Anion activation of γ-glutamyltransferase from fruiting bodies of Lentinus edodes

Activation by different anions of γ-glutamyltransferase obtained in a. particulate form from fruiting bodies of Lentinus edodes has been studied using either L-γ-glutamyl-p-nitroanlide or lentinic acid as substrate. The mushroom transferase was activated by SCN^?, NO₃^?, Cl^?, Br^?, ClO₃^?, Bro₃^?, N₃^?, I^? and F^?, but not those alkali and earth cations previously believed to activate the animal transferase, nor by citrate, claimed to be effective for the kidney bean transferase. Among anions proved hardly to activate the transferase were ClO₄^?, NO₂?, HCO₃^?, H₂PO₄^?, SO₃^2? and SO₄^2?. A high concentration of these anions more or less impeded the halide activation. Kinetic studies revealed that halides function as activators of increasing V_max while keeping K_m constant. These observations appeared least compatible with the possibility that the anion activation might involve a non-specific effect of high solute concentration, viz. dissociation of the enzyme from the supporting structure in the particulates. The activating effect of halides described here probably extends also to the animal enzymes.     

Anion regulation of lupin asparagine synthetase: Chloride activation of the glutamine-utilizing reactions

Small monovalent anions strongly activate glutamine-dependent asparagine synthesis and glutamine hydrolysis catalysed by highly purified asparagine synthetase (EC 6.3.5.4) from cotyledons of Lupinus luteus seedlings. Cl^? and Br^? are most effective, but F^?, I^?, NO₃^? and CN^? also stimulate both reactions. The synthetase reactions with NH₃, or NH₂OH are only slightly stimulated by Cl^? and Br^?, indicating that the anions selectively accelerate the reactions involving glutamine cleavage. In asparagine synthesis Cl^? is a competitive activator vs glutamine and a noncompetitive activator vs MGATP and aspartate. Addition of Cl^? changes the substrate saturation kinetics of glutamine from negatively cooperative to normal hyperbolic and causes a 50-fold increase in the affinity for glutamine. The inherent glutaminase activity of the enzyme is enhanced up to 30-fold by addition of Cl^?, MgATP and aspartate. Thus, ligands of the synthetase reaction act as allosteric activators of the glutaminase step in the enzyme mechanism.     

Fluorescence of proteins in aqueous neutral salt solutions. I. Influence of anions

The effects of anions of neutral salts on the fluorescence emission of six proteins as well as on tryptophan and tyrosine were studied in relation to the structure of proteins. Most anions are good quenchers of tryptophyl and tyrosyl fluorescence, free or in proteins. The results with tryptophan and tyrosine indicate involvement of a collisional quenching mechanism due to agreement with Stern–Volmer law. The deactivation of fluorescence probably occurs because of the transition from singlet state to triplet state. Lehrer's modification of Stern–Volmer law was applied to proteins. The effective quenching constants ([K_Q]eff) and the fraction of fluorescence available ([f_a]eff) to the quencher are also calculated. In contrast to its effect on tryptophan, CH₃COO^? quenches tyrosyl fluorescence and ClO₄^? does not. The effects on fluorescence of ribonuclease and free tyrosine are similar and without any changes in emission maximum. The anions are divided into three groups based on the effect they have on tryptophan-containing proteins. (1) NO₃^?, NO₂^?, Br^?, and I^? have high [K_Q]eff values and readily quench tryptophyl fluorescence of proteins causing a shift of emission maximum to a shorter wavelength. This change is due to the specific quenching of “exposed” tryptophan residues which are accessible to quenchers and the observed residual fluorescence is from the “buried” tryptophyls. (2) ClO₄^? and SCN^? also quench fluorescence of tryptophan in proteins and have lower ([K_Q]eff) values. In their presence the fluorescence maximum is shifted to a longer wavelength, which indicates the unfolding of a protein with [(f_a)eff] = 1. (3) Cl^?, CH₃COO^?, and SO₄? do not have a direct effect on the fluorescence of tryptophan. Besides the “direct” effects, “indirect” effects on fluorophors in protein are also seen, pointing out that the neutral salts can interact in more than one manner with proteins. The effectiveness of anions in quenching fluorescence of proteins follows similar sequences which almost resemble the Hofmeister series, viz., SO₄⁼, CH₃COO^? ? Cl^? < ClO₄^? < SCN^? < Br^? < I^? < NO₃^? < NO₂^?.     

