Effects of parathyroidectomy on tissue calcium,phosphorus, magnesium,and copper concentrations in aluminum-loaded uremic rats

Rats were subjected to a two-stage 5/6 nephrectomy and treated with Al for 2 and 4 wk with a cumulative dose of 4.2 and 8.4 mg of Al, respectively. Other animals were parathyrectomized (PTx) and loaded with 8.4 mg of Al for 4 wk. Total Al, Ca, P, Mg, and Cu contents were analyzed in the liver, kidney, and bone by inductively coupled plasma atomic emission spectrometry (ICP-AES). The results showed that Al given to growing uremic rats significantly increased the content of Al in the liver, kidney, and bone. Moreover, Al treatment increased the liver and kidney Ca levels and decreased the Ca and P values in bone. Previous parathyroidectomy significantly reduced Al accumulation within organs and changes in the Ca and P levels in the bone, liver, and kidney. The result was not influenced by different degrees of renal failure.     

Magnesium ameliorates aluminum rhizotoxicity in soybean by increasing citric acid production and exudation by roots

Superior effectiveness of Mg over Ca in alleviating Al rhizotoxicity cannot be accounted for by predicted changes in plasma membrane Al3+ activity. The influence of Ca and Mg on the production and secretion of citrate and malate, and on Al accumulation by roots was investigated with soybean genotypes Young and PI 416937 which differ in Al tolerance. In the presence of a solution Al3+ activity of 4.6 microM, citrate and malate concentrations of tap root tips of both genotypes increased with additions of either Ca up to 3 mM or Mg up to 50 microM. Citrate efflux rate from roots exposed to Al was only enhanced with Mg additions and exceeded malate efflux rates by as much as 50-fold. Maximum citrate release occurred within 12 h after adding Mg to solution treatments. Adding 50 microM Mg to 0.8 mM CaSO4 solutions containing Al3+ activities up to 4.6 microM increased citrate concentration of tap root tips by 3- to 5-fold and root exudation of citrate by 6- to 9-fold. Plants treated with either 50 microM Mg or 3 mM Ca had similar reductions in Al accumulation at tap root tips, which coincided with the respective ability of these ions to relieve Al rhizotoxicity. Amelioration of Al inhibition of soybean root elongation by low concentrations of Mg in solution involved Mg-stimulated production and efflux of citrate by roots.     

Kinetics of rapid Ca2+ release by sarcoplasmic reticulum. Effects of Ca2+, Mg2+, and adenine nucleotides

A radioisotope flux-rapid-quench-Millipore filtration method is described for determining the effects of Ca2+, adenine nucleotides, and Mg2+ on the Ca2+ release behaviour of "heavy" sarcoplasmic reticulum (SR) vesicles. Rapid 45Ca2+ efflux from passively loaded vesicles was blocked by the addition of Mg2+ and ruthenium red. At pH 7 and 10(-9) M Ca2+, vesicles released 45Ca2+ with a low rate (k = 0.1 s-1). An increase in external Ca2+ concentration to 4 microM or the addition of 5 mM ATP or the ATP analogue adenosine 5'-(beta,gamma-methylenetriphosphate) (AMP-PCP) resulted in intermediate 45Ca2+ release rates. The maximal release rate was observed in media containing 4 microM Ca2+ and 5 mM AMP-PCP and had a first-order rate constant of 30-100 s-1. Mg2+ partially inhibited Ca2+- and nucleotide-induced 45Ca2+ efflux. In the absence of AMP-PCP, 45Ca2+ release was fully inhibited at 5 mM Mg2+ or 5 mM Ca2+. The composition of the release media was systematically varied, and the flux data were expressed in the form of Hill equations. The apparent n values of activation of Ca2+ release by ATP and AMP-PCP were 1.6-1.9. The Hill coefficient of Ca2+ activation (n = 0.8-2.1) was dependent on nucleotide and Mg2+ concentrations, whereas the one of Mg2+ inhibition (n = 1.1-1.6) varied with external Ca2+ concentration. These results suggest that heavy SR vesicles contain a "Ca2+ release channel" which is capable of conducting Ca2+ at rates comparable with those found in intact muscle. Ca2+, AMP-PCP (ATP), and Mg2+ appear to act at noninteracting or interacting sites of the channel.     

