Distribution and characterization of mineral-binding phosphoprotein particles in Bivalvia

Representative species of four bivalve subclasses were examined for the presence of mineral-binding phosphoprotein particles in the physiological fluids. The particles were identified in Heterodont bivalves only, and particles from nine different Heterodont species were isolated and characterized. All phosphoprotein particles are internally cross-linked via histidinoalanine residues. In all species over 80% of the amino acid residues in the particles are aspartic acid, phosphoserine (and/or phosphothreonine), and histidine. These amino acids are probably the only residues directly related to mineral ion binding, since all phosphoprotein particles bind mineral irrespective of the minor amino acid content, which is species dependent. In their native state the phosphoprotein particles contain large amounts of calcium, magnesium, and inorganic phosphate ions (up to 45 metal ions and 8 phosphate ions per 100 amino acid residues) and trace amounts of transition elements. Evidence for the presence of calcium phosphate complexes in the native phosphoprotein particles was obtained by observing a concomitant increase in the inorganic phosphate and calcium ion content of the particles with pH in vivo.     

Effects of ATP and Taurine on Calcium Uptake by Membrane Preparations of the Rat Retina

Abstract: The effects of ATP and taurine on the kinetics of calcium uptake in rat retinal membrane preparations were determined. ATP increased calcium uptake at low calcium ion concentrations. Addition of ATP plus taurine further increased calcium uptake. Cooperative relationships were observed for calcium uptake in the absence of ATP and taurine. In the presence of phosphate ions reciprocal plots demonstrated upward deflections from linear ty, while in the absence of phosphate ions downward deflections were noted. Addition of ATP plus taurine to the incubation system appeared to obliterate the cooperativity. Two uptake systems for calcium were observed.     

Three kinetically different inorganic phosphate entities in bovine casein micelles revealed by isotopic exchange method and compartmental analysis

Much controversy exists concerning the way calcium phosphate is linked to milk phosphoproteins including caseins. Homoionic exchange of inorganic phosphate between micellar calcium phosphates (MCP) of casein micelles and solute phosphates in cows' milk was investigated using H(32)PO(4)(2-) as radiotracer. Compartmental analysis and modelling revealed the presence of three MCP-related inorganic phosphate compartments each representing a separate phosphate entity. The relative phosphate quantities per compartment, i.e. the quantities of kinetically identical phosphate ions per MCP-ion cluster, and their mean residence times are 2:1:1 and 818, 0.24 and 23 h, respectively. Hence each MCP-ion cluster comprises four inorganic phosphate ions divided over three intra-MCP binding sites each characterised by a mean residence time for homomolecular phosphate exchange at solution/MCP interface.     

Model for prebiotic pyrophosphate formation: Condensation of precipitated orthophosphate at low temperature in the absence of condensing or phosphorylating agents

Summary The formation of pyrophosphate (PPi) by condensation of orthophosphate (Pi) at low temperature (37°C) in the absence of condensing or phosphorylating agents could have been an ancient process in chemical evolution. In the present investigation the synthesis of³²PPi from³²Pi was carried out at pH 8.0 and PPi was found in larger amounts in the presence of insoluble Pi (with calcium or manganese ions) than in its absence (with magnesium ions, or with no divalent cations present). After 10 days of incubation in the presence of precipitated calcium phosphate, about 1.6 nmol/ml of PPi was formed (0.057% yield relative to insoluble Pi). The hypothesis that the reaction is dependent on precipitated Pi was reinforced by the effect of adding dimethyl sulfoxide (2.1–9.9 M) in the presence of magnesium ions: the amount of magnesium phosphate precipitated in the presence of the organic solvent was proportional to the amount of PPi formed. The large and negative activation entropies found in aqueous media with calcium ions and in a medium containing 11.3 M dimethyl sulfoxide with magnesium ions suggest that the reaction was favored by a hydrophobic phenomenon at the surface of solid Pi. This reaction could serve as a model for prebiotic formation of PPi.     

Determination of sulfate after chromatography and toluidine blue complex formation

A rapid and simple method for the determination of sulfate involving a complex formation between inorganic sulfate and the dye, toluidine blue O, after chromatography, is presented. The method can be used for the determination of sulfate in the presence of interfering ions such as phosphate and citrate. Most of the ions have a different chromatographic migration in the solvent system employed. An added advantage is the measurement of the labile sulfate of mucopolysaccharides with accuracy.     

