Platinum complexes of nucleotides.

The reaction of [Pt(dien)Cl1Cl (dien = NH2CH2CH2NHCH2CH2NH2) with nucleotides has been studied by nuclear magnetic resonance. It has been found that the CMP (cytidine 5'-monophosp-ate) and GMP (guanosine 5'-monophosphate/coordinate to the platinum atom through N3 and N7, respectively. The reaction of the platinum salt with the nucleotide is complete when one to one ratio of platinum to nucleotide is used and no evidence of phosphate group binding to platinum has been found. No additional binding sites have been detected except the N7 site on the guanylic group of GMP even in the presence of a large excess of [Pt(dien) Cl1Cl. The AMP (adenosine 5'monophosphate] coordinates to the platinum at the N1 and/or N7 sites. The reaction of AMP and platinum is complete is complete at a ratio of four platinum to one AMP.     

Effect of Different Ammonium Salts on Formation of Maize Pollen Embryoid

There are more than 80% embryoids among the induced masses in anther culture; different ammonium salts produced different effects on the formation of embryoids, and the effect of NH4H2PO4 is better than the others, within the range of the concentrations used. When the combinations of KNO3 With NH4H2PO4 act as the nitrogen source the frequencies of formation of embryoids and calli increase with the increase of NO3–/NH4+ ratio, at NO3–/NH4+ = 6:1, the frequency is the highest, but at 8:1, it decreases. In the treatments of lower total nitrogen (22.5–37.5 mN/L), the induction frequency is higher (3%–12%) than those of higher total nitrogen (45–60 mN/L). It has also been discussed that the different materials responded to the same ammonium salt, that the same material responded to different NO3–/NH4+ ratio.     

Effects of Ammonium and Non-Ammonium Salt Additions on Methane Oxidation by Methylosinus trichosporium OB3b and Maine Forest Soils

Additions of ammonium and non-ammonium salts inhibit atmospheric methane consumption by soil at salt concentrations that do not significantly affect the soil water potential. The response of soils to non-ammonium salts has previously raised questions about the mechanism of ammonium inhibition. Results presented here show that inhibition of methane consumption by non-ammonium salts can be explained in part by ion-exchange reactions: cations desorb ammonium, with the level of desorption varying as a function of both the cation and anion added; differential desorption results in differential inhibition levels. Differences in the extent of inhibition among ammonium salts can also be explained in part by the effects of anions on ammonium exchange. In contrast, only minimal effects of cations and anions are observed in liquid cultures of Methylosinus trichosporium OB3b. The comparable level of inhibition by equinormal concentrations of NH(4)Cl and (NH(4))(2)SO(4) and the insensitivity of salt inhibition to increasing methane concentrations (from 10 to 100 ppm) are of particular interest, since both of these patterns are in contrast to results for soils. The greater inhibition of methane consumption for NH(4)Cl than (NH(4))(2)SO(4) in soils can be attributed to increased ammonium adsorption by sulfate; increasing inhibition by non-ammonium salts with increasing methane concentrations can be attributed to desorbed ammonium and a physiological mechanism proposed previously for pure cultures.     

光系统Ⅱ颗粒的毫秒延迟荧光的研究

本文主要报导了具有放氧活性的光系统Ⅱ(PSⅡ)颗粒的毫秒延迟荧光(ms-DF)的特性以及NH_4Cl对它的调节作用.     

