Aluminium speciation in natural water by sorption on a complexing resin

Very stable aluminium complexes may be present in natural waters, which can be detected only using appropriate methods. One of them is the resin titration based on the sorption of aluminium on a strongly sorbing resin, Chelex 100. It was here used to detect strong aluminium complexes, and to characterize them by determining their concentration, and the corresponding stability constant. High and low salinity waters were sampled in different sites in the North of Italy. In all the samples aluminium complexes with high stability constant, up to 10(17.4) M(-1) in the less acidic solution, were detected. The stability constant depends mainly on the solution acidity, increasing with increasing pH up to 7. The concentration of the ligands responsible for the strong complexation is similar to that of aluminium (from 0.5 to 1.5 microM), or somewhat lower in the case of estuarine and sea waters. A small fraction of aluminium (from 0% to 2%) in freshwaters, higher in estuarine and sea waters (14% and 10%, respectively), is present in weakly bound forms which could also be the hydrolysis products. The conditional constants of the strong complexes were determined for the different samples examined. They were found to be slightly lower in the case of the high salinity waters, in which a value of 10(16.1) M(-1) at pH 7.5 was obtained. This is probably due to the higher ionic strength in marine water, which strongly influences the complexation of trivalent metal ions, as seen for example also in the hydrolysis. It could be deduced that similar substances, but at different concentration, would be responsible for the aluminium complexation in the sea and freshwaters here examined. They could be natural organics like fulvic substances, or better some particular complexing sites in this substances with very high affinity for aluminium.     

Aluminium in tea: SEC-ICP-MS speciation studies of infusions and simulated gastrointestinal digests

Abstract

The speciation of aluminium in tea infusions and in vitro gastrointestinal digests of tea infusions has been investigated using size exclusion chromatography coupled to inductively coupled plasma mass spectrometry (SEC-ICP-MS). At pH 2.5, following simulated gastric treatment, Al from tea eluted at a similar retention volume to that obtained for an aqueous Al standard. At pH 5.5, an aqueous Al standard was eluted from an SEC column in Tris buffer (Al recovery ≈ 100%) only in the presence of a complexing agent (NaF), and at a retention volume corresponding to a molecular mass greater than that expected for an ionic species. Aluminium associated with a tea infusion eluted in two fractions: a higher molecular weight fraction corresponding to Al strongly bound to ligands in the tea, and a lower molecular weight fraction probably comprised of labile Al eluting as Al-F complexes. Simulated gastrointestinal digestion produced three Al-bearing fractions, of which the two at higher molecular mass represented ligand-bound Al. The molarity of the Tris buffer strongly influenced the retention volume of the Al fractions, particularly for the ligand-bound Al. After simulated gastrointestinal digestion, the soluble labile fraction (15% of the Al from the tea infusion) was considered to be potentially available for absorption. The actual proportion of the fraction that might be absorbed would depend upon a number of physiological and nutritional factors.     

The effect of beverage preparation method on aluminium content in coffee infusions

Seven types of coffee were prepared by four methods. Three of them – simple coffee infusion, preparation in ibrick and moka pot – are very often used to prepare coffee at home. The fourth one – a single-cup filter is typical for Vietnam. Cookware used for each method was made from glass, aluminium, stainless steel and two types of alloys. Amounts of Al leaching to coffee infusions were determined. On average, the highest amount of Al was in coffee infusions brewed in aluminium single-cup filter, and the lowest in infusions prepared by simple extraction. Other brewing methods in combination with different tool materials resulted in similar Al content. The type of brewing method significantly influences the Al content in final infusion. Aluminium content varies in infusions in relation to the method of choice, especially when using single-cup filter. Despite the fact that coffee is considered to be a poor source of Al for humans, in some cases, Al content in infusions can even reach the values reported for tea infusions.     

The inhibition of ATP-dependent shape change of human erythrocyte ghosts correlates with an inhibition of Mg(2+)-ATPase activity by fluoride and aluminofluoride complexes.