Physicochemical characterization of casein phosphopeptide-amorphous calcium phosphate nanocomplexes

Milk caseins stabilize calcium and phosphate ions and make them available to the neonate. Tryptic digestion of the caseins yields phosphopeptides from their polar N-terminal regions that contain clusters of phosphorylated seryl residues. These phosphoseryl clusters have been hypothesized to be responsible for the interaction between the caseins and calcium phosphate that lead to the formation of casein micelles. The casein phosphopeptides stabilize calcium and phosphate ions through the formation of complexes. The calcium phosphate in these complexes is biologically available for intestinal absorption and remineralization of subsurface lesions in tooth enamel. We have studied the structure of the complexes formed by the casein phosphopeptides with calcium phosphate using a range of physicochemical techniques including x-ray powder diffraction, scanning electron microscopy, transmission electron microscopy, and equilibrium binding analyses. The amorphous nature of the calcium phosphate phase was confirmed by two independent methods: x-ray powder diffraction and selected area diffraction. In solution, the ion activity product of a basic amorphous calcium phosphate phase was the only ion product that was a function of bound phosphate independent of pH, consistent with basic amorphous calcium phosphate being the phase stabilized by the casein phosphopeptides. Detailed investigations of calcium and calcium phosphate binding using a library of synthetic homologues and analogues of the casein phosphopeptides have revealed that although the fully phosphorylated seryl-cluster motif is pivotal for the interaction with calcium and phosphate, other factors are also important. In particular, calcium binding and calcium phosphate stabilization by the peptides was influenced by peptide net charge, length, and sequence.     

Staining of phospho-proteins on acrylamide gel electropherograms

The paper describes a method for staining the phosphate moiety of phospho-proteins after electrophoresis on polyacrylamide gels. The method depends on hydrolysis of the phospho-protein phosphoester linkage using dilute base in the presence of calcium ions. The gel containing the newly formed insoluble calcium phosphate is then treated with ammonium molybdate in dilute nitric acid. The resultant insoluble nitrophospho-molybdate complex is finally stained with the basic dye, methyl green, dissolved in dilute acetic acid. After destaining, a green band is observed on the gel at the locus of the phospho-protein. One nanomole of protein-bound phosphate is detectable.     

The biodegradation of tributyl phosphate by naturally occurring microbial isolates

The biodegradation of tributyl phosphate by a mixed culture of Pseudomonads was demonstrated. Growth and the rate of tributyl phosphate consumption were variable and divisible into rapid and slow rates. Rapidly growing, rapidly tributyl phosphate-utilising cultures contained a 22–24 kb DNA fragment isolated by two methods, which was not visible in the cultures growing slowly. The mixed culture gave five periods of rapid growth interspersed with periods of poor growth during 7 months of weekly subculture, with the 22–24 kb DNA fragment detectable during the rapidly growing periods only. Seven Pseudomonads isolated from the culture grew at the expense of tributyl phosphate as the sole phosphorus source but spontaneously and irreversibly lost this ability after eight serial subcultures.     

Effect of phosphate and acidosis on the calcium sensitivity of the cardiac myofibrils

The treatment of the bundles of rat myocardial fibers with ethyleneglycol-bis(beta-aminoethyl ether)-N,N-tetraacetate (EGTA) made the sarcolemma permeable for ions and small molecules. At the incubation medium pH 7.0 the EGTA-treated fibers developed a half-maximal tension at pCa 5.4, and the maximal tension at pCa 4.8. Inorganic phosphate (10 mM) reduced the maximal tension by 18 +/- 3% and decreased the calcium sensitivity of the myofibrils so that there was a shift of the pCa/tension curve by 0.3 unit to the right. Acidosis (pH 6.6) also decreased significantly the calcium sensitivity, while the presence of 10 mM phosphate produced additional depression of the calcium sensitivity. It is concluded that phosphate accumulation by the ischemic myocardium combined with acidosis may depress the contractility not only due to depletion of the free calcium concentration in the myoplasm but also as a result of the reduced calcium sensitivity of myofibrils.     

Effects of phosphate anions and some divalent metal cations on phosphoenolpyruvate carboxykinase. Comparison of liver and kidney enzyme

The effect of calcium and phosphate anions on rat kidney cytosol phosphoenolpyruvate carboxykinase activity was evaluated using enzyme preparations obtained by two purification procedures. The enzyme activity was not significantly affected by calcium ions at physiological concentration. Phosphate inhibited the enzyme in the presence of Fe2+; the inhibition was overcome by Mn2+. Kidney and liver phosphoenolpyruvate carboxykinases show some qualitative differences in their response to Fe2+ and phosphate.     

The effect of calcium on the properties of charged phospholipid bilayers

We have performed molecular dynamics simulations to investigate the structure and dynamics of charged bilayers as well as the distribution of counterions at the bilayer interface. For this, we have considered the negatively charged di-myristoyl-phosphatidyl-glycerol (DMPG) and di-myristoyl-phosphatidyl-serine (DMPS) bilayers as well as a protonated di-myristoyl-phosphatidyl-serine (DMPSH) bilayer. We were particularly interested in calcium ions due to their important role in biological systems. Simulations performed in the presence of calcium ions (DMPG, DMPS) or sodium ions (DMPS) were run for 45-60 ns. Simulation results for DMPG are compared with fluorescence measurements. The average areas per molecule were 47.4+/-0.5 A2 (DMPG with calcium), 47.3+/-0.5 A2 (DMPS with calcium), 51.3+/-1.0 A2 (DMPS with sodium) and 45.3+/-0.5 A2 (DMPSH). The structure of the negatively charged lipids is significantly affected by the counterions, where calcium ions have a more pronounced effect than sodium ions. Calcium ions were found to be tightly bound to the anionic groups of the lipid molecules and as such appear to constitute an integral part of the membrane interface on nanoseconds time scales. In contrast to sodium ions, calcium ions are localised in a narrow (approximately 10 A) band around the phosphate group. The interaction of calcium with the lipid molecules enhances the molecular packing of the PG and PS lipids. This observation is in good agreement with emission spectra of the membrane partitioning probe Laurdan in DMPG multilamellar vesicles that indicate an increase in the ordering of the DMPG bilayer due to the presence of calcium. Our results indicate that calcium ions, which often function as a second messengers in living cells have a pronounced effect on membrane structures, which may have implications during signal transduction events.     

