Intestinal hydrolysis of pyridoxal 5′-phosphate in vitro and in vivo in the rat

1986 ◽  
Vol 91 (2) ◽  
pp. 343-350 ◽  
Author(s):  
Henry M. Middleton
Keyword(s):  
Hydrolysis Of

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 Catalytic activity and stereoselectivity of engineered phosphotriesterases towards structurally different nerve agents in vitro

2021 ◽  
Author(s):  
Anja Köhler ◽  
Benjamin Escher ◽  
Laura Job ◽  
Marianne Koller ◽  
Horst Thiermann ◽  
...  
Keyword(s):  
Plasma Clearance ◽  
Nerve Agents ◽  
Inhibition Assay ◽  
Ache Inhibition ◽  
Catalytic Activities ◽  
Chiral Lc ◽  
Hydrolysis Of ◽  
Medical Countermeasures

AbstractHighly toxic organophosphorus nerve agents, especially the extremely stable and persistent V-type agents such as VX, still pose a threat to the human population and require effective medical countermeasures. Engineered mutants of the Brevundimonas diminuta phosphotriesterase (BdPTE) exhibit enhanced catalytic activities and have demonstrated detoxification in animal models, however, substrate specificity and fast plasma clearance limit their medical applicability. To allow better assessment of their substrate profiles, we have thoroughly investigated the catalytic efficacies of five BdPTE mutants with 17 different nerve agents using an AChE inhibition assay. In addition, we studied one BdPTE version that was fused with structurally disordered PAS polypeptides to enable delayed plasma clearance and one bispecific BdPTE with broadened substrate spectrum composed of two functionally distinct subunits connected by a PAS linker. Measured kcat/KM values were as high as 6.5 and 1.5 × 108 M−1 min−1 with G- and V-agents, respectively. Furthermore, the stereoselective degradation of VX enantiomers by the PASylated BdPTE-4 and the bispecific BdPTE-7 were investigated by chiral LC–MS/MS, resulting in a several fold faster hydrolysis of the more toxic P(−) VX stereoisomer compared to P(+) VX. In conclusion, the newly developed enzymes BdPTE-4 and BdPTE-7 have shown high catalytic efficacy towards structurally different nerve agents and stereoselectivity towards the toxic P(−) VX enantiomer in vitro and offer promise for use as bioscavengers in vivo.

Enzyme system of Clostridium stercorarium for hydrolysis of arabinoxylan: reconstitution of the in vivo system from recombinant enzymes

Microbiology ◽  
2004 ◽  
Vol 150 (7) ◽  
pp. 2257-2266 ◽  
Author(s):  
Helmuth Adelsberger ◽  
Christian Hertel ◽  
Erich Glawischnig ◽  
Vladimir V. Zverlov ◽  
Wolfgang H. Schwarz
Keyword(s):  
Extracellular Enzymes ◽  
Enzyme System ◽  
Thermophilic Bacterium ◽  
Recombinant Enzymes ◽  
Type Mode ◽  
Complete Set ◽  
First Time ◽  
Hydrolysis Of

Four extracellular enzymes of the thermophilic bacterium Clostridium stercorarium are involved in the depolymerization of de-esterified arabinoxylan: Xyn11A, Xyn10C, Bxl3B, and Arf51B. They were identified in a collection of eight clones producing enzymes hydrolysing xylan (xynA, xynB, xynC), β-xyloside (bxlA, bxlB, bglZ) and α-arabinofuranoside (arfA, arfB). The modular enzymes Xyn11A and Xyn10C represent the major xylanases in the culture supernatant of C. stercorarium. Both hydrolyse arabinoxylan in an endo-type mode, but differ in the pattern of the oligosaccharides produced. Of the glycosidases, Bxl3B degrades xylobiose and xylooligosaccharides to xylose, and Arf51B is able to release arabinose residues from de-esterified arabinoxylan and from the oligosaccharides generated. The other glycosidases either did not attack or only marginally attacked these oligosaccharides. Significantly more xylanase and xylosidase activity was produced during growth on xylose and xylan. This is believed to be the first time that, in a single thermophilic micro-organism, the complete set of enzymes (as well as the respective genes) to completely hydrolyse de-esterified arabinoxylan to its monomeric sugar constituents, xylose and arabinose, has been identified and the enzymes produced in vivo. The active enzyme system was reconstituted in vitro from recombinant enzymes.

