scholarly journals The bromoperoxidase from the lichen Xanthoria parietina is a novel vanadium enzyme

1987 ◽  
Vol 248 (1) ◽  
pp. 277-279 ◽  
Author(s):  
H Plat ◽  
B E Krenn ◽  
R Wever
Keyword(s):  
High Temperatures ◽  
Thermal Denaturation ◽  
Protein Band ◽  
Enzymic Activity ◽  
Single Protein Band ◽  
Xanthoria Parietina ◽  
Single Protein

A novel bromoperoxidase was isolated from the lichen Xanthoria parietina. The enzyme contained vanadium, which is essential for enzymic activity. Under denaturating conditions the preparation showed a single protein band with an Mr of 65,000. Thermal-denaturation studies showed that this bromoperoxidase could tolerate high temperatures. The affinity of the enzyme for its substrate bromide is high; the Km for bromide was 29 microM. Excess halides (50 mM) inhibited enzymic activity considerably.

scholarly journals A murine hybridoma with large cytoplasmic inclusions of kappa light chains.

1987 ◽  
Vol 165 (2) ◽  
pp. 578-583 ◽  
Author(s):  
D C Reason
Keyword(s):  
Cell Line ◽  
Inclusion Bodies ◽  
Protein Band ◽  
Light Chains ◽  
Cytoplasmic Inclusions ◽  
Single Protein Band ◽  
Sds Page ◽  
Single Protein ◽  
Kappa Light Chains

A murine hybridoma cell line has been established that consistently forms large cytoplasmic inclusions. These structures bind antibody specific for mouse kappa L chain when stained in situ. SDS-PAGE analysis of isolated inclusion bodies produce a single protein band of approximately 26,000 Mr that reacts with anti-kappa antibody when transferred to nitrocellulose. No carbohydrate was detected in association with the purified protein. These data are consistent with the intracellular retention and deposition of complete kappa L chain protein.

scholarly journals Purification and photoaffinity labelling of lipid methyltransferase from rat liver

1984 ◽  
Vol 223 (1) ◽  
pp. 61-66 ◽  
Author(s):  
M A Pajares ◽  
S Alemany ◽  
I Varela ◽  
D Marin Cao ◽  
J M Mato
Keyword(s):  
Fatty Acids ◽  
Fatty Acid ◽  
Rat Liver ◽  
Short Wavelength ◽  
Protein Band ◽  
Catalytic Subunit ◽  
Photoaffinity Labelling ◽  
Methyl Donor ◽  
Single Protein Band ◽  
Single Protein

An enzyme that catalyses the three-step methylation of phosphatidylethanolamine to phosphatidylcholine as well as the methylation of fatty acids and that uses S-adenosylmethionine as the methyl donor has been purified about 200-fold from rat liver. Irradiation of the purified enzyme with a short-wavelength u.v. light in the presence of [methyl-3H]8-azido-S-adenosylmethionine followed by electrophoresis results in the incorporation of radioactivity into a single protein band of about 25 kDa. It is concluded that a single catalytic subunit catalyses the conversion of phosphatidylethanolamine into phosphatidylcholine and fatty acid methylation.

scholarly journals Characterization of an EnterotoxigenicEscherichia coli Strain from Africa Expressing a Putative Colonization Factor

1999 ◽  
Vol 67 (8) ◽  
pp. 4019-4026 ◽  
Author(s):  
Sami B. Khalil ◽  
Frederick J. Cassels ◽  
Hind I. Shaheen ◽  
Lewis K. Pannell ◽  
Nemat El-Ghorab ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Immunoblot Analysis ◽  
Protein Band ◽  
Coli Strain ◽  
Spectrometric Analysis ◽  
Dot Blot ◽  
Single Protein Band ◽  
Single Protein

