scholarly journals Refolding of triose phosphate isomerase

1973 ◽  
Vol 135 (1) ◽  
pp. 165-172 ◽  
Author(s):  
Stephen G. Waley
Keyword(s):  
Half Life ◽  
Glycolytic Enzyme ◽  
Enzymic Activity ◽  
Triose Phosphate Isomerase ◽  
Guanidinium Chloride ◽  
Triose Phosphate ◽  
First Order ◽  
Rate Determining Step ◽  
Low Concentrations ◽  
High Concentrations

The refolding and reactivation of the glycolytic enzyme triose phosphate isomerase (EC 5.3.1.1) has been studied. The enzyme, which is a dimer, is disaggregated and unfolded in solutions of guanidinium chloride. Unfolding, followed by changes in E233, took place quite rapidly in 3m-guanidinium chloride (i.e. with a half-life of about 1 min). Refolding also took place rapidly when the solution was diluted about tenfold; two first-order processes could be resolved. Regain of enzymic activity was followed by diluting the solution of the denatured enzyme in guanidinium chloride into assay mixture. The half-life (i.e. the time when the activity was half the final activity) depended markedly on the concentration of protein at low concentrations (about 100ng/ml), but at higher concentrations the half-life became independent of concentration. Thus at low concentrations dimerization was a rate-determining step and this is taken to indicate that the monomers showed little or no activity under these conditions. The rate of regain of enzymic activity was the same as the rate of the slower process of refolding, which was detected spectroscopically. The native enzyme was resistant to proteolysis; high concentrations of subtilisin prevented regain of activity, but at lower concentrations refolding competed with proteolysis.

scholarly journals Akinetic model for the consumption of stabilizer in single base gun propellants

2002 ◽  
Vol 67 (2) ◽  
pp. 103-109 ◽  
Author(s):  
Ljiljana Jelisavac ◽  
Milos Filipovic
Keyword(s):  
Life Time ◽  
Order Reaction ◽  
Zero Order ◽  
Final Phase ◽  
Single Base ◽  
First Order ◽  
Low Concentrations ◽  
Zero Order Reaction ◽  
High Concentrations ◽  
Gun Propellants

A suitable kinetic model for the consumption of stabilizer (diphenylamine) in single base gun propellants was investigated and successfully verified. The model assumes that a reaction of shifting order can be applied for the consumption of diphenylamine in single base gun propellants. It was found that the experimental data were well evaluated by a first-order reaction at high concentrations of diphenylamine in the propellant, but by a zero-order reaction at low concentrations during the final phase of the propellant life time. The mechanism of diphenylamine depletion was discussed with relation to the model and the ageing behavior of the propellants. The kinetic parameters of this model, which permit the calculation of the time up to complete consumption of the diphenylamine, were determined. The results were compared with the kinetic data obtained by a widely accepted model, which combines formally reactions of first and zero order, designated as an "exponential and linear" model. All comparisons gave satisfactory agreement.

scholarly journals The Kinetics of Clearance from the Circulation of Man of Heparin and a Semi-Synthetic Heparin Analogue

1977 ◽  
Author(s):  
D. A. Lane ◽  
R. Michalski ◽  
V. V. Kakkar
Keyword(s):  
High Specificity ◽  
Antithrombotic Agent ◽  
First Order ◽  
First Order Kinetics ◽  
Zero Order Kinetics ◽  
Low Concentrations ◽  
Equal Dose ◽  
Rapid Clearance ◽  
The Difference ◽  
High Concentrations

A study has been made of a low molecular weight semi-synthetic heparin analogue, (SSHA) that may be clinically useful as an antithrombotic agent because of itsreported high specificity for potentiating antithrombin III activity. The clearance from the circulation of both heparin and the analogue has been studied in man following intravenous injection. Heparin obeyed almost zero order kinetics when assayed using a specific anti-Xa assay and first order kinetics when measured with KCCT. At high concentrations the heparin analogue was cleared with first order kinetics when assayed both with the anti-Xa assay and with KCCT. At low concentrations the analogue produced between one half and two-thirds of the anti-Xa activity of an equal dose of heparin, producing only a small prolongation of KCCT. With increasing dose, the more specific anti-Xa potentiating effect of SSHA decreased in part because of the difference in kinetic behaviour between heparin and SSHAbut largely because of a flattening of its anti-Xa dose response curve. Because of the initial more rapid clearance of higher doses of heparin from plasma when it is measured by the KCCT, these results suggest that the use of KCCT can cause a small underestimate of circulating heparin anti-thrombotic activity.