Acid and neutral sphingomyelinases: roles and mechanisms of regulation

2004 ◽  
Vol 82 (1) ◽  
pp. 27-44 ◽  
Author(s):  
Norma Marchesini ◽  
Yusuf A Hannun
Keyword(s):  
Nitric Oxide ◽  
Molecular Mechanisms ◽  
Caveolin 1 ◽  
Niemann Pick Disease ◽  
Extracellular Stimuli ◽  
Niemann Pick ◽  
Hydrolysis Of ◽  
Pick Disease

Ceramide, an emerging bioactive lipid and second messenger, is mainly generated by hydrolysis of sphingomyelin through the action of sphingomyelinases. At least two sphingomyelinases, neutral and acid sphingo myelinases, are activated in response to many extracellular stimuli. Despite extensive studies, the precise cellular function of each of these sphingomyelinases in sphingomyelin turnover and in the regulation of ceramide-mediated responses is not well understood. Therefore, it is essential to elucidate the factors and mechanisms that control the activation of acid and neutral sphingomyelinases to understand their the roles in cell regulation. This review will focus on the molecular mechanisms that regulate these enzymes in vivo and in vitro, especially the roles of oxidants (glu ta thi one, peroxide, nitric oxide), proteins (saposin, caveolin 1, caspases), and lipids (diacylglycerol, arachidonic acid, and ceramide).Key words: sphingomyelinase, ceramide, apoptosis, Niemann-Pick disease, FAN (factor associated with N-SMase activation).

Repeated thyrotrophin stimulation of thyroid secretion: lack of refractoriness in vivo

1985 ◽  
Vol 106 (2) ◽  
pp. 153-157
Author(s):  
N. Bagchi ◽  
T. R. Brown
Keyword(s):  
Adenylate Cyclase ◽  
Thyroid Tissue ◽  
Control Group ◽  
Cyclic Amp Accumulation ◽  
Thyroid Glands ◽  
Rate Of Hydrolysis ◽  
Thyroid Secretion ◽  
Hydrolysis Of

ABSTRACT It has been reported that prior exposure of thyroid tissue to TSH in vitro induces a state of refractoriness to new challenges of the hormone. We have investigated the effect of repeated TSH treatment on thyroid secretion to determine whether such refractoriness exists in vivo. The rate of thyroid secretion was estimated by measuring the rate of hydrolysis of labelled thyroglobulin from mouse thyroid glands in vitro. The thyroid glands were labelled in vivo with 131I and then cultured for 20 h in the presence of mononitrotyrosine, an inhibitor of iodotyrosine deiodinase. The rate of hydrolysis of labelled thyroglobulin was measured as the percentage of radioactivity released as free iodotyrosines and iodothyronines into the gland and the medium at the end of incubation. Thyrotrophin was administered in vivo at hourly intervals for 2–4 injections. The corresponding control group received saline injections every hour except for the last injection when they received TSH. The peak rates of thyroglobulin hydrolysis, measured 2 h following the last injection, were similar in animals receiving two, three or four TSH injections and were not different from those in the control groups. Serum tri-iodothyronine and thyroxine concentrations 2 h after the last injection were higher in the groups receiving multiple TSH injections. Thyroidal cyclic AMP accumulation in response to TSH was markedly depressed in the group receiving multiple injections compared with the group receiving a single injection of TSH in vivo. These data indicate that (1) the stimulatory effect of TSH on thyroidal secretion is not diminished by prior administration of the hormone in vivo, (2) repeated TSH administrations in vivo cause refractoriness of the adenylate cyclase response to TSH and (3) a dichotomy exists between the secretory response and the adenylate cyclase response to repeated administrations of TSH. J. Endocr. (1985) 106, 153–157

scholarly journals Solvolysis of the Tumor-Inhibiting Ru(III)-Complex trans-Tetrachlorobis(Indazole)Ruthenate(III)

2000 ◽  
Vol 7 (4) ◽  
pp. 225-232 ◽  
Author(s):  
Thomas Pieper ◽  
Wolfgang Peti ◽  
Bernhard K. Keppler
Keyword(s):  
Sodium Salt ◽  
Buffer System ◽  
Spectroscopic Techniques ◽  
Tumor Models ◽  
Solubility In Water ◽  
Aqueous Acetonitrile ◽  
Hydrolysis Of

The ruthenium(III) complex Hlnd trans-[RuCl4,(ind)2], with two trans-standing indazole (ind) ligands bound to ruthenium via nitrogen, shows remarkable activity in different tumor models in vitro and in vivo. The solvolysis of the complex trans-[RuCl4,(ind)2]- has been investigated by means of spectroscopic techniques (UV/vis, NMR)in different solvents. We investigated the indazolium as well as the sodium salt, the latter showing improved solubility in water. In aqueous acetonitrile and ethanol the solvolysis results in one main solvento complex. The hydrolysis of the complex is more complicated and depends on the pH of the solution as well as on the buffer system.