ABSTRACT An enterotoxigenic Escherichia coli (ETEC) strain of serotype O114:H− that expressed both heat-labile and heat-stable enterotoxins and tested negative for colonization factors (CF) was isolated from a child with diarrhea in Egypt. This strain, WS0115A, induced hemagglutination of bovine erythrocytes and adhered to the enterocyte-like cell line Caco-2, suggesting that it may elaborate novel fimbriae. Surface-expressed antigen purified by differential ammonium sulfate precipitation and column chromatography yielded a single protein band with M r 14,800 when resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (16% polyacrylamide). A monoclonal antibody against this putative fimbrial antigen was generated and reacted with strain WS0115A and also with CS1-, CS17-, and CS19-positive strains in a dot blot assay. Reactivity was temperature dependent, with cells displaying reactivity when grown at 37°C but not when grown at 22°C. Immunoblot analysis of a fimbrial preparation from strain WS0115A showed that the monoclonal antibody reacted with a single protein band. Electron microscopy and immunoelectron microscopy revealed fimbria-like structures on the surface of strain WS0115A. These structures were rigid and measured 6.8 to 7.4 nm in diameter. Electrospray mass-spectrometric analysis showed that the mass of the purified fimbria was 14,965 Da. The N-terminal sequence of the fimbria established that it was a member of the CFA/I family, with sequence identity to the amino terminus of CS19, a new CF recently identified in India. Cumulatively, our results suggest that this fimbria is CS19. Screening of a collection of ETEC strains isolated from children with diarrhea in Egypt found that 4.2% of strains originally reported as CF negative were positive for this CF, suggesting that it is biologically relevant in the pathogenesis of ETEC.

scholarly journals Glycosidases induced in Aspergillus tamarii. Mycelial α-d-galactosidases

1984 ◽  
Vol 219 (3) ◽  
pp. 849-855 ◽  
Author(s):  
A Civas ◽  
R Eberhard ◽  
P Le Dizet ◽  
F Petek
Keyword(s):  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Deae Cellulose ◽  
Protein Band ◽  
Polyacrylamide Gel ◽  
Aspergillus Tamarii ◽  
Single Protein Band ◽  
Single Protein ◽  
Alpha Galactosidase

Two alpha-D-galactosidases (alpha-D-galactoside galactohydrolase, EC 3.2.1.22) produced by Aspergillus tamarii were purified from the mycelial extract by a procedure including chromatography on hydroxyapatite, DEAE-cellulose and ECTEOLA-cellulose. Each of these enzymes showed a single protein band corresponding to the alpha-D-galactosidase activity when examined by polyacrylamide-gel electrophoresis. They catalysed the hydrolysis of o-nitrophenyl alpha-D-galactoside, melibiose, raffinose and stachyose, but did not attack the galactomannans. Their Mr values were respectively 265000 +/- 5000 and 254000 +/- 5000 by the method of Hedrick & Smith [(1968) Arch. Biochem. Biophys. 126, 155-164]. Polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate in each case showed a single protein band, with Mr 88000 and 77500 respectively. The purified enzymes contained carbohydrate, consisting of N-acetylglucosamine, mannose, glucose and galactose in the estimated molar proportions of 1:9:5:8 in alpha-galactosidase I.

scholarly journals Purification of D-alanine carboxypeptidase from Escherichia coli B on a penicillin-Sepharose column

1975 ◽  
Vol 147 (1) ◽  
pp. 131-137 ◽  
Author(s):  
M Gorecki ◽  
A Bar-Eli ◽  
Y Burstein ◽  
A Patchornik ◽  
E B Chain
Keyword(s):  
Escherichia Coli ◽  
Protein Band ◽  
Enzymic Activity ◽  
Coli Strain ◽  
Sepharose Column ◽  
Absolute Requirement ◽  
Single Protein ◽  
One Step ◽  
Triton X ◽  
Escherichia Coli B

1. A soluble D-alanine carboxypeptidase from Escherichia coli strain B was purified on a p-aminobenzylpenicillin-Sepharose column. This one-step chromatography followed by an (NH4)2SO4 precipitation yielded an enzyme purified 1200-fold and some of its properties are reported. 2. The pure D-alanine carboxypeptidase was devoid of D-alanine carboxypeptidase II activity and migrated as a single protein band on analytical disc gel electrophoresis. 3. Triton X-100 in the purification procedure is an absolute requirement for obtaining a stable enzyme. 4. The enzymic activity of D-alanine carboxypeptidase was greatly affected in solution of high salt concentrations and varied somewhat with the nature of the cation tested.

scholarly journals The isolation and properties of phenylalanine hydroxylase from rat liver

