Characteristics of antioxidant properties of 20-hydroxyecdysone in low density lipoproteins by kinetic parameters of chemiluminescence

20-Hydroxyecdysone is capable to terminate the lipid free-radical oxidation in low density lipoprotein in vitro as displayed by the kinetic chemiluminescence parameters. In concentrations interval from 2 x 10(-6) mol/l up to 8 x 10(-6) mol/l it statistically reliably reduces maximum of the first flash intensity of the low density lipoprotein Fe(2+)-initiated chemiluminescence. It testifies about Fe(2+)-initiated lipid peroxide process decomposition in low density lipoprotein slowing down by 20-hydroxyecdysone. In concentration of 20-hydroxyecdysone 8 x 10(-6) mol/l the statistically reliable reduction of angle tangent of an ascending branch of the second flash of low density lipoprotein Fe(2+)-initiated chemiluminescence was found. This is a result of free-radical lipid oxidation rate reduction in the low density lipoprotein at the 20-hydroxyeodysone presence. The equations of the kinetic parameters linear dependences Fe(2+)-initiated chemiluminescence in low density lipoprotein on the concentration of 20-hydroxyeodysone and hydroquinone have been received. The correlation factors in the interval from 0.8980 up to 0.6789 have been calculated. Thus, the assumption has been forwarded that 20-hydroxyeodysone has antioxidizing properties. However, its antioxidizing activity in free radical lipid oxidation of is low density lipoprotein is as less as for hydroquinone.     

An optical signal correlated with the allosteric transition in Scapharca inaequivalvis HbI

The transient absorbance change within the first 2 mus of photolysis of COHbI (from Scapharca inaequivalvis) reported by Chiancone et al. [Chiancone, E., Elber, R., Royer, W. E., Regan, R., and Gibson, Q. H. (1993) J. Biol. Chem. 268, 5711-5718] has been studied in several mutants. Evidence is presented that this change (rts) is associated with the allosteric transition between R and T states. Two different rts spectra relate to Hb2 and Hb2CO. No rts has been observed for mutants at position 97 (normally Phe). Correlation of ligand binding and rts shows that protein function changes at or near the rates of rts, typically, 2 x 10(6) s-1 (Hb2) and 5 x 10(5) s-1 (Hb2CO). Unique values of allosteric parameters for several mutants have been obtained by combining kinetic and equilibrium data. The effect of mutation on function thus may be assigned to allostery or to a change in intrinsic heme reactivity.     

Effect of mass transfer resistance on the Lineweaver-Burk plots for flocculating microorganisms

It is shown that the mass transfer resistance can significantly distort the linearity of the Lineweaver-Burk plot of the kinetic data for a microbial culture which forms aggregates. For small flocs, the linearity of the Lineweaver-Burk plot is largely retained, but a different slope and intercept will be obtained compared with flocs free from mass transfer resistance. For large flocs, the Lineweaver-Burk plot shows pronounced curvature at high limiting substrate concentrations. Hence, if the true intrinsic kinetic parameters are to be extracted from a highly flocculating microbial culture, sufficient agitation has to be provided to remove the effect of mass transfer resistance. If the behavior of the flocculating microbial culture is to be explored, additional values for some physical parameters, such as the effective diffusion coefficient of the substrate in floc, the floc density, and the mean floc radius, are needed.     

Spectrophotometric assay of immobilized tannase

A procedure for the assay of immobilized tannase with Polyacrylamide gel, collagen and Duolite-S-762 as matrices is described. It is based on the spectrophotometric determination of gallic acid formed by the enzymatic hydrolysis of tannic acid. The kinetic parameters of the enzymatic reaction have been studied and an assay procedure has been formulated. This method appears to be much more accurate than those reported earlier.     

