Purification and kinetic mechanism of 5,10-metliyienetetrahydrofolate reductase from sheep liver

5,10-Methylenetetrahydrofolate reductase (EC 1.1.1.68) was purified from the cytosolic fraction of sheep liver by (NH4)2 SO4 fractionation, acid precipitation, DEAE-Sephacel chromatography and Blue Sepharose affinity chromatography. The homogeneity of the enzyme was established by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, ultracentrifugation and Ouchterlony immunodiffusion test. The enzyme was a dimer of molecular weight 1,66,000 ± 5,000 with a subunit molecular weight of 87,000 ±5,000. The enzyme showed hyperbolic saturation pattern with 5-methyltetrahydrofolate.K 0.5 values for 5-methyltetrahydrofolate menadione and NADPH were determined to be 132 ΜM, 2.45 ΜM and 16 ΜM. The parallel set of lines in the Lineweaver-Burk plot, when either NADPH or menadione was varied at different fixed concentrations of the other substrate; non-competitive inhibition, when NADPH was varied at different fixed concentrations of NADP; competitive inhibition, when menadione was varied at different fixed concentrations of NADP and the absence of inhibition by NADP at saturating concentration of menadione, clearly established that the kinetic mechanism of the reaction catalyzed by this enzyme was ping-pong.

Pen parameters for improving the performance of sheep imported from Australia to Persian Gulf feedlots

Summer in the Persian Gulf region presents physiological challenges for Australian sheep that are part of the live export supply chain coming from the Australian winter. Many feedlots throughout the Gulf have very high numbers of animals during June to August in order to cater for the increased demand for religious festivals. From an animal welfare perspective it is important to understand the necessary requirements of feed and water trough allowances, and the amount of pen space required, to cope with exposure to these types of climatic conditions. This study addresses parameters that are pertinent to the wellbeing of animals arriving in the Persian Gulf all year round. Three experiments were conducted in a feedlot in the Persian Gulf between March 2010 and February 2012, totalling 44 replicate pens each with 60 or 100 sheep. The applied treatments covered animal densities, feed-bunk lengths and water trough lengths. Weights, carcass attributes and health status were the key recorded variables. Weight change results showed superior performance for animal densities of ≥1.2 m2/head during hot conditions (24-h average temperatures greater than 33 °C, or a diurnal range of around 29–37 °C). However the space allowance for animals can be decreased, with no demonstrated detrimental effect, to 0.6 m2/head under milder conditions. A feed-bunk length of ≥5 cm/head is needed, as 2 cm/head showed significantly poorer animal performance. When feeding at 90% ad libitum 10 cm/head was optimal, however under a maintenance feeding regime (1 kg/head/day) 5 cm/head was adequate. A minimum water trough allowance of 1 cm/head is required. However, this experiment was conducted during milder conditions, and it may well be expected that larger water trough lengths would be needed in hotter conditions. Carcass weights were determined mainly by weights at feedlot entry and subsequent weight gains, while dressing percentage was not significantly affected by any of the applied treatments. There was no demonstrated effect of any of the treatments on the number of animals that died, or were classified as unwell. However, across all the treatments, these animals lost significantly more weight than the healthy animals, so the above recommendations, which are aimed at maintaining weight, should also be applicable for good animal health and welfare. Therefore, best practice guidelines for managing Australian sheep in Persian Gulf feedlots in the hottest months (June–August) which present the greatest environmental and physical challenge is to allow feed-bunk length 5 cm/head on a maintenance-feeding program and 10 cm/head for 90% ad libitum feeding, and the space allowance per animal should be ≥1.2 m2/head. Water trough allocation should be at least 1 cm/head with provision for more in the summer when water intake potentially doubles.

A note on the evaluation of dielectric dispersion parameters of non-debije type liquids

A simple method for evaluating dielectric relaxation parameters ie given whioh can be used for analyeing the arelaxation times of a liquid into two absorptions.

Differential scanning calorimetric studies on ammonium perchlorate

Differential scanning calorimetric studies on ammonium perchlorate have been carried out. The enthalpy values for the phase transition endotherm and the two exotherms have been reported in the present communication. A new method has been developed for the estimation of kinetic parameters from DSC the mograms. The values for activation energy as calculated by the above method for low temperature and high temperature exotherms are in close agreement with literature values. The present studies also confirm the presence of small exothermic peaks at the initial stages of high temperature exotherm. Explanation for the same has been given.

