scholarly journals Characterization of Temperature-Dependent Kinetics of Oculocutaneous Albinism-Causing Mutants of Tyrosinase

2021 ◽  
Vol 22 (15) ◽  
pp. 7771
Author(s):  
Samuel A. Wachamo ◽  
Milan H. Patel ◽  
Paul K. Varghese ◽  
Monika B. Dolinska ◽  
Yuri V. Sergeev
Keyword(s):  
Trichoplusia Ni ◽  
Oculocutaneous Albinism ◽  
Size Exclusion ◽  
Temperature Dependent ◽  
Experimental Conditions ◽  
Rate Limiting Step ◽  
Its Gene ◽  
Exclusion Chromatography ◽  
First Time ◽  
Kinetics Of

Human tyrosinase (Tyr) is a glycoenzyme that catalyzes the first and rate-limiting step in melanin production, and its gene (TYR) is mutated in many cases of oculocutaneous albinism type phenotype in patients with OCA1 have only began to be examined and remain to be delineated. Here, we analyze the temperature-dependent kinetics of wild-type Tyr (WT) and two OCA1B mutant variants (R422Q and P406L) using Michaelis–Menten and Van’t Hoff analyses. Recombinant truncated human Tyr proteins (residues 19–469) were produced in the whole insect Trichoplusia Ni larvae. Proteins were purified by a combination of affinity and size-exclusion chromatography. The temperature dependence of diphenol oxidase protein activities and kinetic parameters were measured by dopachrome absorption. Using the same experimental conditions, computational simulations were performed to assess the temperature-dependent association of L-DOPA and Tyr. Our results revealed, for the first time, that the association of L-DOPA with R422Q and P406L followed by dopachrome formation is a complex reaction supported by enthalpy and entropy forces. We show that the WT has a higher turnover number as compared with both R422Q and P406L. Elucidating the kinetics and thermodynamics of mutant variants of Tyr in OCA1B helps to understand the mechanisms by which they lower Tyr catalytic activity and to discover novel therapies for patients.

scholarly journals Human Tyrosinase: Temperature-Dependent Kinetics of Oxidase Activity

2020 ◽  
Vol 21 (3) ◽  
pp. 895 ◽  
Author(s):  
Kenneth L. Young ◽  
Claudia Kassouf ◽  
Monika B. Dolinska ◽  
David Eric Anderson ◽  
Yuri V. Sergeev
Keyword(s):  
Driving Forces ◽  
Autosomal Recessive Disorder ◽  
Oculocutaneous Albinism ◽  
Binding Activity ◽  
Size Exclusion ◽  
Temperature Dependent ◽  
Exclusion Chromatography ◽  
Pigment Biosynthesis ◽  
Kinetics Of ◽  
Human Tyrosinase

Human tyrosinase (Tyr) is involved in pigment biosynthesis, where mutations in its corresponding gene TYR have been linked to oculocutaneous albinism 1, an autosomal recessive disorder. Although the enzymatic capabilities of Tyr have been well-characterized, the thermodynamic driving forces underlying melanogenesis remain unknown. Here, we analyze protein binding using the diphenol oxidase behavior of Tyr and van ’t Hoff temperature-dependent analysis. Recombinant Tyr was expressed and purified using a combination of affinity and size-exclusion chromatography. Michaelis-Menten constants were measured spectrophotometrically from diphenol oxidase reactions of Tyr, using L-3,4-dihydroxyphenylalanine (L-DOPA) as a substrate, at temperatures: 25, 31, 37, and 43 °C. Under the same conditions, the Tyr structure and the L-DOPA binding activity were simulated using 3 ns molecular dynamics and docking. The thermal Michaelis-Menten kinetics data were subjected to the van ‘t Hoff analysis and fitted with the computational model. The temperature-dependent analysis suggests that the association of L-DOPA with Tyr is a spontaneous enthalpy-driven reaction, which becomes unfavorable at the final step of dopachrome formation.

Mechanism and kinetics of inactivation at 40–70°C of the extracellular proteinase from Pseudomonas fluorescens 22F

1998 ◽  
Vol 65 (2) ◽  
pp. 261-272 ◽  
Author(s):  
ERIX P. SCHOKKER ◽  
MARTINUS A. J. S. VAN BOEKEL
Keyword(s):  
Molecular Mass ◽  
Pseudomonas Fluorescens ◽  
Computer Simulations ◽  
Rate Constants ◽  
Size Exclusion ◽  
Temperature Dependent ◽  
Extracellular Proteinase ◽  
Reaction Rate Constants ◽  
Exclusion Chromatography ◽  
Kinetics Of

