scholarly journals The nucleoside triphosphate–ribonucleic acid nucleotidyltransferase (EC 2.7.7.6) of Agrobacterium tumefaciens (Smith and Townsend) Conn. Purification and properties of the enzyme from the tumorigenic strain B6806

1974 ◽  
Vol 143 (3) ◽  
pp. 511-520 ◽  
Author(s):  
U. C. Knopf
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Gradient Centrifugation ◽  
Deae Cellulose ◽  
Nucleoside Triphosphate ◽  
Subunit Structure ◽  
Bacterial Cells ◽  
Protein Subunit ◽  
Ph Optimum ◽  
Protamine Sulphate

The RNA nucleotidyltransferase (RNA polymerase) of the plant-tumorigenic bacterium Agrobacterium tumefaciens was purified. The method involves the disruption of the bacterial cells with glass beads in a Waring Blendor, treatment with DEAE-cellulose, fractionation with (NH4)2SO4, protamine sulphate precipitation, DEAE-cellulose column chromatography and either glycerol-gradient centrifugation or phosphocellulose chromatography. The subunit structure of the highly purified enzyme is similar to, although not identical with, the RNA nucleotidyltransferase of Escherichia coli. It can be described as β′, β, χ1 and α (mol.wts. 160000, 150000, 98000, and 41000±10% respectively). χ1 is the temporary designation for a protein subunit, which might have the same functions as the σ subunit in E. coli. The enzyme of A. tumefaciens is rifampicin-sensitive, has a temperature optimum in vitro of 41±1°C and a pH optimum of 8.2±0.1. Mg2+ and Mn2+ are activators. The enzyme transcribes with different efficiencies artificial, viral, bacterial, plant and animal templates.

scholarly journals Alteration of hepatic glucocorticoid receptor stability and nuclear binding in vitro by citrate

1983 ◽  
Vol 210 (1) ◽  
pp. 259-263 ◽  
Author(s):  
J Hubbard ◽  
M Kalimi
Keyword(s):  
Glucocorticoid Receptor ◽  
Glucocorticoid Receptors ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Deae Cellulose ◽  
Sucrose Density Gradient ◽  
Sucrose Density Gradient Centrifugation ◽  
Nuclear Binding ◽  
Wide Range

Citrate greatly stabilized rat hepatic unbound glucocorticoid receptors in cell-free conditions at 4 degrees C with optimal effectiveness at 5-15 mM. Control receptors were inactivated at 4 degrees C with a half-life of less than 12 h. However, in the presence of 10 mM-citrate, unbound receptors were almost completely stabilized for 48 h at 4 degrees C. Citrate at a concentration of 1-2 mM yielded half-maximal stabilization. The stabilizing effect of citrate was rather specific, as succinate, alpha-oxoglutarate, oxaloacetate, malate and pyruvate had no apparent stabilizing action. Citrate stabilized receptors over a wide range of H+ concentrations, with complete protection between pH 6.5 and 8.5. In addition, citrate appeared to have a significant effect on glucocorticoid-receptor complex activation into a nuclear binding form. Thus 5-10 mM-citrate enhanced nuclear binding, with optimal activation achieved at 10 mM concentration. As analysed by sucrose-density-gradient centrifugation and DEAE-cellulose chromatography, no apparent change was observed in the physical characteristics of the glucocorticoid receptor in the presence of citrate.

scholarly journals Microbial L-phenylalanine ammonia-lyase. Purification, subunit structure and kinetic properties of the enzyme from Rhizoctonia solani

1975 ◽  
Vol 149 (1) ◽  
pp. 65-75 ◽  
Author(s):  
K K Kalghatgi ◽  
P V Subba Rao
Keyword(s):  
Molecular Weight ◽  
Rhizoctonia Solani ◽  
Phenylalanine Ammonia Lyase ◽  
Pathogenic Fungus ◽  
Gradient Centrifugation ◽  
Hill Coefficient ◽  
Velocity Versus ◽  
Kinetic Properties ◽  
Subunit Structure ◽  
Protamine Sulphate

