scholarly journals The properties of a nuclear acidic protein fraction that binds [6,7−3H]oestradiol-17β

1969 ◽  
Vol 114 (3) ◽  
pp. 649-657 ◽  
Author(s):  
R. J. B. King ◽  
J. Gordon ◽  
A W Steggles
Keyword(s):  
Common Property ◽  
Polyacrylamide Gel Electrophoresis ◽  
Convincing Evidence ◽  
Acidic Protein ◽  
Mammary Adenocarcinoma ◽  
Receptor Protein ◽  
Unit Weight ◽  
Partial Purification ◽  
Protamine Sulphate ◽  
Calf Thymus

1. Additional evidence was obtained that the nuclear oestradiol-17β receptor is an acidic protein. Partial purification of the receptor protein was obtained by chromatography on hydroxyapatite and it contains protein-bound phosphate. 2. The nuclear ‘5s’ and cytoplasmic ‘9·5s’ and ‘5s’ receptors from uterus, dimethylbenzanthracene-induced mammary adenocarcinoma and kidney are precipitated together with bound oestradiol-17β by protamine sulphate. This common property suggests that the nuclear and cytoplasmic receptors are related to each other. 3. The properties of two acidic protein fractions from both liver and dimethylbenzanthracene-induced mammary adenocarcinoma are described. Fraction 1 contains two major components and fraction 2 contains one component, as judged from polyacrylamide-gel electrophoresis. Fraction 2 contains RNA and both fractions contain protein-bound phosphate. 4. These fractions form insoluble complexes with calf thymus histone, protamine sulphate and poly-l-lysine. The formation of these complexes is markedly affected by ionic strength and pH. Ionization of both the ∈-amino group of lysine and carboxyl group are involved. RNA and DNA do not appear to be involved. The interaction is not affected by EDTA or 1mm-Na+, -K+, -Ca2+, -Mg2+ or -Mn2+. Per unit weight, whole histone has 4–5 times as many binding sites for the acidic proteins as the latter have for the former. 5. No convincing evidence was obtained for DNA–acidic protein interaction, but, as judged from precipitation experiments, there was competition between DNA and acidic protein for histone. 6. Relatively large amounts of acidic protein partly relieved the histone inhibition of the template activity of DNA for Escherichia coli RNA polymerase (EC 2.7.7.6).

Plasminogen-Activator in Human Early Milk: Its Partial Purification and Characterization

1981 ◽  
Vol 45 (02) ◽  
pp. 121-126 ◽  
Author(s):  
Utako Okamoto ◽  
Noboru Horie ◽  
Yoko Nagamatsu ◽  
Jun-Ichiro Yamamoto
Keyword(s):  
Plasminogen Activator ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Partial Purification ◽  
Order Kinetic ◽  
Diisopropyl Fluorophosphate ◽  
Step Procedure ◽  
Activity Band ◽  
C 50 ◽  
Gel Isoelectric Focusing

SummaryMilk plasminogen-activator was partially purified from human transitional milk collected at about 10 days after delivery, by a five-step procedure involving chloroform treatment, ammonium sulfate precipitation, and column chromatography on Sephadex G-150, CM Sephadex C-50 and DEAE Sephadex A-50. This gave milk-activator with a maximum purification factor of about 2,400-fold with respect to the skimmed milk. The CM Sephadex-step preparation showed, on polyacrylamide gel electrophoresis, a single plasminogen-activator activity band located between the bands of albumin and prealbumin of human serum. This preparation exhibited no kinin forming activity. The activator hydrolyzed acetyl-glycyl-L-lysine methyl ester with similar order kinetic constants to urokinase, and was inhibited strongly by diisopropyl-fluorophosphate. The molecular weight of the activator as estimated by gel filtration was approximately 86,000, the isoelectric points as estimated by gel isoelectric focusing were pH 7.2, 6.9 and 6.6, and the activator activity was not quenched by antiurokinase globulin, indicating that the milk-activator is a different entity from urokinase.

scholarly journals Isolation, partial purification and characterization of phospholipase A2 from Naja Katiensis venom

