gel filtration technique has been found to be 24 000 ± 1000. The amino acid residues occupying the N-terminal positions and the composition of the B- and C-chains of chymotrypsin-P are identical with those found in α-chymotrypsin. Thus the difference between the two enzymes is restricted to the composition of their A-chains.

scholarly journals Characterization of gastric mucoproteins isolated by equilibrium density-gradient centrifugation in caesium chloride

1974 ◽  
Vol 141 (3) ◽  
pp. 633-639 ◽  
Author(s):  
Bryan J. Starkey ◽  
David Snary ◽  
Adrian Allen
Keyword(s):  
Molecular Weight ◽  
Amino Acid ◽  
Gradient Centrifugation ◽  
Gel Filtration ◽  
Equilibrium Density ◽  
Amino Acid Residues ◽  
Gastric Mucus ◽  
Terminal Amino Acid ◽  
Terminal Amino

1. The mucoprotein from pig gastric mucus has been purified by equilibrium centrifugation in a CsCl gradient. 2. This procedure removes the non-covalently bound protein, which is closely associated with the mucoprotein and not easily removed from it by gel filtration. 3. The purified mucoprotein is separable by gel filtration into a high-molecular-weight mucoprotein A (mol.wt. 2.3×106) and a low-molecular-weight mucoprotein B/C (mol.wt. 1.15×106). 4. These two mucoproteins have the same chemical analysis namely fucose 11.3%, galactose 26%, glucosamine 19.5%, galactosamine 8.3% and protein 13.6%. 5. Mucoprotein A contains 3.1% ester sulphate. 6. These mucoproteins are isolated without enzymic digestion and have a higher protein content than the blood-group-substance mucoproteins from proteolytic digestion of gastric mucus. Detailed amino acid analysis shows that the extra protein in the non-enzymically digested material is composed of amino acids other than serine and threonine. 7. Mucoproteins A and B/C contain respectively 130 and 9 half-cystine residues per molecule of which about 78 and 6 residues are involved in disulphide linkages. 8. Cleavage of these disulphide linkages by mercaptoethanol splits both mucoproteins into four equally sized subunits of mol.wt. 5.2×105for mucoprotein A and 2.8×104for mucoprotein B/C. 9. The sole N-terminal amino acid of mucoprotein A is aspartic acid, whereas mucoprotein B/C has several different N-terminal amino acid residues.

scholarly journals Purification and properties of a manganese-stimulated deoxyribonuclease produced during sporulation of Bacillus subtilis

1978 ◽  
Vol 172 (1) ◽  
pp. 69-76 ◽  
Author(s):  
A Akrigg
Keyword(s):  
Molecular Weight ◽  
Bacillus Subtilis ◽  
Metal Ion ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Duplex Dna ◽  
Ph Optimum ◽  
Endonucleolytic Cleavage ◽  
Single Strand Breaks ◽  
Purification And Properties

A DNAase (deoxyribonuclease) was isolated from culture supernatants of sporulating Bacillus subtilis 168. The purified enzyme migrated as a single band during polyacrylamide-gel electrophoresis. The enzyme differs from other DNAases of B. subtilis in molecular weight, metal-ion requirement and mode of action. The enzyme was inactive in the absence of metal ions, and exhibited optimum activity with 10 mM-Mn2+, although Mg2+, Cd2+ and Co2+ could also permit some activity. The pH optimum for the enzyme was pH 7.5, and it degraded linear-duplex DNA or closed-circular-duplex DNA to acid-soluble material. There was little or no activity on single-stranded DNA or rRNA. Sucrose-gradient analysis of the products of DNAase action on bacteriophage T7 DNA showed that endonucleolytic cleavage had occurred by the introduction of single-strand breaks in both strands of the duplex. The molecular weight of the enzyme was determined, by gel filtration on Sephadex G-75, to be 12000.

scholarly journals Purification of a high-molecular-weight somatoliberin (growth-hormone-releasing factor) from pig hypothalami

1983 ◽  
Vol 209 (3) ◽  
pp. 643-651 ◽  
Author(s):  
J E C Sykes ◽  
P J Lowry
Keyword(s):  
Molecular Weight ◽  
Growth Hormone ◽  
Amino Acid ◽  
Response Curve ◽  
Gel Filtration ◽  
Apparent Molecular Weight ◽  
Amino Acid Residues ◽  
Growth Hormone Releasing Factor

Preliminary observations [Sykes & Lowry (1980) J. Endocrinol. 85, 42P-43P] had suggested that the major hypothalamic somatoliberin (growth-hormone-releasing factor) was a larger peptide than the other characterized hypothalamic factors, with an elution position on Sephadex G-50 between those of neurophysin and corticotropin. The present paper reports the isolation and preliminary characterization of pig hypothalamic somatoliberin. Acid extracts of pig stalk median eminence were purified by gel filtration and preparative and analytical high-pressure liquid chromatography to yield a preparation that was specific in the release of somatotropin (growth hormone) in vitro, giving a steep dose-response curve at doses in the range 0.20-3.0 ng. Amino acid analysis revealed a non-cysteine-containing peptide with a high number of glutamate (or glutamine) and aspartate (or asparagine) residues. The peptide had about 56-57 amino acid residues and an apparent molecular weight of 6400, in keeping with its elution position on a column of Sephadex G-50.