Factors controlling spatial distribution of soil acidification and Al forms in forest soils

Soil acidification and Al release in forest soils is controlled by a number of factors, like acid deposition, forest type, parent rock, altitude, etc. This paper studies the principal stand factors affecting spatial distribution of the content of KCl-extractable Al (Al(KCl), mainly exchangeable), Na4P2O7-extractable Al (Al(Na4P2O7), mainly organically bound), and other soil characteristics related to acidification in surface organic (O) and subsurface mineral (B) horizons in the Jizera Mountains region. Geostatistical methods were exploited. The highest Al(KCl) contents in the O horizons were related to high S and N content, low pH and low Ca and Mg content in soil. Liming decreased Al(KCl) contents in the O horizons. Al(Na4P2O7) in the O horizons was more abundant under spruce than under beech; in both horizons it was increased on the immission clear-cut areas populated by grass. Surface horizons are more sensitive to external influence (acid deposition, liming) and their spatial variation is stronger. In the mineral horizons, the effect of pedogenetic processes is more important. The effect of stand factors on Al behaviour is complex and often indirect, mediated for example by organic matter or soil reaction. It is difficult to clearly distinguish the effects of the particular factors.     

Effect of Sodium and Magnesium on Kinetics of Phosphorus Release in Some Calcareous Soils of Western Iran

Knowledge of the rate of release of phosphorus (P) from soils resulting from poor water quality application is essential for long-term planning of crop production while minimizing the impact on groundwater quality. In this study, we examined the effect of sodium adsorption ratio (SAR) and Ca:Mg ratio of water on P release of some calcareous soils from western Iran. Nine different solutions at a total electrolyte concentration of 100 mmol_c l^?1 and three levels of SAR (5, 15, 45) each with Ca:Mg ratios of 1:3, 1:1, or 3:1, prepared using solutions of NaCl, CaCl₂, and MgCl₂, were used to extract P from the soils. The geochemical Visual MINTEQ was used to calculate saturation indices and P species at the initial and end of P release. Significantly different quantities of P were extracted by the solutions. The maximum (average of five soils) (233.6 mg kg^?1) and the minimum (162.9 mg kg^?1) P were extracted by an SAR 45 solution with a Ca:Mg ratio of 1:3 and SAR 15 solution with Ca:Mg ratio of 3:1, respectively. Elovich model adequately described P release. The release rate for SAR 15 with Ca:Mg of 3:1 and SAR 45 with Ca:Mg 1:3 ranges from 16.3 to 31.3 mg kg^?1 h^?1 and from 20.0 to 32.8 mg kg^?1 h^?1, respectively. In the initial stage of P release the solution samples in most soils were saturated with respect to hydroxyapatite, octacalcium phosphate, ß-tricalcium phosphate, and undersaturated with respect to dicalcium phosphate dihydrate, dicalcium phosphate, and mangnesium phosphates. At the end of P release, all solutions were saturated with respect to hydroxyapatite and under saturated with respect to other phosphate minerals. The results imply that P release from soils could be increased during use of saline and sodic irrigation water containing high Mg concentration and that P fertilization management may need modification.     

Characterisation of the mineral fraction in extracellular polymeric substances (EPS) from activated sludges extracted by eight different methods

This work characterises the mineral fraction of EPS extracts obtained using eight different methods from two activated sludges by total mineral content determination, Fourier Transformed Infrared spectrometry and with scanning electron microscopy coupled with an EDX probe. Despite EPS dialysis, the EPS extracts displayed a mineral fraction between 2% and 40% of the EPS dry weight depending on the extraction method used. The main mineral elements found in the EPS extract were Ca, Mg, Na, K, Al, Fe, Mn, P, Si and S, but their contents were strongly affected by the extraction method used. Some of the minerals are associated with the organic molecules within the EPS. The presence of mineral particles of various compositions and structures (clays, quartz or carbonate) in the EPS extract with a wide range in size was clearly demonstrated. Moreover, the association of metallic elements with the mineral particles in the EPS extract was highlighted.     