2021 ◽  
Vol 13 (2) ◽  
pp. 107-112
Author(s):  
C.F. Okechukwu ◽  
P.L. Shamsudeen ◽  
R.K. Bala ◽  
B.G. Kurfi ◽  
A.M. Abdulazeez
Keyword(s):  
Ion Exchange ◽  
Phospholipase A2 ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Partial Purification ◽  
Crude Venom

The most effective and acceptable therapy for snakebite victims is the immediate administration of antivenin which is limited by problems of hypersensitivity reactions in some individuals and its inability to resolve the local effects of the venom. The aim of this study was to isolate, partially purify and characterize phospholipase A2 from Naja Katiensis venom. Phospholipase A2 was partially purified via a two-step process: gel filtration on Sephadex G-75 and ion exchange chromatography using CM Sephadex, and subjected to SDS-PAGE analysis. From the results, the specific activity of the partially purified PLA2 decreased from 0.67μmol/min/mg in crude venom to 0.29μmol/min/mg after ion exchange chromatography with a yield of 5% and purification fold of 0.43. The optimum temperature of the purified PLA2 was found to be 35ºC and optimum p.H of 7. velocity studies for the determination of kinetic constants using L-a-lecithin as substrate revealed a Km  of 1.47mg/ml and Vmax  of 3.32μ moles/min/mg. The sodium dodecyl sulphate polyacrylamide gel electrophoresis of the purified PLA2 showed a distinct band with molecular weight estimated to be 14KDa. In conclusion, the present study shows that phospholipase A2 was isolated, purified and characterized. This may serve as a promising candidate for future development of a novel anti-venin drug.

scholarly journals Biological Properties of IRIM, the Iridium(III) Analogue of (Imidazolium (Bisimidazole) Tetrachlororuthenate) (ICR)

2000 ◽  
Vol 7 (4) ◽  
pp. 195-200 ◽  
Author(s):  
G. Marcon ◽  
A. Casini ◽  
P. Mura ◽  
L. Messori ◽  
A. Bergamo ◽  
...  
Keyword(s):  
Cell Lines ◽  
Biological Effects ◽  
Direct Interaction ◽  
Biological Properties ◽  
Mammary Adenocarcinoma ◽  
Interstrand Crosslinks ◽  
Conformational Effects ◽  
Calf Thymus ◽  
Human Ovarian Carcinoma ◽  
Dna Thermal Denaturation

Some biological aspects of the new complex imidazolium bisimidazole tetrachloro iridate(III)-IRIM- the iridium(III) analogue of ICR, were considered. More in detail the conformational effects produced by IRIM on DNA and the cytotoxic properties of IRIM on some selected human cell lines were measured. Dialysis experiments and DNA thermal denaturation studies are suggestive of poor binding of IRIM to DNA; formation of interstrand crosslinks is not observed. In any case CD measurements suggest that addition of increasing amounts of IRIM to calf thymus DNA results into significant spectral changes, that are diagnostic of a direct interaction with DNA. A number of experiments carried out on the A2780 human ovarian carcinoma, B16 murine melanoma, MCF7 and TS mammary adenocarcinoma tumor cell lines strongly point out that IRIM does not exhibit significant growth inhibition effects within the concentration range 10-4-10-6 M. It is suggested that the lower biological effects of IRIM compared to ICR are a consequence of the larger kinetic inertness of the iridium(III) center with respect to ruthenium(III).

A Dictyostelium mutant with severe defects in alpha-actinin: its characterization using cDNA probes and monoclonal antibodies

1988 ◽  
Vol 90 (1) ◽  
pp. 59-71
Author(s):  
M. Schleicher ◽  
A. Noegel ◽  
T. Schwarz ◽  
E. Wallraff ◽  
M. Brink ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Polypeptide Chain ◽  
Mutant Cell ◽  
Polyacrylamide Gel Electrophoresis ◽  
Cell Extract ◽  
Binding Activity ◽  
Partial Purification ◽  
Wild Type ◽  
Coding Region ◽  
In The Wild