scholarly journals Proteins of the kidney microvillar membrane. Purification and properties of the phosphoramidon-insensitive endopeptidase (‘endopeptidase-2’) from rat kidney

1987 ◽  
Vol 245 (2) ◽  
pp. 515-524 ◽  
Author(s):  
A J Kenny ◽  
J Ingram
Keyword(s):  
Rat Kidney ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Staining Intensity ◽  
Aminopeptidase Activity ◽  
Ph Optimum ◽  
Reducing Conditions ◽  
Purification And Properties ◽  
Relationship Of ◽  
Hydrolysis Of

A second endopeptidase is present in the renal microvillar membrane of rats that can be distinguished from endopeptidase-24.11 by its insensitivity to inhibition by phosphoramidon. The purification of this enzyme, referred to as endopeptidase-2, is described. The enzyme was efficiently released from the membrane by treatment with papain. The subsequent four steps depended on ion-exchange and gel-filtration chromatography. These steps were monitored by the hydrolysis of various substrates: 125I-insulin B chain (the normal assay substrate), benzoyl-L-tyrosyl-p-aminobenzoate (Bz-Tyr-pAB), azocasein and benzyloxycarbonyl-L-phenylalanyl-L-arginine 7-amino-4-methylcoumarylamide (Z-Phe-Arg-NMec). All four assays revealed comparable stepwise increases in activity in the main stages of the purification, although it was apparent that the last-named fluorogenic assay depended on traces of aminopeptidase activity present in the preparation. The Km for 125I-insulin B chain was 16 microM and that for Bz-Tyr-pAB was 4.7 mM. Several experimental approaches confirmed that both peptides were hydrolysed by the same enzyme. The pH optimum was 7.3. Phosphate buffers were inhibitory and shifted the optimum to above pH 9. Zinc was detected in the purified enzyme; EDTA and 1,10-phenanthroline were strongly inhibitory. SDS/polyacrylamide-gel electrophoresis revealed polypeptides of equal staining intensity of Mr 80,000 and 74,000 in reducing conditions. In non-reducing conditions a single band of apparent Mr 220,000 was seen. Gel filtration yielded an Mr of 436,000. These results are consistent with an oligomeric structure in which the alpha and beta chains are linked by disulphide bridges. Endopeptidase-2 hydrolysed a number of neuropeptides. Enkephalins resisted attack, only the heptapeptide [Met]enkephalin-Arg6-Phe7 being susceptible to slow hydrolysis. Luliberin (luteinizing-hormone-releasing hormone) and bradykinin were rapidly hydrolysed. Neurotensin was shown to be slowly attacked at the Tyr3-Glu4 bond. Thus the specificity appears to be limited to the hydrolysis of bonds involving the carboxy group of aromatic residues, provided that this P1 residue is extended by additional residues, at least to the P3′ position. The relationship of this membrane metalloendopeptidase to mouse meprin and human ‘PABA peptidase’ is discussed.

Specificity of the surface aminopeptidase in Moniliformis moniliformis (Acanthocephala)

1986 ◽  
Vol 60 (4) ◽  
pp. 288-292
Author(s):  
Gary L. Uglem ◽  
Mark C. Lewis
Keyword(s):  
Amino Acids ◽  
Glutamic Acid ◽  
Chromatographic Analysis ◽  
Aminopeptidase Activity ◽  
Amino Acid Residues ◽  
Bathing Medium ◽  
Transport Inhibitors ◽  
Moniliformis Moniliformis ◽  
Hydrolysis Of ◽  
Single Enzyme

ABSTRACTThe hydrolysis of various oligopeptides in solution by intact Moniliformis moniliformis was examined using paper chromatographic analysis of the incubation medium. In the presence of transport inhibitors, the respective peptide sub-units and/or amino acid residues accumulated in the bathing medium. Only peptides with serine, methionine, leucine or alanine at the NH2-terminal end of the peptide were hydrolysed. There was no hydrolysis when these amino acids were located internally or at the COOH-terminus indicating genuine aminopeptidase activity of the class, α-aminoacylpeptide hydrolase. Hydrolysis was negligible when the NH2-terminus was arginie, aspartic acid, glutamic acid, glutamic acid, glycine, histidine, lysine, phenylalanine, proline, tryptophan, tyrosine, or valine. In separate experiments, mediated uptake of 0.1 mM3H-leucine by the worms in 2 min was inhibited 100% by 5 mM unlabelled leucine or tri-serine, but only partially inhibited by 5 mM Ser-Gly (66%), 10 mM Ser-Gly (74%), 5 mM Leu-Leu (69%), 10 mM Leu-Leu (70%), 5 mM Leu-Gly (58%) or 5nM Met-Met (69%). Bacause the inhibitions produced by 5 mM Leu-Leu plus 5 mM Met-Met (79%) or 5 mM Leu-Leu plus 5 mM Ser-Gly (76%) were not additive, a single enzyme is indicated. The name serine aminopeptidase is proposed because of its preference for serine.