Mechanism of Stimulation and Inhibition of Tonoplast H-ATPase of Beta vulgaris by Chloride and Nitrate

The H⁺-ATPase of tonoplast vesicles isolated from red beet (Beta vulgaris L.) storage tissue was studied with respect to the kinetic effects of Cl⁻ and NO₃⁻. N-Ethylmaleimide (NEM) was employed as a probe to investigate substrate binding and gross conformational changes of the enzyme. Chloride decreased the K_m of the enzyme for ATP but caused relatively little alteration of the V_max. Nitrate increased K_m only. Michaelis-Menten kinetics applied throughout with respect to ATP concentration. Nitrate yielded similar kinetics of inhibition in both the presence and absence of Cl⁻. Other monovalent anions that specifically increased the K_m of the ATPase for ATP were, in order of increasing K_i, SCN⁻, ClO₄⁻, and ClO₃⁻. Sulfate, although inhibitory, manifested noncompetitive kinetics with respect to ATP concentration. ADP, like NO₃⁻, was a competitive inhibitor of the ATPase but ADP and NO₃⁻ did not interact cooperatively nor did either interfere with the inhibitory action of the other. It is concluded that NO₃⁻ does not show competitive kinetics because of its stereochemical similarity to the terminal phosphoryl group of ATP. NEM was an irreversible inhibitor of the tonoplast ATPase. Both Mg·ADP and Mg·ATP protected the enzyme from inactivation by NEM but Mg·ADP was the more potent of the two. Chloride and NO₃⁻ exerted little or no effect on the protective actions of Mg·ADP and Mg·ATP suggesting that neither Cl⁻ nor NO₃⁻ are involved in substrate binding.     

Effects of Potentiators of Muscular Contraction on Binding of Cations by Sarcoplasmic Reticulum

Anionic (NO₃^-, Br^-, I^-, and SCN^-) and cationic (Zn⁺⁺ and Cd⁺⁺) potentiators of the twitch output of skeletal muscle depress the active binding of Ca by sarcoplasmic reticulum isolated from rabbit skeletal muscle. Zinc and Cd exchange for Ca and Mg at the binding sites of the reticular membranes, whereas the anions effectively induce a replacement by Mg of Ca bound actively in the presence of ATP. In the absence of ATP, the passive binding of both Ca and Mg is increased by the anions tested. Furthermore, the anions increase the total capacity of the membrane fragments for passive cation binding. The Ca-stimulated ATPase activity of the membranes is inhibited by Zn and Cd, but not by the anions. Shifts in cations bound to muscle membrane systems caused by agents that increase the force of contraction developed during the twitch are considered to be the primary event modifying excitation-contraction coupling, and thus leading to potentiation.     

Synthesis and antitumor activity of platinum complexes of protonated diamines

Complexes of the formula cis-[Pt(H$\text{N}\text{+}$^～N)(L)Cl₂], where (H$\text{N}\text{+}$^～N) are the protonated diamines including 3-aminoquinuclidine, N-aminopiperidine, piperazine, N-methylpiperazine, 1,1,4-trimethylpiperazine, and N-methyl-1,4-diazabicyclo [2,2,2] octane (N-methyl-dabco) and L = SCN^?, NO₂^?, Br^?, and F^?, were synthesized from the protonated diamine complexes, [Pt(H$\text{N}\text{+}$^～N)Cl₃]. The antitumor activities of the complexes were evaluated in vitro against L1210 murine leukemia cells, and ID₅₀ values for the L-substituted complexes were compared to values of the parent complexes. In each case it was found that replacement of a chloride ion by SCN^?, NO₂^?, Br^?, or F^?, either reduced or completely eliminated antitumor activity. This effect is explained in terms of the trans-directing ability of the ligand, L, compared to chloride. The NO₂-substituted complex of 3- aminoquinuclidine was tested in vivo and found to exhibit little or no antitumor activity.     