Silver ions trigger Ca2+ release by acting at the apparent physiological release site in sarcoplasmic reticulum

Rapid Ca2+ release from Ca2+ -loaded sarcoplasmic reticulum vesicles (SR) was previously shown to occur upon the addition of micromolar concentrations of heavy metals, and the extent of Ca2+ release was dependent on the binding affinity of the metal to sulfhydryl group(s) on an SR protein (Abramson, J.J., Weden, L., Trimm, J.L., and Salama, G. (1982) Biophys. J. 37, 134a; Abramson, J.J., Trimm, J.L., Weden, L., and Salama, G. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 1526). The nature of this Ca2+ release site was examined further and found to be predominantly distributed in heavy SR (HSR) rather than light SR fractions. Ag+ -induced Ca2+ release from heavy SR was blocked by local anesthetics and ruthenium red which are known to inhibit Ca2+ release in skeletal fibers and in heavy SR, respectively. The rate of Ca2+ efflux from SR triggered by Ag+ was dependent on pH, Mg2+, and ionic strength of the medium. Efflux rates increased by a factor of 4 from pH 6.0 to 7.0 and then decreased in more alkaline reaction mixtures. Efflux rates from actively or passively loaded SR increased by a factor of 2.5 with increasing Mg2+ from 0 to 1 mM and then decreased in the range of 1 to 10 mM Mg2+. ATP-dependent Ca2+ uptake by SR was similar in 100 mM KCl and in 200 mM sucrose solutions, but the extent and rate of Ca2+ efflux induced by Ag+ were dramatically reduced with decreasing ionic strength of the medium. In solutions containing 5 mM Mg2+, the rate of Ca2+ efflux from heavy SR averaged over the first 1.5 s after the addition of Ag+ was 58 nmol of Ca2+/mg of SR/s, a value comparable to the fast initial rate of ATP-dependent Ca2+ uptake. The maximum initial rate of Ag+ -induced Ca2+ efflux from heavy SR in 1 mM Mg2+ may be comparable to the rate of Ca2+ release and tension development in muscle fibers. Our data indicate that Ag+ reacts with a protein or proteins in the SR, probably not the (Ca2+, Mg2+)-ATPase, to induce a rapid release of Ca2+, possibly from the physiological Ca2+ release site.     

The effect of aluminium on the distribution of calcium,magnesium and phosphorus in mycorrhizal and non-mycorrhizal seedlings of Eucalyptus rudis: a cryo-microanalytical study

The distribution of Al, Ca, Mg and P in the lateral roots and leaves of mycorrhizal and non-mycorrhizal seedlings of Eucalyptus rudis grown with and without Al was analysed using energy-dispersive X-ray microanalysis on a cryo-scanning electron microscope. Al accumulated in all tissues of nonmycorrhizal plants: the endodermis was not a barrier to the translocation of Al. In mycorrhizal roots, Al was concentrated within the sheath. The presence of Al reduced the levels of Ca and Mg in both mycorrhizal and non-mycorrhizal roots and shoots in comparison with control plants. The presence of mycorrhizas increased the levels of Ca and Mg in plants grown with Al in comparison with non-inoculated plants, although there was no evidence that mycorrhizas increased the levels of P in plants grown in Al-amended soils. P levels were higher in the mycorrhizal sheath of plants grown with Al than the controls.     

Soil acidity effects on nutrient use efficiency in exotic maize genotypes

Maize (t Zea mays L.) is the third most important cereal grown in the world. In South and Central America, maize is mostly grown on acidic soils. On these soils, yields are limited by deficient levels of available P, Ca, and Mg, and toxic levels of Al and Mn. A greenhouse study was conducted with 22 maize genotypes originating from Africa, Europe, and North, Central, and South America on acid, dark red latosol (Typic Haplorthox) at 2%, 41%, and 64% Al saturation at corresponding pH of 5.6, 4.5, and 4.3. With increasing Al levels, the nutrient efficiency ratios (NER = mgs of dry shoot weight / mg of element in shoot) for K, Ca and Mg increased, and NER for P and Zn tended to decrease. Overall, Al-tolerant genotypes produced higher shoot and root weight and had higher NER for P, Ca Mg, and Fe at 41% Al saturation. Genotypes used in this study showed genetic diversity for growth and NER of essential nutrients. It was concluded that selection of acid-soil-tolerant genotypes and further breeding of acid-soil-tolerant maize cultivars are feasible.     