The effects of elevated pH and high salt concentrations on tubulin

The effects of incubating phosphocellulose-purified bovine tubulin at 4 degrees C in nucleotide-free buffers at alkaline pH or at high concentrations of NaCl, KCl, (NH4)2SO4, or NH4Cl have been studied. At pH greater than or equal to 7.5 or at NaCl concentrations greater than or equal to 0.7 M, tubulin releases bound nucleotides irreversibly and loses, with apparent first-order kinetics, the ability to assemble into microtubules. In 0.1 M 1,4-piperazinediethanesulfonic acid buffer, pH 6.9, in the presence of 1.3 M NH4Cl, tubulin undergoes more rapid loss of capacity to assemble than it does in NaCl and KCl, but 1.3 M (NH4)2SO4 causes no detectable change in tubulin after 1-h incubation. Incubation at high pH or at high neutral salt concentrations also causes an apparently irreversible change in the ultraviolet difference spectrum and in the sedimentation velocity profile of tubulin. At elevated salt concentrations a decrease of approximately 10% in the molar ellipticity within the wavelength range 220-260 nm is observed. The changes that occur during 1-h exposure to pH 8.0 can be completely prevented by including 1 mM guanosine 5'-triphosphate (GTP) or 4 M glycerol in the buffer, but those which occur at pH 9.0 cannot be prevented by these additions. In 1 M NaCl when the ratio of bound guanine nucleotide to tubulin reaches approximately 1.0, tubulin loses the abilities to assemble into microtubules and to bind colchicine. The rate of loss of nucleotide in 2 M NaCl is decreased in the presence of 1 mM GTP, and tubulin is protected almost completely from 1 M NaCl-induced loss of GTP (and retains the ability to exchange [3H]GTP as well) in the presence of bound colchicine. Investigators who anticipate exposing tubulin to buffers of elevated pH or high concentrations of chaotropic salts should be extremely cautious in interpreting the resulting data unless they can demonstrate that irreversible alteration of the protein has not occurred.     

Coregulation of oxidized nicotinamide adenine dinucleotide (phosphate) transhydrogenase and glutamate dehydrogenase activities in enteric bacteria during nitrogen limitation

The relationship between oxidized nicotinamide adenine dinucleotide (phosphate) [NAD(P)+] transhydrogenase (EC 1.6.1.1) and NAD(P)+ glutamate dehydrogenase in several enteric bacteria which differ slightly in their regulation of nitrogen metabolism was studied. Escherichia coli strain K-12 was grown on glucose and various concentrations of NH4Cl as the sole nitrogen source. In the range of 0.5 to 20 mM NH4Cl, the energy-independent transhydrogenase increased two to threefold. Comparable changes occurred in NAD(P)+-linked glutamate dehydrogenase. NH4Cl concentrations of 20 to 60 mM resulted in relatively constant specific activities for both enzymes. Higher exogenous NH4Cl, however, led to a decline in both activities. Isocitrate dehydrogenase, another potential source of cellular NADPH, was insensitive to NH4Cl limitation. Similar studies in the presence of glutamate and different exogenous NH4Cl concentrations again showed concerted effects on both enzymes. Growth on glutamate as the sole nitrogen source led to severe repression of both transhydrogenase and glutamate dehydrogenase. In Salmonella typhimurium, both enzymes were unaffected by limiting NH4Cl or growth on glutamate as the sole nitrogen source. Both were, however, repressed by growth on aspartate, a potential source of cellular glutamate. Coordinate changes in glutamate dehydrogenase and transhydrogenase were also evident in Klebsiella aerogenes, particularly under conditions in which glutamate dehydrogenase was regulated inversely to glutamate synthetase. Coordinate changes in glutamate dehydrogenase and transhydrogenase in enteric bacteria are discussed in terms of the possible involvement of the latter enzyme as a direct source of NADPH in the ammonia assimilation system.     

Regulation of hepatic gluconeogenesis in the guinea pig by fatty acids and ammonia.