Permeant Anions Control Gating of Calcium-dependent Chloride Channels

The effects of external anions (SCN⁻, NO₃⁻, I⁻, Br⁻, F⁻, glutamate, and aspartate) on gating of Ca²⁺-dependent Cl⁻ channels from rat parotid acinar cells were studied using the whole-cell configuration of the patch-clamp technique. Shifts in the reversal potential of the current induced by replacement of external Cl⁻ with foreign anions, gave the following selectivity sequence based on permeability ratios (P_x/P_Cl): SCN⁻>I⁻>NO₃⁻>Br⁻>Cl⁻>F⁻>aspartate>glutamate. Using a continuum electrostatic model we calculated that this lyotropic sequence resulted from the interaction between anions and a polarizable tunnel with an effective dielectric constant of ∼23. Our data revealed that anions with P_x/P_Cl > 1 accelerated activation kinetics in a voltage-independent manner and slowed deactivation kinetics. Moreover, permeant anions enhanced whole-cell conductance (g, an index of the apparent open probability) in a voltage-dependent manner, and shifted leftward the membrane potential-g curves. All of these effects were produced by the anions with an effectiveness that followed the selectivity sequence. To explain the effects of permeant anions on activation kinetics and g_Cl we propose that there are 2 different anion-binding sites in the channel. One site is located outside the electrical field and controls channel activation kinetics, while a second site is located within the pore and controls whole-cell conductance. Thus, interactions of permeant anions with these two sites hinder the closing mechanism and stabilize the channel in the open state.This revised version was published online in August 2005 with a corrected cover date.     

Permeation and Block of the Skeletal Muscle Chloride Channel,ClC-1, by Foreign Anions

A distinctive feature of the voltage-dependent chloride channels ClC-0 (the Torpedo electroplaque chloride channel) and ClC-1 (the major skeletal muscle chloride channel) is that chloride acts as a ligand to its own channel, regulating channel opening and so controlling the permeation of its own species. We have now studied the permeation of a number of foreign anions through ClC-1 using voltage-clamp techniques on Xenopus oocytes and Sf9 cells expressing human (hClC-1) or rat (rClC-1) isoforms, respectively. From their effect on channel gating, the anions presented in this paper can be divided into three groups: impermeant or poorly permeant anions that can not replace Cl⁻ as a channel opener and do not block the channel appreciably (glutamate, gluconate, HCO₃ ⁻, BrO₃ ⁻); impermeant anions that can open the channel and show significant block (methanesulfonate, cyclamate); and permeant anions that replace Cl⁻ at the regulatory binding site but impair Cl⁻ passage through the channel pore (Br⁻, NO₃ ⁻, ClO₃ ⁻, I⁻, ClO₄ ⁻, SCN⁻). The permeability sequence for rClC-1, SCN⁻ ∼ ClO₄ ⁻ > Cl⁻ > Br⁻ > NO₃ ⁻ ∼ ClO₃ ⁻ > I⁻ >> BrO₃ ⁻ > HCO₃ ⁻ >> methanesulfonate ∼ cyclamate ∼ glutamate, was different from the sequence determined for blocking potency and ability to shift the P _open curve, SCN⁻ ∼ ClO₄ ⁻ > I⁻ > NO₃ ⁻ ∼ ClO₃ ⁻ ∼ methanesulfonate > Br⁻ > cyclamate > BrO₃ ⁻ > HCO₃ ⁻ > glutamate, implying that the regulatory binding site that opens the channel is different from the selectivity center and situated closer to the external side. Channel block by foreign anions is voltage dependent and can be entirely accounted for by reduction in single channel conductance. Minimum pore diameter was estimated to be ∼4.5 Å. Anomalous mole-fraction effects found for permeability ratios and conductance in mixtures of Cl⁻ and SCN⁻ or ClO₄ ⁻ suggest a multi-ion pore. Hydrophobic interactions with the wall of the channel pore may explain discrepancies between the measured permeabilities of some anions and their size.     