scholarly journals Studies of triose phosphate isomerase by hydrogen exchange

1974 ◽  
Vol 141 (3) ◽  
pp. 753-760 ◽  
Author(s):  
Christopher A. Browne ◽  
Stephen G. Waley
Keyword(s):  
Hydrogen Exchange ◽  
Marked Effect ◽  
Protein Analysis ◽  
Hollow Fibre ◽  
Triose Phosphate Isomerase ◽  
Rabbit Muscle ◽  
Structural Rearrangements ◽  
Triose Phosphate ◽  
Chicken Muscle ◽  
Low Concentrations

The3H–H exchange of chicken muscle and rabbit muscle triose phosphate isomerases was studied. Their behaviour was mostly very similar. ‘Exchange-in’ (acquisition of radioactivity when protein was incubated in3H2O) was measured at 37°C and at pH7.5, and the rates of exchange of the native and liganded enzymes were compared. Inhibitors and substrates retarded exchange, substrates showing the most marked effect; structural rearrangements in the enzyme may thus play some part in catalysis. The inhibitor phosphoglycollate affected the rabbit enzyme, but had little or no effect on the chicken enzyme. ‘Exchange-out’ (loss of radioactivity from protein previously labelled by incubation in3H2O) was measured by hollow-fibre dialysis. When ligand was removed during the course of dialysis (by replacing buffer that contained ligand with buffer that lacked ligand) there was a prompt decrease in the number of labelled H atoms of the protein. Analysis of the curves provides some information about the number and half-lives of the responsive H atoms. Ligands decrease the motility of the protein and affect about one-fifth of the chain. Low concentrations of glycerol 3-phosphate have an effect that is greater than expected.

scholarly journals The mechanism of folding of globular proteins. Suitability of a penicillinase from Staphylococcus Aureus as a model for refolding studies

1976 ◽  
Vol 155 (2) ◽  
pp. 325-330 ◽  
Author(s):  
B Robson ◽  
R H. Pain
Keyword(s):  
Staphylococcus Aureus ◽  
Enzymic Activity ◽  
Optical Rotatory Dispersion ◽  
Native Conformation ◽  
Guanidinium Chloride ◽  
Tyrosine Residues ◽  
Low Concentrations ◽  
Unfolded State ◽  
Solvent Molecules ◽  
Homogeneous Preparation

1. A homogeneous preparation of penicillinase (penicillin amido-β-lactamhydrolase, EC 3.5.2.6) was isolated and purified from cultures of Staphylococcus aureus by a simple two-stage procedure. 2. The native protein contains 20-30% helix as determined by optical-rotatory-dispersion and circular-dichroism measurements. Some 54(+/-5)% of the 13 tyrosine residues are exposed to solvent molecules of diameter 0.44 and 0.94 nm. 3. Conditions that allow full recovery of enzymic activity and native conformation from the fully unfolded state in 4M-guanidinium chloride were defined. 4. Refolding of the protein was shown to be inhibited by intermolecular interaction, by small changes in ionization and by low concentrations (0.025 M) of phenol.

scholarly journals Thiol stress–dependent aggregation of the glycolytic enzyme triose phosphate isomerase in yeast and human cells

2019 ◽  
Vol 30 (5) ◽  
pp. 554-565 ◽  
Author(s):  
Amy E. Ford ◽  
Catherine Denicourt ◽  
Kevin A. Morano
Keyword(s):  
Cadmium Toxicity ◽  
Hct116 Cell ◽  
Glycolytic Enzyme ◽  
Triose Phosphate Isomerase ◽  
Human Cells ◽  
Protein Fusion ◽  
Triose Phosphate ◽  
Glucose Depletion ◽  
Stress Dependent ◽  
Induced Aggregation