scholarly journals Isolation and characterization of sheep pepsin

1977 ◽  
Vol 161 (2) ◽  
pp. 389-398 ◽  
Author(s):  
P F Fox ◽  
J R Whitaker
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Amino Acid Composition ◽  
Ethyl Ester ◽  
Gel Filtration ◽  
Ph Optimum ◽  
Isolation And Characterization ◽  
Sepharose 4B ◽  
Hydrolysis Of

Sheep pepsin was isolated (approx. 120-fold purification) from aqueous abomasal homogenates by (1) pH fractionation, (2) chromatography on Sepharose 4B-poly-L-lysine columns and (3) gel filtration on Sephadex G-100. The enzyme had mol.wt. approx. 34000, N-terminal valine and C-terminal alanine. The amino acid composition of sheep pepsin was generally similar to that of pig and ox pepsins, with a very low content of basic residues and a high content of acidic and hydroxy-amino acids. The pH optimum for NN-dimethyl-casein and NN-dimethyl-haemoglobin as substrates was approx. 1.8. The Km and kcat. for NN-dimethyl-haemoglobin were 46micronM and 1100min-1 respectively, and for NN-dimethyl-casein the corresponding parameters were 50micronM and 420min-1. These values were generally similar to those for pig and ox pepsins. At the pH optimum of 4.6, the sheep pepsin was about 50% as active on benzyloxycarbonyl-L-histidyl-L-phenyl-alanyl-L-tryptophan ethyl ester as was pig pepsin. The pH optimum for the hydrolysis of N-acetyl-L-phenylalanyl-L-di-iodotyrosine by sheep, ox and pig pepsins was approx. 1.85.

scholarly journals Isolation, properties and amino acid sequences of three neurotoxins from the venom of a sea snake, Aipysurus laevis

1976 ◽  
Vol 153 (1) ◽  
pp. 79-87 ◽  
Author(s):  
N Maeda ◽  
N Tamiya
Keyword(s):  
Molecular Weight ◽  
Amino Acid ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Disc Electrophoresis ◽  
Amino Acid Sequences ◽  
Peptide Sequence ◽  
Amino Acid Residues ◽  
Toxin B ◽  
Almost All

Aipysurus laevis venom was chromatographed on CM-cellulose and Bio-Rex 70 columns. Three neurotoxic components, toxins Aipysurus laevis a, b and c, were isolated. The toxins a, b and c corresponded to 22, 33 and 21% respectively of the proteins in the original venom, and accounted for almost all the lethal activity of the venom. The three toxins a, b and c were monodisperse on disc electrophoresis at pH4; toxins a and b moved at the same velocity and c a little faster. They were monodisperse also on sodium dodecyl sulphate-polyacrylamide-disc-gel electrophoresis, giving a molecular weight of 7600. The molecular weight of toxin b estimated by gel filtration was 7000. The amino acid sequence analyses of these toxins revealed that they consisted of 60 amino acid residues and that Aipysurus laevis b was [25-methionine, 28-arginine] Aipysurus laevis a. Aipysurus laevis c was [28-lysine] Aipysurus laevis a, the tryptic peptide sequence relying on homology. The LD50 values of these toxins for 20g mice were 0.076 μg/g body wt. They inhibited the acetylcholine-induced contracture but did not affect the CKl-induced contracture of the isolated muscle.

Isolation and Characterization of Plasminogen Activator from Pig Leucocytes

1974 ◽  
Vol 31 (01) ◽  
pp. 072-085 ◽  
Author(s):  
M Kopitar ◽  
M Stegnar ◽  
B Accetto ◽  
D Lebez
Keyword(s):  
Molecular Weight ◽  
Plasminogen Activator ◽  
Gel Electrophoresis ◽  
Gel Filtration ◽  
Ph Optimum ◽  
Isolation And Characterization ◽  
Ph Range ◽  
Inhibition Studies ◽  
Final Purification

SummaryPlasminogen activator was isolated from disrupted pig leucocytes by the aid of DEAE chromatography, gel filtration on Sephadex G-100 and final purification on CM cellulose, or by preparative gel electrophoresis.Isolated plasminogen activator corresponds No. 3 band of the starting sample of leucocyte cells (that is composed from 10 gel electrophoretic bands).pH optimum was found to be in pH range 8.0–8.5 and the highest pH stability is between pH range 5.0–8.0.Inhibition studies of isolated plasminogen activator were performed with EACA, AMCHA, PAMBA and Trasylol, using Anson and Astrup method. By Astrup method 100% inhibition was found with EACA and Trasylol and 30% with AMCHA. PAMBA gave 60% inhibition already at concentration 10–3 M/ml. Molecular weight of plasminogen activator was determined by gel filtration on Sephadex G-100. The value obtained from 4 different samples was found to be 28000–30500.

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