scholarly journals Reaction mechanism of chitosanase from Streptomyces sp. N174

1995 ◽  
Vol 311 (2) ◽  
pp. 377-383 ◽  
Author(s):  
T Fukamizo ◽  
Y Honda ◽  
S Goto ◽  
I Boucher ◽  
R Brzezinski
Keyword(s):  
Nmr Spectra ◽  
Time Course ◽  
Product Distribution ◽  
Time Dependent ◽  
Reaction Products ◽  
Streptomyces Sp ◽  
Substrate Degradation ◽  
Time Courses ◽  
Binding Cleft ◽  
Hydrolysis Of

Chitosanase was produced by the strain of Streptomyces lividans TK24 bearing the csn gene from Streptomyces sp. N174, and purified by S-Sepharose and Bio-Gel A column chromatography. Partially (25-35%) N-acetylated chitosan was digested by the purified chitosanase, and structures of the products were analysed by NMR spectroscopy. The chitosanase produced heterooligosaccharides consisting of D-GlcN and GlcNAc in addition to glucosamine oligosaccharides [(GlcN)n, n = 1, 2 and 3]. The reducing- and non-reducing-end residues of the heterooligosaccharide products were GlcNAc and GlcN respectively, indicating that the chitosanase can split the GlcNAc-GlcN linkage in addition to that of GlcN-GlcN. Time-dependent 1H-NMR spectra showing hydrolysis of (GlcN)6 by the chitosanase were obtained in order to determine the anomeric form of the reaction products. The chitosanase was found to produce only the alpha-form; therefore it is an inverting enzyme. Separation and quantification of (GlcN)n was achieved by HPLC, and the time course of the reaction catalysed by the chitosanase was studied using (GlcN)n (n = 4, 5 and 6) as the substrate. The chitosanase hydrolysed (GlcN)6 in an endo-splitting manner producing (GlcN)2, (GlcN)3 and (GlcN)4, and did not catalyse transglycosylation. Product distribution was (GlcN)3 >> (GlcN)2 > (GlcN)4. Cleavage to (GlcN)3 + (GlcN)3 predominated over that to (GlcN)2 + (GlcN)4. Time courses showed a decrease in rate of substrate degradation from (GlcN)6 to (GlcN)5 to (GlcN)4. It is most likely that the substrate-binding cleft of the chitosanase can accommodate at least six GlcN residues, and that the cleavage point is located at the midpoint of the binding cleft.

scholarly journals Stimulation of phosphoinositide hydrolysis by oxytocin and the mechanism by which oxytocin controls prostaglandin synthesis in the ovine endometrium

1986 ◽  
Vol 237 (3) ◽  
pp. 797-805 ◽  
Author(s):  
A P F Flint ◽  
W M F Leat ◽  
E L Sheldrick ◽  
H J Stewart
Keyword(s):  
Arachidonic Acid ◽  
Time Course ◽  
Inositol Phosphates ◽  
Fold Increase ◽  
Prostaglandin Synthesis ◽  
Time Dependent ◽  
Phosphoinositide Hydrolysis ◽  
Hydrolysis Of ◽  
Stimulation Of

Slices of caruncular endometrium from steroid-treated ovariectomized sheep were incubated with myo-[2-3H]inositol to label tissue phosphatidylinositol. Effects of oxytocin were determined on the rate of incorporation of radioactivity into phosphatidylinositol and on the hydrolysis of phosphoinositides to inositol phosphates and diacylglycerol. Incorporation of radioactivity into phosphatidylinositol was linear during 2 h incubations; 10(-7) M (100 nM)-oxytocin caused a 2.8-fold increase in the rate of incorporation. In the presence of Li+, addition of 10(-7) M-oxytocin to slices in which phosphatidylinositol was pre-labelled caused mean increase of 40-fold in the incorporation of radioactivity into inositol mono-, bis- and tris-phosphates. Inositol 1,3,4-trisphosphate was quantitatively the major trisphosphate formed. The action of oxytocin on phosphoinositide hydrolysis was dose- and time-dependent, occurring at concentrations within the range observed in plasma during episodes of secretion in vivo, and with a time course comparable with that of the action of oxytocin on uterine prostaglandin production. The effect of oxytocin on incorporation of radioactivity into inositol phosphates was not affected by inhibitors of prostaglandin synthesis. Diacylglycerol 1- and 2-lipases in caruncular endometrium converted up to 72% of added 2-[3H]arachidonyldiacylglycerol into [3H]arachidonic acid during 30 min incubations at pH 7.0. Caruncular endometrium contained 1.49 mumol of phosphatidylinositol/g, representing approx. 0.2 mumol/g of phosphatidylinositol arachidonic acid. It is proposed that the stimulation of endometrial prostaglandin synthesis by oxytocin is accounted for by increased hydrolysis of phosphoinositides to diacylglycerol and inositol phosphates with subsequent release of arachidonic acid from diacylglycerol.

