scholarly journals Time-dependent inactivation of chick-embryo prolyl 4-hydroxylase by coumalic acid. Evidence for a syncatalytic mechanism

1987 ◽  
Vol 242 (1) ◽  
pp. 163-169 ◽  
Author(s):  
V Günzler ◽  
H M Hanauske-Abel ◽  
R Myllylä ◽  
J Mohr ◽  
K I Kivirikko
Keyword(s):  
Chick Embryo ◽  
Competitive Inhibition ◽  
Enzymic Activity ◽  
Time Dependent ◽  
Alpha Subunit ◽  
Prolonged Incubation ◽  
Dependent Inactivation ◽  
Competitive Inhibitors ◽  
Inactivation Constant ◽  
High Concentrations

From the structure-activity relationships of known competitive inhibitors, coumalic acid (2-oxo-1,2H-pyran-5-carboxylic acid) was deduced to be a potential syncatalytic inhibitor for chick-embryo prolyl 4-hydroxylase. The compound caused time-dependent inactivation, the reaction rate being first-order. The inactivation constant was 0.094 min-1, the Ki 17 mM and the bimolecular rate constant 0.09 M-1 X S-1. Human prolyl 4-hydroxylase and chick embryo lysyl hydroxylase were also inactivated, though to a lesser extent. Inactivation could be prevented by adding high concentrations of 2-oxoglutarate or its competitive analogues to the reaction mixture. In Lineweaver-Burk kinetics, coumalic acid displayed S-parabolic competitive inhibition with respect to 2-oxoglutarate. The inactivation reaction had cofactor requirements similar to those for the decarboxylation of 2-oxoglutarate. Enzymic activity was partially preserved in the absence of iron, but the rescue was incomplete, owing to decreased stability of the enzyme under this condition. Coumalic acid also decreased the electrophoretic mobility of the alpha-subunit, but the beta-subunit was not affected. Prolonged incubation of coumalic acid above pH 6.8 led to loss of its inactivating potency, owing to hydrolysis. It is concluded that the inactivation of prolyl 4-hydroxylase by coumalic acid is due to a syncatalytic mechanism. The data also suggest that the 2-oxoglutarate-binding site of the enzyme is located within the alpha-subunit.

Glucose-6-phosphatase: is activity regulated by phosphorylation–dephosphorylation?

1983 ◽  
Vol 61 (10) ◽  
pp. 1085-1089 ◽  
Author(s):  
Jasbir Singh ◽  
Richard E. Martin ◽  
Robert C. Nordlie
Keyword(s):  
Protein Kinase ◽  
Protein Phosphorylation ◽  
Alternative Explanation ◽  
Potent Inhibitor ◽  
Liver Microsomes ◽  
Time Dependent ◽  
Rat Liver Microsomes ◽  
Phosphoprotein Phosphatase ◽  
Dependent Inactivation ◽  
Competitive Inhibitors

Incubation of rat liver microsomes with ATP and Mg2+ in the absence or presence of an exogenous protein kinase showed no changes in the activity of glucose-6-phosphatase (D-glucose-6-phosphate phosphohydrolase, EC 3.1.3.9). These observations confirm the recent findings of the Burchells and colleagues and refute on methodological grounds the earlier conclusions of Begley and Craft implicating regulation of this enzyme by protein phosphorylation–dephosphorylation. In other studies, the time-dependent inactivation of microsomal glucose-6-phosphatase by incubation with deoxycholate was used to obtain the inactive enzyme which in the presence of a protein kinase, ATP, and Mg2+ could not be restored to its original level. A number of substrates and competitive inhibitors of glucose-6-phosphatase, most notably vanadate which is the most potent inhibitor of the enzyme identified, stabilized this enzyme against its time-dependent inactivation in the presence of detergent as effectively as did fluoride and molybdate which are also effective competitive inhibitors of glucose-6-phosphatase. An alternative explanation to the involvement of a phosphoprotein phosphatase, as discussed by the Burchells, in the time-dependent inactivation of glucose-6-phosphatase is thus suggested.