Octanoate and L-palmitylcarnitine inhibited the synthesis of P-enolpyruvate from alpha-ketoglutarate and malate by isolated guinea pig liver mitochondria. A 50% reduction in P-enolpyruvate formation was obtained with 0.1 to 0.2 mM octanoate or with 0.06 to 0.10 mM L-palmitylcarnitine. At these concentrations, oxidative phosphorylation remained intact and only much higher concentrations of fatty acids altered this process. The addition of NH4Cl in the presence of malate and increasing concentrations of alpha-ketoglutarate (or vice versa) enhanced the formation of glutamate, aspartate, and P-enolpyruvate. The addition of increasing concentrations of NH4Cl in the presence of fixed amounts of malate and alpha-ketoglutarate had a similar effect. Furthermore, the inhibition of P-enolpyruvate synthesis by fatty acids and the reduction of the acetoacetate to beta-hydroxybutyrate ratio were reversed by the addition of NH4Cl. Cycloheximide, which blocks energy transfer at site 1 of the respiratory chain, decreased P-enolpyruvate formation. When cycloheximide and either octanoate or L-palmitylcarnitine were added together, there was an even greater reduction in P-enolpyruvate synthesis from either malate or alpha-ketoglutarate than was noted with either fatty acid alone. Since cycloheximide lowers the rate of ATP synthesis this may in turn reduce P-enolpyruvate formation by a mechanism independent of changes in the mitochondrial NAD+/NADH ratio caused by fatty acids. In the isolated perfused liver metabolizing lactate, the inhibitory effect of octanoate on gluconeogenesis was partially relieved by the addition of 1 mM NH4Cl, but remained unchanged in the presence of 2 mM NH4Cl, despite a highly oxidized NAD+/NADH ratio in the mitochondria. In contrast to glucose synthesis, urea formation was markedly increased during the infusion of 1 mM as well as 2 mM NH4Cl. After cessation of NH4Cl infusion, there was an increase in glucose production, to a rate as high as that observed in the absence of octanoate. This increase was accompanied by the disappearance of alanine, aspartate, and glutamate which had been stored in the liver during NH4Cl infusion. Urea synthesis also decreased progressively. These results indicate that gluconeogenesis in guinea pig liver is regulated, in part, by alterations in the mitochondrial oxidation-reduction state. However, the modulation of this effect by changing the concentrations of intermediates of the aspartate aminotransferase reaction indicates competition for oxalacetate between the aminotransferase reaction and P-enolpyruvate carboxykinase.     

Na+/H+ exchange mechanism in the basolateral membrane of the rat enterocyte

Basolateral membrane vesicles from rat jejunal enterocytes, especially purified of brush-border contamination, were used for Na+ uptake. The basolateral membrane vesicles are osmotically active and under our experimental conditions Na+ binding is much lower than transport. An outwardly directed proton gradient stimulates Na+ uptake at both 5 microM and 5 mM concentrations. The proton gradient effect can be inhibited completely by 2 mM amiloride and partially by either FCCP or NH4Cl (NH3 diffusion). Membrane potential effects can be excluded by having valinomycin plus K+ on both sides of the vesicles. These results suggest that there is an Na+/H+ exchanger in the basolateral membrane of rat enterocytes.     

Polarographic study of oxaloacetate reduction by isolated pea chloroplasts

Suspensions of pea chloroplasts, prepared by differential centrifugation, catalyzed oxaloacetate-dependent O(2) evolution (mean rate of 29 determinations 10.9 micromoles per milligram of chlorophyll per hour, sd 3.2) with the concomitant production of malate. At optimum concentrations of oxaloacetate, both reactions were light-dependent, inhibited by 3-(3,4- dichlorophenyl)-1, 1-dimethylurea and oxalate, and enhanced 2.5- to 4-fold by 10 millimolar NH(4)Cl. At concentrations of oxaloacetate (<50 micromolar), 10 millimolar NH(4)Cl was inhibitory. The ratio of O(2) evolved to malate produced was 0.39 to 0.58. The ratio of O(2) evolved to oxaloacetate supplied was commensurate with the theoretical value of 0.5.Chloroplast suspensions contained both NAD- and NADP-malate dehydrogenase activities. It was concluded from oxalate inhibition studies and the promotion of oxaloacetate-dependent O(2) evolution by shocked chloroplasts by NADPH (but not NADH) that the reaction was mediated via the NADP enzyme.     