The eukaryotic cytosolic proteome is vulnerable to changes in proteostatic and redox balance caused by temperature, pH, oxidants, and xenobiotics. Cysteine-containing proteins are especially at risk, as the thiol side chain is subject to oxidation, adduction, and chelation by thiol-reactive compounds. The thiol-chelating heavy metal cadmium is a highly toxic environmental pollutant demonstrated to induce the heat shock response and recruit protein chaperones to sites of presumed protein aggregation in the budding yeast Saccharomyces cerevisiae. However, endogenous targets of cadmium toxicity responsible for these outcomes are largely unknown. Using fluorescent protein fusion to cytosolic proteins with known redox-active cysteines, we identified the yeast glycolytic enzyme triose phosphate isomerase as being aggregation-prone in response to cadmium and to glucose depletion in chronologically aging cultures. Cadmium-induced aggregation was limited to newly synthesized Tpi1 that was recruited to foci containing the disaggregase Hsp104 and the peroxiredoxin chaperone Tsa1. Misfolding of nascent Tpi1 in response to both cadmium and glucose-depletion stress required both cysteines, implying that thiol status in this protein directly influences folding. We also demonstrate that cadmium proteotoxicity is conserved between yeast and human cells, as HEK293 and HCT116 cell lines exhibit recruitment of the protein chaperone Hsp70 to visible foci. Moreover, human TPI, mutations in which cause a glycolytic deficiency syndrome, also forms aggregates in response to cadmium treatment, suggesting that this conserved enzyme is folding-labile and may be a useful endogenous model for investigating thiol-specific proteotoxicity.

Recent amplification of triose phosphate isomerase related sequences in lettuce

Genome ◽  
1992 ◽  
Vol 35 (4) ◽  
pp. 627-635 ◽  
Author(s):  
Ilan Paran ◽  
Richard V. Kesseli ◽  
Lore Westphal ◽  
Richard W. Michelmore
Keyword(s):  
Large Scale ◽  
Sequence Similarity ◽  
Chromosomal Rearrangements ◽  
Amino Acid Level ◽  
Glycolytic Enzyme ◽  
Triose Phosphate Isomerase ◽  
Rflp Markers ◽  
Restriction Fragments ◽  
Triose Phosphate ◽  
High Level

A random cDNA clone was identified as distinguishing near-isogenic lines for downy mildew resistance in lettuce. The clone detected multiple restriction fragments in genomic Southern blots of lettuce. Restriction fragment length polymorphisms (RFLPs) detected by this clone mapped to separate clusters of resistance genes; therefore, these sequences were studied in a greater detail. Sequence analysis indicated that the cDNA encoded the glycolytic enzyme triose phosphate isomerase (TPI). The lettuce clone shares 85% sequence similarity at the amino acid level with TPI from maize. TPI-related sequences were mapped in lettuce using three crosses. Ten loci were distributed in six linkage groups. Possible mechanisms of amplification and dispersion were investigated. Retrotransposition was excluded, since intron five is retained in all TPI-related genomic sequences. Large scale chromosomal rearrangements were not involved, as RFLP markers flanking TPI loci were not duplicated. A high level of genomic variability was detected by the TPI clone; 37 different restriction fragments were detected in Southern hybridizations to 64 populations of lettuce including 47 cultivars of Lactuca sativa and five wild species. Species distantly related to L. sativa had few TPI loci, indicating that their amplification and dispersion were recent and had occurred after the emergence of the L. serriola complex.Key words: triose phosphate isomerase, gene duplication, lettuce.

Thallic Ion Oxidation of Styrene: Kinetics of Decomposition of the Oxythallation Adduct C6H5—CH(OCH3)—CH2Tl(OAc)2

1974 ◽  
Vol 52 (15) ◽  
pp. 2667-2672 ◽  
Author(s):  
Louise Nadon ◽  
Miklos Zador
Keyword(s):  
Kinetic Parameters ◽  
Reactive Species ◽  
Rate Law ◽  
First Order ◽  
Order Rate ◽  
Rate Determining Step ◽  
Kinetics Of Decomposition ◽  
Low Concentrations ◽  
Kinetics Of

The kinetics of decomposition of the organo-thallic adduct formed in methanol between styrene and Tl(OAc)3, (C6H5—CH(OCH3)—CH2—Tl(OAc)2) has been studied in a water–methanol solvent. The reaction follows a first order rate law. The organo-thallic compound, RTl(OAc)2, is shown to be dissociated at low concentrations yielding two reactive species, RTlOH+ and RTl2+. The influence of acidity on the rate of decomposition shows that RTl2+ is much more reactive than RTiOH+. The kinetic parameters have been determined. The implication of the results on the rate-determining step of Tl(III) oxidation of styrene is discussed.