Non-Stick Sugars: Synthesis of Difluorosugar Fluorides as Potential Glycosidase Inactivators

2009 ◽  
Vol 62 (6) ◽  
pp. 590 ◽  
Author(s):  
Brian P. Rempel ◽  
Stephen G. Withers
Keyword(s):  
Active Site ◽  
Time Dependent ◽  
Enzyme Active Site ◽  
Radical Bromination ◽  
Dependent Inactivation ◽  
Competitive Inhibitors

Four new difluorosugar fluorides, 2-deoxy-2,5-difluoro-α-l-idopyranosyl fluoride, 1,5-difluoro-d-glucopyranosyl fluoride, 1,5-difluoro-l-idopyranosyl fluoride, and 2-deoxy-1,2-difluoro-d-glucopyranosyl fluoride, were synthesized from known precursors by a radical bromination/fluoride displacement sequence, followed by deprotection. The compounds were tested as time-dependent inactivators of the β-glucosidase from Agrobacterium sp. (Abg, EC 3.2.1.21) and, while they were shown to bind to the enzyme active site as reversible competitive inhibitors, the only time-dependent inactivation observed was traced to the presence of an extremely small amount (<0.1%) of a highly reactive contaminating impurity.

In Silico Prediction and Validation of Oxygen-Regulated Protein N-myc Downstream Regulated Gene 3 and Virtual Screening of Competitive Inhibitors of L-Lactate as Therapeutics

2019 ◽  
Vol 16 (6) ◽  
pp. 637-644
Author(s):  
Hongyu Cao ◽  
Yanhua Wu ◽  
Xingzhi Zhou ◽  
Xuefang Zheng ◽  
Ge Jiang
Keyword(s):  
Active Sites ◽  
Competitive Inhibition ◽  
Hydrophobic Effect ◽  
3D Structure ◽  
Amino Acid Residues ◽  
In Silico Prediction ◽  
Hypoxic Response ◽  
Drug Candidates ◽  
Competitive Inhibitors ◽  
Extracellular Regulated Protein Kinases

Background: N-myc downstream regulated gene 3 (NDRG3) is a newly discovered oxygen-regulated protein which will bind with L-Lactate in hypoxia and further activate Raf (rapidly accelerated fibrosarcoma)-ERK (extracellular regulated protein kinases) pathway, promoting cell growth and angiogenesis. Methods: Competitive inhibition on the binding of NDRG3 and L-Lactate may be potentially a useful strategy for the repression of hypoxic response mediated by NDRG3. The threedimensional (3D) structure of NDRG3 was built by using homology modeling for its crystal structure was not available. Then, L-Lactate was docked into NDRG3, from which we knew it bound with amino acid residues Gln69, His183, Asn189, Ala72 and Pro66 of NDRG3 in the most possible active sites. Approximately 3000 compounds have been virtually screened and the 6 topranked compounds were selected as reference molecules to analyze their interaction relationships, which illustrated that some of them might form electrostatic interaction with Glu70 and Asp187, π-&π stack with Phe75 and Tyr180, hydrogen bonds with Gly71 and Asn189, hydrophobic effect with Ala72 and Ile184. Results: Novel molecules were designed through structural optimization of the 6 top-ranked compounds and subsequently their ADMET properties were predicted. Conclusion: These molecules may be potential drug candidates for the suppression of hypoxic response mediated by NDRG3 and targeted therapy for hypoxia-induced diseases.

scholarly journals Effects of Strontium on the Permeation and Gating Phenotype of Calcium Channels in Hair Cells

2008 ◽  
Vol 100 (4) ◽  
pp. 2115-2124 ◽  
Author(s):  
Adrian Rodriguez-Contreras ◽  
Ping Lv ◽  
Jun Zhu ◽  
Hyo Jeong Kim ◽  
Ebenezer N. Yamoah
Keyword(s):  
Hair Cell ◽  
Single Channel ◽  
Single Channels ◽  
Physiological Concentration ◽  
Closed State ◽  
Dependent Inactivation ◽  
Intracellular Ca ◽  
High Concentrations ◽  
Latency Distributions ◽  
Channel Properties

