The influence of pH on kinetic parameters of coleoptile phosphoenolpyruvate carboxylase. Relationship to auxin-stimulated dark fixation

1979 ◽  
Vol 57 (5) ◽  
pp. 543-547 ◽  
Author(s):  
Ceredwyn Smith ◽  
Ahmed Doo ◽  
Alan W. Bown
Keyword(s):  
Binding Affinity ◽  
Catalytic Efficiency ◽  
Enzymic Activity ◽  
Carboxylase Activity ◽  
Pep Carboxylase ◽  
Ph Values ◽  
Dark Fixation ◽  
Rate Limiting ◽  
Cytosol Ph

In vitro phosphoenolpyruvate (PEP) carboxylase activity from Avena coleoptile tissue was investigated over a range of pH values which include cytosol pH values. Increasing the pH from 7.0 to 7.5 increased optimal PEP carboxylase activity (Vmax) by over 100%. In the presence of rate-limiting 0.07 mM PEP, noncompetitive inhibition by 0.1 mM malate decreased from 80% at pH 7.1 to 50% at pH 7.5. The Km for PEP was not influenced by malate, but as the pH was increased from 7.1 to 7.5, the Km decreased from 0.16 to 0.08 mM. Over the same pH rise, the KI for malate inhibition increased from 0.04 mM to 0.09 mM. Fusicoccin had no detectable influence on enzymic activity. These results are discussed in relation to the stimulation of H+ excretion and dark CO2 fixation by indoleacetic acid and fusicoccin. The data indicate that any increase in cytosol pH, resulting from H+ excretion, would stimulate PEP carboxylase activity by promoting catalytic efficiency and binding affinity for PEP and by reducing the binding affinity for the inhibitor malate.

scholarly journals Rational design of a highly efficient catalytic system for the production of 3′-phosphoadenosine-5′-phosphosulfate from ATP

2021 ◽  
Author(s):  
Kaifang Liu ◽  
Xiulai Chen ◽  
Yunlu Zhong ◽  
Jia Liu ◽  
Guipeng Hu ◽  
...  
Keyword(s):  
Catalytic System ◽  
Rational Design ◽  
Specific Activity ◽  
Catalytic Efficiency ◽  
Disodium Salt ◽  
In Vivo Testing ◽  
Dynamics Simulations ◽  
Rate Limiting

The compound 3′-phosphoadenosine-5′-phosphosulfate (PAPS) serves as a sulfate group donor in the production of valuable sulfated compounds, such as glycosaminoglycan and oxamniquine. However, elevated costs and low conversion efficiency limit the industrial applicability of PAPS. Here, we designed and constructed an efficient and controllable catalytic system for the conversion of ATP (disodium salt) into PAPS without inhibition from by-products. In vitro and in vivo testing in Escherichia coli identified adenosine-5′-phosphosulfate kinase from Penicillium chrysogenum (PcAPSK) as the rate-limiting enzyme. Based on analysis of the catalytic steps and molecular dynamics simulations, a mechanism-guided “ADP expulsion” strategy was developed to generate an improved PcAPSK variant (L7), with a specific activity of 48.94 U·mg-1 and 73.27-fold higher catalytic efficiency (kcat/Km) that of the wild-type enzyme. The improvement was attained chiefly by reducing the ADP-binding affinity of PcAPSK, as well as by changing the enzyme’s flexibility and lid structure to a more open conformation. By introducing PcAPSK L7 in an in vivo catalytic system, 73.59 mM (37.32 g·L-1) PAPS was produced from 150 mM ATP in 18.5 h using a 3-L bioreactor. The achieved titer is the highest reported to date and corresponds to a 98.13% conversion rate. The proposed strategy will facilitate industrial production of PAPS as well as the engineering of similar enzymes.

scholarly journals Development of engineered ferredoxin reductase systems for the efficient hydroxylation of steroidal substrates

