Kinetics of the reactions involving CF2 and CF in a pure tetrafluoromethane plasma: II. Production and loss of CF2 and CF in the processes of fluorocarbon polymerization

Mechanisms for the production and loss of CF₂ and CF radicals in a glow discharge in pure CF₄ are investigated by the time-resolved laser-induced fluorescence method. The fluorocarbon polymerization processes are shown to contribute significantly to the production of radicals both in the plasma volume and on the surface of the discharge tube. The effective frequencies of both the volume and surface processes of radical production and loss are determined. An analysis of these frequencies allowed us to study the polymerization mechanism in a CF₄ plasma at a high relative concentration of F atoms and low ion energy. It is shown that, at elevated pressures, when the density of C_xF_y polymer particles in the plasma volume becomes comparable with the density of simple fluorocarbon radicals, the electron-impact dissociation of these particles is the main channel for the production of CF₂ and CF radicals. Another source of CF₂ and CF radicals is related to the reactions of C_nF_2m+1 unsaturated fluorocarbon particles both in the plasma volume and on the surface of a fluorocarbon film arising on the discharge tube wall. The C_xF_y fluorocarbon polymer particles form both in the discharge volume and on the fluorocarbon filmsurface also in the course of the film destruction. At lowered pressures, the main channel for the production of CF₂ and CF is the direct electron-impact dissociation of CF₄ molecules, whereas the loss of these radicals at the tube wall is the main loss channel. The probabilities of the heterogeneous losses of CF₂ and CF radicals on the heavily fluorinated surface of the fluorocarbon film at low ion energies are determined. Under these conditions, the surface recombination of the Fch chemisorbed fluorine atoms and CF _x ^ph physisorbed radicals with the production of an activated complex is shown to be the most probable mechanism for the heterogeneous losses of CF₂ and CF. The approximate activation energies for the production of F_ch · CF ₂ ^ph and F_ch · CF^ph surface complexes are found to be 750±70 K and 1030±100 K, respectively.     

Photoinhibition of chloroplast reactions. I. Kinetics and action spectra

A study was made of photoinhibition of spinach chloroplast reactions. The kinetics and spectral characteristics of the photoinhibition over a range between 230 and 700 mμ have been examined. The decline of activity due to preillumination was independent of wavelength, and dependent upon the number of quanta applied, not upon the rate of application. The effectiveness spectra of photoinhibition indicate that active ultraviolet light is absorbed by a pigment which is not a normal light absorber for photosynthesis and acts with a high quantum efficiency (> 0.1) for photoinhibition.

Active visible light is absorbed by the pigments which sensitize photosynthesis (chlorophyll, carotenoids). A very low quantum efficiency (about 10⁻⁴) was observed for the photoinhibition with visible light.

The action spectrum of the photoinhibition of dye reduction by chloroplasts and lyophylized Anacystis cells indicated that the damage caused by visible light is due to quanta absorbed by photosystem II. However, since system I might not be involved in dye reduction, the spectra may reflect only damage to photosystem II.

     

Carbamoyl-phosphate synthetase I. Kinetics of binding and dissociation of acetylglutamate and of activation and deactivation

The dissociation of the cofactor, acetylglutamate, from the enzyme-cofactor complex formed by carbamoyl-phosphate synthetase I of rat liver in the presence of ATP, Mg2+, K+ and HCO-3 has been studied by centrifugal gel filtration. The rate of its dissociation (k, 0.13 s-1) is considerably slower than the rate of enzyme turnover (approximately equal to 6 s-1) and it is not increased by ammonia, although ammonia reduces the rate of reassociation of the cofactor. Omission of ATP, Mg2+ or K+ from the column buffer leads to virtually complete dissociation of bound acetylglutamate during passage through the column (0.5-2 min), owing to an increase in dissociation and a decrease in reassociation, but reduction of free Mg2+ alone has the opposite action. Dilution of the enzyme-cofactor complex into a large volume of buffer causes a biphasic loss of enzyme activity with a t1/2 of the first phase comparable with that of the dissociation of acetylglutamate. These findings show (a) that acetylglutamate does not dissociate with each turnover of the enzyme; (b) that there are rapid interactions between binding of acetylglutamate and ATPA (ATPA yields Pi in the overall reaction), Mg2+ and K+, suggesting that these ligands bind in close proximity; and (c) that the enzyme transiently retains considerable activity after dissociation of the cofactor.     