The vanadate-sensitive Mg(2+)-dependent ATPase activity of the human erythrocyte ghost is believed to be involved in the shape change events that convert echinocytic ghosts to smoothed forms (biconcave discs and stomatocytes). At physiological salt concentration, pH 7.4, 2 mM ATP, 5 mM Mg2+ and 1 mM EGTA, the Mg(2+)-ATPase activity of ghosts was inhibited strongly by millimolar concentrations of sodium fluoride: I50 = 1.31 +/- 0.23 mM (mean +/- S.D.; n = 12). The addition of aluminium chloride to 15 microM reduced the concentration of NaF required for 50% inhibition to 0.76 +/- 0.21 mM (n = 10). Aluminium alone had only a small inhibitory effect on the ATPase activity (13 +/- 9%; n = 10). Desferrioxamine, a strong chelator of tervalent aluminium ion, failed to reverse the inhibition by fluoride and reversed the inhibition in the presence of aluminium and fluoride back to those values obtained with fluoride alone. Of several metal salts tested only beryllium sulfate was able to replace aluminium as an effective inhibitor in the presence of fluoride. Inhibition of the Mg(2+)-ATPase activity by fluoride and the aluminofluoride complexes correlated with an inhibition of the rate of MgATP-dependent change in red cell ghost shape from echinocytes to smoothed forms. All gross morphological changes of the smoothing process were affected, including the production of discocytes, stomatocytes and endocyctic vesicles.     

Chemical forms of aluminum in xylem sap of tea plants (Camellia sinensis L.)

To identify the chemical forms of aluminum (Al) transported from roots to shoots of tea plants (C. sinensis L.), ²⁷Al-nuclear magnetic resonance and ¹⁹F NMR spectroscopy were used to analyze xylem sap.The concentration of Al in collected xylem sap was 0.29 mM, twice as high as that of F. Catechins were not detected in xylem sap. The concentration of malic acid in xylem sap was higher than that of citric acid, whereas the concentration of oxalic acid was negligible.There were two signals in the ²⁷Al NMR spectra of xylem sap, a larger signal at 11 ppm and a smaller one at −1.5 ppm. The former signal was consistent with the peak for an Al-citrate model solution, suggesting that an Al-citrate complex was present in xylem sap. Although the latter signal at −1.5 ppm was thought to indicate the presence of an Al-F complex (at 1.7 ppm) in xylem sap, there was only one signal at −122 ppm in the ¹⁹F NMR spectrum of xylem sap, indicating that the main F complex in xylem sap was F⁻.These results indicate that Al might be translocated as a complex with citrate, while Al-malate, Al-oxalate and Al-F complexes are not major Al complexes in xylem sap of tea plants.     

Model studies of the precipitation of silica in the presence of aluminium; implications for biology and industry

The unique chemical affinity between the oxides of silicon and aluminium has been cited as a potential route for the amelioration of the detrimental effects of aluminium in the environment and in biological systems. A greater understanding of silicon-aluminium interactions may assist in this endeavour and also provide a means of overcoming silica fouling problems encountered by industry which are exacerbated by the presence of aluminium. It is also conceivable that this increased knowledge may demonstrate a positive use for aluminium in the processing of the silicon dioxide phase. In this study we report the effect of aluminium ions, derived from aluminium chloride, on silicic acid species obtained from potassium catecholato complexes of silicon at circumneutral pH at the molar ratios 1000Si:Al, 100Si:Al and 50Si:Al. Silica and low levels of aluminium-rich silica materials were formed with Si:Al ratios of about 3.5:1 comparable with the element ratios detected in senile plaques and aluminium-rich scale. A kinetic study showed that aluminium in the reaction medium slowed down the rate of formation of one of the silica species formed early in the condensation process, e.g. trimers, but increased the rate at which silicic acid was removed from sub 1 nm diameter particles. The materials precipitated in the presence of aluminium were composed of smaller particles and aggregates with smaller pores (Si100:Al and Si50:Al systems) or larger pores (Si1000:Al) compared to the control. The nature of the interactions responsible for these differences is discussed. The effects described here demonstrate the ability of silica and aluminium to interact under conditions such as those found in biological systems. That silica reacts with aluminium in the presence of catechol supports the protective role assigned to silicon.     

