Ammonium assimilation by young plants of Hordeum vulgare in light and darkness: effects on respiratory oxygen consumption by roots.

Barley plants (Hordeum vutgare L.) grown for 10 d in nitrogen-free hydroponic culture, after a rapid initial phase absorbed supplied NH4 (+) at a constant rate of 15.1 ±1.2 µ mol h(-1) g(-1) f. wt in the light, arid at a rate of 13.81 ± 1.6 µ mol h(-1) g(-1) f. wt in darkness. Ammonium-grown plants assimilated NH4 (+) at a rate of 7.5 ± 0.33 µmol h-1 g(-1) f. wt and at a 50% lower rate in darkness. Nitrogen-free grown plants showed low concentrations of free amino acids in both root and shoot tissues. Supplying NH4 (+) caused an immediate increase in the concentration of free amino in the root tissues of both illuminated and darkened plants over a 120 mm period. The increase in concentration of glutamine then exhibited a lag period of 120 min, after which it resumed, but to a very small extent. Glutamine also accumulated in shoot tissue of illuminated plants at increasing rates, attaining a concentration which, 8 h after NH4 (+) supply, was 1.61-fold greater than that attained in the roots. In shoots of darkened plants, by contrast, the concentration of glutamine increased slowly and was always smaller than that in the root tissue. Overall formation of glutamine (in shoots and roots) occurred at decreasing rates during the first 4 h, and then at increasing rates. The increase was more pronounced in illuminated plants than in darkened plants, liven 24 h after NH4 (+) was supplied, glutamine content in root tissue was lower than that in shoot tissue. However, 48 h later, the concentrations of glutamine in root and shoot were similar, attaining values that were almost 47-fold (in root) and 134-fold (in shoot) greater than initial values. Significant levels of asparagine were detected in the root and in the shoot 24 h after adding NH4 (+) . These increased further during the succeeding period. Ammonium supply caused a transitory drop in the concentration of ATP in root tissue, along with noticeable transitory variations in glucose-6-P concentration. A permanent decrease in free glucose concentration was also detected. Addition of NH4 (+) caused 2- and 1.43-fold increases in respiratory oxygen consumption by roots of illuminated and darkened plants, respectively. Both in the light and in the dark, the root tissue accumulated methylammonium up to a concentration of 55-67 µmol h(-1) g(-1) f. wt. Methylammonium was never found in shoot tissue of either illuminated or darkened plants. Methylammonium stimulated respiration of root barley plants by a factor of 1.2. Regulatory aspects of NH4 (+) metabolism are discussed.

Ionic strength dependence of formation constants-XVIII. The hydrolysis of iron(III) in aqueous KNO(3) solutions.

The hydrolysis of iron(III) was studied potentiometrically at different ionic strengths in KNO(3) aqueous solutions, at 25 degrees C, to determine the dependence of hydrolysis constants on ionic strength (nitrate media), to check the existence of nitrate-ferric ion interactions, and to confirm the formation of high polymeric species. Under the experimental conditions 0.03 I (KNO(3)) 1M, 0.3 C 12 mM, the species Fe(OH)(2+), Fe(2)(OH)(4+)(2), Fe(OH)(+)(2) and Fe(12)(OH)(2+)(34) were found, and the hydrolysis constants log beta(11) = 2.20, log beta(12) = -2.91, log beta(22) = -5.7, log beta(12,34) = -48.9 (I = 0M) were calculated. The ionic strength dependence of hydrolysis constants is quite close to that found for several protonation and metal complex formation constants reported elsewhere.

Protonation and complex formation constants of 3-amino-1,2,4-triazole in NaCl and CaCl(2) media at different temperatures and ionic strengths.

The protonation of 3-amino-1,2,4-triazole was studied potentiometrically (glass electrode) in sodium chloride and calcium chloride solutions, 0.13 I 0.92M, at 10, 25, 37 and 45 degrees . The effect of the background electrolyte on the protonation constants is explained by a complex formation model. The species CaL(+), CaHL(2+) and H(2)LCl (HL = 3-amino-1,2,4-triazole) are proposed. Stability constants, together with their dependence on temperature (DeltaH degrees ) and on ionic strength, are reported for the protonated and complex species.

The determination of formation constants of weak complexes by potentiometric measurements: experimental procedures and calculation methods.

Experimental and calculation procedures for the study of weak complexes by the pH-measurement technique are described. An algorithm for the calculation of formation constants, together with a computer program in FORTRAN and BASIC versions, is reported. The problems of studying weak interactions are discussed. Simulated titration curves and experimental data for K(+)-thiodiacetate complexation were used to check the proposed method.

Ionic strength dependence of formation constants-V Protonation constants of some nitrogen-containing ligands at different temperatures and ionic strengths.

The protonation constants of 2,2'-bipyridyl, 1,10-phenanthroline, 1,3-diaminopropane, l-histidine and histamine have been determined potentiometrically, in the temperature range 10-40 degrees and at ionic strengths ranging from 0.04 to 1 (potassium chloride). The dependence of the protonation constants on ionic strength is described by a simple general equation. The enthalpy changes for the protonation of the ligands have been calculated from the temperature dependence of the protonation constants.

