Characterization of bacteriocin 28 produced by Clostridium perfringens

1982 ◽  
Vol 28 (7) ◽  
pp. 860-873 ◽  
Author(s):  
A. W. Li ◽  
J. A. Verpoorte ◽  
R. G. Lewis ◽  
D. E. Mahony
Keyword(s):  
Clostridium Perfringens ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Active Material ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Hydrophobic Properties ◽  
Isoelectric Points

Bacteriocin 28, produced by Clostridium perfringens, was characterized by gel filtration and sodium dodecyl sulfate – polyacrylamide gel electrophoresis as a glycoprotein with a molecular weight of approximately 100 000. Density gradient centrifugation suggested a lower weight of 84 000. The bacteriocin bound firmly to phenyl-Sepharose CL-4B gel, indicating hydrophobic properties, and elution from this gel with ethylene glycol clearly separated bacteriocin from the alpha and theta toxins of C. perfringens, the latter of which was also hydrophobic. Bacteriocin 28 was immunogenic, inducing neutralizing and precipitating antibodies, and possessed three isoelectric points: 7.37, 7.05, and 5.4. Amino acid and carbohydrate analysis of the active material showed a composition of 15 amino acids and several carbohydrates. The molecule demonstrated instability with increasing purification, and several approaches to purification are described.

scholarly journals Isolation and characterization of lung connective-tissue glycoproteins

1982 ◽  
Vol 203 (3) ◽  
pp. 593-601 ◽  
Author(s):  
C Lafuma ◽  
M Moczar ◽  
L Robert
Keyword(s):  
Proteinase Inhibitors ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Lung Parenchyma ◽  
Isolation And Characterization ◽  
Isoelectric Points ◽  
And Function

1. Glycoproteins of hamster, rat and baboon lung parenchyma were investigated by using [14C]glucosamine incorporation in vitro followed by sequential extraction of the macromolecular components and characterization of the glycoproteins in the extracts. 2. Slices of lung parenchyma maintained in vitro incorporated [U-14C]glucosamine linearly with time into non-diffusible macromolecules for up to 5h. All the macromolecule-associated 14C label was present as [14C]glucosamine. 3. These 14C-labelled macromolecules were extracted from previously delipidated and salt-extracted lung by 5M-guanidinium chloride in the presence of dithiothreitol and proteinase inhibitors before (extract A1) and after (extract A2) hydrolysis of the collagen by collagenase. The [14C]glucosamine-labelled glycoproteins in extracts A1 and A2 contained 55 and 5% respectively of the total [14C]glucosamine incorporated in the lung of all three species studied. 4. The [14C]glucosamine-labelled glycoproteins were analysed by gel-filtration chromatography, sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and isoelectric focusing. The major [14C]glucosamine-labelled glycoproteins of baboon lung parenchyma had apparent mol.wts. of about 400 000, 140 000 and 65 000 with isoelectric points respectively of 4.8, 5.4 and 5.4. The hamster lung glycoproteins with isoelectric points of 4.1 and 5.8 were devoid of hydroxyproline and contained galactose, mannose and N-acetylglucosamine. These experiments indicate that several distinct glycoproteins are synthesized in situ by the cells of pulmonary parenchyma and may well play a role in its structure and function.

scholarly journals A 70000-molecular-weight protein isolated from purified pig gastric mucus glycoprotein by reduction of disulphide bridges and its implication in the polymeric structure

1981 ◽  
Vol 197 (1) ◽  
pp. 155-162 ◽  
Author(s):  
J P Pearson ◽  
A Allen ◽  
S Parry
Keyword(s):  
Density Gradient ◽  
Protein Fraction ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Equilibrium Density ◽  
Gastric Mucus ◽  
Mucus Glycoprotein

The glycoprotein of pig gastric mucus has been isolated free of non-covalently bound protein as judged by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and equilibrium density-gradient centrifugation. After reduction with 0.2 M-mercaptoethanol, protein was released from the glycoprotein, which consisted of a major 70000-mol.wt. component and a minor 60000-mol.wt. component. The 70000-mol.wt. protein fraction was separated from the reduced glycoprotein by either density-gradient centrifugation in CsCl or by gel filtration. Analysis of the 70000-mol.wt. protein fraction showed that, within the limits of the analysis, it was non-glycosylated, and its amino acid analysis was quite different from that of the reduced glycoprotein, which is high in serine, threonine and proline. There was a ratio of one 70000-mol.wt. protein per native glycoprotein molecule of 2 × 10(6) mol.wt. Dissociation of the native glycoprotein into glycoprotein subunits (5 × 10(5) mol.wt.) by reduction or proteolysis results in the release or hydrolysis respectively of the 70000-mol.wt. protein. A similar 70000-mol.wt. protein is demonstrated in human gastric mucus glycoprotein. A structural role for the proteins in these mucus glycoproteins is proposed.

