Competitive Al Inhibition of Net Mg Uptake by Intact Lolium multiflorum Roots : I. Kinetics

Aluminum impairs uptake of Mg²⁺, but the mechanisms of this inhibition are not understood. The depletion technique was used to monitor net Mg²⁺ uptake from nutrient solution by intact, 23-day-old plants of ryegrass (Lolium multiflorum Lam., cv Gulf and Wilo). Activities of Mg²⁺ and monomeric Al species in nutrient solution were calculated and used as the basis for expressing the results. The kinetics of net Mg²⁺ absorption was resolved into (a) a transpiration-dependent uptake component, (b) a metabolically mediated, discontinuous saturable component that is Al³⁺ sensitive and p-chloromercuribenzene sulfonic acid (PCMBS) resistant, and (c) a linear, carbonyl cyanide m-chlorophenylhydrazone resistant, Al³⁺ sensitive component that might be a type of facilitated diffusion. Lowering the pH from 6.0 to 4.2 exerted a noncompetitive inhibition of net Mg²⁺ uptake, while aluminum at 6.6 micromolar Al³⁺ activity exerted competitive inhibition of net Mg²⁺ uptake at pH 4.2. The Al³⁺-induced effect was obvious after 30 minutes. Cultivar-specific ability to retain a higher affinity for Mg²⁺ by postulated transport proteins in the presence of Al³⁺ might be one of the mechanisms of differential Al tolerance among ryegrass cultivars.     

Steryl glucoside and acyl glucoside biosynthesis in maturing pea seeds

Sterol glucosyltransferase activity was found in a particulate fraction of pea seeds. The activity was stimulated by Ca²⁺ and Mg²⁺ and inhibited by Zn²⁺, Cu²⁺, Hg²⁺, EDTA and EGTA. Iodoacetamide was without effect but p-chloromercuribenzoate completely inhibited the enzyme. N -Ethylmaleimide gave 60–70 % inhibition over a wide range of concentrations. The activity was stimulated by ATP in the presence of Mg²⁺. Under such conditions, steryl acyl glucoside was formed. The acyl derivative was barely detectable in the presence of Ca²⁺ either with or without ATP. Both oleyl CoA and palmityl CoA stimulated acyl glucoside synthesis. Of the four nucleoside triphosphates, ATP, GTP, UTP and CTP both ATP and CTP stimulated acylation in the presence of Mg²⁺. The observations suggest that acyl donors other than digalactosyl diglyceride and phospholipids may function in steryl acyl glucoside synthesis in plants.     

Isolation of plasma membrane ofPhytophthora megasperma f. sp.glycinea and some properties of the associated ATPase

Plasma membrane vesicles were isolated fromPhytophthora megasperma f. sp.glycinea using conventional methods of mechanical disruption followed by differential and density gradient centrifugation. The validity of presumed biochemical markers was confirmed using electron microscopy and the phosphotungstic acid-chromic acid staining procedure, which was judged to be specific for plasma membrane when performed under suitable conditions. The plasma membrane fraction showed a peak equilibrium density of 1.14 g/ml and was identified by its vanadate-sensitive Mg²⁺-dependent ATPase with an optimum temperature of 42°C and a pH optimum of 6.0 to 6.5. The activity was weakly stimulated by K⁺ and strongly inhibited by Ca²⁺. The enzyme showed a marked specificity for ATP as a substrate compared to other nucleoside mono-, di-, and triphosphate substrates or other general phosphatase substrates. The divalent cation requirement could be met equally well by Mg²⁺ and Co²⁺ and, to a lesser extent, by Mn²⁺, but not by Ni²⁺, Ba²⁺, Zn²⁺, Sr²⁺, Ca²⁺, Hg²⁺, Cu²⁺, or Fe²⁺ (in decreasing order of preference). Contamination by intact mitochondria (density 1.21 g/ml) or mitochondrial fragments (density 1.16 g/ml) was minimal and could be monitored by measuring cytochromec oxidase or oligomycin-sensitive pH 8.5 ATPase.     