Acetate oxidation to CO2 in anaerobic bacteria via a novel pathway not involving reactions of the citric acid cycle

In several sulfate-reducing bacteria capable of complete oxidation of acetate (or acetyl CoA), the citric acid cycle is not operative. No 2-oxoglutarate dehydrogenase activity was found in these organisms, and the labelling pattern of oxaloacetate excludes its synthesis via 2-oxo-glutarate. These sulfate-reducers contained, however, high activities of the enzymes carbon monoxide dehydrogenase and formate dehydrogenase and catalyzed an isotope exchange between CO₂ and the carboxyl group of acetate (or acetyl CoA), showing a direct C-C-cleavage of activated acetic acid. These findings suggest that in the investigated sulfate-reducers acetate is oxidized to CO₂ via C₁ intermediates. The proposed pathway provides a possible explanation for the reported different fluoroacetate sensitivity of acetate oxidation by anaerobic bacteria, for mini-methane formation, as well as for the postulated anaerobic methane oxidation by special sulfate-reducers.     

Formation of mRNA 3'' termini: stability and dissociation of a complex involving the AAUAAA sequence.

Formation of the 3' termini of mRNAs in animal cells involves endonucleolytic cleavage of a pre-mRNA, followed by polyadenylation of the newly formed end. Here we demonstrate that, during cleavage in vitro, the highly conserved AAUAAA sequence of the pre-mRNA forms a complex with a factor present in a crude nuclear extract. This complex is required for cleavage and polyadenylation. It normally is transient, but is very stable on cleaved RNA to which a single terminal cordycepin residue has been added. The complex can form either during the cleavage reaction, or on a synthetic RNA that ends at the polyadenylation site. Mutations which prevent cleavage also prevent complex formation. The complex dissociates during or after polyadenylation, enabling the released activities to catalyze a second round of cleavage.     

Kinetics of the reactions between streptokinase, plasmin and alpha 2-antiplasmin.

Streptokinase reacts very rapidly with human plasmin (rate constant 5.4 S 10(7) M-1 s-1) forming a 1:1 stoichiometric complex which has a dissociation constant of 5 X 10(-11) M. This plasmin-streptokinase complex is 10(5) times less reactive towards alpha 2-antiplasmin than plasmin, the inhibition rate constant being 1.4 X 10(2) M-1 s-1. The loss of reactivity of the streptokinase-plasmin complex towards alpha 2-antiplasmin is independent of the lysine binding sites in plasmin since low-Mr plasmin, which lacks these sites, and plasmin in which the sites have been blocked by 6-aminohexanoic acid, are both equally unreactive towards alpha 2-antiplasmin on reaction with streptokinase. The plasmin-streptokinase complex binds to Sepharose-lysine and Sepharose-fibrin monomer in the same fashion as free plasmin, showing that the lysine binding sites are fully exposed in the complex. Bovine plasmin is rapidly inhibited by human alpha 2-antiplasmin (k1 = 1.6 X 10(6) M-1 s-1) and similarly loses reactivity towards the inhibitor on complex formation with streptokinase (50% binding at 0.4 microM streptokinase).     

Kinetics of C-14 translocation in soybean: I. Kinetics in the stem

A kinetic study was made of the translocation of ¹⁴C-photosynthate through soybean stems following pulse labeling and during steady state labeling of the first trifoliolate leaf. The translocation profile proceeded down the stem with little or no change in shape. Following pulse labeling, sucrose accounted for 90 to 95% of the radioactivity in the stem at all times up to 2 hours, at which time less than 3% of the activity was in an insoluble form. Kinetic data on the relative specific activities of sucrose in the leaf and petiole indicated that two-thirds of the petiolar sucrose was in the translocation stream and the remaining one-third was in a stationary pool which slowly accumulated sucrose from the translocation stream. With this assumption, the rate of sucrose efflux from the leaf was calculated to be 22 micrograms per minute, which was equivalent to a sucrose mass flux in the sieve tubes of 20 grams per square centimeter per hour.     

