Kinetic analysis

The objective of this study was to develop a method for analysis of MGF in tear fluid that can be further utilised to evaluate the kinetics of pre-cornea drug concentration. For sample collection, lacrimal tear fluid was absorbed into pre-weighed absorbing paper strips, which were then immediately weighed again right after sampling to calculate the sample mass.

12p vihyuga 04-03-2025 0 0   Download

The studies presented in this paper are performed in the general framework of transient coupled calculations with accurate neutron kinetics models able to characterize spatial decoupling in the core. An innovative fission matrix interpolation model has been developed with a correlated sampling technique associated to the Transient Fission Matrix (TFM) approach.

14p christabelhuynh 30-05-2020 8 0   Download

Hepcidin (H25) is a hormone peptide synthesized by the liver that binds to ferroportin and blocks iron export. In this study, H25 was inhibited by administration of single and multiple doses of an anti-H25 monoclonal antibody Ab 12B9m in cynomolgus monkeys. The objective of this analysis was to develop a pharmacodynamic model describing the role of H25 in regulating iron homeostasis and the impact of hepcidin inhibition by Ab 12B9m. Total serum H25 and Ab 12B9m were determined in each animal. Corresponding measurements of serum iron and hemoglobin (Hb) were obtained.

15p caothientrangnguyen 09-05-2020 13 1   Download

To examine the role of the distal His42 residue in the catalytic mechanism of pea cytosolic ascorbate peroxidase, two site-directed variants were prepared in which His42 was replaced with alanine (H42A) or glutamic acid (H42E). Electronic spectra of the ferric derivatives of H42A and H42E (pH 7.0, l ¼ 0.10 M, 25.0 °C) revealed wavelength maxima [kmax (nm): 397, 509, % 540sh, 644 (H42A); 404, 516, % 538sh, 639 (H42E)] consistent with a predominantly fiveco-ordinate high-spin iron. The specific activity of H42E for oxidation of L-ascorbate (8.2 ± 0.

11p system191 01-06-2013 40 4   Download

Cys341 of carboxypeptidase Y, which constitutes one side of the solvent-accessible surface of the S1 binding pocket, was replaced with Gly, Ser, Asp, Val, Phe or His by site-directed mutagenesis. Kinetic analysis, using Cbz-dipeptide substrates, revealed that polar amino acids at the 341 position increased Km whereas hydrophobic amino acids in this position tended to decrease Km.

6p system191 01-06-2013 40 4   Download

Cytidine in the anticodon second position (position 35) and G or U in position 36 of tRNAArg are required for aminoacylation by arginyl-tRNA synthetase (ArgRS) from Escherichia coli. Nevertheless, an arginine-accepting amber suppressor tRNA with a CUA anticodon (FTOR1D26) exhibits suppression activity in vivo [McClain, W.H. & Foss, K. (1988) Science, 241, 1804 –1807]. By an in vitro kinetic study with mutagenized tRNAs, we showed that the arginylation of FTOR1D26 involves C34 and U35, and that U35 can be replaced by G without affecting the activity.

7p system191 01-06-2013 24 3   Download

The involvement of the lysine residue present at the active site of Ehrlich ascites carcinoma (EAC) cell glyceraldehyde-3-phosphate dehydrogenase (Gra3P DH) was investigated by using the lysine specific reagents trinitrobenzenesulfonic acid (TNBS) and pyridoxal phosphate (PP). Both TNBS and PP inactivated EAC cell Gra3P DH with pseudo-first-order kinetics with the rate dependent on modifier concentration. Kinetic analysis, including a Tsou plot, indicated that both TNBS and PP apparently react with one lysine residue per enzyme molecule.

8p system191 01-06-2013 38 3   Download

Institute for Organic Chemistry, University of Karlsruhe, Germany; 2Institute for Organic Chemistry, Budapest University of Technology and Economics, Hungary Elucidation of the 3D structure of histidine ammonia-lyase (HAL, EC 4.3.1.3) from Pseudomonas putida by X-ray crystallography revealed that the electrophilic prosthetic group at the active site is 3,5-dihydro-5-methylidene-4H-i´ midazol-4-one (MIO) [Schwede, T.F., Retey, J., Schulz, G.E. (1999) Biochemistry, 38, 5355 –5361].

9p system191 01-06-2013 50 3   Download

Angiotensin converting enzyme (ACE) was already discovered in insects in 1994, but its physiological role is still enigmatic. We have addressed this problem by purifying four new ACE substrates from the ovaries of the grey fleshfly, Neobellieria bullata. Their primary structures were identified as NKLKPSQWISLSD (Neb-ODAIF-11)13), NKLKPSQWI (Neb-ODAIF-11)9), SLKPSNWLTPSE (Neb-ODAIF-2) and LEQIYHL. Database analysis showed significant homology with amino acid sequence stretches as present in the N-terminal part of several fly yolk proteins....

9p research12 01-06-2013 37 3   Download

Cloning and over-expression of human glucose 6-phosphate dehydrogenase (Glc6P dehydrogenase) has for the first time allowed a detailed kinetic study of a preparation that is genetically homogeneous and in which all the protein molecules are of identical age. The steady-state kinetics of the recombinant enzyme, studied by fluorimetric initial-rate measurements, gave converging linear Lineweaver–Burk plots as expected for a ternary-complex mechanism.

