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Alkaline pH Values (alkaline + ph_value)
Selected AbstractsThe relationship between thermal stability and pH optimum studied with wild-type and mutant Trichoderma reesei cellobiohydrolase Cel7AFEBS JOURNAL, Issue 5 2003Harry Boer The major cellulase secreted by the filamentous fungus Trichoderma reesei is cellobiohydrolase Cel7A. Its three-dimensional structure has been solved and various mutant enzymes produced. In order to study the potential use of T. reesei Cel7A in the alkaline pH range, the thermal stability of Cel7A was studied as a function of pH with the wild-type and two mutant enzymes using different spectroscopic methods. Tryptophan fluorescence and CD measurements of the wild-type enzyme show an optimal thermostability between pH 3.5,5.6 (Tm, 62 ± 2 °C), at which the highest enzymatic activity is also observed, and a gradual decrease in the stability at more alkaline pH values. A soluble substrate, cellotetraose, was shown to stabilize the protein fold both at optimal and alkaline pH. In addition, unfolding of the Cel7A enzyme and the release of the substrate seem to coincide at both acidic and alkaline pH, demonstrated by a change in the fluorescence emission maximum. CD measurements were used to show that the five point mutations (E223S/A224H/L225V/T226A/D262G) that together result in a more alkaline pH optimum [Becker, D., Braet, C., Brumer, H., III, Claeyssens, M., Divne, C., Fagerström, R.B., Harris, M., Jones, T.A., Kleywegt, G.J., Koivula, A., et al. (2001) Biochem. J.356, 19,30], destabilize the protein fold both at acidic and alkaline pH when compared with the wild-type enzyme. In addition, an interesting time-dependent fluorescence change, which was not observed by CD, was detected for the pH mutant. Our data show that in order to engineer more alkaline pH cellulases, a combination of mutations should be found, which both shift the pH optimum and at the same time improve the thermal stability at alkaline pH range. [source] Mechanisms of non-steroid anti-inflammatory drugs action on ASICs expressed in hippocampal interneuronsJOURNAL OF NEUROCHEMISTRY, Issue 1 2008Natalia A. Dorofeeva Abstract The inhibitory action of non-steroid anti-inflammatory drugs was investigated on acid-sensing ionic channels (ASIC) in isolated hippocampal interneurons and on recombinant ASICs expressed in Chinese hamster ovary (CHO) cells. Diclofenac and ibuprofen inhibited proton-induced currents in hippocampal interneurons (IC50 were 622 ± 34 ,M and 3.42 ± 0.50 mM, respectively). This non-competitive effect was fast and fully reversible for both drugs. Aspirin and salicylic acid at 500 ,M were ineffective. Diclofenac and ibuprofen decreased the amplitude of proton-evoked currents and slowed the rates of current decay with a good correlation between these effects. Simultaneous application of acid solution and diclofenac was required for its inhibitory effect. Unlike amiloride, the action of diclofenac was voltage-independent and no competition between two drugs was found. Analysis of the action of diclofenac and ibuprofen on activation and desensitization of ASICs showed that diclofenac but not ibuprofen shifted the steady-state desensitization curve to more alkaline pH values. The reason for this shift was slowing down the recovery from desensitization of ASICs. Thus, diclofenac may serve as a neuroprotective agent during pathological conditions associated with acidification. [source] Inhibitory effect of propolis extract on the growth of Listeria monocytogenes and the mutagenicity of 4-nitroquinoline- N -oxideJOURNAL OF THE SCIENCE OF FOOD AND AGRICULTURE, Issue 6 2006Hsin-Yi Yang Abstract Propolis originates from a resinous substance collected by honeybees from the buds and leaves of trees and plants, which is then mixed with pollen as well as enzymes secreted by the bees. In the present study, the susceptibility of Listeria monocytogenes to the ethanol extract of propolis (EEP) as influenced by EEP concentration, incubation temperature, pH, and cell age was investigated. In addition, the antimutagenic action of EEP against 4-nitroquinoline- N -oxide (4-NQO) was also examined. Results revealed that EEP at a dosage of 7.5 µg mL,1 or higher exerted a bactericidal effect on L. monocytogenes. L. monocytogenes was most susceptible to EEP at 37 °C followed by 25 and 4 °C. At acid pH values, cells of the test