Development of a modified MTT assay for screening antimonial resistant field isolates of Indian visceral leishmaniasis

The semi-automated MTT colorimetric assay has previously been applied on Leishmania promastigotes based on the ability of viable parasites to reduce the tetrazolium salt to an insoluble formazan product. As promastigotes are non-adherent, application of the MTT assay in its original form has a major drawback of a high and variable background absorbance due to incomplete removal of phenol red, a component of most media. We have accordingly optimised a modified MTT assay wherein the absorbance linearity was maintained for cells ranging from 1x10(4) to 1x10(7) being 0.04+/-0.003-2.38+/-0.04. In contrast, the original MTT assay had a narrower linearity range of 1x10(6)-1x10(7) cells, absorbances being 0.05+/-0.005-1.54+/-0.005. The modified MTT assay was effectively applied to study growth kinetics and identification of antimonial resistant field isolates. Considering the growing problem of antimonial unresponsiveness in the Indian subcontinent, this modified MTT assay is a useful tool for Leishmania research.     

A kinetic expression for hydrolysis of soluble starch by glucoamylase

As the hydrolysis of starch by glucoamylase proceeds with stepwise removal of glucose units from the nonreducing ends of the starch chain, the number of available substrate molecules is essentially unchanged in the course of the degradation. In view of this aspect, a simple practical kinetic expression, which consists of a modified Michaelis-Menten form with product inhibition, is presented for the hydrolysis of soluble starch. It is assumed that the values of kinetic parameters V(m) and K(m) vary linearly from the values for starch toward those for maltose. The applicability of this kinetic expression is verified through the simulation with the experimental results for the hydrolysis of two soluble starches with different average molecular weights of 3 x 10(4) and 3 x 10(6).     

On the progenitor cell migration velocity

An attempt is presented to extract cell kinetic information from histomorphological features. It is applicable to rapidly proliferating tissues like the intestinal epithelium. Each replicating tissue has an origin where cells are formed and a periphery toward which cells migrate. The migration path along which they move is denominated as tissue radius on which all cell positions are mapped. Cell migration on the radius is associated with cell proliferation at tissue origin. Each mitosis there is associated with the displacement of all cells distal to it by one cell position. The more mitoses positioned between a cell and tissue origin, the greater its migration velocity. It is possible therefore to derive the cell migration velocity v(x) from the cumulative mitotic distribution on the radius, N(x). v(x) = N(x)/tm (tm = mitotic time). In this form v(x) represents also cell production at any point on the radius and may serve for the computation of other cell kinetic parameters like generation time. These arguments are illustrated on the rat incisor tooth inner enamel epithelium which has been studied in the normal and rapidly erupting tooth.     

Thermodynamics of aminoglycoside and acyl-coenzyme A binding to the Salmonella enterica AAC(6')-Iy aminoglycoside N-acetyltransferase

Kinetic and mechanistic studies on the chromosomally encoded aminoglycoside 6'-N-acetyltransferase, AAC(6')-Iy, of Salmonella enterica that confers resistance toward aminoglycosides have been previously reported [Magnet et al. (2001) Biochemistry 40, 3700-3709]. In the present study, equilibrium binding and the thermodynamic parameters of binding of aminoglycosides and acyl-coenzyme A derivatives to AAC(6')-Iy and of two mutants, C109A and the C109A/C70A double mutant, have been studied using fluorescence spectroscopy and isothermal titration calorimetry (ITC). Association constants for different aminoglycosides varied greatly (4 x 10(4)-150 x 10(4)) while the association constants of several acyl-coenzyme A derivatives were similar (3.2 x 10(4)-4.5 x 10(4)). The association constants and van't Hoff enthalpy changes derived from intrinsic protein fluorescence changes were in agreement with independently measured values from isothermal titration calorimetry studies. Binding of both aminoglycosides and acyl-coenzyme A derivatives is strongly enthalpically driven and revealed opposing negative entropy changes, resulting in enthalpy-entropy compensation. The acetyltransferase exhibited a temperature-dependent binding of tobramycin with a negative heat capacity value of 410 cal mol(-1) K(-1). Isothermal titration studies of acetyl-coenzyme A and tobramycin binding to mutant forms of the enzyme indicated that completely conserved C109 does not play any direct role in the binding of either of the substrates, while C70 is directly involved in aminoglycoside binding. These results are discussed and compared with previous steady-state kinetic studies of the enzyme.     