Potential health risk of selected metals for Polish consumers of oolong tea from the Fujian Province,China

The influence of environmental pollution on the heavy metal content in oolong teas from the Fujian Province, China, and the health risk for the Polish consumers were studied. Average contents of the metals in made oolong tea leaves were (mg kg^?1): Al 1452, Cd 0.08, Co 0.23, Cr 1.59, Cu 10.5, Fe 140, Hg 0.10, Mn 1465, Mo 0.63, Ni 3.45, Pb 1.99, Sb 0.78, Se 5.15, Sn 3.16, Tl 0.28, and Zn 26.9. The metals easily released from leaves to infusions were: Tl, Se, Cd, Sn, Sb, Ni, Al, and Pb. No concentration of As, Hg, Mo, or Pb was found in the first infusion and no As, Co, Hg, or Mo was found in the second one. The hazard quotient values for particular metals found in the infusions and tea leaves and the appraised combined hazard index (HI) amounted to <1. The highest HI values, resulting from the consumption of both infusions, did not exceed 5.88E-03, or 6.94E-04a in the case of tea leaves. No health hazard for the Polish consumers of oolong teas was identified at any of the examined stages of consumption. However, we recommend discarding the first tea infusion to reduce the metal concentrations before consumption.     

Effect of ethanol on troponin calcium binding

The Chelex resin method was found to be suitable for studying drug effects on Ca2+ binding of proteins. In comparison to conventional dialysis techniques, the Chelex method has the following advantages: Ca2+-EGTA buffer is not necessary, free Ca2+ concentration as low as 10(-9) M can be determined directly, and the reaction is complete in 30 min, thus creating fewer problems with protein denaturation at elevated temperatures. Methods to cope with problems inherent to this assay, such as the excluded volume effect of the resin and protein adsorption by the resin are described. The validity of the method was confirmed by the measurements of Ca2+ binding of troponin in the presence and absence of Mg2+. Using this method, it was demonstrated that ethanol concentration as high as 25% does not influence the Ca2+ binding of troponin.     

Kinetics of photosynthetic electron transfer in artificial vesicles reconstituted with purified complexes from Rhodobacter capsulatus. II. Direct electron transfer between the reaction center and the bc1 complex and role of cytochrome c2

1. The cyclic photosynthetic chain of Rhodobacter capsulatus has been reconstituted incorporating into phospholipid liposomes containing ubiquinone-10 two multiprotein complexes: the reaction center and the ubiquinol-cytochrome-c2 reductase (or bc1 complex). 2. In the presence of cytochrome c2 added externally, at concentrations in the range 10-10(4) nM, a flash-induced cyclic electron transfer can be observed. In the presence of antimycin, an inhibitor of the quinone-reducing site of the bc1 complex, the reduction of cytochrome b561 is a consequence of the donation of electrons to the photo-oxidized reaction center. At low ionic strength (10 mM KCl) and at concentrations of cytochrome c2 lower than 1 microM, the rate of this reaction is limited by the concentration of cytochrome c2. At higher concentrations the reduction rate of cytochrome b561 is controlled by the concentration of quinol in the membrane, and, therefore, is increased when the ubiquinone pool is progressively reduced. At saturating concentrations of cytochrome c2 and optimal redox poise, the half-time for cytochrome b561 reduction is about 3 ms. 3. At high ionic stength (200 mM KCl), tenfold higher concentrations of cytochrome c2 are required for promoting equivalent rates of cytochrome-b561 reduction. If the absolute values of these rates are compared with those of the cytochrome-c2-reaction-center electron transfer, it can be concluded that the reaction of oxidized cytochrome c2 with the bc1 complex is rate-limiting and involves electrstatic interactions. 4. A significant rate of intercomplex electron transfer can be observed also in the absence of cytochrome c2; in this case the electron donor to the recation center is the cytochrome c1 of the oxidoreductase complex. The oxidation of cytochrome c1 triggers a normal electron transfer within the bc1 complex. The intercomplex reaction follows second-order kinetics and is slowed at high ionic strength, suggesting a collisional interaction facilitated by electrostatic attraction. From the second-order rate constant of this process, a minimal bidimensional diffusion coefficient for the complexes in the membrane equal to 3 X 10(-11) cm2 s-1 can be evaluated.     