A study of reaction of aromatic polynitro compounds with tributylstannyl hydride

1985 ◽  
Vol 50 (11) ◽  
pp. 2598-2606 ◽  
Author(s):  
Vladimír Macháček ◽  
Antonín Lyčka ◽  
Milan Nádvorník
Keyword(s):  
Nmr Spectra ◽  
Reaction Products ◽  
Tetramethylammonium Bromide ◽  
Composition And Structure

1H, 13C, 15N, and 119Sn NMR spectra have been used to study composition and structure of reaction products from 1,3,5-trinitrobenzene, methyl 2,4,6-trinitrobenzoate, 1-dimethylamino-2,4,6-trinitrobenzene, 1-methoxy-2,4,6-trinitrobenzene, 1-chloro-2,4,6-trinitrobenzene, 2,4,6-trinitrotoluene, 3,5-dinitrobenzonitrile and methyl 3,5-dinitrobenzoate with tributylstannyl hydride in the presence of tetramethylammonium bromide.

A 15N, 13C, and 1H NMR study of reaction products from arylguanidines and chloroformate esters

1991 ◽  
Vol 56 (7) ◽  
pp. 1505-1511 ◽  
Author(s):  
Antonín Lyčka ◽  
Karel Palát
Keyword(s):  
1H Nmr ◽  
Nmr Spectra ◽  
Reaction Products ◽  
H Nmr ◽  
Nmr Study

The 15N, 13C, and 1H NMR spectra of the reaction products from arylguanidines with two mols of chloroformate esters have been measured. With application of the corresponding 15N isotopomer it has been proved that the reaction products have the structures IIIa-IIIc.

Studies on the mechanism of acute inhibition of thyroglobulin hydrolysis by iodine

1985 ◽  
Vol 108 (4) ◽  
pp. 511-517 ◽  
Author(s):  
Nandalal Bagchi ◽  
Birdie Shivers ◽  
Thomas R. Brown
Keyword(s):  
Time Course ◽  
Period 2 ◽  
Iodotyrosine Deiodinase ◽  
Rate Of Hydrolysis ◽  
Underlying Mechanisms ◽  
Excess Iodide ◽  
Sites Of Action ◽  
Hydrolysis Of

Abstract. Iodine in excess is known to acutely inhibit thyroidal secretion. In the present study we have characterized the time course of the iodine effect in vitro and investigated the underlying mechanisms. Labelled thyroid glands were cultured in vitro in medium containing mononitrotyrosine, an inhibitor of iodotyrosine deiodinase. The rate of hydrolysis of labelled thyroglobulin was measured as the proportion of labelled iodotyrosines and iodothyronines recovered at the end of culture and was used as an index of thyroidal secretion. Thyrotrophin (TSH) administered in vivo acutely stimulated the rate of thyroglobulin hydrolysis. Addition of Nal to the culture medium acutely inhibited both basal and TSH-stimulated thyroglobulin hydrolysis. The effect of iodide was demonstrable after 2 h, maximal after 6 h and was not reversible upon removal of iodide. Iodide abolished the dibutyryl cAMP induced stimulation of thyroglobulin hydrolysis. Iodide required organic binding of iodine for its effect but new protein or RNA synthesis was not necessary. The inhibitory effects of iodide and lysosomotrophic agents such as NH4C1 and chloroquin on thyroglobulin hydrolysis were additive suggesting different sites of action. Iodide added in vitro altered the distribution of label in prelabelled thyroglobulin in a way that suggested increased coupling in the thyroglobulin molecule. These data indicate that 1) the iodide effect occurs progressively over a 6 h period, 2) continued presence of iodide is not necessary once the inhibition is established, 3) iodide exerts its action primarily at a post cAMP, prelysosomal site and 4) the effect requires organic binding of iodine, but not new RNA or protein synthesis. Our data are consistent with the hypothesis that excess iodide acutely inhibits thyroglobulin hydrolysis by increasing the resistance of thyroglobulin to proteolytic degradation through increased iodination and coupling.