Bioleaching of Metals from Mineral Waste Product

The possibility of bioleaching Al, K, Na, Ca and Mg using microorganisms of the Thiobacillus thiooxidans group from industrial waste product (IWP) of copper ore flotation from the company MEDET was studied. The aim of the investigations was to establish the possible application of a combined method for processing IWP. The preliminary mechanical activation in combination with bioleaching resulted in a high extent of extraction of useful components. It was established that the removal of useful components from mechanically activated IWP is improved compared to non‐activated IWP. The effect of the concentration of Al‐containing waste product, of incubation time and time of preliminary mechanical activation on the extraction degree (α) [% w/w] of useful elements was investigated. The maximum degree of extraction of Al was achieved on Day 28 and its value reached 71% for industrial waste product mechanically activated for 4 hours. The maximum degrees of extraction of K and Na in the case of industrial waste product mechanically activated for 4 hours were achieved on Day 7 of the incubation period and their values were 78% and 91%, respectively. Under the conditions of bioleaching only Si had a low degree of extraction, accounting for 2.5%. The ability of microorganisms to leach aluminium could be used for the extraction of metals from nonbauxite raw materials and Al‐containing waste product not treatable by means of the BAYER method.     

Aluminium effects on growth, nutrient net uptake and transport in 3 rice (Oryza sativa) cultivars with different sensitivity to aluminium

Varietal differences in net nutrient uptake rate and transport efficiency in the presence of aluminium have seldom been investigated in rice. Therefore, effects of Al on growth, uptake and transport of macronutrients (K, P, Ca, and Mg) and micronutrients (Fe, Zn, Cu, and Mn) were evaluated in 3 rice cultivars (BG35, DA14 and IR45) with different Al sensitivity. The plants were grown in nutrient solution at pH 4.1. An initial growth was completed in the time interval 1 to 5 days immediately before the addition of Al. The final growth period with Al (0, 140, 280 or 560 μ M ) was completed on day 26. With Al, a comparatively high P accumulation occurred in shoots and roots of the Al tolerant cultivar BG35. In contrast, the Al sensitive cultivar IR45 maintained a relatively high Ca accumulation during the Al treatment. A reduced total net uptake rate of P and Ca by IR45 in the time period 5 to 26 days was due to both a reduced root fresh weight and a reduced net uptake rate per g fresh weight of root. Moreover, net Ca transport to the shoots higher than net uptake rate in DA14 and IR45 at > 140 μ M Al during the test period suggests restricted Ca uptake by the roots in combination with a continuous net loss of Ca from the roots to the shoots as time proceeds. In the case of Mg and Mn, there was a general reduction of net uptake rates, irrespective of Al sensitivity of cultivars. With Al treatment, comparatively high accumulation of Fe, Zn and Cu occurred in the roots of IR45, concomitant with a high net Zn and Cu uptake rate. It is concluded that differences in Al sensitivity among rice cultivars BG35, DA14 and IR45 are not primarily linked to the depressed internal Mg or Mn status of the plants but rather to changes in the uptake and distribution of Ca and P.     

Effects of root zone temperature on aluminium toxicity in two cultivars of spring wheat with different resistance to aluminium

Manifestations of aluminium (Al) toxicity in two cultivars of wheat ( Triticum aestivum L. cvs Kadett [relatively Al-resistant] and WW 20299 [relatively Al-sensitive]) were investigated at two root zone temperatures (RZT) that may occur in the field. The plants were grown for 9 days at 10 or 25°C RZT. Mineral nutrients other than CaSO₄ were supplied daily in exponentially increasing amounts to meet the demand of the plants. Al was added as Al₂(SO₄)₃ at the beginning of the culture period at concentrations ranging from 0 to 100 μ M . pH was kept constant at 4.1. Experimental data were analysed for interactions between Al and RZT on a fresh weight basis by the nonlinear Weibull function. Cultivar Kadett, when grown at 25°C RZT, was more resistant to Al than when grown at 10°C RZT. Cultivar WW 20299 was equally sensitive to Al at 10 and 25°C RZT but generally more sensitive to Al than cv. Kadett. It is suggested that cv. Kadett, in contrast to cv. WW 20299, possesses a mechanism for Al resistance that is less effective at 10°C than at 25°C RZT and therefore may be metabolically dependent. In roots, the concentrations of K, P, Mg and Ca were not negatively affected by Al or by RZT. In shoots of both cultivars the concentrations of Ca and Mg became comparatively low when the plants were treated with Al or at low RZT, the effect being larger for Ca than for Mg. At 10°C RZT under Al stress, the Ca concentrations in shoots approached the critical concentration where growth may be inhibited. As no Al was detected in the shoots, it is suggested that Al in the roots inhibits shoot growth by reducing transport of Ca from roots to shoots.     