Factors influencing kinetic and equilibrium behaviour of sodium ion exchange with strong acid cation resin

This study reports an investigation of the ion exchange treatment of sodium chloride solutions in relation to use of resin technology for applications such as desalination of brackish water. In particular, a strong acid cation (SAC) resin (DOW Marathon C) was studied to determine its capacity for sodium uptake and to evaluate the fundamentals of the ion exchange process involved. Key questions to answer included: impact of resin identity; best models to simulate the kinetics and equilibrium exchange behaviour of sodium ions; difference between using linear least squares (LLS) and non-linear least squares (NLLS) methods for data interpretation; and, effect of changing the type of anion in solution which accompanied the sodium species. Kinetic studies suggested that the exchange process was best described by a pseudo first order rate expression based upon non-linear least squares analysis of the test data. Application of the Langmuir Vageler isotherm model was recommended as it allowed confirmation that experimental conditions were sufficient for maximum loading of sodium ions to occur. The Freundlich expression best fitted the equilibrium data when analysing the information by a NLLS approach. In contrast, LLS methods suggested that the Langmuir model was optimal for describing the equilibrium process. The Competitive Langmuir model which considered the stoichiometric nature of ion exchange process, estimated the maximum loading of sodium ions to be 64.7 g Na/kg resin. This latter value was comparable to sodium ion capacities for SAC resin published previously. Inherent discrepancies involved when using linearized versions of kinetic and isotherm equations were illustrated, and despite their widespread use, the value of this latter approach was questionable. The equilibrium behaviour of sodium ions form sodium fluoride solution revealed that the sodium ions were now more preferred by the resin compared to the situation with sodium chloride. The solution chemistry of hydrofluoric acid was suggested as promoting the affinity of the sodium ions to the resin.

Microbial L-phenylalanine ammonia-lyase. Purification, subunit structure and kinetic properties of the enzyme from Rhizoctonia solani

Phenylalanine ammonia-lyase (EC 4.3.1.5) was purified to homogeneity from the acetone-dried powders of the mycelial felts of the plant pathogenic fungus Rhizoctonia solani. 2. A useful modification in protamine sulphate treatment to get substantial purification of the enzyme in a single-step is described. 3. The purified enzyme shows bisubstrate activity towards L-phenylalanine and L-tyrosine. 4. It is sensitive to carbonyl reagents and the inhibition is not reversed by gel filtration. 5. The molecular weight of the enzyme as determined by Sephadex G-200 chromatography and sucrose-density-gradient centrifugation is around 330000. 6. The enzyme is made up of two pairs of unidentical subunits, with a molecular weight of 70000 (alpha) and 90000 (beta) respectively. 7. Studies on initial velocity versus substrate concentration have shown significant deviations from Michaelis-Menten kinetics. 8. The double-reciprocal plots are biphasic (concave downwards) and Hofstee plots show a curvilinear pattern. 9. The apparent Km value increases from 0.18 mM to as high as 5.0 mM with the increase in the concentration of the substrate and during this process the Vmax, increases by 2-2.5-fold. 10. The value of Hill coefficient is 0.5. 11. Steady-state rates of phenylalanine ammonia-lyase reaction in the presence of inhibitors like D-phenylalanine, cinnamic, p-coumaric, caffeic, dihydrocaffeic and phenylpyruvic acid have shown that only one molecule of each type of inhibitor binds to a molecule of the enzyme. These observations suggest the involvement of negative homotropic interactions in phenylalanine ammonia-lyase. 12. The enzyme could not be desensitized by treatment with HgCl2, p-chloromercuribenzoic acid or by repeated freezing and thawing.

Study of third-order non-linear systems- Variation of parameters approach

In this paper, the transient response of a third-order non-linear system is obtained by first reducing the given third-order equation to three first-order equations by applying the method of variation of parameters. On the assumption that the variations of amplitude and phase are small, the functions are expanded in ultraspherical polynomials. The expansion is restricted to the constant term. The resulting equations are solved to obtain the response of the given third-order system. A numerical example is considered to illustrate the method. The results show that the agreement between the approximate and digital solution is good thus vindicating the approximation.