Comparative study of the interaction of polyuridylic acid with 30S subunits and 70S ribosomes of Escherichia coli.

Fractionated polyuridylic acid with an average chain length of 55 nucleotides forms binary complexes with 30S subunits with a stoichiometry of I:I. These complexes are heterogeneous in stability. The more stable one is characterized by an association constant K2 - 5.5xI09 M-I, and the less stable-by KI = I06xM-I, at 20 mM Mg2+, 200 mM NH4(+) and 0 degrees C. The main reason for this heterogeneity is the presence or absence of the ribosomal protein SI in the presence or absence of the ribosomal protein SI in the subunits. Decrease of Mg2+ concentration down to 5 mM hardly changes the K2 values but reduction of the NH4(+) concentration to 50 mM results in a 25-fold increase of K2. Association constants K2 for the stable complex, i.e. in the presence of SI protein, were measured at different temperatures (0 - 30 degrees C) and the thermodynamic parameters of binding (delta H degrees, delta S degrees, delta G degrees) were determined. Analogous experiments were made with 70S ribosomes. K2 values as well as delta H degrees, delta S degrees, delta G degrees appeared the same both for 30S and 70S ribosomes in all conditions examined. This is strong evidence that the 50S subunits do not contribute to the interaction of poly(U) with the complete 70S ribosomes.     

Antifungal activity of aluminium‐containing salts against the development of carrot cavity spot and potato dry rot

As an alternative to the use of synthetic chemical fungicides to control plant disease, aluminium‐containing salts were evaluated for their effects on the mycelial growth of various fungal or fungus‐like pathogens and their ability to control carrot cavity spot (Pythium sulcatum) and potato dry rot (Fusarium sambucinum). Results showed that various aluminium‐containing salts provided strong inhibition of all the tested pathogens (Alternaria solani, Botrytis cinerea, F. sambucinum, P. sulcatum and Rhizopus stolonifer) with minimal inhibitory concentration of 1–10 mM. Aluminium chloride and aluminium sulphate were generally the most effective, inhibiting mycelial growth of pathogens by as much as 47% and 100%, respectively, at a salt concentration of 1 mM. Applied at 5 mM, aluminium sulphate also provided 28% and 100% inhibition of dry rot and cavity spot, respectively. Aluminium chloride (5 mM) reduced dry rot by 25% whereas aluminium lactate (5 mM) decreased cavity spot lesions by 86%. These results indicate that various aluminium‐containing salts may provide an alternative to the use of synthetic fungicides to control these pathogens.     

Protonation of nitrite is an obligatory stage in the generation of nitric oxide from nitrite in biological systems

The yield of nitric oxide from 1 mM sodium nitrite differs 200 times when the process was initiated by 10 mM sodium dithionite in the solution of 5 or 150 mM HEPES-buffer (pH 7.4). Dithionite acted both as a strong reductant and an agent that induced a local acidification of solutions without notable change in pH value. The amount of nitric oxide was estimated by the EPR method by measuring the incorporation of nitric oxide to water-soluble complexes of Fe with N-methyl-D-glucamine dithiocarbamate (MGD), which led to the formation of EPR-detectable mononitrosyl iron complexes with MGD (MNIC-MGD). Ten seconds after dithionite addition, the concentration of MNIC - MGD complexes reached 2 microM in 5 mM HEPES-buffer in contrast to 0.01 microM in 150 mM HEPES-buffer. The difference was suggested to be due to a higher life-time of zones with decreased pH values in a weaker weak buffer solution. The life-time was high enough to ensure the protonation of a part of nitrite. The resulting nitrous acid was decomposed to form nitric oxide. The difference in the formation of nitric oxide from nitrite was also observed in weak and strong buffer solutions in the presence of hemoglobin (0.3 mM) or serum albumin (0.5 mM). However, the ratios of nitric oxide yields in weak and strong buffer did not exceed 3-4 times. The increase in the formation of nitric oxide from nitrite was characteristic for the solutions containing both proteins. Large amounts of nitric oxide formed from nitrite was observed in mouse liver preparation subjected to freezing-thawing procedure followed by incubation in 150 mM HEPES-buffer (pH 7.4) and addition of dithionite. The proposition was made that the presence of zones with low pH value in cells and tissues can ensure the predominant operation of the acid mechanism formation of nitric oxide from nitrite. The contribution of the formation of nitric oxide from nitrite catalyzing with heme-containing proteins nitrite reductases can be minor one under these conditions.     