Na-K-ATPase activity along the rabbit, rat, and mouse nephron

1979 ◽  
Vol 237 (2) ◽  
pp. F114-F120 ◽  
Author(s):  
A. I. Katz ◽  
A. Doucet ◽  
F. Morel
Keyword(s):  
Atpase Activity ◽  
Thick Ascending Limb ◽  
Sensitivity Limit ◽  
Henle's Loop ◽  
Collecting Tubule ◽  
Pars Recta ◽  
Hydrolysis Of ◽  
Total Enzyme

Na-K-ATPase activity along the rabbit, rat, and mouse nephron was determined with a micromethod that measures directly labeled phosphate released by the hydrolysis of [gamma-32P]ATP. Na-K-ATPase activity was highest in the rat, intermediate in the mouse, and lowest in the rabbit nephron. With the exception of rabbit cortical thick ascending limb, the enzyme profile was similar in the three species: Na-K-ATPase activity per millimeter tubule length was highest in the distal convoluted tubule and thick ascending limb of Henle's loop, intermediate in the proximal convoluted tubule, and lowest in the pars recta and collecting tubule. The enzyme was present in the thin limbs of Henle's loop, but its activity was very low and measurements were close to the sensitivity limit of the method. Both the absolute activity and the fraction of the total enzyme represented by Na-K-ATPase were severalfold higher than in kidney homogenates. Finally, the Na-K-ATPase activity measured in certain segments of the rat and rabbit nephron in this study seems sufficient to account in theory for the active component of the net sodium transport found in the corresponding region of the nephron with either in vivo or in vitro single tubule microperfusion techniques.

Differences between in vitro and in vivo degradation of LHRH by rat brain and other organs

1987 ◽  
Vol 253 (3) ◽  
pp. E317-E321 ◽  
Author(s):  
F. A. Carone ◽  
M. A. Stetler-Stevenson ◽  
V. May ◽  
A. LaBarbera ◽  
G. Flouret
Keyword(s):  
Degradation Products ◽  
Venous Blood ◽  
Simultaneous Action ◽  
Pituitary Cells ◽  
Time Intervals ◽  
Luteinizing Hormone Releasing Hormone ◽  
Hydrolysis Of ◽  
The Brain

Homogenates of brain, pituitary, liver, lung, ovary, and testes were incubated with [pyro Glu1-3,4-3H]luteinizing hormone-releasing hormone ([3H]LHRH), and the profiles of metabolites generated as a function of time were determined. After 5 min of incubation, 5 was the predominant metabolite in most homogenates. Although the profiles of metabolites varied at different time intervals, metabolites 2, 3, 4, and 5, and in some instances 7 and 9, appeared to form simultaneously and were detectable at 10 min. Neither metabolite 6 nor other larger metabolites formed initially as dominant degradation products. The findings suggest cleavage of LHRH by the simultaneous action of several endopeptidases. After a single vascular transit of [3H]LHRH, metabolites were determined in the venous blood of liver, lung, and brain of rats in vivo. There were no metabolites of [3H]LHRH in venous blood of liver and lung; however, metabolites 2-4 were present in venous blood of the brain. Incubation of rat anterior pituitary cells with [3H]LHRH yielded metabolites 1-4 but not metabolites 5 or 9 as in homogenates. Incubation of [3H]LHRH with porcine follicular granulosa cells resulted in the generation of metabolites 2-7 and 9, similar to the profile in homogenates. Thus, since homogenates contain enzymes of disrupted cells, they do not always reflect mechanisms for in vivo hydrolysis of circulating LHRH. Brain degraded 12.1% of LHRH during a single vascular transit and may account for substantial degradation of the circulating hormone.

scholarly journals Epoxide hydrolase 3 (Ephx3) gene disruption reduces ceramide linoleate epoxide hydrolysis and impairs skin barrier function

2020 ◽  
pp. jbc.RA120.016570
Author(s):  
Matthew L. Edin ◽  
Haruto Yamanashi ◽  
William E. Boeglin ◽  
Joan P. Graves ◽  
Laura M. DeGraff ◽  
...  
Keyword(s):  
Epoxide Hydrolase ◽  
Skin Barrier ◽  
Skin Barrier Function ◽  
Potential Contribution ◽  
Wild Type ◽  
Lipoxygenase Pathway ◽  
Physiological Relevance ◽  
Hydrolysis Of