Transecting the gustatory branches of the facial nerve impairs NH(4)Cl vs. KCl discrimination in rats

Ammonium and potassium chloride share a common taste quality and an amiloride-insensitive route of transduction. An amiloride-sensitive pathway might also be partially activated by these salts, although very few studies have reported effects of amiloride on nonsodium salt perception. This experiment was designed to determine 1) whether rats could discriminate KCl from NH(4)Cl and, if discrimination was evident, whether performance was impaired with 2) amiloride or 3) gustatory nerve transection. Rats were trained to discriminate KCl from NH(4)Cl (n = 8) and NaCl from NH(4)Cl (n = 8). Amiloride (100 microM) impaired NaCl vs. NH(4)Cl but not KCl vs. NH(4)Cl performance, whereas both groups showed significant impairments after transection of the chorda tympani (CT) and greater superficial petrosal (GSP) branches of the facial nerve. This suggests that rats can discriminate between KCl and NH(4)Cl and that this discrimination does not rely on an amiloride-sensitive mechanism but does depend on the CT and/or GSP nerves. This experiment supports the hypothesis that the facial nerve is important for salt taste recognition and discrimination.     

Kinetic analysis of the preserved rat liver by isolated perfusion with ammonium chloride as a load

Oxygen consumption and urea synthesis from ammonium chloride (NH4Cl) were investigated in the liver preserved in University of Wisconsin solution at 4 degrees C for 24 hours using an isolated rat liver perfusion system in which the perfusate contained five different concentrations of NH4Cl. When a Michaelis-Menten equation was applied to oxygen consumption and urea synthesis against NH4Cl concentration, the preserved liver showed smaller increase in oxygen consumption rate and larger Km of urea synthesis for NH4Cl than the fresh liver. The ratio of respiration velocity without any substrate to maximal velocity (v/Vmax), which reflects the mitochondrial functional reserve, was 55.9 +/- 4.1% and 41.5 +/- 4.8% in the preserved and fresh liver, respectively (p less than 0.05). From the viewpoint of work-cost relationship, it was shown that the mitochondrial function in the preserved liver was deteriorated. On the other hand, conventional mitochondrial study after rewarming and reoxygenation but before NH4Cl load revealed no deterioration of mitochondrial function after preservation. These results indicate that it is necessary to take the metabolic load on the reperfused liver into account when assessing graft viability, and that high v/Vmax suggests decrease in the reserve of mitochondrial function under consideration of the metabolic load.     

Effect of ammonium chloride on the growth and metabolism of bovine ovarian granulosa cells and the development of ovine oocytes matured in the presence of bovine granulosa cells previously exposed to ammonium chloride

Three experiments determined first, the effect of increasing ammonium chloride (NH(4)Cl) concentrations on the growth and metabolism of bovine granulosa cells isolated from small and medium-sized bovine ovarian follicles; secondly, whether the changes in granulosa cell growth and metabolism induced by NH(4)Cl were reversible; and thirdly, whether granulosa cells, previously conditioned with NH(4)Cl, were able to support maturation of oocytes in vitro. In Experiment 1, using a 2 (follicle size class) x 5 (NH(4)Cl concentration) factorial design, granulosa cells from small or medium-sized ovarian follicles were incubated for 96 h with 0, 0.2, 0.4, 0.8 or 1.6 micromol NH(4)Cl/ml. Experiment 2 used a split plot factorial design where granulosa cells were incubated for 96 h in the presence or absence of 1 micromol/ml NH(4)Cl and then incubated in the absence or presence of 1 micromol/ml NH(4)Cl for a further 48 h. Finally in Experiment 3, ovine oocytes were matured on layers of bovine granulosa cells which had not been conditioned with NH(4)Cl or conditioned with 0.5 or 1.0 micromol/ml NH(4)Cl and development of embryos to the blastocyst stage followed and blastocyst quality assessed. In Experiment 1, incubation of granulosa cells in increasing concentrations of NH(4)Cl reduced cell growth, increased cell protein concentrations and increased the amounts of MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) oxidised and oestradiol and progesterone produced per 10(5) cells. Cells from medium-sized follicles were more sensitive to NH(4)Cl concentration and oxidised more MTT and produced less progesterone at high NH(4)Cl concentrations than cells from small-sized follicles. When, in Experiment 2, NH(4)Cl was removed from cell culture after 96 h incubation, cells previously exposed to NH(4)Cl grew at a slower rate during the subsequent 48 h, contained more cellular protein, oxidised more MTT and produced more oestradiol and progesterone than cells not previously exposed to NH(4)Cl. Maturation of ovine oocytes in coculture with bovine granulosa cells not exposed to NH(4)Cl (Experiment 3) increased egg cleavage rate and the proportion of cleaved eggs which developed to the blastocyst stage. Conditioning of granulosa cells with NH(4)Cl supported egg cleavage and development to the blastocyst stage at rates similar to those observed in the absence of granulosa cells. In conclusion, these experiments showed that the in vitro growth and metabolism of granulosa cells were altered by concentrations of NH(4)Cl similar to ammonium ion concentrations measured in follicular fluid and that these effects were not immediately reversible. Furthermore, the ability of granulosa cells conditioned with NH(4)Cl to support in vitro maturation of oocytes was impaired.     