scholarly journals Purification and Characterization of 2,6-β-d-Fructan 6-Levanbiohydrolase fromStreptomyces exfoliatus F3-2

2000 ◽  
Vol 66 (1) ◽  
pp. 252-256 ◽  
Author(s):  
Katsuichi Saito ◽  
Kazuya Kondo ◽  
Ichiro Kojima ◽  
Atsushi Yokota ◽  
Fusao Tomita
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Extracellular Enzyme ◽  
Molecular Weights ◽  
Sds Page ◽  
Monomeric Structure ◽  
Ph Range ◽  
Hydrolysis Of

ABSTRACT Streptomyces exfoliatus F3-2 produced an extracellular enzyme that converted levan, a β-2,6-linked fructan, into levanbiose. The enzyme was purified 50-fold from culture supernatant to give a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The molecular weights of this enzyme were 54,000 by SDS-PAGE and 60,000 by gel filtration, suggesting the monomeric structure of the enzyme. The isoelectric point of the enzyme was determined to be 4.7. The optimal pH and temperature of the enzyme for levan degradation were pH 5.5 and 60°C, respectively. The enzyme was stable in the pH range 3.5 to 8.0 and also up to 50°C. The enzyme gave levanbiose as a major degradation product from levan in an exo-acting manner. It was also found that this enzyme catalyzed hydrolysis of such fructooligosaccharides as 1-kestose, nystose, and 1-fructosylnystose by liberating fructose. Thus, this enzyme appeared to hydrolyze not only β-2,6-linkage of levan, but also β-2,1-linkage of fructooligosaccharides. From these data, the enzyme from S. exfoliatus F3-2 was identified as a novel 2,6-β-d-fructan 6-levanbiohydrolase (EC 3.2.1.64 ).

scholarly journals Biochemical and Genetic Characterization of an FK506-Sensitive Peptidyl Prolyl cis-trans Isomerase from a Thermophilic Archaeon, Methanococcus thermolithotrophicus

1998 ◽  
Vol 180 (2) ◽  
pp. 388-394 ◽  
Author(s):  
Masahiro Furutani ◽  
Toshii Iida ◽  
Shigeyuki Yamano ◽  
Kei Kamino ◽  
Tadashi Maruyama
Keyword(s):  
Amino Acid ◽  
Genomic Library ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Ppiase Activity ◽  
Methanococcus Thermolithotrophicus ◽  
Fk506 Binding Protein ◽  
Thermophilic Archaeon

ABSTRACT A peptidyl prolyl cis-trans isomerase (PPIase) was purified from a thermophilic methanogen, Methanococcus thermolithotrophicus. The PPIase activity was inhibited by FK506 but not by cyclosporine. The molecular mass of the purified enzyme was estimated to be 16 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 42 kDa by gel filtration. The enzyme was thermostable, with the half-lives of its activity at 90 and 100°C being 90 and 30 min, respectively. The catalytic efficiencies (k cat/Km ) measured at 15°C for the peptidyl substrates,N-succinyl-Ala-Leu-Pro-Phe-p-nitroanilide andN-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide, were 0.35 and 0.20 μM−1 s−1, respectively, in chymotrypsin-coupled assays. The purified enzyme was sensitive to FK506 and therefore was called MTFK (M. thermolithotrophicusFK506-binding protein). The MTFK gene (462 bp) was cloned from anM. thermolithotrophicus genomic library. The comparison of the amino acid sequence of MTFK with those of other FK506-binding PPIases revealed that MTFK has a 13-amino-acid insertion in the N-terminal region that is unique to thermophilic archaea. The relationship between the thermostable nature of MTFK and its structure is discussed.

scholarly journals Characterization of Biosynthetic Enzymes for Ectoine as a Compatible Solute in a Moderately Halophilic Eubacterium, Halomonas elongata