Kinetics of Mg2+-dependent CF1-ATPase in the presence of stimulators

A kinetic study of ATP hydrolysis by CF1-ATPase from chloroplasts in the presence of optimal concentrations of the stimulators, sodium sulfite and ethyl alcohol, has been carried out. At MgCl2/ATP ratios more than 1 the reaction kinetics obey the Michaelis--Menten equation. At ATP excess the kinetics are of the second order with respect to Mg2+. The data obtained are consistent with the hypothesis on the formation of an enzyme substrate Mg.CF1-MgATP complex containing beside Mg-ATP substrate Mg2+. The dependence of the maximal rate of the reaction on pH was studied. Two active groups with pK of 6.3 and 8.9 were revealed. The group responsible for Mg+2 binding to the enzyme has a pK of 8.3. The possible nature of the active groups of the enzyme is discussed.     

Kinetics of reactions involving C-550 and cytochrome b559 in chloroplasts at low temperature. Evidence for two photoreactions

The kinetics of reduction of C-550 and of oxidation of cytochrome b₅₅₉ are studied with spinach chloroplasts, at ?170°, under light-limited conditions, at different light intensities. The rate of reduction of C-550 is proportional to the light intensity I; the rate of oxidation of b₅₅₉ is 2–3 times slower and not proportional to I. We propose that two light reactions occur at the reaction center of Photosystem-II (RC-II) at low temperature.     

Binding of ethidium to the nucleosome core particle. 1. Binding and dissociation reactions

We have examined binding properties of and dissociation induced by the intercalating dye ethidium bromide when it interacts with the nucleosome core particle under low ionic strength conditions. Ethidium binding to the core particle results in a reversible dissociation which requires the critical binding of 14 ethidium molecules. Under low ionic strength conditions, dissociation is about 90% completed in 5 h. The observed ethidium binding isotherm was corrected for the presence of free DNA due to particle dissociation. The corrected curve reveals that the binding of ethidium to the core particle itself is a highly cooperative process characterized by a low intrinsic binding constant of KA = 2.4 X 10(4) M-1 and a cooperativity parameter of omega = approximately 140. The number of base pairs excluded to another dye molecule by each bound dye molecule (n) is 4.5. Through the use of a chemical probe, methidiumpropyl-EDTA (MPE), we have localized the initial binding sites of ethidium in the core particle to consist of an average of 27 +/- 4 bp of DNA that are distributed near both ends of the DNA termini. MPE footprint analysis has also revealed that, prior to dissociation, the fractional population of core particles which bind the dye (f) may be as low as 50%. Comparison of the binding and dissociation data showed that the cooperative maximum of the binding curve occurred at or near the critical value, i.e., at the point where dissociation began. The data were used to generate a detailed model for the association of ethidium with chromatin at the level of the nucleosome.     

Nonlocal nature of the electron energy spectrum in a glow discharge in pure O2: I. Nonlocal character of the electron distribution function

Abstrac A study is made of the nonlocal nature of the electron energy distribution function in the positive column of a glow discharge in a tube filled with pure oxygen. The distribution function and the axial (E _z) and radial (E _r) electric fields as functions of radius are measured using an array of mobile probes. The experimentally obtained spatial profiles of the distribution function are used to test the applicability of the two-term approximation to the distribution function of the electrons with a nonlocal energy spectrum. The distribution function in a specified electric field E=E _z +E _r (where E _z ⊥ E _r) is calculated by solving the coordinate-dependent Boltzmann equation in the two-term approximation and by directly integrating the equations of electron motion using the Monte Carlo method. A comparison between the experimental data and the results of simulations carried out for a broad parameter range shows that, in the case of a highly nonlocal electron energy spectrum, the two-term approximation makes it possible to calculate the electron distribution function with a fairly good accuracy, in which case, however, in imposing the boundary conditions, the electron losses at the plasma surface should be treated in the kinetic approximation. It is shown that using the reflection coefficient of the plasma surface for electrons instead of the loss cone in space makes it possible to accurately calculate the electron energy distribution function over the entire parameter range under consideration, including the transient region in which the electron-energy relaxation length is comparable to the characteristic plasma dimension. __________ Translated from Fizika Plazmy, Vol. 26, No. 11, 2000, pp. 1038–1045. Original Russian Text Copyright ? 2000 by Ivanov, Klopovskii, Lopaev, Rakhimov, Rakhimova.     