AMMONIUN AND NITRATE UPTAKE BY THE MARINE MACROPHYTES HYPNEA MUSVUFORMIS (RHODOPHYTA) AND MACROCYSTIS PYRIFERA (PHAEOPHYTA)1, 2

NH₄⁺ and NO₃^? uptake were measured by continuous sampling with an autoanalyzer. For Hypnea musciformis (Wulfen) Lamouroux, NO₃^?up take followed saturable kinetics (K₂=4.9 μg-at N t^?1, V_max= 2.85 μg- at N, g(wet)^?1. h^?1. The ammonium uptake data fit a trucatd hyperbola, i.e., saturation was not reach at the concentrations used. NO₃^? uptake was reduced one-half in the presence of NH₄⁺, but presence of NO₃^? had no effect on NH₄⁺ uptake. Darkness reduced both NO₃^? and NH₄⁺ uptake by one-third to one-half. For Macrocystis pyrufera (L) C. Agardh, NO₃^? uptake followed saturable kinetices: K₂=13.1 μg-at N. l^?1. V_max=3.05 μg-at N. g(wet)^?1. h^?1.NH₄⁺ uptake showed saturable kinetics at concentration below 22 μg-at N l -1 (K₂=5.3 μg-at N.1–1, V_max= 2.38 μg-at N G (wet)^?1.h^?1: at higher concentration uptake increased lincarly with concentrations. NO₃^?and NH₄⁺ were taken up simulataneously: presence of one form did not affect uptake of the other.     

The effect of neutral salt anions on the oxidative deamination activity of plant glutamate dehydrogenase

Increasing concentrations of anions of the Hofmeister series decrease the activity of highly purified glutamate dehydrogenase (EC 1.4.1.2.) from Pisum sativum L. The extent of the inactivation, as estimated by the ion concentration which causes a 50% transformation of the native form to the low activity form of the enzyme (approximately halfmaximal activity), follows the ranking Cl^–––3 ^– –. Sulfate has a slightly activating effect. At salt concentrations higher than 1 M (with SCN^– higher than 200 mM), the activity decreases to a value from 3–6% of the initial activity and remains then stable over a wide range of higher anion concentrations. From kinetic investigations it is seen that the treatment of the enzyme with anions decreases the affinity for the cosubstrate NAD⁺ and the substrate L-glutamate (K _M-values increased) and also increases the dissociation constant for NAD⁺. The salt induced inactivation is reversible by dilution. From a mathematical treatment of the kinetic data of the inactivation, it is seen that increasing concentrations of the anions exert cooperative effects on the inactivation process.     

Constitutive and inducible aspects of nitrate-nitrogen uptake by Chlamydomonas reinhardtii

Similarly to higher plant root systems, Chlamydomonas reinhardtii Dangeard (UTEX 90) cells exhibited biphasic NO₃^? uptake kinetics. The uptake pattern was similar in cells cultured in 10 mM NO₃^? (NO₃^?-grown), 0.25 mM NO₃^? (N-limited) or 10 mM NO₃^? followed by an 18-h period of N-deprivation (N-starved). In all cell types there was an apparent phase transition in uptake at 1.1 mM NO₃^?, although there were variations in the uptake V_max of both isotherms. The rate of uptake via isotherm 0 ([NO₃^?]<1.1 mM) in N-limited cells was higher than that of either NO₃^?-grown or N-starved cells. In contrast, NO₃^?-grown and N-limited cells exhibited comparable V_max values when supplied with 1.1 to 1.8 mM NO₃^? (isotherm 1). When supplied with 1.6 mM NO₃^?, both N-limited and N-starved cells exhibited enhanced linear uptake after 60 min of incubation. We ascribed this to an induction phenomenon. This trend was not observed when NO₃^?-grown cells were supplied with 1.6 mM NO₃^?, or when N-limited and N-starved cells were supplied with 0.6 mM NO₃^?. The ‘inducible’ aspect of uptake by N-limited cells was blocked by cycloheximide (10 mg l^?1), but not by actinomycin D (5 mg l^?1), thus indicating the involvement of a translational or post-translational event. To investigate this phenomenon further, we analysed the cell proteins of N-limited cells supplied with either 0.6 or 1.6 mM NO₃^? for 90 min, using two-dimensional gel electrophoresis. Comparison of protein profiles enabled the identification of a single cell membrane-associated polypeptide (21 kDa, pI ca 5.5) and ten soluble fraction polypeptides (17–73 kDa, pI ca 5.0 to 7.1) unique to the high NO₃^? treatment. We propose that the ‘inducible’ portion of NO₃^? uptake may provide the means by which C. reinhardtii cells regulate uptake in accordance with assimilatory capacity.     