Induction of complete and incomplete chromosome aberrations by bleomycin in human lymphocytes

Bleomycin (BLM) is a clastogenic compound, which due to the overdispersion in the cell distribution of induced dicentrics has been compared to the effect of high-LET radiation. Recently, it has been described that in fibroblast derived cell lines BLM induces incomplete chromosome elements more efficiently than any type of ionizing radiation. The objective of the present study was to evaluate in human lymphocytes the induction of dicentrics and incomplete chromosome elements by BLM. Peripheral blood samples have been treated with different concentrations of BLM. Two cytogenetic techniques were applied, fluorescence plus Giemsa (FPG) and FISH using pan-centromeric and pan-telomeric probes. The observed frequency of dicentric equivalents increases linearly with the BLM concentration, and for all BLM concentrations the distribution of dicentric equivalents was overdispersed. In the FISH study the ratio between total incomplete elements and multicentrics was 0.27. The overdispersion in the dicentric cell distribution, and the linear BLM-concentration dependence of dicentrics can be compared to the effect of high-LET radiation, on the contrary the ratio of incomplete elements and multicentrics is similar to the one induced by low-LET radiation (~0.40). The elevated proportion of interstitial deletions in relation to total acentric fragments, higher than any type of ionizing radiation could be a characteristic signature of the clastogenic effect of BLM.     

Extraction of proteins from Saccharomyces cerevisiae ribosomes under nondenaturing conditions

The differential sensitivity of ribosomal proteins to removal by salts has been studied. Proteins were extracted from the large and small subunits of cytoplasmic ribosomes from Saccharomyces cerevisiae by washing the individual subunits with a series of solutions containing increasing concentrations of NH4Cl (0.74-3.56 M) for a defined time (20 min) at 0 degrees C. The molar ratio of magnesium to ammonium ions of 1:40 was maintained to protect the ribosomal subparticles from complete disassembly. Proteins extracted under each salt condition were analyzed for composition by two-dimensional polyacrylamide gel electrophoresis. The relative quantity of each protein was determined. Most proteins were not removed from the ribosomal particle completely by any one condition, but were preferentially enriched in a single fraction. Whereas most proteins could be solubilized, several proteins remained predominantly or exclusively with the final core particle. The kinetics of protein release from both subunits at a single NH4Cl concentration (0.74 M) were also studied. Release of protein was time dependent, i.e., longer extraction generally removed more of the same proteins. However, prolonged treatment (240 min) of subunits, even at the same salt concentration, resulted in removal of additional species of proteins in varying amounts. Among the ribosomal RNA species, only the 5 S RNA species was released from the ribosomal particles upon treatment.     

Internal pH can regulate Ca2+ uptake and the acrosome reaction in sea urchin sperm