scholarly journals Optimizing Chitin Depolymerization by Lysozyme to Long-Chain Oligosaccharides

Marine Drugs ◽  
2021 ◽  
Vol 19 (6) ◽  
pp. 320
Author(s):  
Arnaud Masselin ◽  
Antoine Rousseau ◽  
Stéphanie Pradeau ◽  
Laure Fort ◽  
Rodolphe Gueret ◽  
...  
Keyword(s):  
Time Course ◽  
Water Soluble ◽  
Organic Fertilizers ◽  
Symbiotic Associations ◽  
Egg White Lysozyme ◽  
Hen Egg White ◽  
Ecological Transition ◽  
Optimized Conditions ◽  
Hydrolysis Of ◽  
Long Chain

Chitin oligosaccharides (COs) hold high promise as organic fertilizers in the ongoing agro-ecological transition. Short- and long-chain COs can contribute to the establishment of symbiotic associations between plants and microorganisms, facilitating the uptake of soil nutrients by host plants. Long-chain COs trigger plant innate immunity. A fine investigation of these different signaling pathways requires improving the access to high-purity COs. Here, we used the response surface methodology to optimize the production of COs by enzymatic hydrolysis of water-soluble chitin (WSC) with hen egg-white lysozyme. The influence of WSC concentration, its acetylation degree, and the reaction time course were modelled using a Box–Behnken design. Under optimized conditions, water-soluble COs up to the nonasaccharide were formed in 51% yield and purified to homogeneity. This straightforward approach opens new avenues to determine the complex roles of COs in plants.

Chitosanase from Streptomyces sp. strain N174: a comparative review of its structure and function

1997 ◽  
Vol 75 (6) ◽  
pp. 687-696 ◽  
Author(s):  
Tamo Fukamizo ◽  
Ryszard Brzezinski
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Substrate Binding ◽  
Structure And Function ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Amino Acid Residues ◽  
Streptomyces Sp ◽  
Binding Cleft ◽  
And Function

Novel information on the structure and function of chitosanase, which hydrolyzes the beta -1,4-glycosidic linkage of chitosan, has accumulated in recent years. The cloning of the chitosanase gene from Streptomyces sp. strain N174 and the establishment of an efficient expression system using Streptomyces lividans TK24 have contributed to these advances. Amino acid sequence comparisons of the chitosanases that have been sequenced to date revealed a significant homology in the N-terminal module. From energy minimization based on the X-ray crystal structure of Streptomyces sp. strain N174 chitosanase, the substrate binding cleft of this enzyme was estimated to be composed of six monosaccharide binding subsites. The hydrolytic reaction takes place at the center of the binding cleft with an inverting mechanism. Site-directed mutagenesis of the carboxylic amino acid residues that are conserved revealed that Glu-22 and Asp-40 are the catalytic residues. The tryptophan residues in the chitosanase do not participate directly in the substrate binding but stabilize the protein structure by interacting with hydrophobic and carboxylic side chains of the other amino acid residues. Structural and functional similarities were found between chitosanase, barley chitinase, bacteriophage T4 lysozyme, and goose egg white lysozyme, even though these proteins share no sequence similarities. This information can be helpful for the design of new chitinolytic enzymes that can be applied to carbohydrate engineering, biological control of phytopathogens, and other fields including chitinous polysaccharide degradation. Key words: chitosanase, amino acid sequence, overexpression system, reaction mechanism, site-directed mutagenesis.

scholarly journals Pseudo-streaming potentials in Necturus gallbladder epithelium. II. The mechanism is a junctional diffusion potential.