The conversion of 3-deoxyarabinoheptulosonate 7-phosphate to 3-dehydroquinate by sorghum seedling preparations

Partially purified preparations from etiolated sorghum seedlings catalyzed the conversion of DAHP to DHQ. The reaction catalysed by DHQ synthetase was stimulated by 0.1 μM to 0.1 mM NAD in the presence O-0.5 mM Co²⁺. NADH at 1 μM stimulated the reaction as much as 50% but became inhibitory at 100μM. Co²⁺ at 0.5mM stimulated enzyme activity 3-fold; Mg²⁺, Mn²⁺, Cu²⁺, and Zn²⁺ were not stimulatory. EDTA at 5 mM inhibited the reaction 95% but its effects were reversed by equal concentrations of Co²⁺. Phe, Tyr, Trp, t-cinnamate, several hydroxylated cinnamates, DHS, quinate, and shikimate at 0.3 mM failed to affect enzyme activity but slight inhibition occurred with DHQ and protocatechuic acid at 0.3 mM, inhibition being 14 % and 22 %, respectively. DHQ synthetase activity also was detected in spinach leaves and potato tuber tissue. Synthetase activity appeared to increase in response to injury of potato tuber and sweet potato root tissues.     

The Effect of cAMP on Phosphohydrolase Activity of the Barley Root Cells

We studied the effects of cAMP on phosphohydrolase activity in the microsomal fraction and its 0.5 M KCl extract. In both preparations, cAMP inhibited the activity of acid phosphatases. In the microsomal fraction, a 50% inhibition of acid cation-dependent (Mg²⁺, Cu²⁺) phosphatase was induced at pH 6.0 by 10 M cAMP; whereas, for cation-independent phosphatase, this effect was exerted only by 50 M cAMP. In the KCl extract, the sensitivity of both cation-dependent and cation-independent acid phosphatases to cAMP was higher than in the microsomal fraction. Cyclic AMP did not reduce the activity of nucleosidases. Selectivity in the cAMP action on acid phosphatases is assumed to be one of the mechanisms controlling the biochemical activity outside the barley root cells.     

Stimulation of ethylene production in the mung bean hypocotyls by cupric ion, calcium ion, and kinetin

The synergistic stimulation of ethylene production by kinetin and Ca²⁺ in hypocotyl segments of mung bean (Phaseolus aureus Roxb.) seedling was further studied. The requirement for Ca²⁺ in this system was specific. Except for Sr²⁺, which mimicked the effect of Ca²⁺, none of the following divalent cations, including Ba²⁺, Mg⁶⁺, Cu²⁺, Hg²⁺, Co²⁺, Ni²⁺, Sn²⁺, and Zn²⁺, showed synergism with kinetin on ethylene production. Fe²⁺, however, showed a slight synergism with kinetin. Some of them (Hg²⁺, Co²⁺, and Ni²⁺) had a strong inhibitory effect, while others (Zn²⁺, Mg²⁺, Sn²⁺, and Ba²⁺) had a slight or no inhibitory effect on ethylene production in the absence or presence of kinetin.     

Mg<Superscript>2+ </Superscript>Decreases Arrhenius Energies of Activation for High Temperature Catalysis of Phosphatases in <Emphasis Type="Italic">Thermoactinomyces vulgaris</Emphasis>

The nonspecific acid and alkaline phosphatases of Thermoactinomyces vulgaris were found to be optimally active at 65°C and 70°C, respectively, indicating the thermophilic nature of these enzymes in this obligate thermophile. Mg²⁺, when added in the assay mixture (in the form of MgCl₂), increased the specific activities of these enzymes without affecting their respective temperature optima. This divalent cation decreased the Arrhenius energies of activation (E _A ) of both acid and alkaline phosphatases, as substantiated by Mg²⁺-dependent decrease in the slopes of their Arrhenius plots, which were found to be linear. Thus, Mg²⁺-dependent stimulation of high temperature catalysis of T. vulgaris phosphatases appeared to be accomplished by the decrease in their E _A values by this divalent cation, and such unique feature of these enzymes might be associated with their evolutionary adaptation in this thermophilic actinomycete to support its growth at elevated temperatures. The catalytic role of Mg²⁺ in enhancing the phosphatase activities was specified by the fact that this metal ion was able to recover the enzyme activities inhibited by dialysis and EDTA.     