2020 ◽  
Author(s):  
Zhangliang Zhu ◽  
Xin Gao ◽  
Zhan Song ◽  
Chao Li ◽  
Fuping Lu ◽  
...  
Keyword(s):  
Electron Transfer ◽  
Catalytic Efficiency ◽  
Nadh Regeneration ◽  
Whole Cell ◽  
Rate Limiting Step ◽  
Engineering Strategy ◽  
Whole Cell Catalysis ◽  
Rate Limiting ◽  
Insight Into

Abstract Background 9α-hydroxy-4-androstene-3,17-dione (9OHAD), catalyzed by 3-ketosteroid-9-hydroxylase (KSH) using 4-androstene-3,17-dione (AD) as a substrate, is an important precursor for the synthesis of adrenocortical hormones. Whole-cell catalyst in microorganisms with this KSH system for desirable hydroxylated steroids in high purity and productivity have rarely been successful. Results The rate-limiting step for the biosynthesis of 9OHAD is catalyzed by the reductase KshB, which is an important component of electrons donor. A sufficient supply system of the cofactor NADH was constructed on KshB by introducing the formate dehydrogenase (FDH) gene. Several reductases were then screened to find a TDO reductase containing a ferredoxin, which showed a maximal NADH activity with a catalytic efficiency (kcat/Km) of 0.43 s− 1 µM− 1 and 54.8% of 9OHAD yield via multienzyme cascade catalysis in vitro. TDO mutagenesis was further performed via a protein engineering strategy, resulting in a 2.25-fold improvement in activity and a 74.8% 9OHAD yield. The modification of a Rieske [2Fe-2S] cluster in KshB and TDO showed 9OHAD yields of 56.1% and 74.5% higher than wild-type ones, which implied Rieske [2Fe-2S] ferredoxin strengthening for electrons transferring. The biosynthesis of 9OHAD was further optimized in a whole-cell catalysis system with FDH, KshA, and TDO_M9 mutant with the Rieske [2Fe-2S] ferredoxin (BLKA-RMT-F), resulting in a final production of 5.24 g/L 9OHAD, a considerable yield of 99.3% of theoretical without by-products. Conclusion An efficient whole-cell catalyst was constructed with considerable production and yield by modification of a Rieske [2Fe-2S] cluster in TDO with increased the efficiency of electron transfer. This research provided comprehensive insight into the electron transfer system for these steroid hydroxylation reactions and NADH regeneration systems.

METABOLISM OF 3β-HYDROXY-5-ENE STEROIDS BY TESTES OF RATS ACCLIMATIZED TO A HOT ENVIRONMENT

1973 ◽  
Vol 58 (2) ◽  
pp. 207-217 ◽  
Author(s):  
E. BEDRAK ◽  
V. SAMOILOFF ◽  
U. A. SOD-MORIAH
Keyword(s):  
Prolonged Exposure ◽  
Enzymic Activity ◽  
Seminiferous Tubules ◽  
Continuous Exposure ◽  
Heat Acclimatization ◽  
Adult Rats ◽  
In Vitro Synthesis ◽  
Hot Environment ◽  
Rate Limiting

SUMMARY The metabolism of 3β-hydroxysteroids, in the presence of NADPH, by testicular homogenates of adult rats acclimatized to a hot environment (33–35 °C, 25–40% relative humidity), was compared with that of control and surgically produced cryptorchid testes. Prolonged exposure to a hot environment stimulated the transformation of 3β-hydroxysteroids to 3-oxo-4-ene metabolites, so that relatively large amounts of the latter accumulated. Pregnenolone was metabolized rapidly to progesterone, 17α-hydroxyprogesterone, androstenedione and testosterone. A similar trend was observed in the metabolism of 17α-hydroxypregnenolone. In-vitro synthesis of testosterone in the rat apparently takes place primarily by the 4-ene pathway. The 5-ene-17,20-lyase reaction appears to be rate-limiting. Heat acclimatization does not seem to affect this step. It does, however, seem to enhance the conversion of dehydroepiandrosterone via 5-androstene-3β,17β-diol to testosterone. Enzymic activity was much lower in cryptorchid than in heat-acclimatized testes, where it actually increased, except for 17β-hydroxysteroid oxidoreductase. Continuous exposure of rats to a high ambient temperature for 4–6 months tended to decrease testosterone concentration in peripheral blood, and retarded growth rate and gonadal size. Histological examination showed atrophied areas in the testes where necrobiosis of the germinal epithelium had occurred. These necrotic foci, unlike those found in cryptorchid testes, were randomly scattered among intact seminiferous tubules in which active spermatogenesis was taking place.