1999 ◽  
Vol 181 (1) ◽  
pp. 91-99 ◽  
Author(s):  
Hisayo Ono ◽  
Kazuhisa Sawada ◽  
Nonpanga Khunajakr ◽  
Tao Tao ◽  
Mihoko Yamamoto ◽  
...  
Keyword(s):  
Molecular Mass ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Apparent Molecular Mass ◽  
Optimum Temperature ◽  
Sds Page ◽  
Halomonas Elongata ◽  
Optimum Ph

ABSTRACT 1,4,5,6-Tetrahydro-2-methyl-4-pyrimidinecarboxylic acid (ectoine) is an excellent osmoprotectant. The biosynthetic pathway of ectoine from aspartic β-semialdehyde (ASA), in Halomonas elongata, was elucidated by purification and characterization of each enzyme involved. 2,4-Diaminobutyrate (DABA) aminotransferase catalyzed reversively the first step of the pathway, conversion of ASA to DABA by transamination with l-glutamate. This enzyme required pyridoxal 5′-phosphate and potassium ions for its activity and stability. The gel filtration estimated an apparent molecular mass of 260 kDa, whereas molecular mass measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was 44 kDa. This enzyme exhibited an optimum pH of 8.6 and an optimum temperature of 25°C and had Km s of 9.1 mM forl-glutamate and 4.5 mM for dl-ASA. DABA acetyltransferase catalyzed acetylation of DABA to γ-N-acetyl-α,γ-diaminobutyric acid (ADABA) with acetyl coenzyme A and exhibited an optimum pH of 8.2 and an optimum temperature of 20°C in the presence of 0.4 M NaCl. The molecular mass was 45 kDa by gel filtration. Ectoine synthase catalyzed circularization of ADABA to ectoine and exhibited an optimum pH of 8.5 to 9.0 and an optimum temperature of 15°C in the presence of 0.5 M NaCl. This enzyme had an apparent molecular mass of 19 kDa by SDS-PAGE and a Km of 8.4 mM in the presence of 0.77 M NaCl. DABA acetyltransferase and ectoine synthase were stabilized in the presence of NaCl (>2 M) and DABA (100 mM) at temperatures below 30°C.

Purification and Characterization of an Acetyl Xylan Esterase from Bacillus pumilus

1998 ◽  
Vol 64 (2) ◽  
pp. 789-792 ◽  
Author(s):  
Giuliano Degrassi ◽  
Benedict C. Okeke ◽  
Carlo V. Bruschi ◽  
Vittorio Venturi
Keyword(s):  
Gel Electrophoresis ◽  
Bacillus Pumilus ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Acetyl Xylan Esterase ◽  
Purification And Characterization ◽  
Michaelis Constant ◽  
Naphthyl Acetate

ABSTRACT Bacillus pumilus PS213 was found to be able to release acetate from acetylated xylan. The enzyme catalyzing this reaction has been purified to homogeneity and characterized. The enzyme was secreted, and its production was induced by corncob powder and xylan. Its molecular mass, as determined by gel filtration, is 190 kDa, while sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed a single band of 40 kDa. The isoelectric point was found to be 4.8, and the enzyme activity was optimal at 55°C and pH 8.0. The activity was inhibited by most of the metal ions, while no enhancement was observed. The Michaelis constant (Km ) andV max for α-naphthyl acetate were 1.54 mM and 360 μmol min−1 mg of protein−1, respectively.

scholarly journals Studies on the assembly of the rat lens capsule. Biosynthesis and partial characterization of the collagenous components

1978 ◽  
Vol 176 (1) ◽  
pp. 283-294 ◽  
Author(s):  
J G Heathcote ◽  
C H J Sear ◽  
M E Grant
Keyword(s):  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Lens Capsule ◽  
Time Dependent ◽  
Lower Molecular Weight Species ◽  
Direct Incorporation ◽  
Isolated Rat

1. Isolated rat lens capsules synthesized hydroxy[3H]proline-containing polypeptides when incubated with [3H]proline. 2. The collagenous polypeptides synthesized during a 2 h incubation were analyzed by both gel-filtration chromatography and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and shown to have an apparent mol.wt. of approx. 180,000. 3. No evidence was obtained for conversion of these polypeptides into a lower-molecular-weight species in experiments where capsules were labelled for 2 h and chased with non-radioactive proline for up to 22 h. However, a time-dependent incorporation of the 180,000-mol.wt. species into a larger collagenous component was observed and this could be prevented by the inclusion of beta-aminopropionitrile in the incubation medium. 4. The radioactive components synthesized by the capsules correspond to subunits of the intact lens capsule and the direct incorporation of the polypeptide of mol.wt. 180,000 into deoxycholate-insoluble basement membrane was demonstrated.