Inhibition of Na+/K+-ATPase and Mg2+-ATPase by metal ions and prevention and recovery of inhibited activities by chelators

Kinetics and inhibition of Na⁺/K⁺-ATPase and Mg²⁺-ATPase activity from rat synaptic plasma membrane (SPM), by separate and simultaneous exposure to transition (Cu²⁺, Zn²⁺, Fe²⁺ and.Co²⁺) and heavy metals (Hg²⁺and Pb²⁺) ions were studied. All investigated metals produced a larger maximum inhibition of Na⁺/K⁺-ATPase than Mg²⁺-ATPase activity. The free concentrations of the key species (inhibitor, MgATP^{2 ?} , MeATP^{2 ?} ) in the medium assay were calculated and discussed. Simultaneous exposure to the combinations Cu²⁺/Fe²⁺ or Hg²⁺/Pb²⁺caused additive inhibition, while Cu²⁺/Zn²⁺ or Fe²⁺/Zn²⁺ inhibited Na⁺/K⁺-ATPase activity synergistically (i.e., greater than the sum metal-induced inhibition assayed separately). Simultaneous exposure to Cu²⁺/Fe²⁺ or Cu²⁺/Zn²⁺ inhibited Mg²⁺-ATPase activity synergistically, while Hg²⁺/Pb²⁺ or Fe²⁺/Zn²⁺ induced antagonistic inhibition of this enzyme. Kinetic analysis showed that all investigated metals inhibited Na⁺/K⁺-ATPase activity by reducing the maximum velocities (V_max) rather than the apparent affinity (K_m) for substrate MgATP^2-, implying the noncompetitive nature of the inhibition. The incomplete inhibition of Mg²⁺-ATPase activity by Zn²⁺, Fe²⁺ and Co²⁺ as well as kinetic analysis indicated two distinct Mg²⁺-ATPase subtypes activated in the presence of low and high MgATP^{2 ?} concentration. EDTA, L-cysteine and gluthathione (GSH) prevented metal ion-induced inhibition of Na⁺/K⁺-ATPase with various potencies. Furthermore, these ligands also reversed Na⁺/K⁺-ATPase activity inhibited by transition metals in a concentration-dependent manner, but a recovery effect by any ligand on Hg²⁺-induced inhibition was not obtained.     

Function of Teichoic Acids and Effect of Novobiocin on Control of Mg<Superscript>2+</Superscript> at the Bacterial Membrane

ALTHOUGH the occurrence of both wall and membrane teichoic acids in Gram-positive bacteria has been known for a considerable time and it is believed that they are essential for normal cellular activity, their main function has been somewhat obscure. Confirmatory evidence for the proposal¹ that teichoic acids participate in ion-exchange in the outer regions of the bacterial cell has been described recently². It has been shown that the phosphate groups of the wall teichoic acid are responsible for the capacity of isolated walls to bind magnesium ions; but whole cells of Gram-positive bacteria also invariably contain a poly-glycerol phosphate-teichoic acid located in the region between the wall and the cytoplasmic membrane³ and it is believed that this must be able to bind Mg²⁺ as does the wall polymer. These two regions of anionic polymer might thus constitute an integrated cation-exchange system between the exterior of the cell and the cytoplasmic membrane, where relatively high concentrations of Mg²⁺ are required for a variety of processes. We report here experiments with a membrane-bound enzyme system that requires Mg²⁺, obtained from a broken cell preparation and in which the close contact between the outer layers of the cell is preserved. In this preparation the enzyme system displays maximum activity in the presence of Mg²⁺ bound to the endogenous teichoic acid and is insensitive to changes in the concentration of added Mg²⁺, in marked contrast to the behaviour of the enzyme system in isolated cytoplasmic membrane. These results provide the first direct demonstration of the function of teichoic acids in concentrating Mg²⁺at the cytoplasmic membrane. They lead to the conclusion that failure of teichoic acid biosynthesis in the whole cell would cause inhibition of membrane function through magnesium starvation. In view of this the effect of novobiocin, an antibiotic shown to inhibit teichoic acid biosynthesis in vitro^4–6, is discussed.     