The egg jelly-induced acrosome reaction of sea urchin sperm is accompanied by intracellular alkalinization and Ca2+ entry. We have previously shown that in the absence of egg jelly, NH4Cl, which increases intracellular pH (pHi), induces Ca2+ uptake and the acrosome reaction in sperm of the sea urchin, Strongylocentrotus purpuratus. Here we show that at a constant concentration of NH4Cl (20 mM) in seawater, sperm react less as external pH is lowered from the normal 8 to 7.25. The pH dependence of the NH4Cl response is not very sensitive to temperatures between 12 and 17 degrees C. NH4Cl (15-50 mM) stimulates Ca2+ uptake and acrosome reactions in sperm suspended in Na+-free seawater, a condition known to inhibit the inductive effect of jelly. Jelly does not further stimulate Ca2+ uptake of sperm preincubated in NH4Cl, indicating that once the permeability to Ca2+ is increased by raising the pHi, the jelly has no further effect. We have used the membrane potential-sensitive dye 3,3'-dipropylthiadicarbocyanine iodide to follow the membrane potential change that occurs when NH4Cl is added. Depolarization (25 mV) is associated with the acrosome reaction when either the natural inducer, egg jelly, or NH4Cl is added to sperm. Response to both inducers is inhibited under conditions known to abolish the acrosome reaction, i.e., low-pH seawater and nisoldipine. These results indicate that the NH4Cl-induced depolarization that accompanies the reaction is probably due to the opening of channels that allow Ca2+ to enter the cell and not to the depolarization by NH4+ ions. High-K+ seawater, which depolarizes sperm, and tetraethylammonium, a K+ channel blocker, inhibit the jelly-induced depolarization and the acrosome reaction, but do not inhibit NH4Cl-induced changes. It has already been shown that nigericin promotes Ca2+ entry and the acrosome reaction in sea urchin sperm. We found that the action of this ionophore depends on the pH of normal seawater. In the absence of external Na+ (replaced by choline), nigericin does not induce the reaction and does not stimulate Ca2+ uptake.     

Reciprocal effects of energy utilization on palmitate oxidation and esterification in hepatocytes of fed rats

The effects of the energy-dependent process of urea synthesis from NH4Cl on the partition of [1-14C]palmitate between oxidation and esterification were examined in hepatocytes of fed rats. A high rate of urea formation from NH4Cl resulted in stimulation of total palmitate oxidation by 25 and 15% at 0.2 and 1 mM fatty acid, respectively. The stimulation of palmitate oxidation was reciprocally correlated with diminished palmitate incorporation into lipids, mainly triacylglycerols. This relationship was almost stoichiometric. NH4Cl increased the palmitate oxidation/esterification ratio from 0.72 to 1.13 and from 0.94 to 1.36 in the presence of 0.2 mM and 1 mM palmitate, respectively. The transaminase inhibitor, aminooxyacetate, strongly inhibited urea synthesis from NH4Cl, had little effect on the low beta-hydroxybutyrate/acetoacetate ratio in the presence of NH4Cl, completely reversed the changes in palmitate metabolism caused by NH4Cl and did not affect palmitate metabolism in the absence of NH4Cl. Therefore, the increased utilization of energy for urea synthesis was the causative factor by which NH4Cl stimulated total palmitate oxidation and led in consequence to its decreased esterification into lipids. Accordingly, these observations indicate that in liver cells the rate of ATP utilization is one of the determinants of triacylglycerol synthesis.     

Effects of ammonia and norvaline on lactate metabolism by hepatocytes from starved rats. The use of 14C-labelled lactate in studies of hepatic gluconeogenesis

1. Hepatocytes from starved rats were incubated with l-lactate and NH(4)Cl or norvaline, and the rates of the tricarboxylic acid cycle and of gluconeogenesis were calculated from changes in metabolite concentrations or from radioisotopic data from incubations with labelled lactate or propionate. 2. Gluconeogenesis was stimulated by the addition of 10mm-NH(4)Cl, 5mm-norvaline or 1mm-oleate by 27, 45 and 59% respectively. NH(4)Cl or norvaline also increased lactate uptake. Norvaline inhibited urea synthesis from NH(4)Cl by 85%. 3. The effects of NH(4)Cl and norvaline were not additive. However, NH(4)Cl inhibited and norvaline was without effect on gluconeogenesis from pyruvate, indicating that the two compounds act by different mechanisms. 4. The tricarboxylic acid-cycle flux was increased 80% by lactate, and NH(4)Cl caused a further 25% stimulation. Norvaline had no effect on the tricarboxylic acid-cycle flux. NH(4)Cl and norvaline tripled and doubled, respectively, flux through pyruvate dehydrogenase. 5. Total ATP formation was calculated to range from 470 to 830mumol/h per 100mg of protein, of which the basic metabolic activity accounted for 400-450mumol/h per 100mg of protein. ATP formation does not seem to be rate-limiting for gluconeogenesis. 6. Pyruvate recycling was estimated from the (14)C yield from [1-(14)C]propionate in lactate and glucose to be 10-30% of the flux of phosphoenolpyruvate to glucose. The further addition of NH(4)Cl more than doubled the recycling of pyruvate. 7. [1,4-(14)C]Succinate was rapidly metabolized by hepatocytes. About 20% of the radioactivity was recovered in glucose, indicating that succinate is also metabolized by intact (non-damaged) hepatocytes. 8. It is concluded that the metabolism of lactate by the liver is too complex to allow simple rate measurements with labelled compounds.     