scholarly journals The biochemistry of fatty liver and kidney syndrome. Biotin-mediated restoration of hepatic gluconeogenesis in vitro and its relationship to pyruvate carboxylase activity

1976 ◽  
Vol 156 (1) ◽  
pp. 167-173 ◽  
Author(s):  
D W Bannister
Keyword(s):  
Fatty Liver ◽  
Nutrient Medium ◽  
Pyruvate Carboxylase ◽  
Control Valve ◽  
Protein Synthesis Inhibitors ◽  
Hepatic Gluconeogenesis ◽  
Carboxylase Activity ◽  
Liver And Kidney ◽  
Rate Limiting

Liver slices from chicks affected by the fatty liver and kidney syndrome display an extremely low extent of hepatic gluconeogenesis which is associated with decreased activities of certain rate-limiting gluconeogenic enzymes. Pyruvate carboxylase activity is particularly severely affected, being less than 4% of control values. Incubation of affected slices in a biotin-containing nutrient medium restores both gluconeogenesis and pyruvate carboxylase actiivity (the latter to approx. 35% of the control valve). Activities of the other enzymes studied were not greatly affected by this treatment. Restoration of gluconeogenesis did not occur if biotin was excluded from the nutrient medium, nor was it prevented by protein-synthesis inhibitors. It is concluded that the syndrome involves the lack of available biotin in the liver rather than suppression of apocarboxylase synthesis.

Binding of Tissue Plasminogen Activator to Vascular Grafts

1989 ◽  
Vol 61 (01) ◽  
pp. 131-136 ◽  
Author(s):  
Richard A Harvey ◽  
Hugh C Kim ◽  
Jonathan Pincus ◽  
Stanley Z Trooskin ◽  
Josiah N Wilcox ◽  
...  
Keyword(s):  
Plasminogen Activator ◽  
Tissue Plasminogen Activator ◽  
Fibrinolytic Activity ◽  
Polymer Surface ◽  
Enzymic Activity ◽  
Vascular Prostheses ◽  
Chemically Modified ◽  
Insoluble Surfactant ◽  
Tissue Plasminogen

SummaryTissue plasminogen activator labeled with radioactive iodine (125I-tPA) was immobilized on vascular prostheses chemically modified with a thin coating of water-insoluble surfactant, tridodecylmethylammonium chloride (TDM AC). Surfactant- treated Dacron, polytetrafluoroethylene (PTFE), silastic, polyethylene and polyurethane bound appreciable amounts of 125I- tPA (5-30 μg 125I-tPA/cm2). Upon exposure to human plasma, the amount of 125I-tPA bound to the surface shows an initial drop during the first hour of incubation, followed by a slower, roughly exponential release with a t½ of appoximately 75 hours. Prostheses containing bound tPA show fibrinolytic activity as measured both by lysis of clots formed in vitro, and by hydrolysis of a synthetic polypeptide substrate. Prior to incubation in plasma, tPA bound to a polymer surface has an enzymic activity similar, if not identical to that of the native enzyme in buffered solution. However, exposure to plasma causes a decrease in the fibrinolytic activity of both bound tPA and enzyme released from the surface of the polymer. These data demonstrate that surfactant-treated prostheses can bind tPA, and that these chemically modified devices can act as a slow-release drug delivery system with the potential for reducing prosthesis-induced thromboembolism.