Phosphatases from pollen of Brassica campestris and Lilium regale

Soluble and wall-bound acid phosphatases isolated from rape seed pollen showed similar properties except for the pH optimum curve which was elevated for the cell wall enzyme. About 50 % of the phosphatase activity of washed pollen wall preparations could be solubilized with Triton X-100, compared with only ca 20% for the corresponding preparation from lily pollen. A comparison of the wall-bound acid phosphatase of rape seed and lily pollen showed a marked difference in specificity towards fructose-6-phosphate and glucose-6-phosphate. A Mg²⁺-dependent alkaline pyrophosphatase was obtained from rape seed pollen but this activity could not be detected in cell wall preparations.     

The low-molecular-weight acid phosphatase from bovine liver: Isolation,amino acid composition,and chemical modification studies

The smallest of the three molecular weight forms of acid phosphatase from bovine liver was purified to a specific activity of 100 μmol min^?1 mg^?1 (measured at pH 5.5 and 37 °C with p-nitrophenyl phosphate). Using several chromatographie and electrophoretic methods, no evidence of heterogeneity was detected. The enzyme was characterized with respect to its stability as a function of pH, molecular weight, amino acid composition, steady-state kinetic parameters in the pH range 4–7 and inhibition by common acid phosphatase inhibitors at pH 5.5. The amino acid composition differed somewhat from a previous literature report. The enzyme was stoichiometrically inactivated upon incubation with Hg²⁺, Ag⁺, and iodoacetate. Inactivation also occurred upon photoinactivation in the presence of Rose Bengal but no inactivation occurred with diethyl pyrocarbonate. The alkylation of one of five cysteine residues by iodoacetate was shown to cause complete inactivation of the enzyme. This alkylation was prevented by the presence of phosphate ion. A tryptic dipeptide containing this essential cysteine was isolated following inactivation with iodo[2-¹⁴C]acetate.     

Fluorescence properties of terbium-alkaline phosphatase.

The addition of Tb³⁺ to apoalkaline phosphatase at pH 8.0 results in the formation of a metalloprotein with an enhanced Tb³⁺ fluorescence at 492, 545, and 580 nm. The Tb³⁺ excitation spectrum is most consistent with a process in which energy is transferred from one or more tyrosyl chromophores to the bound lanthanide. An analysis of the fluorescence data under equilibrium conditions yields one Tb³⁺ binding site per enzyme dimer with a K_n = 0.16 ± 0.02 μm. The Tb³⁺-alkaline phosphatase complex is not catalytically active nor does it incorporate covalently bound phosphate, but the specific activity of Zn²⁺-alkaline phosphatase is significantly enhanced in the presence of Tb³⁺ indicating that this lanthanide mimics Mg²⁺ in stabilizing the structure of alkaline phosphatase. The fluorescence of the Tb³⁺-enzyme is found to be quite sensitive to conformational changes which occur upon addition of Zn²⁺ or substrates.     

Effect of inhibitors on the sigmoidicity of the calcium ion transport kinetics in rat liver mitochondria

The kinetic plot (initial rate of Ca²⁺ transport versus concentration) of mitochondrial Ca²⁺ transport is hyperbolic in a sucrose medium. The plot becomes sigmoidal in the presence of competitive inhibitors of Ca²⁺ binding to low affinity sites of the membrane surface such as Mg²⁺ and K⁺. The plot also becomes sigmoidal in the presence of Ba²⁺. Ba²⁺ is a competitive inhibitor of both Ca²⁺ transport and Ca²⁺ binding to the low affinity sites. The K_i for the inhibition of Ca²⁺ transport by Ba²⁺ increases in the presence of K⁺ and Mg²⁺, which suggests a competition for the low affinity sites between the cations. The plot is still hyperbolic in the presence of La³⁺, which inhibits Ca²⁺ transport competitively. Ruthenium red which is a pure non-competitive inhibitor of mitochondrial Ca²⁺ transport, does not affect the shape of the kinetic plot. These results indicate that the surface potential, which depends on the ions bound to the low affinity sites, determines whether the kinetics of Ca²⁺ uptake in mitochondria is sigmoidal or hyperbolic.     