Interaction and inhibitory effect of ammonium cation in the oxygen evolving center of photosystem II

Photosynthetic O(2) evolution takes place at the Mn cluster in photosystem II (PSII) by oxidation of water. It has been proposed that ammonia, one of water analogues, functions as an inhibitor of O(2) evolution at alkaline pH. However, the detailed mechanism of inhibition has not been understood yet. In this study, we investigated the mechanism of ammonia inhibition by examining the NH(4)Cl-induced inhibition of O(2) evolution in a wide pH range (pH 5.0-8.0) and by detecting the interaction site using Fourier transform infrared (FTIR) spectroscopy. In addition to intact PSII membranes from spinach, PSII membranes depleted of the PsbP and PsbQ extrinsic proteins were used as samples to avoid the effect of the release of these proteins by salt treatments. In both types of samples, oxygen evolution activity decreased by approximately 40% by addition of 100 mM NH(4)Cl in the range of pH 5.0-8.0. The presence of inhibition at acidic pH without significant pH dependence strongly suggests that NH(4)(+) cation functions as a major inhibitor in the acidic pH region, where neutral NH(3) scarcely exists in the buffer. The NH(4)Cl treatment at pH 6.5 and 5.5 induced prominent changes in the COO(-) stretching regions in FTIR difference spectra upon the S(1) → S(2) transition measured at 283 K. The NH(4)Cl concentration dependence of the amplitude of the spectral changes showed a good correlation with that of the inhibition of O(2) evolution. From this observation, it is proposed that NH(4)(+) cation interacts with carboxylate groups coupled to the Mn cluster as direct ligands or proton transfer mediators, causing inhibition of the O(2) evolving reaction.     

Relative role of anions and cations in the stabilization of halophilic malate dehydrogenase.

Halophilic malate dehydrogenase unfolds at low salt, and increasing the salt concentration stabilizes, first, the folded form and then, in some cases, destabilizes it. From inactivation and fluorescence measurements performed on the protein after its incubation in the presence of various salts in a large range of concentrations, the apparent effects of anions and cations were found to superimpose. A large range of ions was examined, including conditions that are in general not of physiological relevance, to explore the physical chemistry driving adaptation to extreme environments. The order of efficiency of cations and anions to maintain the folded form is, for the low-salt transition, Ca(2+) approximately Mg(2+) > Li(+) approximately NH(4)(+) approximately Na(+) > K(+) > Rb(+) > Cs(+), and SO(4)(2)(-) approximately OAc(-) approximately F(-) > Cl(-), and for the high-salt transition, NH(4)(+) approximately Na(+) approximately K(+) approximately Cs(+) > Li(+) > Mg(2+) > Ca(2+), and SO(4)(2)(-) approximately OAc(-) approximately F(-) > Cl(-) > Br(-) > I(-). If a cation or anion is very stabilizing, the effect of the salt ion of opposite charge is limited. Anions of high charge density are always the most efficient to stabilize the folded form, in accordance with the order found in the Hofmeister series, while cations of high charge density are the most efficient only at the lower salt concentrations and tend to denature the protein at higher salt concentrations. The stabilizing efficiency of cations and anions can be related in a minor way to their effect on the surface tension of the solution, but the interaction of ions with sites only present in the folded protein has also to be taken into account. Unfolding at high salt concentrations corresponds to interactions of anions of low charge density and cations of high charge density with the peptide bond, as found for nonhalophilic proteins.