Covalent complexes formed between plasma gelsolin and actin with a zero-length cross-linking compound

Actin and plasma gelsolin were covalently cross-linked with the zero-length cross-linker 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide. Two major intermolecularly linked products were identified on polyacrylamide gels. By use of 14C-labeled actin and 125I-labeled gelsolin, these were shown to be the 1:1 and 2:1 complexes of actin with gelsolin, respectively. The higher molecular weight complex predominated under all conditions tested including the presence and absence of Ca2+. In titration experiments in which actin at different concentrations was reacted with a fixed concentration of gelsolin, end points were obtained for the formation of both cross-linked species at about two actins per gelsolin, implying that a 2:1 noncovalent complex is cross-linked. In 0.1 mM Ca2+, the extent of cross-linking was independent of protein concentration down to 50 nM gelsolin. At low Ca2+ concentrations (less than 10(-8)M), the extent of cross-linking was very much reduced at micromolar gelsolin and fell to zero at about 100 nM gelsolin. The binding of actin to gelsolin to give a cross-linkable complex is therefore very strong at 0.1 mM Ca2+ but much weaker at low Ca2+ concentrations.     

Electron spin resonance study on the formation of ascorbate free radical from ascorbate: The effect of dehydroascorbic acid and ferricyanide

Summary Ascorbate free radical is considered to be a substrate for a plasma membrane redox system in eukaryotic cells. Moreover, it might be involved in stimulation of cell proliferation. Ascorbate free radical can be generated by autoxidation of the ascorbate dianion, by transition metal-dependent oxidation of ascorbate, or by an equilibrium reaction of ascorbate with dehydroascorbic acid. In this study, we investigated the formation of ascorbate free radical, at physiological pH, in mixtures of ascorbate and dehydroascorbic acid by electron spin resonance spectroscopy. It was found that at ascorbate concentrations lower than 2.5 mM, ascorbate-free radical formation was not dependent on the presence of dehydroascorbic acid. Removal of metal ions by treatment with Chelex 100 showed that autoxidation under these conditions was less than 20%. Therefore, it is concluded that at low ascorbate concentrations generation of ascorbate free radical mainly proceeds through metal-ion-dependent reactions. When ascorbate was present at concentrations higher than 2.5 mM, the presence of dehydroascorbic acid increased the ascorbate free-radical signal intensity. This indicates that under these conditions ascorbate free radical is formed by a disproportionation reaction between ascorbate and dehydroascorbic acid, having aK _equil of 6 × 10^–17 M. Finally, it was found that the presence of excess ferricyanide completely abolished ascorbate free-radical signals, and that the reaction between ascorbate and ferricyanide yields dehydroascorbic acid. We conclude that, for studies under physiological conditions, ascorbate free-radical concentrations cannot be calculated from the disproportionation reaction, but should be determined experimentally.Abbreviations AFR ascorbate free radical - DHA dehydroascorbic acid - EDTA ethylenediaminetetraacetic acid - DTPA diethylenetri-aminepentaacetic acid - TEMPO 2,2,6,6-tetramethylpiperidinoxy     

Nitrite protonation as a necessary stage in the generation of nitric oxide from nitrite in biological systems