1. Phenylalanine ammonia-lyase (EC 4.3.1.5) was purified to homogeneity from the acetone-dried powders of the mycelial felts of the plant pathogenic fungus Rhizoctonia solani. 2. A useful modification in protamine sulphate treatment to get substantial purification of the enzyme in a single-step is described. 3. The purified enzyme shows bisubstrate activity towards L-phenylalanine and L-tyrosine. 4. It is sensitive to carbonyl reagents and the inhibition is not reversed by gel filtration. 5. The molecular weight of the enzyme as determined by Sephadex G-200 chromatography and sucrose-density-gradient centrifugation is around 330000. 6. The enzyme is made up of two pairs of unidentical subunits, with a molecular weight of 70000 (α) and 90000 (β) respectively. 7. Studies on initial velocity versus substrate concentration have shown significant deviations from Michaelis-Menten kinetics. 8. The double-reciprocal plots are biphasic (concave downwards) and Hofstee plots show a curvilinear pattern. 9. The apparent Km value increases from 0.18 mM to as high as 5.0 mM with the increase in the concentration of the substrate and during this process the Vmax, increases by 2-2.5-fold. 10. The value of Hill coefficient is 0.5. 11. Steady-state rates of phenylalanine ammonia-lyase reaction in the presence of inhibitors like D-phenylalanine, cinnamic, p-coumaric, caffeic, dihydrocaffeic and phenylpyruvic acid have shown that only one molecule of each type of inhibitor binds to a molecule of the enzyme. These observations suggest the involvement of negative homotropic interactions in phenylalanine ammonia-lyase. 12. The enzyme could not be desensitized by treatment with HgCl2, p-chloromercuribenzoic acid or by repeated freezing and thawing.

scholarly journals Biosynthesis of respiratory-tract mucins. Incorporation of radioactive precursors into glycoproteins by canine tracheal explants in vitro

1973 ◽  
Vol 136 (4) ◽  
pp. 837-844 ◽  
Author(s):  
Daniel B. Ellis ◽  
Glenn H. Stahl
Keyword(s):  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Deae Cellulose ◽  
Sialic Acids ◽  
Equilibrium Density ◽  
Agarose Gels ◽  
Cscl Gradient ◽  
Tracheal Explants

1. Canine tracheal explants, cultured in medium 199, actively incorporated radioactive precursors into secreted macromolecules in vitro. 2. Puromycin, 6-diazo-5-oxo-l-norleucine and ouabain markedly inhibited the incorporation of these precursors. 3. Exogenous glucosamine at concentrations above 20mm caused a greater than 50% inhibition of the incorporation of l-[G-3H]fucose and l-[U-14C]serine. 4. Carbohydrate content of the purified secretions was approximately 50% and consisted principally of galactose, N-acetylglucosamine, N-acetylgalactosamine, fucose and sialic acids. 5. Chromatography on DEAE-cellulose and Bio-Gel A-150m and equilibrium density-gradient centrifugation in a CsCl gradient confirmed the presence of mucous glycoproteins. 6. Electrophoresis on 1% agarose gels gave profiles that were identical with canine respiratory mucus obtained in vivo. 7. These results support the utility of the explant system for studies of respiratory secretions.

scholarly journals The degradation of l-histidine in the rat. The formation of imidazolylpyruvate, imidazolyl-lactate and imidazolylpropionate

1973 ◽  
Vol 136 (3) ◽  
pp. 649-658 ◽  
Author(s):  
Alan V. Emes ◽  
Harold Hassall
Keyword(s):  
Deae Cellulose ◽  
Substrate Affinity ◽  
Significant Activity ◽  
Anaerobic Conditions ◽  
Ph Optimum ◽  
Oxidoreductase Activity ◽  
Molecular Weights ◽  
Optimum Stability ◽  
Nad Oxidoreductase