An approximate mathematical procedure for the determination of characteristic parameters for a general case in three dimensional photoelasticity

In cases whazo zotatLon of the seoondazy pztncipal 8tzo,ae axes along tha light path ,exists, it is always poaeible to detezmlna two dizactions along which plane-polazlaad light ,antazlng the model ,amerCe8 as plene-pela~l,aed light fzom the model. Puzth,az the nat zstazdatton Pot any light path is dlff,azant Prom the lntsgtatad zetazd,ation Pat the l£ght path nogZsctlng the ePfsct or z,atation.

Structural and kinetic studies on the activators of succinate dehydrogenase

1. 1. Diverse classes of compounds such as dicarboxylates, pyrophosphates, quinols and nitrophenols are known to activate mitochondrial succinate dehydrogenase (EC 1.3.99.1). Examples in each class — malonate, pyrophosphate, ubiquinol and 2,4-dinitrophenol — are selected for comparative studies on the kinetic constants and structural relationship. 2. 2. The activated forms of the enzyme obtained on preincubating mitochondria with the effectors exhibited Michaelian kinetics and gave doublereciprocal plots which are nearly parallel to that of the basal form. On activation, Km for the substrate also increased along with V. The effectors activated the enzyme at low concentrations and inhibited, in a competitive fashion, at high concentrations. The binding constant for activation was lower than that for inhibition for each effector. 3. 3. These compounds possess ionizable twin oxygens separated by a distance of Image and having fractional charges in the range of −0.26 to −0.74 e. The common twin-oxygen feature of the substrate and the effectors suggested the presence of corresponding counter charges in the binding domain. The competitive nature of effectors with the substrate for inhibition further indicated the close structural resemblance of the activation and catalytic sites.

Kinetics of hydrogen evolution on a stainless steel electrode

The kinetic parameters for the hydrogen evolution reaction on a stainless steel substrate have been obtained from a study of the steady-state polarization curves as well as the galvanostatic transients. The high Tafel slope obtained in the steady-state polarization measurements was ascribed to the presence of an oxide film present on the surface of the stainless steel electrode.

Analysis of human chorionic gonadotropin-monoclonal antibody interaction in BIAcore

Kinetic studies of macromolecular ligand-ligate interaction have generated ample interest since the advent of plasmon resonance based instruments like BIAcore. Most of the studies reported in literature assume a simple 1 : 1 Langmuir binding and complete reversibility of the system. However we observed that in a high affinity antigen-antibody system [human chorionic gonadotropin-monoclonal antibody (hCG-mAb)] dissociation is insignificant and the sensogram data cannot be used to measure the equilibrium and kinetic parameters. At low concentrations of mAb the complete sensogram could be fitted to a single exponential. Interestingly we found that at higher mAb concentrations, the binding data did not conform to a simple bimolecular model. Instead, the data fitted a two-step model, which may be because of surface heterogeneity of affinity sites. In this paper, we report on the global fit of the sensograms. We have developed a method by which a single two-minute sensogram can be used in high affinity systems to measure the association rate constant of the reaction and the functional capacity of the ligand (hCG) immobilized on the chip. We provide a rational explanation for the discrepancies generally observed in most of the BIAcore sensograms

On the relationship of thermodynamic parameters with the buried surface area in protein-ligand complex formation

Prediction of thermodynamic parameters of protein-protein and antigen-antibody complex formation from high resolution structural parameters has recently received much attention, since an understanding of the contributions of different fundamental processes like hydrophobic interactions, hydrogen bonding, salt bridge formation, solvent reorganization etc. to the overall thermodynamic parameters and their relations with the structural parameters would lead to rational drug design. Using the results of the dissolution of hydrocarbons and other model compounds the changes in heat capacity (DeltaCp), enthalpy (DeltaH) and entropy (DeltaS) have been empirically correlated with the polar and apolar surface areas buried during the process of protein folding/unfolding and protein-ligand complex formation. In this regard, the polar and apolar surfaces removed from the solvent in a protein-ligand complex have been calculated from the experimentally observed values of changes in heat capacity (DeltaCp) and enthalpy (DeltaH) for protein-ligand complexes for which accurate thermodynamic and high resolution structural data are available, and the results have been compared with the x-ray crystallographic observations. Analyses of the available results show poor correlation between the thermodynamic and structural parameters. Probable reasons for this discrepancy are mostly related with the reorganization of water accompanying the reaction which is indeed proven by the analyses of the energetics of the binding of the wheat germ agglutinin to oligosaccharides.