Investigation on the micro-mechanisms of Al3+ interfering the reactivities of aspartic acid and its biological processes with Mg2+

Density functional theory (DFT) has been applied to study the micro-mechanisms of Al³⁺ interfering the reactivities of aspartic acid (H₂asp) and its biological processes with Mg²⁺. All the 46 stable conformers of Hasp^- and 3 of asp²⁻ have been determined at the B3LYP/6-311++G** level, showing that the 7 most stable conformers of Hasp⁻ all present a very strong and linear O–H···O H-bond between carboxyl and carboxylic acid groups with the bond energy high up to 162 kJ mol⁻¹. The reaction thermodynamics and micro-mechanism between Al³⁺ and Hasp⁻ (or asp²⁻) in aqueous phase have been investigated by the combined application of supramolecular model and polarizable continuum IEFPCM solvent model, firstly revealing Al³⁺ interfering in the biological processes of aspartic acid. The substitution thermodynamics and mechanisms of Mg²⁺ by Al³⁺ in the biological processes between the species of aspartic acid and Mg²⁺ in aqueous phase were probed, revealing the facile displacement of Mg²⁺ by Al³⁺. These results may provide a reasonable mechanism of Al³⁺ biological toxicity at the microscopic level.     

Amdxidation and demethylation of N,N-dimethylaniline by rabbit liver and lung microsomes effects of age and metals

Lung N-oxidase enzyme activity was about three times higher than liver N-oxidase at the pH optimum, about pH 8.9, whereas the activities were nearly the same at more physiological ranges of pH. The lung N-oxidase was also stimulated about 2-fold by 100 mM Mg²⁺ and by 0.1 mM Hg²⁺, whereas liver N-oxidase activity was inhibited by these concentrations of ions. The difference in response of liver and lung enzymes to Mg²⁺ and Hg²⁺ was not altered by preparing the microsomes in the presence of 50 mM ethylenediamine tetraacetic acid (EDTA) in 0.1 M Tris (hydroxymethyl) amino methane (Tris) buffer or 50 mM EDTA in 0.1 M KPO₄ buffer, both at pH 7.6, indicating that the differences are probably not due to the presence of endogenous metals. The difference between the liver and lung N-oxidase systems may be due to the tissue environment rather than to the enzyme itself since mercury stimulation of lung N-oxidation began to disappear upon partial purification of the N-oxidase enzymes. In contrast to the effects of Hg²⁺ and Mg²⁺, 1 mM Ni²⁺ enhanced liver N-oxidase activity about 30% and 5 mM Ni²⁺ stimulated lung enzyme activity about 30% whereas concentrations above 10 mM were inhibitory to both N-oxidases. Both liver and lung demethylase activities were inhibited by these concentrations of Mg²⁺, Hg²⁺ and Ni²⁺.Various suifhydryl reagents were also tested for their effects on these enzymes. The mercurials, para-chloromercurybenzoate (pCMB) and phenylmercuryacetate (PMA) at concentrations of 0.1 mM had the same effect as HgCl₂ inhibiting both demethylases and liver N-oxidase, but stimulating lung N-oxidase activity. However, 0.1 mM to 1 mMN-ethylmaleimide (NEM) and iodoacetamide had little if any effect on either liver or lung N-oxidase. It was also shown that Hg²⁺ effects on N-oxidase activity could be overcome by dilution.Changes in N,N-dimethyl aniline (DMA) metabolism with age were followed in rabbits from 4 days old to adult. There was a steady increase in lung demethylase activity and N-oxidase activity in the liver and lung to adult levels. However, the liver demethylase had a sharp increase in activity between 2 weeks and 1 month much like that seen with benzphetamine demethylase in rabbit liver.Activities of N-demethylase in liver and lung, and N-oxidr.se in liver from new-born rabbits were from 10 to 20 % of adult levels. However, in lung, N-oxidase activities in the newborn were about 50 % of adult levels. Microsomal N-oxidation in lungs from 2-day-old rabbits was stimulated by 0.1 mM mercury just as in the adult.     