1992 ◽  
Vol 99 (3) ◽  
pp. 317-338 ◽  
Author(s):  
L Reuss ◽  
B Simon ◽  
C U Cotton
Keyword(s):  
Time Course ◽  
Bathing Solution ◽  
Intercellular Spaces ◽  
Similar Time ◽  
Streaming Potentials ◽  
Osmotic Water ◽  
Lateral Intercellular Spaces ◽  
Transepithelial Voltage ◽  
Time Courses ◽  
Good Agreement

The mechanisms of apparent streaming potentials elicited across Necturus gallbladder epithelium by addition or removal of sucrose from the apical bathing solution were studied by assessing the time courses of: (a) the change in transepithelial voltage (Vms). (b) the change in osmolality at the cell surface (estimated with a tetrabutylammonium [TBA+]-selective microelectrode, using TBA+ as a tracer for sucrose), and (c) the change in cell impermeant solute concentration ([TMA+]i, measured with an intracellular double-barrel TMA(+)-selective microelectrode after loading the cells with TMA+ by transient permeabilization with nystatin). For both sucrose addition and removal, the time courses of Vms were the same as the time courses of the voltage signals produced by [TMA+]i, while the time courses of the voltage signals produced by [TBA+]o were much faster. These results suggest that the apparent streaming potentials are caused by changes of [NaCl] in the lateral intercellular spaces, whose time course reflects the changes in cell water volume (and osmolality) elicited by the alterations in apical solution osmolality. Changes in cell osmolality are slow relative to those of the apical solution osmolality, whereas lateral space osmolality follows cell osmolality rapidly, due to the large surface area of lateral membranes and the small volume of the spaces. Analysis of a simple mathematical model of the epithelium yields an apical membrane Lp in good agreement with previous measurements and suggests that elevations of the apical solution osmolality elicit rapid reductions in junctional ionic selectivity, also in good agreement with experimental determinations. Elevations in apical solution [NaCl] cause biphasic transepithelial voltage changes: a rapid negative Vms change of similar time course to that of a Na+/TBA+ bi-ionic potential and a slow positive Vms change of similar time course to that of the sucrose-induced apparent streaming potential. We conclude that the Vms changes elicited by addition of impermeant solute to the apical bathing solution are pseudo-streaming potentials, i.e., junctional diffusion potentials caused by salt concentration changes in the lateral intercellular spaces secondary to osmotic water flow from the cells to the apical bathing solution and from the lateral intercellular spaces to the cells. Our results do not support the notion of junctional solute-solvent coupling during transepithelial osmotic water flow.

scholarly journals Sodium and calcium currents in dispersed mammalian septal neurons.

1991 ◽  
Vol 97 (2) ◽  
pp. 303-320 ◽  
Author(s):  
A Castellano ◽  
J López-Barneo
Keyword(s):  
Time Constant ◽  
Time Course ◽  
Calcium Currents ◽  
Closing Time ◽  
Patch Clamp Technique ◽  
Average Value ◽  
Time To Peak ◽  
Time Courses ◽  
Fast Activation ◽  
Septal Neurons

Voltage-gated Na+ and Ca2+ conductances of freshly dissociated septal neurons were studied in the whole-cell configuration of the patch-clamp technique. All cells exhibited a large Na+ current with characteristic fast activation and inactivation time courses. Half-time to peak current at -20 mV was 0.44 +/- 0.18 ms and maximal activation of Na+ conductance occurred at 0 mV or more positive membrane potentials. The average value was 91 +/- 32 nS (approximately 11 mS cm-2). At all membrane voltages inactivation was well fitted by a single exponential that had a time constant of 0.44 +/- 0.09 ms at 0 mV. Recovery from inactivation was complete in approximately 900 ms at -80 mV but in only 50 ms at -120 mV. The decay of Na+ tail currents had a single time constant that at -80 mV was faster than 100 microseconds. Depolarization of septal neurons also elicited a Ca2+ current that peaked in approximately 6-8 ms. Maximal peak Ca2+ current was obtained at 20 mV, and with 10 mM external Ca2+ the amplitude was 0.35 +/- 0.22 nA. During a maintained depolarization this current partially inactivated in the course of 200-300 ms. The Ca2+ current was due to the activity of two types of conductances with different deactivation kinetics. At -80 mV the closing time constants of slow (SD) and fast (FD) deactivating channels were, respectively, 1.99 +/- 0.2 and 0.11 +/- 0.03 ms (25 degrees C). The two kinds of channels also differed in their activation voltage, inactivation time course, slope of the conductance-voltage curve, and resistance to intracellular dialysis. The proportion of SD and FD channels varied from cell to cell, which may explain the differential electrophysiological responses of intracellularly recorded septal neurons.