Depression of nitrate reductase in the presence of excess ammonium in a unicellular alga growing under conditions of phosphate limitation.

Chemostat cultures of the unicellular alga Cyanidium caldarium have shown that under conditions of phosphate limitation nitrate reductase is completely derepressed even in cells growing in a large excess of ammonium, but that it occurs mainly in a catalytically inactive form. It is hypothesized that phosphate limitation contributes to maintaining intracellular level of glutamine suitable to stimulate inactivation but not repression of nitrate reductase. It is not excluded that in addition to variations in the intracellular level of glutamine, there are other metabolic events of the cell by which repression and inactivation of nitrate reductase could be differently influenced.

Ionic strength dependence of formation constants-I Protonation constants of organic and inorganic acids.

The protonation constants of formic, acetic, benzoic, oxalic, phthalic, maleic, malonic, succinic, dl-malic, dl-tartaric, aminoacetic, citric, nitrilotriacetic, ethylenediaminetetra-acetic, sulphuric and orthophosphoric acids have been determined from pH measurements, in tetraethylammonium iodide solution, at various ionic strengths in the range 0.01-1.0M (for phosphoric and sulphuric acids 0.01-0.5M). For each acid the dependence of the protonation constants on ionic strength was determined and an equation, valid for all the acids studied, to describe this was derived. The use of tetraethyl-ammonium salts as background to avoid ion-pair formation is discussed.

Active and inactive nitrate reductase. Effects of mild treatment with denaturing agents of protein.

Nitrate reductase (NAD(P)H:nitrate oxidoreductase, EC 1.6.6.2) of the unicellular alga Cyanidium caldarium can exist in two interconvertible forms; one catalytically active and one inactive. The inactive nitrate reductase can be activated by mild treatment with denaturing agents of protein. By treatment with urea or mersalyl, activation of both the NADPH and benzyl viologen activities can be realized under mild conditions, whereas by treatment with heat, the activation of benzyl viologen activity is concomitant with loss of the NADPH activity. On the other hand, both activities are activated and destroyed concomitantly by ethylene glycol. In the present of FAD, either activation of benzyl viologen activity or loss of NADPH activity upon heating occur only at higher temperatures. The existence of a controlling region in the nitrate reductase molecule is postulated.

A non-linear least-squares approach to the refinement of all parameters involved in acid-base titrations.

A non-linear least-squares computer program has been written for the refinement of the parameters involved in potentiometric acid-base titrations. The program ACBA (ACid-BAse titrations) is applicable under quite general conditions to solutions containing one or more acids or bases. The method of refinement used gives the program several advantages over the other programs described previously.

Studies on utilization of 2-ketoglutarate, glutamate and other amino acids by the unicellular alga Cyanidium caldarium.

Two strains of Cyandium caladarium which possess different biochemical and nutritional characteristics were examined with respect to their ability to utilize amino acids or 2-ketoglutarate as substrates. One strain utilizes alanine, glutamate or aspartate as nitrogen sources, and glutamate, alanine, or 2-ketoglutarate as carbon and energy sources for growth in the dark. The growth rate in the dark on 2-ketoglutarate is almost twice as high or higher than that on glutamate or alanine. During growth or incubation of this alga on amino acids, large amounts of ammonia are formed; however, ammonia formation is strongly inhibited by 2-ketoglutarate. The capacity of the alga and develops fully only when the cells are grown or incubated in the presence of glutamate.

Observations on enzymes of ammonia assimilation in two different strains of Cyanidium caldarium.

Two strains of Cyanidium caldarium, one able to utilize nitrate as a substrate, and the other not, were tested for the presence of enzymes of ammonia assimilation. The nitrate-assimilating strain exhibits glutamate dehydrogenase activity. By contrast, the other strain lacks glutamate dehydrogenase; it possesses high alanine dehydrogenase and L-alanine aminotransferase activities which suggest that this strain may incorporate ammonia through reductive amination of pyruvate and may form glutamate from 2-ketoglutarate by a transamination reaction with alanine. Neither strain reveals glutamate synthase activity. Both strains contain similar levels of glutamine synthetase.

Electron donors and inhibitors of nitrate reductase from Cyanidium caldarium.

Studies on nitrate reductase (NAD(P)H:nitrate oxidoreductases EC 1.6.6.2) of Cyanidium caldarium revealed that the enzyme is inhibited by excess of electron donor, NADPH, reduced benzylviologen and FMN. Also dithionite, used to reduce benzylviologen and FMN, inactivates nitrate reductase: however, FMN at an optimal concentration and nitrate, added before the dithionite, protect the enzyme against this inactivation. Cyanide, cyanate and carbamyl phosphate inhibit the enzyme competitively with respect to nitrate, and Ki values are reported. Organic mercurials, 0.1 mM, act preferentially on NADPH activity, whereas Ag+ and Hg-2+ at the same concentration inactivate 80--90% of the benzylviologen and FMN activities. ADP is very poor inhibitor. Urea 4 M in 2 h destroys 90% of the NADPH activity and only 30% of the benzylviologen and FMN activities. The apparent Km values for NADPH, benzylviologen, FMN and nitrate have been determined.