Partial reactivation of impaired immune competence in aged mice by synthetic thymus factors

The stability of mouse uterine cytosol receptor-[³H]estradiol complex was evaluated in the presence of neutral salts of the Hoffmeister series. Marked increases in the rate of dissociation of the complex were observed with the more chaotropic anions (SCN^?, $\text{ClO}{}_4{}^{\mathrm{?}}$, $\text{NO}{}_3{}^{\mathrm{?}}$, Br^?), and the effects of these ions were greater at lower temperatures, where water assumes a more rigid structure. At higher temperatures F^? and CH₃COO^?, which tend to stabilize water structure, led to retardation of the rate of dissociation of the hormone-receptor complex. There was essentially no change in steroid specificity in the presence of the markedly chaotropic salts. The perturbation of water structure adjacent to the steroid binding site is a factor to be considered in the isolation of steroid receptor complexes.     

Lysis of halophilic Vibrio alginolyticus and Vibrio costicolus induced by chaotropic anions

High concentration (1.0 M) of KSCN, but not of NaSCN, induced lysis of slightly halophilic Vibrio alginolyticus and moderately halophilic Vibrio costicolus, and the decrease in absorbance of the cell suspension was complete after 30 min at 25°C. Replacement of K⁺ with Na⁺ effectively prevented the lysis by SCN⁻. K⁺ salts of NO₃⁻, Br⁻, however, induced no significant lysis. In electron micrographs, a prolonged exposure of the cells of V. alginolyticus to 1.0 M KSCN displaced the nucleoplasm to maintain close contact with the cell membranes. After 40 min of interaction, 50% of the cellular protein, 96% of RNA and 94% of DNA were recovered in the lysed cells. In contrast to lysis in hypotonic conditions, the lysis induced by KSCN is due mainly to a partial release of protein from the cells. V. costicolus was more susceptible to SCN⁻ than V. alginolyticus, whereas nonhalophilic Escherichia coli was resistant to 1.0 M KSCN. Thus, lysis by SCN⁻ is characteristic of halophilic bacteria and cell membranes of more halophilic bacteria are more susceptible to chaotropic anions. The protective effect of Na⁺ observed here was considered to be manifested by specific interactions of Na⁺ with components of cell membranes, thereby rendering their structures resistant to the action of chaotropic anions.     

Evidence for an arginine residue at the allosteric sites of spinach leaf ADPglucose pyrophosphorylase

The covalent modification of spinach leaf ADPglucose pyrophosphorylase leads to inactivation of both activator-stimulated and -unstimulated activity. Inactivation can be prevented if either the activator 3PGA or the inhibitor Pi are present during the modification. Pi proved to be more effective at protecting the enzyme from inactivation as it afforded 50% protection at 51 µM compared to 50% protection by 405 µM 3PGA. Partial modification of the enzyme using [¹⁴C]-phenylglyoxal leads to a decrease in bothV _max,A _0.5 and a decrease in the ability of the 3PGA to stimulate the enzyme's activity. Modification increased the enzyme's susceptibility to inhibition by Pi and completely abolished the cooperative binding of Pi seen in the unmodified enzyme in the presence of 3PGA. Thus, phenylglyoxal appears to interfere, with the normal allosteric regulation of ADPglucose pyrophosphorylase from spinach leaf. Greater than 90% of the enzyme's activity is lost when 7.2 mol [¹⁴C]-phenylglyoxal are bound per mole of tetramer and this label is present in both the larger and small subunits. In addition, inactivation appears to involve two different arginine residues having different rates of modification.     