HPLC size exclusion chromatography experiments showed that during inactivation at 40–70°C of the extracellular proteinase from Pseudomonas fluorescens 22F small molecular mass fragments were formed, indicating that autoproteolysis was at least one of the major causes of inactivation. The formation of small molecular mass fragments and the reaction order indicated that intermolecular autoproteolysis was more likely than intramolecular autodigestion. This was confirmed by computer simulations. The rate constants and the activation enthalpy (ΔH[Dagger]) and entropy (ΔS[Dagger]) for the reactions of the intermolecular autoproteolysis model were derived from computer simulations. ΔH[Dagger] and ΔS[Dagger] of the unfolding reaction were 504 kJ mol−1 and 1252 J mol K−1 respectively. ΔH[Dagger] and ΔS[Dagger] of the refolding reaction were strongly temperature dependent. The estimates for the enthalpy (ΔH0) and entropy (ΔS0) difference between the folded and unfolded state as derived from the reaction rate constants of unfolding and refolding were subject to large deviations, owing to accumulation of errors in the estimation of the kinetic characteristics.

scholarly journals Fragmentation of Chitosan by Acids

2013 ◽  
Vol 2013 ◽  
pp. 1-11 ◽  
Author(s):  
Mohammad Reza Kasaai ◽  
Joseph Arul ◽  
Gérard Charlet
Keyword(s):  
Aqueous Solution ◽  
Molecular Weight ◽  
Kinetic Data ◽  
Chemical Structure ◽  
Size Exclusion ◽  
Experimental Conditions ◽  
First Order ◽  
H Nmr ◽  
Exclusion Chromatography ◽  
Kinetics Of

Fragmentation of chitosan in aqueous solution by hydrochloric acid was investigated. The kinetics of fragmentation, the number of chain scissions, and polydispersity of the fragments were followed by viscometry and size exclusion chromatography. The chemical structure and the degree of N-acetylation (DA) of the original chitosan and its fragments were examined by1H NMR spectroscopy and elemental analysis. The kinetic data indicates that the reaction was of first order. The results of polydispersity and the DA suggest that the selected experimental conditions (temperature and concentration of acid) were appropriate to obtain the fragments having the polydispersity and the DA similar to or slightly different from those of the original one. A procedure to estimate molecular weight of fragments as well as the number of chain scissions of the fragments under the experimental conditions was also proposed.

Kinetic and structural analysis of the ultrasensitive behaviour of cyanobacterial ADP-glucose pyrophosphorylase

2000 ◽  
Vol 350 (1) ◽  
pp. 139-147 ◽  
Author(s):  
Diego F. GÓMEZ CASATI ◽  
Miguel A. AON ◽  
Alberto A. IGLESIAS
Keyword(s):  
Conformational Changes ◽  
Tertiary Structure ◽  
Fluorescence Emission ◽  
Maximal Activity ◽  
Size Exclusion ◽  
Exclusion Chromatography ◽  
Adp Glucose Pyrophosphorylase ◽  
Kinetics Of ◽  
Allosteric Regulators

The kinetic and (supra)molecular properties of the ultrasensitive behaviour of ADP-glucose pyrophosphorylase (AGPase) from Anabaena PCC 7120 (a cyanobacterium) were exhaustively studied. The response of the enzyme toward the allosteric activator 3-phosphoglycerate (3PGA) occurs with ultrasensitivity as a consequence of the cross-talk with the inhibitor Pi. Molecular ‘crowding’renders AGPase more sensitive to the interplay between the allosteric regulators and, consequently, enhances the ultrasensitive response. In crowded media, and when orthophosphate is present, the activation kinetics of the enzyme with 3PGA proceed with increased co-operativity and reduced affinity toward the activator. Under conditions of ultrasensitivity, the enzyme's maximal activation takes place in a narrow range of 3PGA concentrations. Moreover, saturation kinetics of the enzyme with respect to its substrates, glucose 1-phosphate and ATP, were different at low or high 3PGA levels in crowded media. Only under the latter conditions did AGPase exhibit discrimination between low or high levels of the activator, which increased the affinity toward the substrates and the maximal activity reached by the enzyme. Studies of fluorescence emission of tryptophan residues, fourth-derivative spectroscopy and size-exclusion chromatography indicated that the ultrasensitive behaviour is correlated with intramolecular conformational changes induced in the tertiary structure of the homotetrameric enzyme. The results suggest a physiological relevance of the ultrasensitive response of AGPase in vivo, since the enzyme could be subtly sensing changes in the levels of allosteric regulators and substrates, and thus determining the flux of metabolites toward synthesis of storage polysaccharides.

Investigating the propagation kinetics of a novel class of nitrogen-containing methacrylates via PLP-SEC

2016 ◽  
Vol 7 (26) ◽  
pp. 4342-4351 ◽  
Author(s):  
Katrin B. Kockler ◽  
Friederike Fleischhaker ◽  
Christopher Barner-Kowollik
Keyword(s):  
Size Exclusion Chromatography ◽  
Rate Coefficient ◽  
Pulsed Laser ◽  
Propagation Rate ◽  
Size Exclusion ◽  
Arrhenius Parameters ◽  
Exclusion Chromatography ◽  
Kinetics Of ◽  
Propagation Rate Coefficient ◽  
Pulsed Laser Polymerization