Cells of a Dictyostelium discoideum mutant deficient in binding a monoclonal antibody to alpha-actinin have previously been shown to grow and develop similarly to the wild type and to exert unimpaired chemotaxis as well as patching and capping of membrane proteins. Here we show that the normal 3.0 kb message for alpha-actinin is replaced in the mutant by two RNA species of approximately 3.1 and 2.8 kb. The 3.1 kb RNA was recognized by DNA fragments from all parts of the coding region, while the 2.8 kb RNA hybridized to all but a 3′-terminal fragment. Proteins synthesized in the mutant were analysed using four monoclonal antibodies that in the wild type specifically recognize the 95 × 10(3) Mr polypeptide of alpha-actinin. Cleavage mapping indicated that the binding sites of these antibodies are distributed over a region comprising more than half of the alpha-actinin polypeptide chain. In the mutant, three of the antibodies faintly labelled two polypeptides of 95 × 10(3) Mr and 88 × 10(3) Mr; the fourth antibody, which binds closest to one end of the polypeptide chain, faintly labelled the 95 × 10(3) Mr polypeptide only. The 88 × 10(3) Mr polypeptide most probably lacks the C-terminal portion of alpha-actinin. The binding of an antibody that recognized both polypeptides was quantified by a radio-immuno competition assay using wild-type alpha-actinin as a reference. In a mutant cell extract containing total soluble proteins the antibody binding activity was decreased to 1.1% when compared with wild-type extract. After their partial purification and SDS-polyacrylamide gel electrophoresis the mutant 95 × 10(3) Mr and 88 × 10(3) Mr polypeptides were barely detectable as Coomassie Blue-stained bands, indicating that in the mutant not only certain epitopes of alpha-actinin were altered but the entire molecule is almost completely lacking. When the fitness of mutant cells relative to wild type was determined during growth in nutrient medium, a slight disadvantage for the mutant was indicated, by finding selection coefficients between 0.03 and 0.05.

Partial purification and characterisation of α-amylases from the digestive tract of the Indian major carp Labeo rohita (Hamilton, 1822)

2020 ◽  
Vol 67 (2) ◽  
Author(s):  
Umalatha . ◽  
N. Sridhar ◽  
Jairam Prasad Kushwaha ◽  
Vadlapudi Kumar
Keyword(s):  
Digestive Tract ◽  
Incubation Temperature ◽  
Amylase Activity ◽  
Labeo Rohita ◽  
Polyacrylamide Gel Electrophoresis ◽  
Exchange Chromatography ◽  
Partial Purification ◽  
Indian Major Carp ◽  
Native Polyacrylamide Gel Electrophoresis ◽  
Acetone Fractionation

Partial purification of α-amylases from the digestive tract of the Indian major carp Labeo rohita (Hamilton, 1822) through acetone fractionation and ion exchange chromatography (DEAE-SephadexA-50) resulted in 8-fold purification with 86% recovery. Characterisation of amylase activity revealed two pH optima at 4.5 and 6.5. Activity was stable over wide pH ranges of 3.5 to 4.5 and 7 to 12. Optimum incubation temperature was 35°C. The enzyme lost 91% activity at 60°C within 15 min and was inhibited by Amylase inhibitor Type-1 (wheat); 1, 10 Phenanthroline, Ethylene diamine tetra-acetate (EDTA) and Phenyl methyl sulphonyl fluoride (PMSF). Heavy metal ions Hg++ and Cu++ strongly inhibited the enzyme activity, while Zn++ and Bi++ inhibited to a lesser extent. Native polyacrylamide gel electrophoresis of the purified amylase fractions revealed four bands, with corresponding molecular weights of 43.59, 52.36, 55.42 and 54.01 kDa. α-Amylase activity from L. rohita exhibited linear hydrolysis of starch upto 7% concentration in 60 min.