To minimize the effects of Ca2+ buffering and signaling, this study sought to examine single Ca2+ channel properties using Sr2+ ions, which substitute well for Ca2+ but bind weakly to intracellular Ca2+ buffers. Two single-channel fluctuations were distinguished by their sensitivity to dihydropyridine agonist (L-type) and insensitivity toward dihydropyridine antagonist (non-L-type). The L- and non-L-type single channels were observed with single-channel conductances of 16 and 19 pS at 70 mM Sr2+ and 11 and 13 pS at 5 mM Sr2+, respectively. We obtained KD estimates of 5.2 and 1.9 mM for Sr2+ for L- and non-L-type channels, respectively. At Ca2+ concentration of ∼2 mM, the single-channel conductances of Sr2+ for the L-type channel was ∼1.5 and 4.0 pS for the non-L-type channels. Thus the limits of single-channel microdomain at the membrane potential of a hair cell (e.g., −65 mV) for Sr2+ ranges from 800 to 2,000 ion/ms, assuming an ECa of 100 mV. The channels are ≥4-fold more sensitive at the physiological concentration ranges than at concentrations >10 mM. Additionally, the channels have the propensity to dwell in the closed state at high concentrations of Sr2+, which is reflected in the time constant of the first latency distributions. It is concluded that the concentration of the permeant ion modulates the gating of hair cell Ca2+ channels. Finally, the closed state/s that is/are altered by high concentrations of Sr2+ may represent divalent ion-dependent inactivation of the L-type channel.

Activités cholinestérasiques dans le foie de Poulet Rôle de l'endoderme dans l'apparition d'activités cholinestérasiques dans la composante mésenchymateuse du tissu hépatique

Development ◽  
1971 ◽  
Vol 26 (3) ◽  
pp. 481-495
Author(s):  
Par Elisabeth Houssaint ◽  
Nicole Le Douarin
Keyword(s):  
Endothelial Cells ◽  
Chick Embryo ◽  
Cholinesterase Activity ◽  
Enzymic Activity ◽  
The Body ◽  
Hepatic Tissue ◽  
Experimental Procedure ◽  
Septum Transversum ◽  
Cholinesterase Activities

Cholinesterases in the chick liver. The role of the endoderm in the appearance of the activity of cholinesterases in the hepatic mesenchyme The histochemical method of Koelle & Friedenwald (1949), as modified by Gerebtzoff (1953), has been used to investigate the distribution of cholinesterases in the chick embryonic and adult liver. Non-specific cholinesterases and, in a lower proportion acetylcholinesterase, have been detected in the endothelial cells of blood sinusoids of both adult and embryonic hepatic tissue. The hepatocytes do not show any cholinesterase activity. Cholinesterases appear precociously in the liver mesenchyme, since they already occur in the septum transversum of the 3-day-old chick embryo. An experimental procedure preventing the invasion of the hepatic mesenchymal Anlage by the endodermic cords has been used. The experimentally isolated hepatic mesenchyme shows an important cholinesterase activity; therefore this activity does not depend on the presence of the hepatocytes. The grafting of the determined hepatic endodern in the somatopleura of the 3-day-old chick embryo results in the development of hepatic tissue in the body wall. In this experimentally produced liver, cholinesterase activities are present in the endothelial cells which have arisen from somatopleura mesenchymal cells, though normally somatopleural mesenchyme does not possess these enzymes. The role of the endoderm in the appearance of this enzymic activity in the somatopleural mesenchyme is discussed.

FURTHER STUDIES ON THE DUAL REQUIREMENT FOR PHENYLALANINE AND TYROSINE IN TISSUE CULTURE

1961 ◽  
Vol 39 (5) ◽  
pp. 925-932 ◽  
Author(s):  
Helen J. Morton ◽  
Joseph F. Morgan
Keyword(s):  
Amino Acids ◽  
Tissue Culture ◽  
Chick Embryo ◽  
Heart Cells ◽  
Present System ◽  
Chick Heart ◽  
High Concentrations ◽  
Chick Embryo Heart ◽  
Embryo Heart ◽  
Related Compounds

Seventeen structurally related compounds were tested for their ability to substitute for phenylalanine or tyrosine in the nutrition of chick embryo heart fragments. DL-Alanyl-DL-phenylalanine replaced phenylalanine. All other compounds had negligible effects, and most were toxic at high concentrations. β-Phenylserine, a phenylalanine antagonist, actually prolonged the survival of chick heart cells but only if both phenylalanine and tyrosine were present. Similarly, optimal reversal of β-phenylserine toxicity was dependent on the presence of both amino acids. Although phenylalanine and tyrosine are not interconvertible in the present system, it has been shown that three phenylalanine antagonists, p-fluorophenylalanine, β-2-thienylalanine, and β-phenylserine, can be identified by their relationship to tyrosine, rather than to phenylalanine.