Alleviation of soil acidity in Ultisol and Oxisol for corn growth

Malaysian Ultisols and Oxisols are characterized by low pH, high soil solution Al concentration and Ca and/or Mg deficiencies, which are limiting to corn growth. An experiment was conducted to determine the changes in solid and soil solution phase properties of a representative Ultisol and Oxisol following applications of ground magnesium limestone (GML), gypsum and their combinations, and their effects on corn growth. A plot of pAl against lime potential (pH-1/2 pCa) showed that the points were mostly positioned between the theoretical lines for kaolinite-quartz and gibbsite equilibrium, reflecting the kaolinitic-oxidic mineralogy of the Ultisol and Oxisol. Gypsum application increased Al concentration in the soil solutions of the Ultisol, but had no significant effect on that of the Oxisol. The increase in Al concentration in the Ultisol was due to an increase in ionic strength. Gypsum application increased soil solution pH of the Oxisol due to release of OH as a result of ligand exchange between SO₄ and OH ions on the oxides of Fe and/or Al. Exchangeable Al in both soils was reduced by gypsum application. The reduction was associated with solid phase immobilization through alunite formation; the soil solutions of soil samples treated with 2 and 4 t gypsum ha⁻¹ were supersaturated with respect to alunite. Application of GML at 2 t ha⁻¹ together with 1–2t gypsum ha⁻¹ gave high top weight of corn. Relative top weight of corn was positively correlated with a soil solution Mg and Ca/Al concentration ratio, but negatively correlated with soil solution Al concentration. Foliar Al corn was positively correlated with soil solution Al concentration. Soil solution Al and Mg concentrations, and Ca/Al concentration ratio can be used as indices of soil acidity in Ultisols and Oxisols. ei]{gnB E}{fnClothier}     

Studies on an activator of the (Ca2+ plus Mg2+)-ATPase of human erythrocyte membranes.

1. An activator of the (Ca2+ plus Mg2+)-stimulated ATPase present in the human erythrocytes (membrane) has been isolated in soluble form from hemolysates of these cells. Partial purification has been achieved through use of carboxymethyl-Sephadex chromatography. The resulting activator fraction contained no hemoglobin and only 0.3% of the total adenylate kinase activity of the cell. 2. Whereas the activator was released from erythrocytes subjected to hemolysis in 20 miosM buffer at pH 7.6 or at pH 5.8, only the membranes prepared at pH 7.6 were affected by it. 2. Whereas the activator was released from erythrocytes subjected to hemolysis in 20 miosM buffer at pH 7.6 or at pH 5.8, only the membranes prepared at pH 7.6 were affected by it. 3. When (Ca2+ plus Mg2+)-ATPase activity was measured by 32Pi release from (gamma-32P)ATP, freeze-thawed erythrocytes, as well as membranes prepared at pH 5.8 and at pH 7.6, expressed lower values than noted by assay for total Pi release. When ADP instead of ATP was used as substrate, significant amount of Pi were released by these erythrocyte preparations. Further study revealed (a) production of ATP and AMP from ADP with membranes and hemolysate alone, and (b) exchange of the gamma-and B-position phosphate on (gama-32P)ATP in the presence of membranes plus hemolysates. These observations established the presence of adenylate kinase activity in the (membrane-free) hemolysates and in membranes. It further supports the conclusion that Pi release from ADP by human erythrocytes (freeze-thawed) and by their isolated membranes is due to formation of ATP by adenylate kinase and hydrolysis of this generated ATP by (Ca2+ plus Mg2+)-ATPase. 4. The following points were also established: (a) absence of an ADPase in human erythrocytes; (b) the (Ca2+ plus Mg2+)-ATPase activator enhanced cleavage only of the gama-position of ATP and (c) the (Ca2+ plus Mg2+)-ATPase activator is neither adenylate kinase nor hemoglobin.     

Magnesium binding to DM-nitrophen and its effect on the photorelease of calcium

The effect of Mg(2+) on the process of Ca(2+) release from the caged Ca(2+) compound DM-nitrophen (NP) was studied in vitro by steady light UV photolysis of NP in the presence of Ca(2+) and Mg(2+). Ca(2+) release during photolysis and its relaxation/recovery after photolysis were monitored with the Ca(2+)-sensitive dye fura-2. Mg(2+) speeds the photorelease of Ca(2+) during photolysis and slows the relaxation of Ca(2+) to new steady-state levels after photolysis. Within the context of a model describing NP photolysis, we determined the on and off rates of Mg(2+) binding to unphotolyzed NP (k(on) = 6.0 x 10(4) M(-1) s(-1); k(off) = 1.5 x 10(-1) s(-1)). Furthermore, to fully account for the slow postphotolysis kinetics of Ca(2+) in the presence of Mg(2+) we were forced to add an additional photoproduct to the standard model of NP photolysis. The additional photoproduct is calculated to have a Ca(2+) affinity of 13.3 microM and is hypothesized to be produced by the photolysis of free or Mg(2+)-bound NP; photolysis of Ca(2+)-bound NP produces the previously documented 3 mM Ca(2+) affinity photoproduct.     