Guanosine 3’:5’-Cyclic Monophosphate -Dependent Particulate Protein Kinase Activity from Yeast (Saccharomyces cerevisiae)

1999 ◽  
Vol 54 (1-2) ◽  
pp. 84-93 ◽  
Author(s):  
Hans Eckstein ◽  
Birgit Flügge
Keyword(s):  
Protein Kinase ◽  
Polyacrylamide Gel Electrophoresis ◽  
Deae Cellulose ◽  
Temperature Tolerance ◽  
Maximum Activity ◽  
Partial Purification ◽  
Protein Kinase Activity ◽  
Ph Optimum ◽  
Yeast Saccharomyces Cerevisiae ◽  
High Concentrations

Continuing our studies on cGMP in growing yeast we detected a particulate cGMPdependent protein kinase (Pk-G), which was solubilized by detergents and NaCl. It achieves maximum activity at 25 °C and pH = 6.8, high concentrations of substrate proteins or cGMP produce saturation. Casein and histones are appropriate substrates, phosphatase-pretreated histone H-2a provokes outstandingly high activity. Pk-G differs from cAMP-dependent protein kinase (Pk-A) with respect to pH optimum, temperature tolerance above 50 °C, and stability. Partial purification is achieved by chromatography with DEAE-cellulose, Sepharose, and cGMP-substituted Sepharose. The latter step also markedly removes Pk-A. At least three proteins with Pk-G-activity and high cGMP-affinity are separated by polyacrylamide-gel-electrophoresis. Their apparent molecular masses, as deduced from comigrating marker proteins, differ considerably from those of other Pk-G’s, but also of Pk-A’s

scholarly journals Characterization of a Host-Specific Protein Toxin (Ptr ToxB) from Pyrenophora tritici-repentis

1999 ◽  
Vol 12 (8) ◽  
pp. 728-732 ◽  
Author(s):  
Stephen E. Strelkov ◽  
Lakhdar Lamari ◽  
G. Murray Ballance
Keyword(s):  
Amino Acid ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Specific Protein ◽  
Tan Spot ◽  
Partial Purification ◽  
Toxic Activity ◽  
Pyrenophora Tritici Repentis ◽  
Protein Toxin ◽  
Cation Exchange Column

Pyrenophora tritici-repentis, the causal agent of tan spot of wheat, differentially induces tan necrosis and/or chlorosis in wheat. A chlorosis-inducing, host-specific toxin, termed Ptr ToxB (formerly Ptr chlorosis toxin), was purified from the culture filtrates of a race 5 isolate of P. tritici-repentis. Partial purification was performed by 25 to 80% ammonium sulfate precipitation and passage through a carboxy-methyl-Sephadex C-25 cation exchange column. Final purification was performed by fast performance liquid chromatography, with a Mono S HR 5/5 cation exchanger, followed by size fractionation on a Superose 12 HR 10/30 column. The toxin was shown to be proteinaceous in nature, and purity was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular mass of Ptr ToxB was determined to be 6.61 kDa. The amino acid composition and partial N terminus amino acid sequence of the toxin were also obtained. Ptr ToxB was found to be heat stable, maintaining full toxic activity after 1 h at 55°C. Infiltration of toxin concentrations as low as 14 nM produced chlorosis on susceptible cultivars.

THE BINDING OF ANIONS BY LYSOZYME, CALF THYMUS HISTONE SULPHATE, AND PROTAMINE SULPHATE

1952 ◽  
Vol 30 (4) ◽  
pp. 320-331 ◽  
Author(s):  
J. Ross Colvin
Keyword(s):  
Protein Binding ◽  
Temperature Coefficient ◽  
Adsorption Isotherms ◽  
Large Series ◽  
Orange Ii ◽  
General Process ◽  
Protamine Sulphate ◽  
Calf Thymus ◽  
Pass Through ◽  
Positively Charged

The adsorption of a large series of anions by positively charged lysozyme, calf thymus histone sulphate, and protamine sulphate has been studied by dialysis equilibrium. One group of anions was not adsorbed by lysozyme while the adsorption isotherms for Orange I, Orange II, and 2, 4-dinitro-1-naphthol-7-sulphonic acid were S-shaped. These anomalous isotherms, which were also obtained with histone sulphate and protamine sulphate, have an appreciable temperature coefficient at intermediate dye concentrations. It is emphasized that this type of adsorption is inconsistent with the exclusively electrostatic view of protein binding of anions. It is also shown that −ΔF, −ΔH, and ΔS per mole of anion bound must pass through a maximum for intermediate free anion concentrations in such systems. The lack of binding of some anions and the anomalous isotherms for others is interpreted in terms of a solvation sheath about the charged protein. Their significance for the general process of binding of ions by proteins is discussed.

