Effects of oxygen and chloramphenicol on Frankia nitrogenase activity

The kinetics of asymbiotic nitrogenase activity in three strains of the actinomycete Frankia were studied. Decay rates for enzyme activity were determined by adding chloramphenicol to active acetylene-reducing cells and measuring the time required for all activity to cease. Synthesis rates were measured by bubbling oxygen through actively-reducing cells (which totally destroyed all activity) and then measuring the time required for activity to return to normal. Decay rates (t _1/2) for these three strains were approximately 30 to 40 min. Synthesis rates were slower and initial nitrogenase activities were recorded about 110 min (DDB 011610) or 210 min (DDB 020210 and WgCc1.17) after return to air-equilibrated cultures. Frankia strain WgCc1.17 showed a greater sensitivity to oxygen and nitrogenase activity was totally lost when cells were bubbled only with atmospheric concentrations of oxygen. The results presented here indicate that nitrogenase activity turnover time is relatively rapid, on the order of minutes rather than hours or days. However, regulation of nitrogenase activity will differ from one strain to another and asmmbiotic characterization will be useful for understanding nitrogenase regulation in the bacterial-plant symbiosis.Contribution no. 879 from the Battelle-Kettering Laboratory     

Kinetics of ionic transport across frog skin: Two concentration-dependent processes

Summary Sodium and chloride influxes across the nonshort-circuited isolated skin ofRana esculenta were measured at widely varying external ionic concentrations.The curve describing sodium transport has two Michaelis-Menten components linked at an inflection point occurring at an external sodium concentration of about 7 meq. Chloride transport can also be represented by two saturating components. A possible explanation of these kinetics is discussed.At sodium concentrations lower than 4 meq it is possible to define a component of the sodium transport mechanism as having a high affinity for sodium and which is independent of the nature of the external anion. A high affinity for chloride of the chloride transport system functioning at low external concentrations is also found but is significantly different from that of sodium. These systems show the physiological characteristics of the countertransports (Na _ext ⁺ /H _int ⁺ ; Cl _ext ^– /HCO _3int ^– ) functioning at low external concentrations.At external concentrations higher than 4 meq a low affinity transporting system in which chloride and sodium are linked superimpose on the high affinity components.The physiological significance of these results is discussed.     

The fluorescence decay kinetics of in vivo chlorophyll measured using low intensity excitation

We report fluorescence lifetimes for in vivo chlorophyll a using a time-correlated single-photon counting technique with tunable dye laser excitation. The fluorescence decay of dark-adapted chlorella is almost exponential with a lifetime of 490 ps, which is independent of excitation from 570 nm to 640 nm.Chloroplasts show a two-component decay of 410 ps and approximately 1.4 ns, the proportion of long component depending upon the fluorescence state of the chloroplasts. The fluorescence lifetime of Photosystem I was determined to be 110 ps from measurements on fragments enriched in Photosystem I prepared from chloroplasts with digitonin.     

Change in kinetic regime of protein aggregation with temperature increase. Thermal aggregation of rabbit muscle creatine kinase

Creatine kinase thermal aggregation kinetics has been studied in 30 mM Hepes-NaOH buffer, pH 8.0, at two temperatures: 50.6 and 60°C. Aggregation kinetics was analyzed by measuring the growth of apparent absorption (A) at 400 nm. It was found that the limiting value of apparent absorption (A _lim) is proportional to protein concentration at both temperatures. The first order rate constant (k _I) does not depend on protein concentration in the range 0.05–0.2 mg/ml at temperature 50.6°C, but at temperature 60°C it increases with the growth of protein concentration in the range 0.1–0.4 mg/ml. Kinetic curves, shown in coordinates {A/A _lim; t}, in experiments at 50.6°C fuse to a common curve, which coincides with the theoretical curve of creatine kinase denaturation calculated using the denaturation rate constant determined from differential scanning calorimetry. At temperature 60°C, half-transformation time t _1/2 = ln2/k _I decreases when protein concentration grows. We conclude that when temperature increased from 50.6 to 60°C, change in the kinetic regime of thermal creatine kinase aggregation took place: at 50.6°C aggregation rate is limited by the stage of protein molecule denaturation, but at 60°C it is limited by the stage of protein aggregate growth, which proceeds as a reaction of pseudo-first order. Small heat shock protein Hsp 16.3 Mycobacterium tuberculosis suppresses the creatine kinase aggregation. Published in Russian in Biokhimiya, 2006, Vol. 71, No. 3, pp. 408–416.     

Seeded isothermal batch crystallization of lysozyme

The kinetics of lysozyme crystallization under seeded isothermal batch conditions was followed by measurement of the decline in solution concentration versus time. Kinetics were measured for five different values of the seed crystal mass. The data were analyzed using a recently proposed mathematical model. For each seed mass, the model fit the kinetic data well. Growth rate constants determined using the model were approximately constant over a sixfold increase in the seed crystal mass, and fell well within the range of values reported in the literature, but obtained using entirely different experimental techniques. These results confirmed the utility of the proposed model. The proposed model can be used to analyze crystallization kinetics using absorbance measurements only, without the need to characterize the crystal size, thus avoiding the need for expensive laser light scattering and digital microscopy instrumentation. Thus, the model offers a low-cost straightforward method to analyze and simulate the effects of changes in operating parameters such as the seed crystal mass, solution volume, initial protein concentration, pH, temperature, salt concentration, and time.     

