Reaction rates of glutathione and ascorbate with alkyl radicals are too slow for protection against protein peroxidation in vivo

2017 ◽  
Vol 633 ◽  
pp. 118-123 ◽  
Author(s):  
Thomas Nauser ◽  
Janusz M. Gebicki
Keyword(s):  
Reaction Rates ◽  
Alkyl Radicals ◽  
Protein Peroxidation

scholarly journals mTORC1 controls phase-separation and the biophysical properties of the cytoplasm by tuning crowding

2017 ◽  
Author(s):  
M. Delarue ◽  
G.P. Brittingham ◽  
S. Pfeffer ◽  
I.V. Surovtsev ◽  
S. Ping-lay ◽  
...  
Keyword(s):  
Phase Separation ◽  
Fluorescent Protein ◽  
Effective Diffusion ◽  
Reaction Rates ◽  
Biophysical Properties ◽  
Cell Interior ◽  
Profound Impact ◽  
Mtorc1 Pathway

Summary (Abstract)Macromolecular crowding has a profound impact on reaction rates and the physical properties of the cell interior, but the mechanisms that regulate crowding are poorly understood. We developed Genetically Encoded Multimeric nanoparticles (GEMs) to dissect these mechanisms. GEMs are homomultimeric scaffolds fused to a fluorescent protein. GEMs self-assemble into bright, stable fluorescent particles of defined size and shape. By combining tracking of GEMs with genetic and pharmacological approaches, we discovered that the mTORC1 pathway can tune the effective diffusion coefficient of macromolecules ≥15 nm in diameter more than 2-fold without any discernable effect on the motion of molecules ≥5 nm. These mTORCI-dependent changes in crowding and rheology affect phase-separation both in vitro and in vivo. Together, these results establish a role for mTORCI in controlling both the biophysical properties of the cytoplasm and the phase-separation of biopolymers.

scholarly journals Molecular immunocytochemistry of the CuZn superoxide dismutase in rat hepatocytes.

1988 ◽  
Vol 107 (6) ◽  
pp. 2169-2179 ◽  
Author(s):  
L Y Chang ◽  
J W Slot ◽  
H J Geuze ◽  
J D Crapo
Keyword(s):  
Superoxide Dismutase ◽  
Rat Liver ◽  
Oxidant Stress ◽  
Rat Hepatocytes ◽  
Reaction Rates ◽  
Cytoplasmic Matrix ◽  
Cytosolic Proteins ◽  
Cuzn Superoxide Dismutase ◽  
O2 Production

The distribution of CuZn superoxide dismutase (SOD) molecules in subcellular organelles in rat liver hepatocytes was studied using integrated biochemical, stereological, and quantitative immunocytochemical techniques. A known concentration of purified CuZn SOD in 10% gelatin was embedded alongside the liver tissue for the calculation of CuZn SOD concentrations in hepatocyte organelles and total CuZn SOD in the rat liver. Most of the CuZn SOD was located in the cytoplasmic matrix (73.1%) and in the nucleus (11.9%) with concentrations of 1.36 and 0.71 mg/cm3, respectively. Lysosomes contained the highest concentration (5.81 mg/cm3). Only low concentrations were measured in mitochondria (0.21 mg/cm3). Membrane-bound spaces of rough endoplasmic reticulum (ER), smooth ER, and the Golgi system did not contain significant concentrations of the enzyme. By adding up the concentrations in all subcellular compartments, a total liver content of CuZn SOD was established from the immunocytochemical measurements (0.386 +/- 0.028 mg/gm liver) that agreed closely with those obtained by biochemical assays (0.380 +/- 0.058 mg/gm liver). The average distances between two CuZn SOD molecules can be calculated from enzyme concentrations. It was determined that CuZn SOD molecules in the cytoplasmic matrix and nucleus were 34 and 42 nm apart, respectively. In peroxisomes and mitochondria, average intermolecular distance increased to approximately 60 nm and increased to 136 nm in smooth ER. CuZn SOD is a relatively abundant protein in the cytosol of hepatocytes and its distribution overlaps with major sites of O2- production. The efficiency of protection CuZn SOD can provide to cytosolic proteins from attacks by superoxide anion depends on the rate of O2- production, distribution of CuZn SOD relative to cytosolic proteins, and the relative reaction rates between O2- with both cytosolic proteins and CuZn SOD. Future studies of these substrate-enzyme relationships in vivo can lead to a greater understanding of how cells handle oxidant stress.