Trans-sialidase Associated with Atherosclerosis: Defining the Identity of a Key Enzyme Involved in the Pathology

2019 ◽  
Vol 20 (9) ◽  
pp. 938-941
Author(s):  
Victor Y. Glanz ◽  
Veronika A. Myasoedova ◽  
Andrey V. Grechko ◽  
Alexander N. Orekhov
Keyword(s):  
Acid Metabolism ◽  
Research Subject ◽  
Disease Pathogenesis ◽  
Sialidase Activity ◽  
Ph Values ◽  
Ldl Particles ◽  
Optimum Ph ◽  
Key Enzyme ◽  
St6gal I

Atherosclerosis is associated with the increased trans-sialidase activity, which can be detected in the blood plasma of atherosclerosis patients. The likely involvement in the disease pathogenesis made this activity an interesting research subject and the enzyme that may perform such activity was isolated and characterized in terms of substrate specificity and enzymatic properties. It was found that the enzyme has distinct optimum pH values, and its activity was enhanced by the presence of Ca2+ ions. Most importantly, the enzyme was able to cause atherogenic modification of lowdensity lipoprotein (LDL) particles in vitro. However, the identity of the discovered enzyme remained to be defined. Currently, sialyltransferases, mainly ST6Gal I, are regarded as major contributors to sialic acid metabolism in human blood. In this mini-review, we discuss the possibility that atherosclerosis- associated trans-sialidase does, in fact, belong to the sialyltransferases family.

Computational Evaluation and In Vitro Validation of New Epidermal Growth Factor Receptor Inhibitors

2020 ◽  
Vol 20 (18) ◽  
pp. 1628-1639
Author(s):  
Sergi Gómez-Ganau ◽  
Josefa Castillo ◽  
Andrés Cervantes ◽  
Jesus Vicente de Julián-Ortiz ◽  
Rafael Gozalbes
Keyword(s):  
Cell Proliferation ◽  
Epidermal Growth Factor Receptor ◽  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Binding Affinity ◽  
Cell Lines ◽  
Growth Factor Receptor ◽  
Significant Activity ◽  
Epidermal Growth

Background: The Epidermal Growth Factor Receptor (EGFR) is a transmembrane protein that acts as a receptor of extracellular protein ligands of the epidermal growth factor (EGF/ErbB) family. It has been shown that EGFR is overexpressed by many tumours and correlates with poor prognosis. Therefore, EGFR can be considered as a very interesting therapeutic target for the treatment of a large variety of cancers such as lung, ovarian, endometrial, gastric, bladder and breast cancers, cervical adenocarcinoma, malignant melanoma and glioblastoma. Methods: We have followed a structure-based virtual screening (SBVS) procedure with a library composed of several commercial collections of chemicals (615,462 compounds in total) and the 3D structure of EGFR obtained from the Protein Data Bank (PDB code: 1M17). The docking results from this campaign were then ranked according to the theoretical binding affinity of these molecules to EGFR, and compared with the binding affinity of erlotinib, a well-known EGFR inhibitor. A total of 23 top-rated commercial compounds displaying potential binding affinities similar or even better than erlotinib were selected for experimental evaluation. In vitro assays in different cell lines were performed. A preliminary test was carried out with a simple and standard quick cell proliferation assay kit, and six compounds showed significant activity when compared to positive control. Then, viability and cell proliferation of these compounds were further tested using a protocol based on propidium iodide (PI) and flow cytometry in HCT116, Caco-2 and H358 cell lines. Results: The whole six compounds displayed good effects when compared with erlotinib at 30 μM. When reducing the concentration to 10μM, the activity of the 6 compounds depends on the cell line used: the six compounds showed inhibitory activity with HCT116, two compounds showed inhibition with Caco-2, and three compounds showed inhibitory effects with H358. At 2 μM, one compound showed inhibiting effects close to those from erlotinib. Conclusion: Therefore, these compounds could be considered as potential primary hits, acting as promising starting points to expand the therapeutic options against a wide range of cancers.