8p research12 01-06-2013 66 4   Download

Cytochrome P450foxy (P450foxy, CYP505) is a fused protein of cytochrome P450 (P450) and its reductase isolated from the fungus Fusarium oxysporum, which catalyzes the subterminal (x-1x-3) hydroxylation of fatty acids. Here, we produced, purified and characterized a fused recombinant protein (rP450foxy) using the Escherichia coli expression system. Purified rP450foxy was catalytically and spectrally indistinguishable from the native protein, but most of the rP450foxy was recovered in the soluble fraction of E. coli cells unlike the membrane-bound native protein....

8p research12 01-06-2013 60 4   Download

A cDNA encoding 6-phosphofructo-2-kinase/fructose-2,6bisphosphatase was isolated from a Spinacia oleracea leaf library and used to express a recombinant enzyme in Escherichia coli and Spodoptera frugiperda cells. The insoluble protein expressed in E. coli was purified and used to raise antibodies. Western blot analysis of a protein extract from spinach leaf showed a single band of 90.8 kDa. Soluble protein was purified to homogeneity from S. frugiperda cells infected with recombinant baculovirus harboring the isolated cDNA. ...

11p research12 01-06-2013 43 5   Download

The Rv0948c gene fromMycobacterium tuberculosisH37Rv encodes a 90 amino acid protein as the natural gene product with chorismate mutase (CM) activity. The protein, 90-MtCM, exhibits Michaelis–Menten kinetics with akcat of 5.5 ± 0.2 s )1 and a Kmof 1500 ± 100lmat 37C and pH 7.5. The 2.0 A˚ X-ray structure shows that 90-MtCM is an alla-helical homodimer (Protein Data Bank ID: 2QBV) with the topology ofEscheri-chia coliCM (EcCM), and that both protomers contribute to each catalytic site.

12p research12 29-04-2013 41 3   Download

The kinetics and thermodynamics of the urea-induced unfolding of ¯avodoxin and apo¯avodoxin fromDesulfo-vibrio vulgariswere investigated by measuring changes in ¯avin and protein ¯uorescence. The reaction of urea with ¯avodoxin is up to 5000 times slower than the reaction with the apoprotein (0.67 s )1 in 3 Murea in 25 mMsodium phosphate at 25°C), and it results in the dissociation of FMN.

12p research12 29-04-2013 59 3   Download

The present study reports the first purification and kinetic characterization of two plant arogenate dehydrogenases (EC 1.3.1.43), an enzyme that catalyses the oxidative decarboxylation of arogenate into tyrosine in presence of NADP. The twoArabidopsis thalianaarogenate dehy-drogenases TyrAAT1 and TyrAAT2 were overproduced in Escherichia coliand purified to homogeneity. Bio-chemical comparison of the two forms revealed that at low substrate concentration TyrAAT1 is four times more efficient in catalyzing the arogenate dehydrogenase reac-tion than TyrAAT2. ...

9p research12 23-04-2013 50 4   Download

The kinetics of the reversible thermal unfolding, irreversible thermal unfolding, and reductive unfolding processes of bovine pancreatic ribonuclease A (RNase A) were investi-gated in NaCl/Pi solutions. Image parameters including Shannon entropy, Hamming distance, mutual information and correlation coefficient were used in the analysis of the CD and 1D NMR spectra. The irreversible thermal unfolding transition of RNase A was not a cooperative process, pretransitional structure changes occur before the main thermal denaturation....

9p tumor12 22-04-2013 46 4   Download

The mechanism by which the fatty acid (1,4)-desaturase of Calendulaofficinalisproduces calendicacid fromlinoleicacid has been probed through the use of kinetic isotope effect (KIE) measurements. This was accomplished by incubating appropriate mixtures of linoleate and regiospecifically dideuterated isotopomers with a strain ofSaccharomyces cerevisiaeexpressing a functional (1,4)-desaturase.

6p tumor12 22-04-2013 24 2   Download

Liver microsomal preparations are routinely used to predict drug interactions that can occurin vivo as a result of inhi-bition of cytochrome P450 (CYP)-mediated metabolism. However, the concentration of free drug (substrate and inhibitor)at its intrahepatic site of action, a variable that cannot be directly measured, may be significantly different from that in microsomal incubation systems.

10p tumor12 20-04-2013 48 4   Download

Penicillin acylase catalyses the hydrolysis and synthesis of semisyntheticb-lactam antibiotics via formation of a cova-lent acyl-enzyme intermediate. The kinetic and mechanistic aspects of these reactions were studied. Stopped-flow experiments with the penicillin and ampicillin analogues 2-nitro-5-phenylacetoxy-benzoic acid (NIPAOB) and D-2-nitro-5-[(phenylglycyl)amino]-benzoic acid (NIPGB) showed that the rate-limiting step in the conversion of penicillin G and ampicillin is the formation of the acyl-enzyme....

9p tumor12 20-04-2013 55 2   Download

In this article, we report the results of an analysis of the glycolytic enzyme enolase (2-phospho-D-glycerate hydro-lase) of Trypanosoma brucei. Enolase activity was detected in both bloodstream-form and procyclic insect-stage try-panosomes, although a 4.5-fold lower specific activity was found in the cultured procyclic homogenate. Subcellular localization analysis showed that the enzyme is only pre-sent in the cytosol.

9p tumor12 20-04-2013 30 3   Download