Fusion proteins of an enoate reductase and a Baeyer-Villiger monooxygenase facilitate the synthesis of chiral lactones

2017 ◽  
Vol 398 (1) ◽  
pp. 31-37 ◽  
Author(s):  
Christin Peters ◽  
Florian Rudroff ◽  
Marko D. Mihovilovic ◽  
Uwe T. Bornscheuer
Keyword(s):  
Fusion Protein ◽  
Fusion Proteins ◽  
Reaction Rates ◽  
Cascade Reactions ◽  
Product Formation ◽  
Similar Reaction ◽  
Enzyme Cascade ◽  
Enoate Reductase ◽  
Chiral Lactones

Abstract Nature uses the advantages of fusion proteins for multi-step reactions to facilitate the metabolism in cells as the conversion of substrates through intermediates to the final product can take place more rapidly and with less side-product formation. In a similar fashion, also for enzyme cascade reactions, the fusion of biocatalysts involved can be advantageous. In the present study, we investigated fusion of an alcohol dehydrogenase (ADH), an enoate reductase (ERED) and a Baeyer-Villiger monooxygenase (BVMO) to enable the synthesis of (chiral) lactones starting from unsaturated alcohols as substrates. The domain order and various linkers were studied to find optimal conditions with respect to expression levels and enzymatic activities. Best results were achieved for the ERED xenobiotic reductase B (XenB) from Pseudomonas putida and the cyclohexanone monooxygenase (CHMO) from Acinetobacter sp., whereas none of the ADHs studied could be fused successfully. This fusion protein together with separately supplied ADH resulted in similar reaction rates in in vivo biocatalysis reactions. After 1.5 h we could detect 40% more dihydrocarvone lactone in in vivo reactions with the fusion protein and ADH then with the single enzymes.

scholarly journals Introductory remarks to the second session

1981 ◽  
Vol 293 (1063) ◽  
pp. 63-64
Keyword(s):  
Steady State ◽  
Enzymatic Catalysis ◽  
Transition States ◽  
Phosphoglycerate Kinase ◽  
Reaction Rates ◽  
Steady State Rate ◽  
And Function ◽  
Enzyme Structure And Function ◽  
And Control

The four presentations in this session cover studies on different aspects of enzyme structure and function. They effectively illustrate how one has to combine different approaches to arrive at an understanding of enzymatic catalysis and control. Nowadays, the molecular description of an enzyme is hardly credible without detailed crystallographic information. Thus, Dr Blake’s paper on the structure of phosphoglycerate kinase is particularly relevant to our understanding of phosphate-transfer mechanisms. The question of the relation between structure in the solid and solution is still with us and n.m.r. has proved to be the best way to study the differences and similarities. M any enzyme substrate complexes have been ‘mapped’ in solution, by using the perturbation of the n.m.r. spectra by param agnetic centres as a measure of interatomic distances. How such results can lead to both structural and mechanistic information will be discussed by Dr Mildvan. To understand mechanisms we must also get some information about the nature of transition states. Here, stereochemical observations play an important role and Dr Lowe will describe some elegant work on the use of chiral phosphates in approaching this problem. Finally, it is important to describe structures, intermediates and transition states in terms of the kinetic behaviour of the enzyme and Dr Dalziel will give us an example of both steady-state and pre-steady-state rate studies. Measurements of reaction rates ultimately link studies on the isolated enzyme to their behaviour in vivo . As this last step in the sequence is not covered by the formal presentations at this meeting I should like to show briefly how n.m.r. can now be used to obtain fluxes of enzyme catalysed reactions in vivo both in the steady state and at equilibrium.

Effects of surfaces and macromolecular crowding on bimolecular reaction rates

2019 ◽  
Author(s):  
Steven S. Andrews
Keyword(s):  
Reaction Rates ◽  
Bimolecular Reaction ◽  
Complex Environments ◽  
Perfect Agreement ◽  
Confined Spaces ◽  
Separate Model ◽  
Planar Membranes ◽  
Reaction Rate Equation

AbstractBiological cells are complex environments that are densely packed with macromolecules and subdivided by membranes, both of which affect the rates of chemical reactions. It is well known that crowding reduces the volume available to reactants, which increases reaction rates, and also inhibits reactant diffusion, which decreases reaction rates. This work investigates these effects quantitatively using analytical theory and particle-based simulations. A reaction rate equation based on only these two processes turned out to be inconsistent with simulation results. However, accounting for diffusion inhibition by the surfaces of nearby obstacles, which affects access to reactants, led to perfect agreement for reactions near impermeable planar membranes and improved agreement for reactions in crowded spaces. A separate model that quantified reactant occlusion by crowders, and extensions to a thermodynamic “cavity” model proposed by Berezhkovskii and Szabo (J. Phys. Chem. B 120:5998, 2016), were comparably successful. These results help elucidate reaction dynamics in confined spaces and improve prediction of in vivo reaction rates from in vitro ones.