Synthesis, Antimicrobial Evaluation and Docking Study of Novel Thiosemicarbazone clubbed with 1,2,3-Triazoles

2020 ◽  
Vol 16 ◽  
Author(s):  
Adinath D. Badar ◽  
Shubham M. Sulakhe ◽  
Mahesh B. Muluk ◽  
Naziya N. M. A. Rehman ◽  
Prashant P. Dixit ◽  
...  
Keyword(s):  
Binding Affinity ◽  
Biological Activities ◽  
Dna Gyrase ◽  
Docking Study ◽  
Antimicrobial Activities ◽  
Diffusion Method ◽  
Molecular Docking Study ◽  
Triazole Derivatives ◽  
Antibacterial And Antifungal

Background: Thiosemicarbazone, 1,2,3-triazole and their derivatives received great pharmaceutical importance due to their prominent biological activities. In the present study, the molecular hybrid thiosemicarbazone-1,2,3-triazoles derivatives were synthesized and screened for their antimicrobial activities. Methods: A series of thiosemicarbazone clubbed with 1,2,3-triazole derivatives were synthesized via click chemistry approach in good yields. The structures of synthesized compounds were assigned by their spectral data. The in vitro antimicrobial activity was performed by the agar well diffusion method. A molecular docking study was performed to identify the possible mode of action of synthesized derivatives. Results: The compounds 5d, 5h, 5i and 5k have exhibited excellent antimicrobial activities against both antibacterial and antifungal pathogens. The active thiosemicarbazone-1,2,3-triazole derivatives have shown excellent binding affinity towards DNA gyrase. Conclusion: The molecular hybrid thiosemicarbazone-1,2,3-triazole derivatives were synthesized. The newly synthesized compounds were evaluated for their antimicrobial activities. Few of the thiosemicarbazone-1,2,3-triazoles derivatives have exhibited good antimicrobial activities. They have been shown excellent binding affinity towards DNA gyrase.

scholarly journals Comparative Analysis of Different Local Anesthetic Solutions Available in Market: An In Vitro and Clinical Study

2021 ◽  
Author(s):  
Eisha Imran ◽  
Faisal Moeen ◽  
Beenish Abbas ◽  
Bakhtawar Yaqoob ◽  
Mehreen Wajahat ◽  
...  
Keyword(s):  
Comparative Analysis ◽  
Local Anesthetic ◽  
Compositional Analysis ◽  
Bacterial Activity ◽  
Similar Composition ◽  
Ph Values ◽  
Significant Difference ◽  
The Mean ◽  
The Difference

Abstract Objectives The study aimed to evaluate and compare various commercially available local anesthetic solutions. Materials and Methods A total of 150 commercially available local anesthetic cartridges of similar composition (2% lidocaine with epinephrine 1:100,000) were randomly collected and divided into 3 groups. The designations of groups were selected from their product names such that each group consisted of 60 cartridges. Group S (Septodont, France) Group M (Medicaine, Korea) and Group H (HD-Caine, Pakistan). The samples were divided into five sub-groups, each consisting of 10 cartridges from each group to investigate each parameter. Results The acquired data was statistically analyzed and compared (using SPSS version 12). Compositional analysis revealed a non-significant (P>0.05) difference when the three Groups were compared with standard lidocaine and epinephrine solutions. The mean pH values of samples from group S, M and H respectively fell within the range of pH values of commercially available solutions. Non-significant difference in EPT values of Group S and H was found when efficacy was compared (p = 0.3), however a significant difference (p < 0.01) was observed in contrast to Group M. Anti-bacterial activity was observed in all the group and a non-significant difference in cell viability values of Group S and M was found (p = 0.6), while the difference was significant in comparison to Group H. Conclusion Within the limitations of these investigations, it appears that the properties of different manufacturers fall within the recommended ranges as mentioned in literature and do not appear to be statistically different in the variables we have tested.

scholarly journals Assembly of the B subunit pentamer of Escherichia coli heat-labile enterotoxin. Kinetics and molecular basis of rate-limiting steps in vitro.

1996 ◽  
Vol 271 (43) ◽  
pp. 27188
Author(s):  
Lloyd W. Ruddock ◽  
Jeremy J.F. Coen ◽  
Caroline Cheesman ◽  
Robert B. Freedman ◽  
Timothy R. Hirst
Keyword(s):  
Escherichia Coli ◽  
Molecular Basis ◽  
B Subunit ◽  
Heat Labile ◽  
Rate Limiting
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