Effect of lipid phase transition on the binding of anions to dimyristoylphosphatidylcholine liposomes

Temperature dependence of the electrophoretic mobility of multilamellar liposomes prepared from dimyristoylphosphatidylcholine was measured in the presence of salts with different anions in aqueous solutions. It was established that specific binding of anions to liposome surface induced a pronounced zeta potential (electrostatic potential at the hydrodynamic plane of shear). A combination of Langmuir, Gouy-Chapman, and Boltzmann equations was used to describe the dependence of the zeta potential on the concentration of anions. The values of binding constants (K) and maximum numbers of binding sites per unit area (σ_max) were determined by this method. The sequence for anion affinities to liposome surface was found to be as follows: trinitrophenol >ClO₄⁻ >I⁻ >SCN⁻ >Br⁻ >NO₃⁻ >Cl⁻ SO₄²⁻. A sharp increase in the negative zeta potential was detected at the temperature of phase transition of the lipid from the gel to liquid-crystalline state. It was found that the parameter K did not change at lipid phase transition and the shifts in zeta potential might be due to alterations of σ_max. The binding sites were considered as defects in the package of lipid molecules in membranes.     

Probing the role of active site histidine residues in the catalytic activity of lacrimal gland peroxidase

The role of active site histidine residues in SCN^– oxidation by lacrimal gland peroxidase (LGP) has been probed after modification with diethylpyrocarbonate (DEPC). The enzyme is irreversibly inactivated following pseudo-first order kinetics with a second order rate constant of 0.26 M^–1 sec^–1 at 25°C. The pH dependent rate of inactivation shows an inflection point at 6.6 indicating histidine derivatization. The UV difference spectrum of the modified versus native enzyme shows a peak at 242 nm indicating formation of N-carbethoxyhistidine. Carbethoxyhistidine formation and associated inactivation are reversed by hydroxylamine indicating histidine modification. The stoichiometry of histidine modification and the extent of inactivation show that out of five histidine residues modified, modification of two residues inactivates the enzyme. Substrate protection with SCN^– during modification indicates that although one histidine is protected, it does not prevent inactivation. The spectroscopically detectable compound II formation is lost due to modification and is not evident after SCN^– protection. The data indicate that out of two histidines, one regulates compound I formation while the other one controls SCN^– binding. SCN^– protected enzyme is inactive due to loss of compound I formation. SCN^– binding studies by optical difference spectroscopy indicate that while the native enzyme binds SCN^– with the K_d of 15 mM, the modified enzyme shows very weak binding with the K_d of 660 mM. From the pH dependent binding of SCN^–, a plot of log K_d vs. pH shows a sigmoidal curve from which the involvement of an enzyme ionizable group of pKa 6.6 is ascertained and attributed to the histidine residue controlling SCN^– binding. LGP has thus two distinctly different essential histidine residues – one regulates compound I formation while the other one controls SCN^– binding.     

Cooperation of Three Denitrifying Bacteria in Nitrate Removal of Acidic Nitrate- and Uranium-Contaminated Groundwater

In uranium-contaminated aquifers co-contaminated with nitrate, denitrifiers play a critical role in bioremediation. Six strains of denitrifying bacteria belonging to Rhizobium, Pseudomonas, and Castellaniella were isolated from the Oak Ridge Integrated Field Research Challenge Site (OR-IFRC), where biostimulation of acidic (pH 3.5–6.5), nitrate-contaminated (up to 140 mM) groundwater occurred. Three isolates were characterized in regards to nitrite tolerance, denitrification kinetic parameters, and growth on different denitrification intermediates. Kinetic and growth experiments showed that Pseudomonas str. GN33#1 reduced NO^? ₃ most rapidly (V_max = 15.8 μmol e^?·min^?1·mg protein^?1) and had the fastest generation time (gt) on NO^? ₃ (2.6 h). Castellaniella str. 4.5A2 was the most low pH and NO^? ₂ tolerant and grew rapidly on NO^? ₂ (gt = 4.0 h). Rhizobium str. GN32#2 was also tolerant of low pH values and reduced NO^? ₂ rapidly (V_max = 10.6 μmol e^?·min^?1·mg protein^?1) but was far less NO^? ₂ tolerant than Castellaniella str. 4.5A2. Growth of and denitrification by these three strains incubated together and individually were measured in OR-IFRC groundwater at pHs 5 and 7 to determine whether they cooperate or compete during denitrification. Mixed assemblages reduced NO^? ₃ more rapidly and more completely than any individual isolate over the course of the experiment. The results described in this article demonstrate 1) that this synthetic assemblage comprised of three physiologically distinct denitrifying bacterial isolates cooperate to achieve more complete levels of denitrification and 2) the importance of pH- and nitrite-tolerant bacteria such as Castellaniella str. 4.5A2 in minimizing NO^? ₂ accumulation in high-NO^? ₃ groundwater during bioremediation. Supplemental materials are available for this article. Go to the publisher's online edition of Geomicrobiology Journal to view the free supplemental files.     