scholarly journals Formation of peroxides in amino acids and proteins exposed to oxygen free radicals

1993 ◽  
Vol 289 (3) ◽  
pp. 743-749 ◽  
Author(s):  
S Gebicki ◽  
J M Gebicki
Keyword(s):  
Amino Acids ◽  
Free Radicals ◽  
Oxygen Free Radicals ◽  
Radiation Energy ◽  
Protonated Form ◽  
Isoleucine Leucine ◽  
Protein Peroxidation ◽  
Dilute Aqueous Solutions ◽  
Kinetics Of

Dilute aqueous solutions of BSA or lysozyme gave positive tests for peroxides after exposure to reactive oxygen species. The reactive species were generated by gamma-irradiation, reduction of H2O2 with Fe2+ ions or thermal decomposition of an azo compound. Peroxides were assayed by an iodometric method. Identification of the new groups as hydroperoxides was confirmed by their ability to oxidize a range of compounds and by the kinetics of their reaction with iodide. The hydroperoxide groups were bound to the proteins and their yields (G values) corresponded to 1.2 -OOH groups per 100 eV of radiation energy absorbed for BSA, and 0.8 for lysozyme. The oxygen free radicals effective in protein peroxidation were the hydroxyl and organic peroxyl, but not superoxide or its protonated form. The efficiency of BSA peroxidation initiated by the hydroxyl radicals was 40%. Protein peroxides decayed spontaneously with a half-life of about 1.5 days at 20 degrees C. Exposure of the common amino acids to hydroxyl free radicals showed that six of them (glutamate, isoleucine, leucine, lysine, proline and valine) were peroxidized with similar efficiency to the proteins, whereas the rest were inert or much less susceptible. These results suggest that some proteins may be peroxidized by a variety of agents in vivo and that their subsequent reactions with protective agents, such as ascorbate or glutathione, may decrease the antioxidant potential of cells and tissues.

scholarly journals Four-angle saturation transfer (FAST) method for measuring creatine kinase reaction rates in vivo

2002 ◽  
Vol 47 (5) ◽  
pp. 850-863 ◽  
Author(s):  
Paul A. Bottomley ◽  
Ronald Ouwerkerk ◽  
Ray F. Lee ◽  
Robert G. Weiss
Keyword(s):  
Creatine Kinase ◽  
Reaction Rates ◽  
Fast Method ◽  
Saturation Transfer ◽  
Kinase Reaction ◽  
Creatine Kinase Reaction

scholarly journals Recent Advances in Heterologous Synthesis Paving Way for Future Green-Modular Bioindustries: A Review With Special Reference to Isoflavonoids

2021 ◽  
Vol 9 ◽  
Author(s):  
Moon Sajid ◽  
Shane Ramsay Stone ◽  
Parwinder Kaur
Keyword(s):  
Genetic Factors ◽  
Plant Secondary Metabolites ◽  
Reaction Rates ◽  
Recent Time ◽  
Commercial Application ◽  
Biosynthetic Pathways ◽  
Market Demand ◽  
Metabolism Regulation ◽  
Multiple Signaling

Isoflavonoids are well-known plant secondary metabolites that have gained importance in recent time due to their multiple nutraceutical and pharmaceutical applications. In plants, isoflavonoids play a role in plant defense and can confer the host plant a competitive advantage to survive and flourish under environmental challenges. In animals, isoflavonoids have been found to interact with multiple signaling pathways and have demonstrated estrogenic, antioxidant and anti-oncologic activities in vivo. The activity of isoflavonoids in the estrogen pathways is such that the class has also been collectively called phytoestrogens. Over 2,400 isoflavonoids, predominantly from legumes, have been identified so far. The biosynthetic pathways of several key isoflavonoids have been established, and the genes and regulatory components involved in the biosynthesis have been characterized. The biosynthesis and accumulation of isoflavonoids in plants are regulated by multiple complex environmental and genetic factors and interactions. Due to this complexity of secondary metabolism regulation, the export and engineering of isoflavonoid biosynthetic pathways into non-endogenous plants are difficult, and instead, the microorganisms Saccharomyces cerevisiae and Escherichia coli have been adapted and engineered for heterologous isoflavonoid synthesis. However, the current ex-planta production approaches have been limited due to slow enzyme kinetics and traditionally laborious genetic engineering methods and require further optimization and development to address the required titers, reaction rates and yield for commercial application. With recent progress in metabolic engineering and the availability of advanced synthetic biology tools, it is envisaged that highly efficient heterologous hosts will soon be engineered to fulfill the growing market demand.

Sign in / Sign up

Export Citation Format

Share Document