1974 ◽  
Vol 139 (3) ◽  
pp. 731-739 ◽  
Author(s):  
Shirley Su Gillam ◽  
Savio L. C. Woo ◽  
Louis I. Woolf
Keyword(s):  
Rat Liver ◽  
Phenylalanine Hydroxylase ◽  
Protein Band ◽  
Enzymic Activity ◽  
Hydroxylation Reaction ◽  
Single Protein ◽  
Thiol Binding ◽  
Filtration Step ◽  
Preliminary Filtration ◽  
Haem Iron

Phenylalanine hydroxylase was prepared from rat liver and purified 200-fold to about 90% purity. All the enzymic activity of the liver appeared in a single protein of mol.wt. approx. 110000, but omission of dithiothreitol and of a preliminary filtration step to remove lipids resulted in partial conversion into a second enzymically active protein of mol.wt. approx. 250000. The Km and Vmax. values of the enzyme for phenylalanine, p-fluorophenylalanine and dimethyltetrahydropterin were measured; p-chlorophenylalanine inhibited the enzyme by competing with phenylalanine. Disc gel electrophoresis at pH7.2 showed a single protein band containing all the enzymic activity, but at pH8.7 the enzyme dissociated into two inactive fragments of similar but not identical molecular weight. The molecule of phenylalanine hydroxylase contained two atoms of iron, one atom of copper and one molecule of FAD; molybdenum was absent. Treatment with chelating agents showed that both non-haem iron and copper were necessary for enzymic activity. The molecule contained five thiol groups, and thiol-binding reagents inhibited the enzyme. Catalase or peroxidase enhanced enzymic activity fivefold; it is postulated that catalase (or other peroxidase) plays a part in the hydroxylation reaction independent of the protection by catalase of enzyme and cofactor from inactivation by a hydroperoxide.

Purification of p-hydroxyphenylpyruvate hydroxylase from rat liver—requirements for cofactors

1976 ◽  
Vol 54 (5) ◽  
pp. 423-431 ◽  
Author(s):  
Kun-Tsan Lin ◽  
John C. Crawhall
Keyword(s):  
Molecular Weight ◽  
Enzyme Activity ◽  
Rat Liver ◽  
Gel Filtration ◽  
Protein Band ◽  
Final Concentration ◽  
Assay Method ◽  
Enzyme Extract ◽  
Crude Enzyme Extract ◽  
Single Protein

Theenzyme p-hydroxyphenylpyruvate hydroxylase (EC 1.13.11.27)from rat liver was studied with the assay method which measures the release of 14CO2 from p-hydroxyphenyl [carboxyl-,14C]pyruvate. Extensive dialysis of the crude enzyme extract against Tris buffer or purification involving ammonium sulfate, gel filtration, and ion exchange results in loss of enzyme activity that can be reactivated by Fe2+, dichlorophenolindophenol, and various other agents. The effect of these activators depends critically on their final concentration in the assay media.A 70-fold purification of the enzyme fraction yielded a preparation which behaved as a single protein band in Sephadex G-150. It had an isoelectric point at 5.85 and molecular weight of 63 000. The enzyme obtained appears to be different in some respects from those described by other workers from the liver of dog, human, chicken, and frog.

scholarly journals Nanomaterials Enhanced Gene Expression in Yeast Cells

2008 ◽  
Vol 2008 ◽  
pp. 1-9 ◽  
Author(s):  
Su-Fang Chien ◽  
Shi-Hui Chen ◽  
Chhiu-Tsu Lin
Keyword(s):  
Gene Expression ◽  
Ag Nanoparticles ◽  
Product Yield ◽  
Protein Band ◽  
Yeast Cells ◽  
Vis Spectroscopy ◽  
Yeast Chromosome ◽  
Single Protein ◽  
Enhance Gene Expression ◽  
Metal Nanomaterials