1. Soluble and mitochondrial forms of histidine–pyruvate aminotransferase were separated from rat liver preparations by chromatography on DEAE-cellulose. 2. These enzymes were characterized with respect to substrate specificity, substrate affinity, pH optimum, stability and molecular weight by chromatography on Sephadex G-200. 3. Each enzyme has a relatively broad specificity showing significant activity towards l-phenylalanine and l-tyrosine and catalysing transamination with a number of monocarboxylic 2-oxo acids. 2-Oxoglutarate is not a substrate for either enzyme. 4. The molecular weights of the two enzymes, by chromatography on Sephadex G-200, are in the range 130000–150000. 5. The formation in vitro of imidazolyl-lactate from imidazolylpyruvate and NADH was demonstrated by using liver preparations. 6. From a study of imidazolyl-lactate–NAD+oxidoreductase activity after electrophoresis of liver preparations on polyacrylamide gel, and from an examination of the activity of l-lactate–NAD+oxidoreductase (EC 1.1.1.27) towards imidazolylpyruvate, it is concluded that this latter enzyme is responsible for the formation of imidazolyl-lactate in the liver. 7. Preparations of bacteria obtained from rat faeces form imidazolylpropionate from l-histidine and urocanate without further subculture. The amount of imidazolylpropionate formed is increased under anaerobic conditions and more so in an atmosphere of H2. It is suggested that the gut flora of the rat contribute largely, if not exclusively, to the formation of imidazolylpropionate normally found in the urine.

scholarly journals An NAD+-dependent alanine dehydrogenase from a methylotrophic bacterium

1987 ◽  
Vol 244 (3) ◽  
pp. 565-570 ◽  
Author(s):  
E Bellion ◽  
F Tan
Keyword(s):  
Native Enzyme ◽  
Methylotrophic Bacterium ◽  
Subunit Structure ◽  
Pseudomonas Sp ◽  
Polyacrylamide Gels ◽  
Limited Extent ◽  
Ph Optimum ◽  
Alanine Dehydrogenase

A study was made of the NAD+-dependent alanine dehydrogenase (EC 1.4.1.1) elaborated by the methylotrophic bacterium Pseudomonas sp. strain MA when growing on succinate and NH4Cl. This enzyme was purified 400-fold and was found to be highly specific for NH3 and NAD+; however, hydroxypyruvate and bromopyruvate, but not alpha-oxoglutarate or glyoxylate, could replace pyruvate to a limited extent. The Mr of the native enzyme was shown to be 217,000, and electrophoresis in SDS/polyacrylamide gels revealed a minimum Mr of 53,000, suggesting a four-subunit structure. The enzyme, which has a pH optimum of 9.0, operated almost exclusively in the aminating direction in vitro. It was induced by NH3 or by alanine, and was repressed by growth on methylamine or glutamate. It is suggested that this enzyme has two roles in this organism, namely in NH3 assimilation and in alanine catabolism.

scholarly journals Purification and subunit analysis of wheat-germ ribonucleic acid polymerase II

1974 ◽  
Vol 139 (3) ◽  
pp. 771-777 ◽  
Author(s):  
Jerome J. Jendrisak ◽  
Wayne M. Becker
Keyword(s):  
Enzyme Activity ◽  
Rna Polymerase ◽  
Wheat Germ ◽  
Sucrose Gradient ◽  
Gradient Centrifugation ◽  
Deae Cellulose ◽  
Protamine Sulphate ◽  
Molecular Weights ◽  
Molar Ratios ◽  
High Salt Buffer

A procedure is described for the purification of the α-amanitin-sensitive DNA-dependent RNA polymerase [EC 2.7.7.6] from wheat germ. Solubilization of the enzyme activity was achieved by sonication of a crude extract in a high-salt buffer. Purification involved precipitation with protamine sulphate and (NH4)2SO4, chromatography on DEAE-cellulose and phosphocellulose, and sucrose gradient centrifugation. Under denaturing conditions the enzyme dissociated into five polypeptides with molecular weights and molar ratios of 220000 (0.9), 170000 (0.1), 140000 (1.0), 45000 (0.2), and 40000 (0.4). Approx. 1mg of purified RNA polymerase was obtained as a routine from 100g of starting material.

scholarly journals Antibody-dependent lymphocyte-mediated granulocyte cytotoxicity in man