The yields of nitric oxide from 1 mM and 10 mM sodium dithionite in 5 or 150 mM solutions of HEPES buffer (pH 7.4) differed by a factor of 200. Dithionite acted as both a strong reducing agent and an agent responsible for local acidification of the solutions without significant changes in pH. The concentration of nitric oxide was estimated by electron paramagnetic resonance (EPR) by monitoring its incorporation into water-soluble complexes of Fe with N-methyl-D-glucamine dithiocarbamate (MGD), which resulted in the formation of EPR-detectable mononitrosyl complexes of iron. Ten seconds after dithionite addition, the concentration of mononitrosyl iron complexes reached 2 μM, whereas it did not become greater than 0.01 μM in 5 mM HEPES buffer. It has been suggested that this difference results from a longer lifetime of a localized decrease in pH in a weaker buffer solution. This time could be long enough for the protonation of some nitrite molecules. Nitrous acid thus formed decomposed to nitric oxide. A difference in nitric oxide formation from nitrite in weak and strong buffer solutions was also observed in the presence of hemoglobin (0.3 mM) or serum albumin (0.5 mM). However, in the weak buffer the nitric oxide yield was only three-four times greater than in the strong buffer. An increase in the nitric oxide yield from nitrite was observed in solutions containing both proteins. A significant amount of nitric oxide from nitrite was formed in mouse liver preparation subjected to freezing and thawing procedure followed by slurrying in 150 mM HEPES buffer (pH 7.4) and dithionite addition (10 mM). We suggest that the presence of zones with lowered pH values in cells and tissues may be responsible for the predominance of the acidic mechanism of nitric oxide formation from nitrite. The contribution of nitric oxide formation from nitrite catalyzed by heme-containing proteins as nitrite reductases may be minor under these conditions.     

Alleviation of Aluminum Stress and Stimulation of Tea Pollen Tube Growth by Fluorine

Aluminum (Al) and fluorine (F) were found to affect tea pollentube growth on an agar medium. Not only was the growth stronglyrepressed by increasing Al content of the medium but it wasalso distinctly affected by declining pH from 5.2 to 4.4. Additionof 0.2 mM Al as Al₂(SO₄)₃ to a pH 4.6 medium containing 1.2%agar, 8% sucrose and 17 ppm boron remarkably repressed tea pollentube growth. However, NaF added to medium containing Al clearlyalleviated the growth inhibition. This effect was observed with0.2 mM and 0.4 HIM NaF, and the presence of 0.6 mM and 1.2 DIMNaF with 0.2 mM Al even produced a stimulatory effect. Treatmentwith NaF alone significantly stimulated growth at pH 4.6 and5.2. These results indicate that Al-F complexes have a favorablerather than adverse effect on tea pollen tube growth. (Received November 22, 1982; Accepted April 20, 1983)     

A comparison of the in vitro activity of DNA-armed and all-RNA hammerhead ribozymes.

Hammerhead ribozymes targeted against two unrelated RNA substrates have been prepared. For each substrate, four ribozymes, differing in their hybridising arm length and composition (DNA or RNA), have been synthesised and kinetically characterised. The presence of DNA in the hybridising arms had little effect on the overall cleavage rate when the cleavage step was rate determining. Shortening each of the hybridising arms of ribozymes from 10 to 6 nucleotides generally resulted in modest changes in rate constants for cleavage of the same 13mer substrate. In one case the presence of long RNA hybridising arms significantly impeded the cleavage reaction. Cleavage rates displayed first order dependence on hydroxide ion concentration at low pHs. At higher pH, some ribozymes deviated from this first order dependence because of a change in the rate-determining step, possibly due to a requirement for a conformation change in the ribozyme-substrate complex prior to cleavage. Ribozyme cleavage was strongly dependent on temperature in the range 5-45 degrees C, with an activation energy for the reaction of approximately 60 kJ mol-1. The ribozymes displayed biphasic dependence on magnesium ion concentration; evidence of strong apparent binding (Kd approximately 10 mM) as well as a looser interaction was observed for all ribozymes.     

A study of the kinetics of iron and copper binding to hen ovotransferrin.