scholarly journals Biological Control of Fusarium Wilt of Cucumber by Chitinolytic Bacteria

1999 ◽  
Vol 89 (1) ◽  
pp. 92-99 ◽  
Author(s):  
Pushpinder Paul Singh ◽  
Yong Chul Shin ◽  
Chang Seuk Park ◽  
Young Ryun Chung
Keyword(s):  
Fusarium Wilt ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Hydrolytic Enzymes ◽  
Partial Purification ◽  
Streptomyces Sp ◽  
Chitinolytic Bacteria ◽  
Potting Medium ◽  
Paenibacillus Sp ◽  
Molecular Masses

Two chitinolytic bacterial strains, Paenibacillus sp. 300 and Streptomyces sp. 385, suppressed Fusarium wilt of cucumber (Cucumis sativus) caused by Fusarium oxysporum f. sp. cucumerinum in nonsterile, soilless potting medium. A mixture of the two strains in a ratio of 1:1 or 4:1 gave significantly (P < 0.05) better control of the disease than each of the strains used individually or than mixtures in other ratios. Several formulations were tested, and a zeolite-based, chitosan-amended formulation (ZAC) provided the best protection against the disease. Dose-response studies indicated that the threshold dose of 6 g of formulation per kilogram of potting medium was required for significant (P < 0.001) suppression of the disease. This dose was optimum for maintaining high rhizosphere population densities of chitinolytic bacteria (log 8.1 to log 9.3 CFU/g dry weight of potting medium), which were required for the control of Fusarium wilt. The ZAC formulation was suppressive when added to pathogen-infested medium 15 days before planting cucumber seeds. The formulation also provided good control when stored for 6 months at room temperature or at 4°C. Chitinase and β-1,3-glucanase enzymes were produced when the strains were grown in the presence of colloidal chitin as the sole carbon source. Partial purification of the chitinases, followed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and activity staining, revealed the presence of five bands with molecular masses of 65, 62, 59, 55, and 52 kDa in the case of Paenibacillus sp. 300; and three bands with molecular masses of 52, 38, and 33 kDa in the case of Streptomyces sp. 385. Incubation of cell walls of F. oxysporum f. sp. cucumerinum with partially purified enzyme fractions led to the release of N-acetyl-D-glucosamine (NAGA). NAGA content was considerably greater when pooled enzyme fractions (64 to 67) from Paenibacillus sp. were used, because they contained high β-1,3-glucanase activity in addition to chitinase activity. Suppression of Fusarium wilt of cucumber by a combination of these two bacteria may involve the action of these hydrolytic enzymes.

Partial purification and characterization of isopenicillin N epimerase activity from Streptomyces clavuligerus

1983 ◽  
Vol 29 (11) ◽  
pp. 1526-1531 ◽  
Author(s):  
Susan E. Jensen ◽  
Donald W. S. Westlake ◽  
Saul Wolfe
Keyword(s):  
Pyridoxal Phosphate ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Streptomyces Clavuligerus ◽  
Enzymic Activity ◽  
Partial Purification ◽  
Major Protein ◽  
Major Protein Band ◽  
Isopenicillin N

Epimerase activity, which converts isopenicillin N to penicillin N, has been partially purified from cell-free extracts of Streptomyces clavuligerus. No stimulating cofactors of this activity were found, and neither EDTA nor anaerobic incubation caused significant inhibition of activity. Although pyridoxal phosphate did not stimulate epimerase activity, the presence of this cofactor was necessary for the stabilization of enzymic activity during the purification process. Epimerase activity was purified 35.5-fold by a combination of salt precipitation, gel filtration, and ion exchange chromatography. Gel filtration indicated that the epimerase has a molecular weight of 60 000 and sodium dodecyl sulphate – polyacrylamide gel electrophoresis of the 35.5-fold purified epimerase showed a major protein band running near that location. Pyridoxal phosphate antagonists did not uniformly inhibit epimerase activity, but the inhibitory effect of hydroxylamine could be partially reversed by pyridoxal phosphate.

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