Enzymatic hydrolysis of soluble starch with an alpha-amylase from Bacillus licheniformis

The enzymatic hydrolysis of soluble starch with an alpha-amylase from Bacillus licheniformis (commercial enzyme Termamyl 300 L Type DX) have been experimentally studied at pH 7.5, within the temperature range of 37-75 degrees C, at initial substrate concentrations of between 0.25 and 2.00 g/L, and enzyme concentrations of between 0.575 x 10(-4) and 13.8 x 10(-4) g/L. To follow the reaction a procedure based on the iodometric method for measuring alpha-amylase activity was used. The kinetics of the enzymatic hydrolysis was fitted to the Michaelis-Menten equation using the integral method, taking into account that the thermal deactivation of the enzyme follows a second-order kinetic. These parameters were fitted to the Arrhenius equation obtaining activation energies of 24.4 and 41.7 kJ/mol and preexponential factors of 734.9 g/L and 1.74 x 10(8) min(-1) for K(M) and k, respectively.     

Development of a kinetic model for the alcoholic fermentation of must

We Propose a kinetic expression which accounts for the temperature dependence of ethanol yield losses in batch alcoholic fermentation. Moreover, the characteristic parameters of the microbial growth equation have been calculated for Saccharomyces cerevisiae under typical wine industry conditions. A substrate consumption equation is established which minimizes possible model deviations in the latter process stages. Experimental data were obtained in the laboratory and the proposed equations were then applied at an industrial level (2.5 x 10(4) L) where they described the data well.     

Novel inhibition mechanism of Bacillus cereus sphingomyelinase by beryllium fluoride

Phosphate analogs have been known to inhibit competitively various phosphatases and phospholipase C and D. We found for the first time that only beryllium fluoride (BeF(x)) among the phosphate analogs studied inhibits Bacillus cereus sphingomyelinase (SMase) activity. The active inhibitory species proved to be not BeF(3)(-) but BeF(2) by the measurement of SMase activity and of (19)F NMR spectroscopy in the presence of a fixed concentration of BeCl(2) and different concentrations of NaF, although both the species have been reported for other kinds of enzymes. The result of kinetic experiment also indicated that the BeF(x) binds in the vicinity of the essential binding site for the substrate and that the Mg(2+) binding to SMase is essential for the binding of BeF(x) to the enzyme.     

Immobilization of glucose oxidase on sepharose by UV-initiated graft copolymerization

The performance of a new method of enzyme immobilization based on photochemically initiated direct graft copolymerization was recently investigated. The immobilization reaction can be carried out in a simple way and by carefully selecting the reaction conditions, the enzyme-graft copolymer can be obtained as the main reaction product. Coupling efficiency of glucose oxidase has been found to depend only on the amount of photocatalyst (FeCl(3)) fixed on Sepharose used as polysaccharide support. Small quantities of glycidymethacrylate (GMA) (0.25 g/g dry Sepharose) are sufficient but necessary to achieve the best enzyme coupling efficiency (20-40%). Enzyme immobilization occurs very rapidly and the entire reaction occurs within 60 min. Reaction patterns and physicochemical characteristics of the obtained enzyme-graft copolymers exclude the glucose oxidase entrapment: therefore a covalent attachment mechanism may be proposed. The kinetic parameters of immobilized glucose oxidase (K(m)' = 2.0 x 10(-2)M) are quite similar to those of free enzyme (K(m) = 1.93 x 10(-2)M), and no diffusion limitation phenomena are evidenced in samples having different enzyme or polymer content. Lyophilization, thermostability, and long-term continuous operation also have been investigated. The advantages of this method over that using vinylenzyme copolymerization are discussed.     