organism were more sensitive to EEP than at neutral pH, while most resistant at alkaline pH values. Cell age was also found to affect the susceptibility of L. monocytogenes to EEP. Cells in the mid-exponential phase showed the highest susceptibility, followed by cells in the late-exponential phase and stationary phase. EEP caused cell leakage of the test organism. A marked increase in the absorbance at 260 nm, UV-absorbing material in the supernatant of cell suspension, and irregularly shaped materials around the cell surface were noted after cells of L. monocytogenes were exposed to EEP. Furthermore, EEP at a dosage of 7.5,60.0 µg per plate was found to suppress 4-NQO-induced mutation by 17.6,88.8%. Copyright © 2006 Society of Chemical Industry [source] Stimuli-responsive properties of aminophenylboronic acid-carrying thermosensitive copolymersPOLYMER INTERNATIONAL, Issue 5 2003Abstract Thermosensitive copolymers of N -isopropylacrylamide (NIPA) and N -acryloxysuccinimide (NASI) were obtained by solution polymerization using azobisisobutyronitrile as the initiator in a tetrahydrofuran,toluene mixture at 65,°C. A boronic acid-carrying ligand, m -aminophenylboronic acid (APBA) was covalently attached to the thermosensitive copolymer via the reaction between amino and succinimide groups. APBA-coupled thermosensitive copolymer exhibited both temperature and pH sensitivity. Thermally reversible phase transitions were observed both in the acidic and alkaline pH region for the APBA-modified copolymers obtained with different NASI feed concentrations. In our study, ribonucleic acid (RNA) was selected as a biomolecule having reactive groups which could potentially interact with the boronic acid functionality. The response of boronic acid-carrying thermosensitive copolymer against RNA was investigated in aqueous media in the pH range 4,9. In the acidic pH region, an increase was observed in the lower critical solution temperature (LCST) of the APBA-coupled thermosensitive copolymer with increasing RNA concentration. However, LCST decreased with increasing RNA concentration at both neutral and alkaline pH values. The LCST of the APBA-attached copolymer varied linearly with the RNA concentration at pH of 3, 4 and 7. © 2003 Society of Chemical Industry [source] Elucidation of the mechanism and end products of glutaraldehyde crosslinking reaction by X-ray structure analysisBIOTECHNOLOGY & BIOENGINEERING, Issue 3 2007Yariv Wine Abstract Glutaraldehyde has been used for several decades as an effective crosslinking agent for many applications including sample fixation for microscopy, enzyme and cell immobilization, and stabilization of protein crystals. Despite of its common use as a crosslinking agent, the mechanism and chemistry involved in glutaraldehyde crosslinking reaction is not yet fully understood. Here we describe feasibility study and results obtained from a new approach to investigate the process of protein crystals stabilization by glutaraldehyde crosslinking. It involves exposure of a model protein crystal (Lysozyme) to glutaraldehyde in alkaline or acidic pH for different incubation periods and reaction arrest by medium exchange with crystallization medium to remove unbound glutaraldehyde. The crystals were subsequently incubated in diluted buffer affecting dissolution of un-crosslinked crystals. Samples from the resulting solution were subjected to protein composition analysis by gel electrophoresis and mass spectroscopy while crosslinked, dissolution resistant crystals were subjected to high resolution X-ray structural analysis. Data from gel electrophoresis indicated that the crosslinking process starts at specific preferable crosslinking site by lysozyme dimer formation, for both acidic and alkaline pH values. These dimer formations were followed by trimer and tetramer formations leading eventually to dissolution resistant crystals. The crosslinking initiation site and the end products obtained from glutaraldehyde crosslinking in both pH ranges resulted from reactions between lysine residues of neighboring protein molecules and the polymeric form of glutaraldehyde. Reaction rate was much faster at alkaline pH. Different reaction end products, indicating different reaction mechanisms, were identified for crosslinking taking place under alkaline or acidic conditions. Biotechnol. Bioeng. 2007;98:711,718. © 2007 Wiley Periodicals, Inc. [source] | |||||||||||||