Blood ◽  
1978 ◽  
Vol 51 (1) ◽  
pp. 97-108 ◽  
Author(s):  
GL Logue ◽  
R Kurlander ◽  
P Pepe ◽  
W Davis ◽  
H Silberman
Keyword(s):  
Gradient Centrifugation ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Normal Subjects ◽  
Exchange Chromatography ◽  
Phagocytic Cells ◽  
Igg Antibody ◽  
Microtiter Plates ◽  
Granulocyte Antibodies

Abstract Sera from two patients with granulocytopenia associated with collagen vascular disease caused the destruction of normal human granulocytes by autologous lymphocytes in vitro. Granulocyte cytotoxicity was measured by the release of 51Cr during incubation with test sera and lymphocytes in microtiter plates. Between 8% and 46% granulocytoxicity was produced in granulocytes from 8 normal donors by the sera from these two patients. Less than 6% granulocytotoxicity was seen with the sera from 14 normal subjects and 29 patient controls. Treatment of lymphocyte preparations with carbonyl iron and magnetic separation to remove phagocytic cells or treatment with complement-coated red cells followed by repeated gradient centrifugation to remove complement receptor- bearing lymphocytes did not reduce the granulocytotoxicity. There was a dose-response relationship between the concentration of positive sera and granulocytotoxicity. When these sera were fractionated by Sephadex G-200 gel filtration and by ion-exchange chromatography with DEAE- cellulose, the active component appeared in the IgG-containing fractions. Thus, IgG antibody-dependent, lymphocyte-mediated granulocyte cytotoxicity represents a means of detecting human granulocyte antibodies and is a possible mechanism of autoimmune neutropenia in these two patients.

scholarly journals Guanosine cyclic monophosphate-dependent protein kinase from foetal calf heart. Purification, general properties and catalytic subunit

1977 ◽  
Vol 161 (2) ◽  
pp. 213-221 ◽  
Author(s):  
M Shoji ◽  
J G Patrick ◽  
C W Davis ◽  
J F Kuo
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Cyclic Gmp ◽  
Deae Cellulose ◽  
Catalytic Subunit ◽  
Subunit Structure ◽  
Dependent Protein Kinase ◽  
Ph Optimum ◽  
General Properties ◽  
Dependent Protein

Cyclic GMP-dependent protein kinase was purified from foetal calf hearts, and its general properties and subunit structure were studied. The enzyme was purified over 900-fold from the heart extract by pH 5.3-isoelectric precipitation, DEAE-cellulose chromatography, Sephadex G-200 filtration and hydroxyapatite treatment. The purified myocardial enzyme, free from cyclic AMP-dependent protein kinase contamination, exhibited an absolute requirement of stimulatory modulator (or crude modulator containing the stimulatory modulator component) for its cyclic GMP-stimulated activity. Inhibitory modulator (protein inhibitor) of cyclic AMP-dependent protein kinase could not stimulate nor inhibit the cyclic GMP target enzyme. The enzyme had Ka values of 0.013, 0.033 and 3.0 micronM for 8-bromo cyclic GMP, cyclic GMP and cyclic AMP respectively. The cyclic GMP-dependent enzyme required Mg2+ and Co2+ for its activity, with optimal concentrations of about 30 and 0.5 mM respectively. The pH optimum for the enzyme activity ranged from 6 to 9. Histones were generally effective substrate proteins. The enzyme exhibited a greater affinity for histones than did the cyclic AMP-dependent class of protein kinase. The holoenzyme (apparent mol.wt. 150 000) of the myocardial cyclic GMP-dependent protein kinase was dissociated into a cyclic GMP-independent catalytic subunit (apparent mol.wt. 60 000) by cyclic GMP and histone. The catalytic subunit required the stimulatory modulator for its activity, as in the case of the holoenzyme in the presence of cyclic GMP.

scholarly journals Human acetyl-coenzyme A:α-glucosaminide N-acetyltransferase. Kinetic characterization and mechanistic interpretation