The kinetics of iron and copper binding to hen's-egg apo-ovotransferrin were studied by using citrate chelates of these metals at pH9.3 in borate buffer in the presence of bicarbonate. The kinetics of the absorbance change associated with the formation of the final product show a fast process, which is pseudo-first-order, where the reagents are in excess with respect to the protein, and the citrate concentration is higher than 25mM. At lower citrate concentration, the progress curves are clearly biphasic. There is marked dependence of the rate of the reaction on bicarbonate concentration, which may be interpreted as a displacement reaction of the ligand-metal-protein ternary complex. The kinetics have been interpreted in the framework of a reaction scheme which involves bimolecular reaction of a metal chelate to the protein and subsequent colour development by displacement of the chelator by bicarbonate. The pH-dependence of this reaction supports the belief that tyrosine residues are involved in the process of iron-binding. The overall similarity of kinetics for iron and copper binding, notwithstanding their different co-ordination preferences, suggests that the process of metal-binding or chromophore development for the two metal complexes must be similar.     

UDP-N-acetylmuramoyl-L-alanyl-D-glutamyl-L-lysine synthetase from Bacillus sphaericus: activation by potassium phosphate

During the course of purification of UDP-N-acetylmuramoyl-L-alanyl-D-glutamyl-L-lysine synthetase, we observed a marked stimulation of the enzymatic activity in the presence of phosphate ions. This activation effect was studied with enzyme purified 979-fold from Bacillus sphaericus. Each salt tested stimulated the activity of the synthetase. The order of activation by different anions was HPO4(2-) greater than Cl- greater than SO4(2-). In every case, the potassium salt gave higher activity than the corresponding sodium salt. The activation in the presence of phosphate was quite pronounced (almost sevenfold with K2HPO4) and occurred at a relatively low concentration. The Ka for K2HPO4 was found to be 3.4 mM and the Hill coefficient was calculated to be 1.0. This would suggest that there is one phosphate-binding site per active centre. The presence of phosphate did not affect either the pH optimum of this enzyme or the optimum concentration of Mg2+ required. The presence of phosphate has little or no effect on the Km of any of the substrates. Thus, it appears that the presence of phosphate changes the enzyme conformation to a catalytically more active form. The activation of this enzyme in the presence of phosphate anion is all the more interesting because phosphate is a product of the reaction catalyzed by this enzyme.     

Regulation of liver glycogen synthase phosphatase activity by ATP-Mg

The kinetics of a synthase phosphatase reaction inhibited by ATP-Mg in a liver glycogen particle preparation were complex. In the presence of a physiological concentration of ATP-Mg, synthase phosphatase activity in the glycogen particle follows a biphasic course. Initially, the reaction was inhibited but later the reaction rate accelerated. The reaction was inhibited but the rate was constant in the presence of ATP-Mg with the addition of a physiological concentration of glucose 6-phosphate (Glc 6-P). Therefore, in most subsequent experiments Glc 6-P was added. The concentration of ATP-Mg at which 50% maximal inhibition (I0.5) occurred was approximately 0.1 mM in preparations obtained from rats given glucagon prior to being killed. In preparations from animals given glucose, the I0.5 was increased to 2.0 mM. The maximum inhibition was little changed in preparations from glucose- or glucagon-treated animals. Thus, administration of glucose in vivo reduced the sensitivity of the synthase phosphatase to ATP-Mg inhibition. Complexes of ATP with paramagnetic ions such as Co2+ and Mn2+ were less inhibitory than complexes with diamagnetic ions, including Ca2+ and Mg2+. Magnesium complexes of adenosine tetraphosphate and 5'-adenylimidodiphosphate also were inhibitory. Inhibition was independent of phosphorylase a and not a nonspecific, polyvalent anion effect. The best explanation for the distinctive effects of ATP-Mg in preparations from glucagon- and glucose-treated animals is that the respective treatments promote and stabilize different forms of synthase D or possibly synthase phosphatase with different affinities for ATP-Mg. These forms are interconvertible, as previously suggested, in studies employing EDTA (20).