scholarly journals Mechanisms of autoantibody production in autoimmune MRL mice.

1980 ◽  
Vol 152 (5) ◽  
pp. 1302-1310 ◽  
Author(s):  
D S Pisetsky ◽  
G A McCarty ◽  
D V Peters
Keyword(s):  
Antibody Response ◽  
Time Course ◽  
Fundamental Difference ◽  
Autoantibody Production ◽  
Quantitative Expression ◽  
Autoantibody Response ◽  
Time Courses ◽  
Antibody Levels

The quantitative expression of anti-DNA and anti-Sm antibodies has been investigated in autoimmune MRL-lpr/lpr and MRL-+/+ mice. Anti-Sm antibodies were detected in sera from 21/23 lpr/lpr and 10/16 +/+ mice, with individual animals showing striking variation in the time-course and magnitude of this autoantibody response. The peak antibody levels of the responding animals of each substrain did not differ significantly. For anti-DNA antibody, a different pattern of responsiveness was observed. Individual animals of each substrain produced very similar responses in terms of the magnitude and time-course of serum anti-DNA antibody. The differences in the peak levels of the two substrains were highly significant, with lpr/lpr mice demonstrating a much greater anti-DNA antibody response than +/+ mice. In lpr/lpr mice tested for both autoantibody systems, serum anti-DNA and anti-Sm antibodies showed distinct time-courses. These studies indicate that anti-DNA and anti-Sm antibodies are expressed independently in MRL mice, with the expression of anti-DNA, but not anti-Sm antibody markedly influenced by the presence of the 1pr gene. A fundamental difference in the mechanisms involved in the generation of anti-DNA and anti-Sm antibodies is suggested by the quantitative pattern of the two responses.

scholarly journals Study of the mode of action of a polygalacturonase from the phytopathogen Burkholderia cepacia

2007 ◽  
Vol 407 (2) ◽  
pp. 207-217 ◽  
Author(s):  
Claudia Massa ◽  
Mads H. Clausen ◽  
Jure Stojan ◽  
Doriano Lamba ◽  
Cristiana Campa
Keyword(s):  
Mode Of Action ◽  
Burkholderia Cepacia ◽  
Time Course ◽  
Glycoside Hydrolase Family ◽  
Enzymatic Mechanism ◽  
Processive Enzyme ◽  
Chain Lengths ◽  
Methylation Patterns ◽  
Hydrolysis Of ◽  
Hydrolase Family

We have recently isolated and heterologously expressed BcPeh28A, an endopolygalacturonase from the phytopathogenic Gram-negative bacterium Burkholderia cepacia. Endopolygalacturonases belong to glycoside hydrolase family 28 and are responsible for the hydrolysis of the non-esterified regions of pectins. The mode of action of BcPeh28A on different substrates has been investigated and its enzymatic mechanism elucidated. The hydrolysis of polygalacturonate indicates that BcPeh28A is a non-processive enzyme that releases oligomers with chain lengths ranging from two to eight. By inspection of product progression curves, a kinetic model has been generated and extensively tested. It has been used to derive the kinetic parameters that describe the time course of the formation of six predominant products. Moreover, an investigation of the enzymatic activity on shorter substrates that differ in their overall length and methylation patterns sheds light on the architecture of the BcPeh28A active site. Specifically the tolerance of individual sites towards methylated saccharide units was rationalized on the basis of the hydrolysis of hexagalacturonides with different methylation patterns.

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