Changes in Ca2+ affinity related to conformational transitions in the phosphorylated state of soluble monomeric Ca2+-ATPase from sarcoplasmic reticulum

Changes in Ca2+ binding after phosphorylation of membranous or detergent-solubilized preparations of sarcoplasmic reticulum Ca2+-ATPase with ATP were followed spectrophotometrically by the use of murexide. Distinct Ca2+ release from the two high-affinity translocation sites was observed, particularly at alkaline pH and at low Ca2+/Mg2+ concentration ratios. Phosphorylation also induced additional binding of Ca2+ at a third site in competition with Mg2+. Ca2+ release was increased after solubilization of Ca2+-ATPase in predominantly monomeric form with the nonionic detergent octaethyleneglycol monododecyl ether. At 0 degree C, chemical-quench studies with [32P]ATP indicated that release of Ca2+ is correlated with the level of ADP-insensitive phosphoenzyme (2 mol of Ca2+ released per mol of E2P formed), both for membranous and detergent solubilized Ca2+-ATPase. Ca2+ release was also found to be accompanied by changes in intrinsic fluorescence. Analysis of the data at 20 degrees C, pH 8.0, showed that binding of Ca2+ to transport sites on E2P occurs with a half-saturation constant of 0.7 mM and a Hill coefficient of 1.8. This is consistent with a drastic decrease in Ca2+ affinity following conversion of ADP-sensitive E1P to ADP-insensitive E2P. The similarity between membranous and detergent-solubilized Ca2+-ATPase supports the view that not more than a single Ca2+-ATPase polypeptide chain is required to complete the conformational transitions which are the basis for active transport of Ca2+.     

The kinetic mechanism of yeast inorganic pyrophosphatase

The kinetic mechanism of yeast inorganic pyrophosphatase (PPase) was examined by carrying out initial velocity studies. Ca2+ and Rh(H2O)4(methylenediphosphonate) (Rh(H2O)4PCP) were used as dead-end inhibitors to study the order of binding of Cr(H2O)4PP to the substrate site and Mg2+ to the "low affinity" activator site on the enzyme. Competitive inhibition was observed for Ca2+ vs Mg2+ (Kis = 0.93 +/- 0.03 mM), for Rh(H2O)4PCP vs Cr(H2O)4PP (Kis = 0.25 +/- 0.07 mM), and for RH(H2O)4PCP vs Mg2+ (Kis = 0.38 +/- 0.03 mM). Uncompetitive inhibition was observed for Ca2+ vs Cr(H2O)4PP (Kii = 0.49 +/- 0.01). On the basis of these results a rapid equilibrium ordered mechanism in which Cr(H2O)4PP binding precedes Mg2+ ion binding is proposed. The inert substrate analog, Mg(imidodiphosphate) (MgPNP) was shown to induce Mg2+ inhibition of the PPase-catalyzed hydrolysis of MgPP. The Mg2+ inhibition observed was competitive vs MgPP and partial. These results suggest that Mg2+/MgPNP release from the enzyme occurs in preferred rather than strict order and that the Mg2+/MgPP-binding steps are at steady state. Zn2+, Co2+, and Mn2+ (but not Mg2+) displayed activator inhibition of the PPase-catalyzed hydrolysis of PPi (this study) and of Cr(H2O)4PP (W.B. Knight, S. Fitts, and D. Dunaway-Mariano, (1981) Biochemistry 20, 4079). These findings suggest that cofactor release from the low affinity cofactor site on the enzyme must precede product release and that Zn2+, Mn2+, and Co2+ (but not Mg2+) have high affinities for the cofactor sites on both the PPase.M.MPP and PPase.M.M(P)2 complexes. The role of the metal cofactor in determining PPase substrate specificity was briefly explored by testing the ability of the Mg2+ complex of tripolyphosphate (PPPi) (a substrate for the Zn2+-activated enzyme but not the Mg2+-activated enzyme) to induce Mg2+ inhibition of PPase-catalyzed hydrolysis of MgPP. MgPPP was shown to be as effective as MgPNP in inducing competitive Mg2+ inhibition (vs MgPP). This result suggests that the low affinity Mg2+ cofactor-binding site present in the enzyme-MgPP complex is maintained in the enzyme-MgPPP complex. Thus, failure of Mg2+ to bind to the enzyme-MgPPP complex was ruled out as a possible explanation for the failure of the Mg2+-activated enzyme to catalyze the hydrolysis of MgPPP.     