Competitive Al Inhibition of Net Mg Uptake by Intact Lolium multiflorum Roots : II. Plant Age Effects

Rhizotoxicity of Al is more pronounced in younger plants. Effects of Al on nutrient uptake by plants of different age are poorly understood. The depletion technique was used to monitor net Mg²⁺ uptake from nutrient solutions by intact 15- and 35-day-old plants of two ryegrass (Lolium multiflorum Lam.) cultivars. Lowering the pH from 6.0 to 4.2 decreased the maximum net ion influx without affecting K_m. Aluminum at 6.6 micromolar Al³⁺ activity increased K_m indicating competitive inhibition. The effects of pH and 6.6 micromolar Al³⁺ on net Mg²⁺ uptake were much larger in 15- than in 35-day-old plants. Aluminum at 26 micromolar Al³⁺ activity competitively inhibited net Mg²⁺ uptake by 35-day-old plants, while causing time- and external Mg²⁺ activity-dependent net Mg²⁺ efflux from 15-day-old plants. The equilibrium constant (K_i) of a reversible combination of postulated plasmalemma Mg²⁺ transporter and Al³⁺ was calculated to be 2 and 5 micromolar Al³⁺ activity for 15-day-old plants of Wilo and Gulf ryegrass, respectively, and 21 micromolar Al³⁺ activity for 35-day-old plants of both cultivars. The Al³⁺-mediated increase in K_m was larger for 15-day-old plants of the Al-sensitive cultivar `Wilo' than of the more Al-tolerant cultivar `Gulf,' while Al³⁺ affected 35-day-old plants of both cultivars to the same extent.     

A comparative study of 5’nucleotidase and alkaline phosphatase in human placenta during development

Activities and a few properties of alkaline phosphatase and 5’-nucleotidase were compared in the developing human placenta. Both the enzymes were mostly membrane-bound and displayed similar developmental patterns with the highest activities at 24/26 weeks of the placenta. L-Phenylalanine, L-tryptophan and L-leucine were inhibitors of alkaline phosphatase, whereas they had no effect on the 5’-nucleotidase. Alkaline phosphatase from a late stage of gestation appeared to be almost heat-stable. An appreciable part of 5’-nucleotidase was also resistant to heat inactivation and this fraction varied with gestational age of the tissue. For both the enzymes, V_max changed without alteringK _m values with periods of gestation. Ca²⁺, Mg²⁺ and Mn²⁺ ions stimulated the alkaline phosphatase activity and Hg²⁺, Zn²⁺, Cu²⁺, Ni²⁺ were inhibitory. 5’-Nucleotidase was not activated by any of these cations. EDTA and Concanavalin A inhibited both the enzymes, although the extent of inhibition was different and also varied with gestation.     

The effect of calmodulin and troponin-C on activities of homogeneous liver phosphoprotein phosphatases

The effect of calmodulin was determined on activities of two homogeneous liver phosphoprotein phosphatases with phosphorylase a and phosphorylated histones as substrates. Calmodulin in the absence or presence of calcium had no effect on the dephosphorylation of phosphorylase a by either phosphatases. However, calmodulin inhibited the dephosphorylation of histones catalyzed by both phosphatases. No difference was found whether the reactions were carried out in the absence or presence of calcium. The effect of calmodulin on histone dephosphorylation was variable depending on (i) the absence or presence of KCl and Mg²⁺, and (ii) the concentration of histone in the reaction mixture. In the presence of KCl and Mg²⁺ at a histone concentration of 0.1 mg/ml, calmodulin inhibited the enzyme activity. At 1 mg/ml histone, lower concentrations of calmodulin activated whereas higher concentrations of calmodulin inhibited the enzyme activity. Similar, but relatively less, effect was observed with troponin-C. In the absence of KCl and Mg²⁺, calmodulin as well as troponin-C activated the enzyme activity. The optimal concentration of calmodulin (or troponin-C) was approximately 30–50% of histone concentration in the reaction mixture. Calcium alone or with calmodulin (or troponin-C) had no additional effect on enzyme activities. DNA and RNA, two negatively charged nucleic acids, also showed similar effects on histone dephosphorylation. Depending on whether KCl and Mg²⁺ were absent or present in the reaction mixtures, both nucleic acids either activated or inhibited the dephosphorylation of histones.