CO(2) Metabolism in Corn Roots. I. Kinetics of Carboxylation and Decarboxylation

The kinetics of ¹⁴CO₂ carboxylation and decarboxylation in corn root tips were determined to ascertain the sequence of product formation and subsequent utilization, and to obtain further evidence to predict the enzymes mediating the carboxylation and decarboxylations. The carboxylation data indicated that the first product was oxaloacetate followed by malate and aspartate. Malate was the first stable product which could be detected. Decarboxylation data indicated that a large fraction of the ¹⁴CO₂ release and turnover of ¹⁴C was accountable for by a decrease in malate: however, essentially all labeled amino acids turned over rapidly and at a greater rate than organic acids. The data generally support the hypothesis that CO₂ fixation in corn root tips is via P-enolpyruvate carboxylase and malic dehydrogenase and that subsequent malate metabolism is for the most part by direct decarboxylation, possibly by the malic enzyme.     

Experimental dissociation of food intake and plasma beta-endorphin following 2-deoxy-D-glucose in rats

The present studies were undertaken to further assess the role of plasma beta-endorphin (beta-EP) in the hyperphagia induced by the glucose antimetabolite, 2-deoxy-D-glucose (2-DG). Plasma concentrations of immunoreactive beta-EP (ir-beta-EP) were measured at the end of the first hour of feeding in all animals treated with 400 mg/kg 2-DG. Previous studies had shown a consistent, positive association between 2-DG hyperphagia and plasma ir-beta-EP concentrations, but the present data revealed dissociations between hyperphagia and plasma ir-beta-EP. Dexamethasone administration blocked the 2-DG-induced rise in plasma ir-beta-EP, but had no effect on the 2-DG hyperphagia measured at 1 hour. Forced drinking of a 2% NaCl solution decreased 2-DG hyperphagia, but not the 2-DG induced rise in plasma ir-beta-EP. Thus, elevations in plasma ir-beta-EP are not necessary for the full expression of 2-DG-induced hyperphagia in dexamethasone-treated rats. Furthermore, decreased feeding responses to 2-DG could coexist with increased levels of plasma ir-beta-EP in NaCl-treated normal rats. Elevations in plasma ir-beta-EP do not appear to be the critical opiate link in 2-DG induced hyperphagia.     

Kinetics of calcium dissociation from calmodulin and its tryptic fragments. A stopped-flow fluorescence study using Quin 2 reveals a two-domain structure

The kinetics of calcium dissociation from bovine testis calmodulin and its tryptic fragments have been studied by fluorescence stopped-flow methods, using the calcium indicator Quin 2. Two distinct rate processes, each corresponding to the release of two calcium ions are resolved for calmodulin at both low and high ionic strength. The effect of 0.1 M KCl is to accelerate the slow process from 9.1 +/- 1.5 s-1 to 24 +/- 6.0 s-1 and to reduce the rate of the fast process from 650 s-1 to 240 +/- 50 s-1 at 25 degrees C. In the presence of 0.1 M KCl it was possible to determine activation parameters for the fast process: delta H# = 41 +/- 5 kJ mol-1 and delta S# = -63 +/- 17 J K-1 mol-1. These values are in good agreement with those obtained by 43Ca NMR. Studies of the tryptic fragments TR1C and TR2C, comprising the N-terminal or C-terminal half of calmodulin, clearly identified Ca2+-binding sites I and II as the low-affinity (rapidly dissociating) sites and sites III and IV as the high-affinity (slowly dissociating) sites. The kinetic properties of the two proteolytic fragments are closely similar to the fast and slowly dissociating sites of native calmodulin, supporting the idea that calmodulin is constructed from two largely independent domains. The presence of the calmodulin antagonist trifluoperazine markedly decreased the Ca2+ dissociation rates from calmodulin. One of the two high-affinity trifluoperazine-binding sites was found to be located on the N-terminal half and the other on the C-terminal half of calmodulin. The affinity of the C-terminal site is at least one order of magnitude greater.     

Photoenzymatic Repair of Ultraviolet Damage in DNA : I. Kinetics of the reaction

As previously reported, ultraviolet-inactivated bacterial transforming DNA can be restored to activity by an enzyme-like agent from bakers' yeast which requires light for its activity. Kinetics of this reaction, in the presence and absence of inhibitors, are found consistent with the Michaelis-Menten reaction scheme, with the sites of ultraviolet damage on the DNA serving as substrate and the repaired structure as product. Kinetic studies with different light intensities suggest that the necessary illumination causes photolysis of the enzyme-substrate complex with concurrent repair of the DNA. Competitive inhibition of irradiated transforming DNA repair, which occurs when irradiated non-transforming DNA is present in the same reaction mixture, permits ultraviolet damage (of the kind capable of being photoreactivated) to be detected in any type of DNA.