Influence of Ca2+ and Mg2+ on the turnover of the phosphomonoester group of phosphatidylinositol 4-phosphate in human erythrocyte membranes.

In isolated erythrocyte membranes, increasing the free Mg2+ concentration from 0.5 to 10 mM progressively activates the membrane-bound phosphatidylinositol (PtdIns) kinase and leads to the establishment of a new equilibrium with higher phosphatidylinositol 4-phosphate (PtdIns4P) and lower PtdIns concentrations. The steady-state turnover of the phosphomonoester group of PtdIns4P also increases at high Mg2+ concentrations, indicating a simultaneous activation of PtdIns4P phosphomonoesterase by Mg2+. Half-maximum inhibition of PtdIns kinase occurs at 10 microM free Ca2+ in the presence of physiological free Mg2+ concentrations. Increasing free Mg2+ concentrations overcome Ca2+ inhibition of PtdIns kinase. In the presence of Ca2+, calmodulin activates Ca2+-transporting ATPase 5-fold, but does not alter pool size and radiolabelling of PtdIns4P. In intact erythrocytes, adding EGTA or EGTA plus Mg2+ and the ionophore A23187 to the external medium does not exert significant effects on concentration and radiolabelling of polyphosphoinositides when compared with controls in the presence of 1.4 mM free Ca2+.     

The polyphosphoinositide phosphodiesterase of erythrocyte membranes

1. A new assay procedure has been devised for measurement of the Ca(2+)-activated polyphosphoinositide phosphodiesterase (phosphatidylinositol polyphosphate phosphodiesterase) activity of erythrocyte ghosts. The ghosts are prepared from cells previously incubated with [(32)P]P(i). They are incubated under appropriate conditions for activation of the phosphodiesterase and the released (32)P-labelled inositol bisphosphate and inositol trisphosphate are separated by anion-exchange chromatography on small columns of Dowex-1 (formate form). When necessary, phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate can be deacylated and the released phosphodiesters separated on the same columns. 2. The release of both inositol bisphosphate and inositol trisphosphate was rapid in human ghosts, with half of the labelled membrane-bound phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate broken down in only a few minutes in the presence of 0.5mm-Ca(2+). For both esters, optimum rates of release were seen at pH6.8-6.9. Mg(2+) did not provoke release of either ester. 3. Ca(2+) provoked rapid polyphosphoinositide breakdown in rabbit erythrocyte ghosts and a slower breakdown in rat ghosts. Erythrocyte ghosts from pig or ox showed no release of inositol phosphates when exposed to Ca(2+). 4. In the presence of Mg(2+), the inositol trisphosphate released from phosphatidylinositol 4,5-bisphosphate was rapidly converted into inositol bisphosphate by phosphomonoesterase activity. 5. Neomycin, an aminoglycoside antibiotic that interacts with polyphosphoinositides, inhibited the breakdown of both phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate, with the latter process being appreciably more sensitive to the drug. Phenylmethanesulphonyl fluoride, an inhibitor of serine esterases that is said to inhibit phosphatidylinositol phosphodiesterase, had no effect on the activity of the erythrocyte polyphosphoinositide phosphodiesterase. 6. These observations are consistent with the notion that human, and probably rabbit and rat, erythrocyte membranes possess a single polyphosphoinositide phosphodiesterase that is activated by Ca(2+) and that attacks phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate with equal facility. Inhibition of this activity by neomycin seems likely to be due to interactions between neomycin and the polyphosphoinositides, with the greater inhibition of phosphatidylinositol 4,5-bisphosphate breakdown consistent with the greater affinity of the drug for this lipid. In addition, erythrocyte membranes possess Mg(2+)-dependent phosphomonoesterase that converts inositol 1,4,5-triphosphate into inositol bisphosphate.