Humic acid differentially improves nitrate kinetics under low‐ and high‐affinity systems and alters the expression of plasma membrane H+‐ATPases and nitrate transporters in rice

Humic acids (HAs) have a major effect on nutrient uptake, metabolism, growth and development in plants. Here, we evaluated the effect of HA pretreatment applied with a nutrient solution on the uptake kinetics of nitrate nitrogen (N‐NO₃^?) and the metabolism of nitrogen (N) in rice under conditions of high and low NO₃^? supply. In addition, the kinetic parameters of NO₃^? uptake, N metabolites, and nitrate transporters (NRTs) and the plasma membrane (PM) H⁺‐ATPase gene expression were examined. The plants were grown in a growth chamber with modified Hoagland and Arnon solution until 21 days after germination (DAG), and they were then transferred to a solution without N for 48 h and then to another solution without N and with and without the addition of HAs for another 48 h. After this period of N deprivation, the plants received new nutrient solutions containing 0.2 and 2.0 mM N‐NO₃^?. Treatment of rice plants with HA promoted the induction of the genes OsNRT2.1‐2.2/OsNAR2.1 and some isoforms PM H⁺‐ATPase in roots. The application of HAs differentially modified the parameters of the uptake kinetics of NO₃^? under both concentrations. When grown with 0.2 mM NO₃^?, the plants pretreated with HA had lower K_m and C_min values as well as a higher V_max/K_m ratio. When grown with 2 mM NO₃^?, the plants pretreated with HA had a higher V_max value, a greater root and shoot mass, and a lower root/shoot ratio. The N fractions were also altered by pretreatment with HA, and a greater accumulation of NO₃^? and N‐amino was observed in the roots and shoots, respectively, of plants pretreated with HA. The results suggest that pretreatment with HA modifies root morphology and gene expression of PM H⁺‐ATPases and NO₃^? transporters, resulting in a greater efficiency of NO₃^? acquisition by high‐ and low‐affinity systems.     

Equilibrium dialysis binding studies of 1,N6-ethenoadenosine diphosphate (epsilon ADP) to myosin, heavy meromyosin, and subfragment one

The binding of the fluorescent analog of adenosine diphosphate (ADP)¹, 1,N⁶-ethenoadenosine diphosphate (εADP) to myosin and its subfragments, heavy meromyosin (HMM) and subfragment one (S1), has been studied under analagous conditions to those previously used in comparable studies on the binding of ADP to these molecules. The results indicate that there are two binding sites for εADP on myosin and HMM, and one site on S1. The dissociation constants for all had an identical value, within experimental error, of 2.0 (^± .5) × 10^?5 M^?1. This is identical to the values found by Young (J. Biol. Chem., $\text{242}$, 2790 (1967)) for ADP. In addition, the kinetics of hydrolysis of εATP versus ATP by S1 were studied. Values of V_max and K_m were 25 μM phosphate sec^?1 (gm protein)^?1 and 5 × 10^?5 M^?1 for ATP, and 80 μN phosphate sec^?1 (gm protein)^?1 and 45 × 10^?5 M^?1 for εATP. The results indicate that the increased V_max that occurs when εATP is used as a substitute for ATP is not due to either an increased binding affinity of ATP for myosin and its subfragments, nor due to a decreased binding affinity of εATP versus ADP. This in turn suggests that the increase in V_max may be due to an increased hydrolytic rate of εATP vs ATP in the enzyme substrate complex.