Differential Time-Dependent Inactivation of P450 3A4 and P450 3A5 by Raloxifene: A Key Role for C239 in Quenching Reactive Intermediates

2007 ◽  
Vol 20 (12) ◽  
pp. 1778-1786 ◽  
Author(s):  
Josh T. Pearson ◽  
Jan L. Wahlstrom ◽  
Leslie J. Dickmann ◽  
Santosh Kumar ◽  
James R. Halpert ◽  
...  
Keyword(s):  
Reactive Intermediates ◽  
Time Dependent ◽  
Dependent Inactivation

Le transport de la phénylalanine et de la tyrosine chez Brevibacterium linens : spécificité et incorporation dans les protéines

1984 ◽  
Vol 30 (4) ◽  
pp. 430-438 ◽  
Author(s):  
P. Boyaval ◽  
Evelyne Moreira ◽  
M. J. Desmazeaud
Keyword(s):  
Amino Acids ◽  
Protein Synthesis ◽  
Cellular Protein ◽  
Cellular Proteins ◽  
Resting Cells ◽  
Brevibacterium Linens ◽  
Competitive Inhibitors ◽  
High Concentrations ◽  
Cellular Protein Synthesis

The specificity of phenylalanine and tyrosine carriers was investigated using actively metabolizing cells of Brevibacterium linens. The cellular protein synthesis of resting cells was very weakly inhibited, even with high concentrations of chloramphenicol or tetracycline. The nonaromatic amino acids were weak inhibitors for these carriers, while fluorinated analogues of phenylalanine and tyrosine were very potent competitive inhibitors. In practice these analogues cannot be used to replace amino acids to evaluate transport without incorporation because they are incorporated in cellular proteins.

scholarly journals Plasma glutathione status as indicator of pre-analytical centrifugation delay

2020 ◽  
Author(s):  
Tomin Tamara ◽  
Natalie Bordag ◽  
Elmar Zuegner ◽  
Abdullah Al-Baghdadi ◽  
Maximilian Schinagl ◽  
...  
Keyword(s):  
Plasma Concentrations ◽  
Time Dependent ◽  
Dependent Manner ◽  
Prolonged Incubation ◽  
Glutathione Status ◽  
Potential Marker ◽  
Analytical Centrifugation ◽  
Plasma Glutathione ◽  
Downstream Analysis

Prolonged incubation of blood prior to plasma preparation can significantly influence the quality of the resulting data. Different markers for this pre-clinical variability have been proposed over the years but with limited success. In this study we explored the usefulness of glutathione (GSH) status, namely ratio of reduced to oxidized glutathione (GSH/GSSG), as potential marker of plasma preparation delay. For that purpose, blood from 20 healthy volunteers was collected into tubes with a cysteine quencher (N-ethylmaleimide; NEM) for GSH stabilization. Plasma preparation was delayed at room temperature for up to 3 hours and every hour, a plasma sample was prepared and the GSH/GSSG ratio measured. We report that over the course of the investigation, plasma concentrations of both GSH and GSSG increased linearly (R2 = 0.99 and 0.98, respectively). Since GSH increased at a much faster rate compared to GSSG, the GSH/GSSG ratio also increased linearly in a time dependent manner (R2 = 0.99). As GSH is an intracellular antioxidant, we speculated that this might stem from ongoing blood hemolysis, which was confirmed by the time dependent rise in lactate dehydrogenase (LDH) activity in the plasma samples. Moreover, we demonstrate that the addition of the thiol alkylating reagent NEM directly to the blood tubes does not seem to influence downstream analysis of clinical parameters. In conclusion we propose that the glutathione status could be used as an indicator of the centrifugation delay prior to plasma preparation.

Sign in / Sign up

Export Citation Format

Share Document