The Mark–Houwink–Kuhn–Sakurada parameters as well as Arrhenius parameters of the propagation rate coefficient for a new group of nitrogen-containing methacrylates were determined via triple detector SEC and pulsed laser polymerization–size exclusion chromatography, respectively.

scholarly journals Kinetics of the diamine oxidase reaction

1973 ◽  
Vol 131 (3) ◽  
pp. 459-469 ◽  
Author(s):  
William G. Bardsley ◽  
M. James C. Crabbe ◽  
Julian S. Shindler
Keyword(s):  
Diamine Oxidase ◽  
Experimental Conditions ◽  
Ph Values ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Deuterium Substitution ◽  
Effects Of Temperature ◽  
Rate Limiting ◽  
Kinetics Of ◽  
Oxidase Reaction

1. The oxidation of p-dimethylaminomethylbenzylamine was followed spectrophotometrically by measuring the change in E250 caused by the p-dimethylaminomethylbenzaldehyde produced under a wide variety of experimental conditions. 2. The effect of variations in concentrations of both substrates (amine and oxygen) and all products (aminoaldehyde, hydrogen peroxide and ammonia) on this reaction was studied and the results used to develop a formal mechanism. 3. The nature of the rate-limiting step was elucidated by studying the effects of alterations in ionic strength, dielectric constant and deuterium substitution on the velocity of the forward reaction. 4. Thermodynamic activation energy parameters were obtained at several pH values from the effects of temperature on the reaction.

scholarly journals Extraction and Isolation of Antineoplastic Pristimerin from Mortonia greggii (Celastraceae)

2015 ◽  
Vol 10 (11) ◽  
pp. 1934578X1501001
Author(s):  
Luis Alberto Mejía-Manzano ◽  
Bertha A. Barba-Dávila ◽  
Janet A. Gutierrez-Uribe ◽  
Edgardo J. Escalante-Vázquez ◽  
Sergio O. Serna-Saldívar
Keyword(s):  
Size Exclusion ◽  
Root Extract ◽  
Root Bark ◽  
Extraction Temperature ◽  
Experimental Conditions ◽  
Plant Weight ◽  
Exclusion Chromatography ◽  
Solvent Systems ◽  
Solid Liquid ◽  
Solvent Volume

The aim of this research was to identify, extract and isolate pristimerin in leaves, stems and roots of the Mexican plant Mortonia greggii ( Celastraceae). The principal objective was to determine the best laboratory experimental conditions for the extraction and isolation of this powerful natural anticancer agent from the root tissue. Six experimental factors in solid-liquid pristimerin extraction were analyzed: solvent systems, number of extractions, ratio of plant weight (g)/solvent volume (mL) used, time of extraction, temperature and agitation. A mathematical model was generated for pristimerin purity and yield. Ethanol, first extraction, 0.5 ratio of plant weight/solvent volume (g/mL), 0.5 h, 200 rpm and 49.7°C were optimal conditions for the extraction of this phytochemical. The degree of purification of pristimerin root extract was studied by size-exclusion chromatography (SEC) using Sephadex LH-20 reaching fractions with purification indexes (PI) greater than 2 and recoveries of 28.3%. When fractions with purification indices higher than 1 and less than 2 were accumulated, the recovery of pristimerin increased by about 73.6%. By combining the optimum extracts and SEC purification protocols, an enriched fraction containing 245.6 mg pristimerin was obtained from 100 g of root bark, representing about 14.4%, w/w, pristimerin from the total solids presented in the fraction.

Host-finding in Echinostoma caproni: miracidia and cercariae use different signals to identify the same snail species

Parasitology ◽  
2000 ◽  
Vol 120 (5) ◽  
pp. 479-486 ◽  
Author(s):  
B. HABERL ◽  
M. KÖRNER ◽  
Y. SPENGLER ◽  
J. HERTEL ◽  
M. KALBE ◽  
...  
Keyword(s):  
Amino Acids ◽  
Molecular Weight ◽  
Total Concentration ◽  
Host Finding ◽  
Snail Host ◽  
Size Exclusion ◽  
Snail Species ◽  
Echinostoma Caproni ◽  
Exclusion Chromatography ◽  
First Time

The snail host signals releasing host-finding responses in miracidia and cercariae of Echinostoma caproni were analysed by fractionation of snail-conditioned water (SCW). Cercariae responded non-specifically to organic and hydrophilic, low molecular weight components of SCW showing their typical turning response. Hydrolysis of peptides in SCW had no effect on cercarial responses. An artificial mixture of amino acids in concentrations determined from SCW as well as glycine alone in a concentration corresponding to the total concentration of amino acids in SCW showed nearly the same efficacy as SCW itself. Miracidia responded to a high molecular weight glycoprotein fraction, which could be isolated from SCW by ion-exchange and size-exclusion chromatography. In contrast to an Egyptian Schistosoma mansoni strain, the echinostome miracidia were not able to differentiate between different snail species. The results show for the first time that miracidia and cercariae of the same species may use different signals to identify the same snail host species. This indicates an independent evolution of host-finding mechanisms in the two parasite stages.

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