scholarly journals Purification of the enzyme NADPH: protochlorophyllide oxidoreductase

1981 ◽  
Vol 195 (1) ◽  
pp. 83-92 ◽  
Author(s):  
N S Beer ◽  
W T Griffiths
Keyword(s):  
Specific Activity ◽  
Gradient Centrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
High Specific Activity ◽  
Sucrose Density Gradient ◽  
Sucrose Density Gradient Centrifugation ◽  
Triton X ◽  
Etioplast Membranes

A procedure for the purification of the enzyme NADPH:protochlorophyllide oxidoreductase is described. This involves fractionation of sonicated oat etioplast membranes by discontinuous-sucrose-density-gradient centrifugation, which gives membranes in which the enzyme is present at a high specific activity. The enzyme is solubilized from the membranes with Triton X-100, followed by gel filtration of the extract; enzyme activity is eluted in fractions corresponding to a mol.wt of approx. 35000. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of the enzyme-containing fractions from gel filtration shows two peptides, of mol.wts. approx. 35000 and 37000.

Purification and preliminary characterization of 2-monoacylglycerol acyltransferase from rat intestinal villus cells

1985 ◽  
Vol 63 (5) ◽  
pp. 341-347 ◽  
Author(s):  
F. Manganaro ◽  
A. Kuksis
Keyword(s):  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Cell Protein ◽  
Racemic Mixture ◽  
Small Intestinal Mucosa ◽  
Small Intestinal ◽  
Monoacylglycerol Acyltransferase

We have purified the monoacylglycerol acyltransferase from rat small intestinal mucosa to homogeneity by a combination of hydrophobic absorption, guanidine dissociation, and gel filtration. The purified enzyme gives a single band of 37 000 daltons on sodium dodecyl sulphate – polyacrylamide gel electrophoresis. The enzyme has a specific activity of about 5900 nmol/mg per hour and represents 0.12% of total cell protein, corresponding to about a 600-fold purification. The enzyme does not acylate diacylglycerols to triacylglycerols, which is consistent with the separate physical existence of the mono- and di-acylglycerol acyltransferases. The enzyme acylates the 2-monoacylglycerols to yield an essentially racemic mixture of diacylglycerols. It does not acylate glycerol 3-phosphate.

scholarly journals Biochemical characterization of guanidinium chloride-soluble dentine collagen from lathyritic-rat incisors

1979 ◽  
Vol 181 (3) ◽  
pp. 667-676 ◽  
Author(s):  
M Wohllebe ◽  
D J Carmichael
Keyword(s):  
Biochemical Characterization ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Neutral Salt ◽  
Guanidinium Chloride ◽  
Lysine Residues ◽  
Cross Links ◽  
Lysine Hydroxylation

alpha- and beta-Chains were isolated by sequential ion-exchange and gel-filtration chromatography of guanidinium chloride-soluble dentine collagen obtained from Tris/NaCl-extracted EDTA-demineralized lathyritic-rat incisors. The alpha-chains were identified as alpha 1 I and alpha 2 by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and amino acid analysis of the intact chains and their CNBr peptides. The dentine alpha-chains exhibited higher lysine hydroxylation and phosphate content, but lower hydroxylysine glycosylation, than alpha-chains from skin. Increased lysine hydroxylation was observed in the helical sequences. The alpha 1 I/alpha 2 ratio was approx. 3:1, and was presumably due to the presence of (alpha 1 I)3 molecules along with (alpha 1 I)2 alpha 2 molecules as shown recently for neutral-salt-soluble dentine collagen [Wohllebe & Carmichael (1978) Eur. J. Biochem. 92, 183–188]. In the borohydride-reduced beta 11- and beta 12-chains from guanidinium chloride-soluble dentine collagen, the reduced cross-links hydroxylysinohydroxynorleucine and hydroxylysinonorleucine were present. A higher proportion of hydroxylysinonorleucine in the reduced beta 12-chain probably reflects differences in extent of hydroxylation of specific lysine residues of the alpha 1 I- and alpha 2-chains.

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