Effect of breed and sperm concentration on the changes in structural, functional and motility parameters of ram-lamb spermatozoa during storage at 4 degrees C

The objectives of this study were (1) to determine the changes in structural, functional and motility parameters of ram-lamb semen stored at two different concentrations at 4 degrees C for 8 days in egg-yolk based extender and (2) to determine the effect of breed of ram-lambs on the changes in structural, functional and motility parameters of ram-lamb semen from different breeds stored at two different concentrations at 4 degrees C for 8 days in egg-yolk based extender. Two different concentrations suitable for laparoscopic and cervical insemination were employed in this experiment. A total of 14 ram-lambs (Polled Dorset-5, Suffolk-5, Katahdin-4) with satisfactory breeding potential were selected. Semen samples were collected by electro-ejaculation. Semen samples were extended to 50 and 200 million sperm per ml with a commercial egg yolk based extender (Triladyl, Minitube of America, Verona, WI, USA) at room temperature and were stored at 4 degrees C. The sperm DNA fragmentation index (DFI), percentages of high mitochondrial membrane potential (hMMP) and plasma membrane integrity (PMI) were assessed using flow cytometry as part of structural and functional parameters on Days 0, 1, 4, 6, and 8. A computer assisted sperm analyser (HTM-IVOS, Version 10.8, Hamilton Thorne Research, Beverly, MA, USA) was used to assess the sperm motility parameters on Days 0, 1, 4, 6, and 8. PROC MIXED procedure was used to determine the effect of days of storage, concentration and breed. The concentration and days of storage significantly affected the sperm structural, functional and motility parameters (P<0.0001). Significant concentration x days of storage interaction was found for all structural and functional parameters. There was a significant concentration x days of storage interaction for average path velocity, curvilinear velocity, straightness and linearity. Overall changes in the sperm structural, functional and sperm motility parameters over the storage period were less dramatic in the 200 x 10(6) ml(-1) concentration when compared to 50 x 10(6) ml(-1) concentration. The hMMP and total progressive motility were influenced by breed. In conclusion, the quality of structural, functional and motility parameters declined as days of storage were increased and the magnitude of changes in the parameters was less dramatic at the higher concentration.     

Flow-force relationships for a six-state proton pump model: intrinsic uncoupling, kinetic equivalence of input and output forces, and domain of approximate linearity

General flow-force relations have been determined, by the Hill diagram method, for a six-state proton pump model with and without intrinsic uncoupling (molecular slipping). A computer-aided analysis of the resulting sigmoidal flow-force curves has been performed by using a set of physically meaningful rate constants. It is shown that gating effects and apparent irreversibility can arise from sigmoidicity. The regions of approximate linearity in the vicinity of inflection points, which may be far from equilibrium, have been examined with a view to characterization in terms of linear phenomenological equations, with due regard to the problems of kinetic equivalence of the forces and symmetry. The determination of thermodynamic parameters such as the degree of coupling, the phenomenological stoichiometry, and the efficiency in these regions is discussed, and their meaning is analyzed in relation to the parameters characterizing the Onsager domain close to equilibrium. The application of the phenomenological equations of near-equilibrium nonequilibrium thermodynamics to such regions is at best a simplification to be treated with great caution. A knowledge of the distance from equilibrium of the flow-controlling ranges of the forces (i.e., the ranges of approximate linearity) turns out to be crucial for the interpretation of thermodynamic parameters determined by manipulating one of the forces while the other remains constant, as well as for the interpretation of measurements of force ratios at static head. The latter approaches can give good estimates of the magnitude of the mechanistic stoichiometry and of the constant force if the pumps are highly coupled and are operating not far from equilibrium. The force-flow relationships are shown to be modified by intrinsic uncoupling, reflecting the regulatory influence of the forces on the extent and nature of the slip. Thus reaction slip increases, for example, as the force against which the proton pump operates increases. The possible physiological significance of regulated intrinsic uncoupling is discussed.     

Ultrasound assisted intensification of enzyme activity and its properties: a mini-review

Over the last decade, ultrasound technique has emerged as the potential technology which shows large applications in food and biotechnology processes. Earlier, ultrasound has been employed as a method of enzyme inactivation but recently, it has been found that ultrasound does not inactivate all enzymes, particularly, under mild conditions. It has been shown that the use of ultrasonic treatment at appropriate frequencies and intensity levels can lead to enhanced enzyme activity due to favourable conformational changes in protein molecules without altering its structural integrity. The present review article gives an overview of influence of ultrasound irradiation parameters (intensity, duty cycle and frequency) and enzyme related factors (enzyme concentration, temperature and pH) on the catalytic activity of enzyme during ultrasound treatment. Also, it includes the effect of ultrasound on thermal kinetic parameters and Michaelis–Menten kinetic parameters (k_m and V_max) of enzymes. Further, in this review, the physical and chemical effects of ultrasound on enzyme have been correlated with thermodynamic parameters (enthalpy and entropy). Various techniques used for investigating the conformation changes in enzyme after sonication have been highlighted. At the end, different techniques of immobilization for ultrasound treated enzyme have been summarized.     