The mammalian epoxide hydrolase EPHX3 is known from in vitro experiments to efficiently hydrolyze the linoleate epoxides 9,10-epoxyoctadecamonoenoic acid (EpOME) and epoxyalcohol 9R,10R-trans-epoxy-11E-13R-hydroxy-octadecenoate to corresponding diols and triols, respectively. Herein we examined the physiological relevance of EPHX3 to hydrolysis of both substrates in vivo.  Ephx3-/- mice show no deficiency in EpOME-derived plasma diols, discounting a role for EPHX3 in their formation, whereas epoxyalcohol-derived triols esterified in acylceramides of the epidermal 12R-lipoxygenase pathway are reduced. Although the Ephx3-/- pups appear normal, measurements of trans-epidermal water loss detected a modest and statistically significant increase compared to the wild-type or heterozygote mice, reflecting a skin barrier impairment that was not evident in the knockouts of mouse microsomal epoxide hydrolase (EPHX1/mEH) or soluble epoxide hydrolase (EPHX2/sEH). This barrier phenotype in the Ephx3-/- pups was associated with a significant decrease in the covalently bound ceramides in the epidermis (40% reduction, p<0.05), indicating a corresponding structural impairment in the integrity of the water barrier. Quantitative LC-MS analysis of the esterified linoleate-derived triols in the murine epidermis revealed a marked and isomer-specific reduction (~85%) in the Ephx3-/- epidermis of the major trihydroxy isomer 9R,10S,13R-trihydroxy-11E-octadecenoate. We conclude EPHX3 (and not EPHX1 or EPHX2) catalyzes hydrolysis of the 12R-LOX/eLOX3-derived epoxyalcohol esterified in acylceramide, and may function to control flux through the alternative and crucial route of metabolism via the dehydrogenation pathway of SDR9C7. Importantly, our findings also identify a functional role for EPHX3 in transformation of a naturally esterified epoxide substrate, pointing to its potential contribution in other tissues.

Serum interspecies differences in metabolic pathways of bradykinin and [des-Arg9]BK: influence of enalaprilat

1996 ◽  
Vol 271 (4) ◽  
pp. H1340-H1347 ◽  
Author(s):  
A. Decarie ◽  
P. Raymond ◽  
N. Gervais ◽  
R. Couture ◽  
A. Adam
Keyword(s):  
Metabolic Pathways ◽  
Neutral Endopeptidase ◽  
Thrombin Inhibitor ◽  
Rat Serum ◽  
Highly Sensitive ◽  
Significant Difference ◽  
Hydrolysis Of

Among the different enzymes responsible for the metabolism of bradykinin (BK), three peptidases look relevant in vivo: kininase I (KI), which transforms BK into its active metabolite, [des-Arg9]BK; kininase II (KII); and neutral endopeptidase, which inactivate BK and [des-Arg9]BK. The in vitro incubation of BK and [des-Arg9]BK in the serum of four species with or without enalaprilat and the quantification of the immunoreactivity of both peptides at different time intervals allowed the measurement of the kinetic parameters characterizing their metabolic pathways. Highly sensitive chemiluminescent enzyme immunoassays were used to measure the residual concentrations of BK and [des-Arg9]BK. Half-life (t1/2) of BK showed significant difference among species: rats (10 +/- 1 s) = dogs (13 +/- 1 s) < rabbits (31 +/- 1 s) < humans (49 +/- 2 s). t1/2 values of [des-Arg9]BK were also species dependent: rats (96 +/- 6 s) < < rabbits (314 +/- 6 s) = dogs (323 +/- 11 s) = humans (325 +/- 12 s). Enalaprilat significantly prevented the rapid BK and [des-Arg9]BK degradation in all species except that of [des-Arg9]BK in rat serum. Relative amount of BK hydrolyzed by serum KII was given as follows: rabbits (93.7 +/- 14.8%) = rats (83.6 +/- 6.7%) = humans (76.0 +/- 7.5%) > dogs (50.0 +/- 3.9%). Its importance in the hydrolysis of [des-Arg9]BK was 5.2 +/- 0.5% in rats < < 33.9 +/- 1.5% in humans < 52.0 +/- 1.1% in rabbits < 65.1 +/- 3.4% in dogs. The participation of serum KI in the transformation of BK into [des-Arg9]BK was dogs (67.2 +/- 5.3%) > > humans (3.4 +/- 1.2%) = rabbits (1.8 +/- 0.2%) = rats (1.4 +/- 0.3%). Finally, no significant difference on t1/2 values for BK and [des-Arg9]BK could be demonstrated between serum and plasma treated with either sodium citrate or a thrombin inhibitor. These results revealed striking species differences in the serum metabolism of kinins that could address at least partially some of the controversial data related to the cardioprotective role of kinins.

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