Comparative effects of limited tryptic hydrolysis on physicochemical and structural features of seed 11S globulins

The effect of the limited proteolysis by trypsin on selected seed storage 11S globulins (broad bean and pea legumins, glycinin and helianthinin) was studied by high-sensitive differential scanning calorimetry, fluorescence spectroscopy and analysis of proteolysis kinetics. Different behaviour of glycinin and helianthinin, on one hand, and broad bean and pea legumins, on the other, were observed: in the first group changes in the physicochemical characteristics of the proteins due to their limited proteolysis are more pronounced in comparison with the second one, in relation with the extent of primary structure modifications. The differences observed have been evaluated in relation with the amino acid sequence features of the four 11S globulin studied and agree with the literature data concerning the protein structural changes in the course of the limited proteolysis.     

Carbon monoxide uptake kinetics in unamended and long-term nitrogen-amended temperate forest soils

The effect of nitrogen (N) additions on the dynamics of carbon monoxide consumption in temperate forest soils is poorly understood. We measured soil CO profiles, potential rates of CO consumption and uptake kinetics in temperate hardwood and pine control plots and plots amended with 50 and 150 kg N ha-1 year-1 for more than 15 years. Soil profiles of CO concentrations were above atmospheric levels in the high-N plots of both stands, suggesting that in these forest soils the balance between consumption and production may be shifted so that either production is increased or consumption decreased. Highest rates of CO consumption were measured in the organic horizon and decreased with soil depth. In the N-amended plots, CO consumption increased in all but one soil depth of the hardwood stand, but decreased in all soil depths of the pine stand. CO enzyme affinities increased with soil depth in the control plots. However, enzyme affinities in the most active soil depths (organic and 0-5 cm mineral) decreased in response to low levels of N in both stands. In the high-N plots, affinities dramatically-increased in the hardwood stand, but decreased in the organic horizon and increased slightly in the 0-5 cm mineral soil in the pine stand. These findings indicate that long-term N addition either by fertilization or deposition may alter the size, composition and/or physiology of the community of CO consumers so that their ability to act as a sink for atmospheric CO has changed. This change could have a substantial effect on the lifetime of greenhouse gases such as CH4 and therefore the future of Earth's climate.     

Folding and misfolding mechanisms of the p53 DNA binding domain at physiological temperature

p53 modulates a large number of cellular response pathways and is critical for the prevention of cancer. Wild-type p53, as well as tumorigenic mutants, exhibits the singular property of spontaneously losing DNA binding activity at 37 degrees C. To understand the molecular basis for this effect, we examine the folding mechanism of the p53 DNA binding domain (DBD) at elevated temperatures. Folding kinetics do not change appreciably from 5 degrees C to 35 degrees C. DBD therefore folds by the same two-channel mechanism at physiological temperature as it does at 10 degrees C. Unfolding rates, however, accelerate by 10,000-fold. Elevated temperatures thus dramatically increase the frequency of cycling between folded and unfolded states. The results suggest that function is lost because a fraction of molecules become trapped in misfolded conformations with each folding-unfolding cycle. In addition, at 37 degrees C, the equilibrium stabilities of the off-pathway species are predicted to rival that of the native state, particularly in the case of destabilized mutants. We propose that it is the presence of these misfolded species, which can aggregate in vitro and may be degraded in the cell, that leads to p53 inactivation.     

Preparation and in vitro evaluation of polystyrene-coated diltiazem-resin complex by oil-in-water emulsion solvent evaporation method

The purpose of this study was to examine the suitability of polystyrene-coated (PS-coated) microcapsules of drug-resin complex for achieving prolonged release of diltiazem-HCl, a highly water-soluble drug, in simulated gastric and intestinal fluid. The drug was bound to Indion 254, a cation-exchange resin, and the resulting resinate was microencapsulated with PS using an oil-in-water emulsion-solvent evaporation method. The effect of various formulation parameters on the characteristics of the microcapsules was studied. Mean diameter and encapsulation efficiency of the microcapsules rose with an increase in the concentration of emulsion stabilizer and the coat/core ratio, while the same characteristics tended to decrease with an increase in the volume of the organic disperse phase. The desorption of drug from the uncoated resinate was quite rapid and independent of the pH of the dissolution media. On the other hand, the drug release from the microcapsules was prolonged for different periods of time depending on the formulation parameters and was also found to be independent of the pH of the dissolution media. Both the encapsulation efficiency and the retardation of drug release were found to be dependent on the uniformity of coating, which in turn was influenced by the formulation parameters. Kinetic studies revealed that the desorption of drug from the resinate obeyed the typical particle diffusion process, whereas the drug release from the microencapsulated resinate followed the diffusion-controlled model in accordance with the Higuchi equation. PS appeared to be a suitable polymer to provide prolonged release of diltiazem independent of the pH of the dissolution media.     

Improved purification protocol for wild-type and mutant human foamy virus proteases

Wild-type and an active site mutant (S25T) human foamy virus (HFV) proteases were expressed in fusion with maltose binding protein in Escherichia coli. The mutant enzyme contained a Ser to Thr mutation in the -Asp-Ser-Gly- active site triplet of the enzyme, which forms the "fireman's grip" between the two subunits of the homodimeric enzyme. The fusion proteins were purified by affinity chromatography on amylose resin, cleaved with factor Xa, and the processed enzymes were purified by gel filtration under denaturing condition. Refolding after purification resulted in active enzymes with comparable yields. Furthermore, both enzymes showed similar catalytic activities in an oligopeptide substrate representing an HFV Gag cleavage site. However, the S25T mutant showed increased stability in urea unfolding experiment, in a good agreement with the suggested role of the Thr residue of fireman's grip.