Metal nanomaterials are shown to enhance gene expression for rice -galactosidase gene (-Gal) in yeast cells. Au and Ag nanoparticles and their nanocomposites, silica-Au and silica-Ag, were prepared and characterized by UV-vis spectroscopy and TEM technique. The rice -galactosidase gene was cloned into the yeast chromosome, where the cloned cells were precultured and induced into a medium containing each of the testing nanomaterials. The nanomaterials were observed to incorporate inside the cells, and no cell death has been detected during the course of gene expression. The enzyme activity was determined by a synthetic substrate, p-nitrophenyl--D-galctopyranoside, and the yellow product yield was recorded in a spectrophotometer at 400 nm. When Au and Ag nanoparticles were incorporated with the culture, a 3–5 fold enhancement in -galactosidase was observed for intracellular activity as well as the secreted activity into the medium. The secreted protein was analyzed to have a pure form and displayed as a single protein band in the SDS-gel electrophoresis. The effects of size and chemical nature of nanomaterials on gene expression for the rice -galactosidase gene in yeast cells are discussed.

scholarly journals β-Defensin 22 is a major component of the mouse sperm glycocalyx

Reproduction ◽  
2008 ◽  
Vol 136 (6) ◽  
pp. 753-765 ◽  
Author(s):  
Ashley I Yudin ◽  
Theodore L Tollner ◽  
Cathy A Treece ◽  
Robert Kays ◽  
Gary N Cherr ◽  
...  
Keyword(s):  
Vas Deferens ◽  
Ultrastructural Level ◽  
Protein Band ◽  
Seminal Vesicles ◽  
Major Site ◽  
Western Blots ◽  
Sperm Surface ◽  
Single Protein ◽  
Cauda Epididymidis ◽  
Purified Antigen

Surface components of sperm isolated from the cauda epididymides were stabilized by whole sperm fixation for immunization of rabbits. The resulting immunoglobulins (Igs) recognized a single protein of 130 kDa (non-reduced) or 54–57 kDa (reduced) on western blots of cauda sperm. Igs recognized the same 54–57 kDa protein band on whole tissue blots of the corpus and cauda epididymidis and vas deferens. No immunoreactive bands were detected on blots of the prostate, seminal vesicles, testes, caput epididymis, or any of various non-reproductive tissues. Removal of sperm from the vas deferens prior to blotting eliminated the detection of the sperm antigen. Antibodies raised to synthetic peptides, identical in amino acid sequence to two unique spans of DEFB22, recognized the same 130/54–57 kDa antigen on western blots of both caudal sperm and the purified antigen isolated with the anti-sperm Ig. From indirect immunofluorescence, both the anti-sperm and anti-peptide Igs appeared to localize to the entire sperm surface, a pattern confirmed at the ultrastructural level. Real-time PCR identified the corpus epididymides as the major site of expression of DEFB22, with negligible expression in the testes, caput epididymides, and vas deferens. Immunostaining of epididymal sections showed DEFB22 being released into the lumen at the distal caput/proximal corpus, with sperm becoming intensely coated with DEFB22 as they reached the distal corpus. Most uterine sperm recovered from mice 4 h following copulation exhibited DEFB22 coating the entire sperm surface. By contrast, some sperm recovered from the oviduct and cumulus extracellular matrix showed loss of DEFB22 from the sperm head.

scholarly journals Physiochemical properties of a heat-stable enterotoxin produced by Escherichia coli of human origin

1980 ◽  
Vol 28 (3) ◽  
pp. 1051-1053
Author(s):  
G L Madsen ◽  
F C Knoop
Keyword(s):  
Escherichia Coli ◽  
Isoelectric Focusing ◽  
Gel Electrophoresis ◽  
Molecular Sieve ◽  
Protein Band ◽  
Biologically Active ◽  
Enterotoxigenic Escherichia Coli ◽  
Physiochemical Properties ◽  
Heat Stable ◽  
Single Protein

Concentrated cell-free filtrates, prepared from a human strain of enterotoxigenic Escherichia coli, were subjected to isoelectric focusing, molecular sieve chromatography, and polyacrylamide disc gel electrophoresis. Isoelectric focusing in a pH 3 to 5 gradient resulted in two biologically active peaks, I and II, that electrofocused at pI 1.5 and 3.8, respectively. Molecular sieve chromatography of the major enterotoxic peak (II) at pH 3.8 indicated a molecular weight of 2,500. Polyacrylamide disc gel electrophoresis of ST revealed a single protein band containing enterotoxic activity.

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