In situ kinetic analysis of glyoxalase I and glyoxalase II in Saccharomyces cerevisiae.

The kinetics of glyoxalase I [(R)-S-lactoylglutathione methylglyoxal-lyase; EC 4.4.1.5] and glyoxalase II (S-2-hydroxyacylglutathione hydrolase; EC 3.1.2.6) from Saccharomyces cerevisiae was studied in situ, in digitonin permeabilized cells, using two different approaches: initial rate analysis and progress curves analysis. Initial rate analysis was performed by hyperbolic regression of initial rates using the program HYPERFIT. Glyoxalase I exhibited saturation kinetics on 0.05-2.5 mM hemithioacetal concentration range, with kinetic parameters Km 0.53 +/- 0.07 mM and V (3.18 +/- 0.16) x 10(-2) mM.min(-1). Glyoxalase II also showed saturation kinetics in the SD-lactoylglutathione concentration range of 0.15-3 mM and Km 0.32 +/- 0.13 mM and V (1.03 +/- 0.10) x 10(-3) mM.min(-1) were obtained. The kinetic parameters of both enzymes were also estimated by nonlinear regression of progress curves using the raw absorbance data and integrated differential rate equations with the program GEPASI. Several optimization methods were used to minimize the sum of squares of residuals. The best parameter fit for the glyoxalase I reaction was obtained with a single curve analysis, using the irreversible Michaelis-Menten model. The kinetic parameters obtained, Km 0.62 +/- 0.18 mM and V (2.86 +/- 0.01) x 10(-2) mM.min(-1), were in agreement with those obtained by initial rate analysis. The results obtained for glyoxalase II, using either the irreversible Michaelis-Menten model or a phenomenological reversible hyperbolic model, showed a high correlation of residuals with time and/or high values of standard deviation associated with Km. The possible causes for the discrepancy between data obtained from initial rate analysis and progress curve analysis, for glyoxalase II, are discussed.     

Estimation of effective eosinopoiesis and bone marrow eosinophil reserve capacity in normal man.

The eosinophil reserve capacity of the post-mitotic granulocyte compartment in the bone marrow and the effective eosinopoiesis in three haematologically normal men have been quantified by means of kinetic parameters of [3H]thymidine flash-labelled peripheral blood eosinophils. From (a) the time of the appearance in the blood of labelled eosinophils after the tracer injection, (b) the inflow characteristics of the labelled eosinophils in the blood and (c) the magnitude of the eosinophil granulocyte pool in the venous blood, the effective eosinopoiesis (i.e. the eosinophil turnover) was calculated to range between 0.014 and 0.031 x 10(9) cells/kg body weight per day (mean 0.22 x 10(9) cell/kg per day). The post-mitotic eosinophil reserve capacity of the bone marrow ranged from 0.09 to 0.20 x 10(9) cells/kg body weight (mean 0.14 x 10(9) cells/kg). The large reserve pool and the high turnover rate may contribute to sudden rises of the peripheral blood oesinophil counts in some cases of eosinophilia.     

Kinetics of inhibition of thermolysin-catalyzed peptide synthesis by alcohols in aqueous organic one-phase system

The kinetic patterns and parameters of 12 alcoholic organic solvents of different classes inhibiting thermolysin-catalyzed synthesis of N-(benzyloxycarbonyl)-L-phenylalanyl-L-phenylalanine methyl ester (Z-Phe-Phe-OMe) in aqueous organic one-phase reaction system have been determined. All alcohols showed a linear mixed type inhibition. A kinetic model of inhibition is suggested. It was presumed that alcohols interact with substrate in the active site of thermolysin.