A kinetic study of the chlorination of ketones by chloramine-T: Novel concentration dependences on the chlorinating agent and the ketones

1976 ◽  
Vol 29 (7) ◽  
pp. 1449 ◽  
Author(s):  
V Balasubramanian ◽  
V Thiagarajan
Keyword(s):  
Acetic Acid ◽  
Kinetic Study ◽  
Acidic Medium ◽  
Aqueous Acetic Acid ◽  
Acetic Acid Medium ◽  
Rate Determining Step ◽  
Low Concentrations ◽  
Concentration Dependences ◽  
Chloramine T ◽  
High Concentrations

The chlorination of acetone in aqueous acidic medium in the presence of acetic acid and dimethylformamide was subjected to kinetic study. Although the kinetics follows the traditional mechanism at high concentrations of chloramine-T (cat) and in aqueous acetic acid in the presence of sodium acetate, a term in the rate law independent of ketone concentration is reported for the first time in strongly acidic medium at low concentrations of chloramine-T. Generation of the chlorinating species is considered as the rate-determining step (Scheme 1). The mechanistic changes which occur on addition of chloride and on changing the structure of the ketone are reported. The effect of acetate on the rate in aqueous acetic acid medium is rationalized by invoking a prior equilibrium formation of enolate followed by a rate-controlling chlorination by chlorinium acetate. The retardation in rate and the consequent changes in mechanism in the case of p-bromo- and p-nitroacetophenone are accounted for by invoking a complex between the enol and the positive chlorine species similar to that in the mechanism for the chlorination of phenols.

scholarly journals Molecular dynamics simulations of the interactions between triose phosphate isomerase and sulfonamides

2020 ◽  
Vol 2 ◽  
pp. e13
Author(s):  
Neville Y. Forlemu ◽  
Joseph Sloop
Keyword(s):  
Active Site ◽  
Malaria Incidence ◽  
Antimalarial Drugs ◽  
Glycolytic Enzyme ◽  
Triose Phosphate Isomerase ◽  
Van Der Waals Interactions ◽  
Dimer Interface ◽  
Triose Phosphate ◽  
Solvation Energies ◽  
Active Site Region

Malaria is a disease with debilitating health and negative economic impacts in regions at high risk of infection. Parasitic resistance and side effects of current antimalarial drugs are major setbacks to the successful campaigns that have reduced malaria incidence by 40% in the last decade. The parasite’s dependence on glycolysis for energy requirements makes pathway enzymes suitable targets for drug development. Specifically, triose phosphate isomerase (TPI) from Plasmodium falciparum (pTPI) and human (hTPI) cells show striking structural features that can be used in development of new antimalarial agents. In this study MD simulations were used to characterize binding sites on hTPI and pTPI interactions with sulfonamides. The molecular mechanics Poisson–Boltzmann surface area (MM–PBSA) method was used to estimate the interaction energies of four sulfonamide-TPI docked complexes. A unique combination of key residues at the dimer interface of pTPI is responsible for the observed selective affinity to pTPI compared to hTPI. The representative sulfonamide; 4-amino-N-(3,5-dimethylphenyl)-3-fluorbenzenesulfonamide (sulfaE) shows a strong affinity with pTPI (dimer interface, −42.91 kJ/mol and active site region, −71.62 kJ/mol), hTPI (dimer interface, −41.32 kJ/mol and active site region, −84.40 kJ/mol). Strong and favorable Van der Waals interactions and increases in non-polar solvation energies explain the difference in affinity between pTPI with sulfaE compared to hTPI at the dimer interface. This is an indication that the dimer interface of TPI glycolytic enzyme is vital for development of sulfonamide based antimalarial drugs.

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