1995 ◽  
Vol 308 (1) ◽  
pp. 327-333 ◽  
Author(s):  
P J Meikle ◽  
A M Whittle ◽  
J J Hopwood
Keyword(s):  
Ternary Complex ◽  
Gradient Centrifugation ◽  
Heparan Sulphate ◽  
Random Order ◽  
Lysosomal Membrane ◽  
Complex Mechanism ◽  
Acetyl Coa ◽  
Ph Optimum ◽  
Inhibition Studies

Acetyl-CoA: alpha-glucosaminide N-acetyltransferase (N-acetyltransferase) is an integral lysosomal membrane protein which catalyses the transfer of acetyl groups from acetyl-CoA on to the terminal glucosamine in heparin and heparan sulphate chains within the lysosome. In vitro, the enzyme is capable of acetylating a number of mono- and oligo-saccharides derived from heparin, provided that a non-reducing terminal glucosamine is present. We have prepared highly enriched lysosomal membrane fractions from human placenta by a combination of differential centrifugation and density-gradient centrifugation in Percoll. This preparation was used to investigate the kinetics of the enzyme with three acetyl-acceptor substrates, i.e. glucosamine and a disaccharide and a tetrasaccharide derived from heparin, each containing a terminal glucosamine residue. The enzyme showed a pH optimum at 6.5, extending to 8.0 for the mono- and di-saccharide substrates but falling off sharply above pH 6.5 for the tetrasaccharide substrate. We identified two distinct Km values for the glucosamine substrate at both pH 7.0 and pH 5.0, whereas the tetrasaccharide substrate displayed only a single Km value at each pH. The Km values were found to be highly pH-dependent, and at pH 5.0 the values for the acetyl-acceptor substrates showed a decreasing trend as the size of the substrate increased, suggesting that the enzyme recognizes an extended region of the non-reducing terminus of the heparin or heparan sulphate polysaccharides. Double-reciprocal analysis, isotope exchange between N-acetylglucosamine and glucosamine, and inhibition studies with desulpho-CoA indicate that the enzyme operates by a random-order ternary-complex mechanism. Product inhibition studies display a complex pattern of dead-end inhibition. Taken in context with what is known about lysosomal utilization and physiological levels of acetyl-CoA, these results suggest that in vivo the enzyme operates via a random-order ternary-complex mechanism which involves the utilization of cytosolic acetyl-CoA to transfer acetyl groups on to the terminal glucosamine residues of heparin within the lysosome.

Low Anticoagulant Activity of High Sulphated Heparan Sulphates

1990 ◽  
Vol 63 (03) ◽  
pp. 488-492 ◽  
Author(s):  
J Kovensky ◽  
B Sassetti ◽  
A Fernández Cirelli ◽  
L Kordich
Keyword(s):  
Molecular Weight ◽  
Anticoagulant Activity ◽  
Heparan Sulphate ◽  
Deae Cellulose ◽  
Gel Chromatography ◽  
Sulphate Content ◽  
Protamine Sulphate ◽  
Wide Variability ◽  
L1 And L2

SummaryTwo high sulphated heparin-like polysaccharides (LI, MW 16,000 and L2, MW 11,700) were isolated from rat liver tissues, after DEAE-cellulose chromatography. Heparan sulphates from heart and lung tissues were isolated for comparison and fractionated according to their molecular weight. The anticoagulant activities in vitro were studied using clotting antifactor Xa, antifactor IIa, and APTT assay methods, falling in a narrow range (5-44 IU/mg) although the wide variability m molecular weight and sulphate content. The heparan sulphate nature of fractions L1 and L2 (sulphate/disaccharide ratio 2.05 and 2.48, respectively) has been verified by: a) low iduronic/glucuronic acid ratio; b) nitrous acid degradation followed by gel chromatography; c) heparinase treatment followed by gel chromatography; d) electrophoretic behaviour. Native proteoglycans have been isolated and the glycosidic chains compared with L1 and L2. Their anticoagulant activities in vitro and the fact that antiXa clotting activity was not neutralized by protamine sulphate are in accordance with the results of structural studies.

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