Binding of calcium and phosphate ions to dentin phosphophoryn

The concomitant binding of calcium and inorganic phosphate ions by the highly phosphorylated rat dentin phosphophoryn (HP) was measured in the pH range of 7.4-8.5 by an ultrafiltration procedure. HP binds almost exclusively the triply charged PO4(3-) ion, and for each PO4(3-) ion bound, the protein binds about 1.5 additional Ca2+ ions. Therefore, the protein-mineral ion complex can be described as a protein with two different ligands, Ca2+ ions and calcium phosphate clusters having a stoichiometry of about Ca1.5PO4. Empirically the binding of calcium and phosphate can best be described as a function of a neutral ion activity product in which 2.5-10% of the phosphate is HPO4(2-). The stoichiometry of the bound clusters is similar to that of amorphous calcium phosphate, and it is clear that the protein does not sequester crystal embryos of octacalcium phosphate or hydroxyapatite. The protein-mineral ion complex is amorphous by electron diffraction analysis and does not catalyze the formation of a crystalline phase when aged in contact with its solution. About 15% of the bound phosphate is buried in protected domains, and it is stable with respect to dissociation for extended periods in phosphate-free calcium buffers. The buried mineral maintains the protein in an aggregated state even at calcium ion concentrations which are too low for the aggregation of unmineralized HP. In vivo HP should be ineffective in the nucleation of a crystalline mineral phase, if it is secreted in a mineralized aggregated state similar to casein and the bivalve phosphoprotein.     

INFLUENCE OF THE CLAY TYPES ILLITE AND MONTMORILLONITE ON THE UPTAKE OF65 Zn BY SCENEDESMUS OBLIQUUS.

SUMMARY

The stimulatory effect of the clays illite and montmorillonite on zinc uptake by Scenedesmus obliquus is discussed. The competitive effects of hydrogen, phosphate, magnesium and copper ions on zinc uptake are illustrated. This competition is reduced in the presence of illite and montmorillonite and zinc uptake is stimulated. Ferrous ions have no effect on zinc uptake, while calcium ions antagonize zinc uptake at high concentrations. The latter effect was remarkable in the presence of montmorillonite.     

On the state of calcium ions in isolated rat liver mitochondria. II. Effects of phosphate and pH on Ca2+-induced Ca2+ release

At high K+ concentration, the effect of phosphate on Ca2+ uptake and release was studied in isolated rat liver mitochondria. Phosphate stimulated uptake at moderately high Ca2+ concentration, and inhibited release at high pH. At low pH, phosphate accelerated Ca2+ release. Ca2+ was released after a lag phase. The time of onset and the velocity of Ca2+ release depended on Ca2+ concentration. Ca2+ release was associated with mitochondrial swelling and destruction of the permeability barrier for sucrose and for chloride. Mg2+ inhibited Ca2+ release and the accompanying events. Ruthenium red and EGTA protected mitochondria from the destructive Ca2+ release and induced an immediate, slow release of Ca2+ and phosphate. Destructive Ca2+ release depended on the time of preincubation of respiration-inhibited mitochondria in the presence of Ca2+, prior to respiration-initiated Ca2+ uptake. The presence of phosphate and mitochondrial energization antagonized the destructive effect of calcium ions. Ca2+ release by acetoacetate also depended on pH. At pH 6.8, phosphate-stimulated Ca2+ release by acetoacetate, while it inhibited the acetoacetate effect at pH 7.6. The results suggest that an essential cause for the destruction of mitochondrial integrity is an increase in the intramitochondrial concentration of free calcium ions under the influence of phosphate.     

Enzyme mechanism and catalytic property of beta propeller phytase

BACKGROUND: Phytases hydrolyze phytic acid (myo-inositol-hexakisphosphate) to less-phosphorylated myo-inositol derivatives and inorganic phosphate. Phytases are used in animal feed to reduce phosphate pollution in the environment. Recently, a thermostable, calcium-dependent Bacillus phytase was identified that represents the first example of the beta propeller fold exhibiting phosphatase activity. We sought to delineate the catalytic mechanism and property of this enzyme. RESULTS: The crystal structure of the enzyme in complex with inorganic phosphate reveals that two phosphates and four calcium ions are tightly bound at the active site. Mutation of the residues involved in the calcium chelation results in severe defects in the enzyme's activity. One phosphate ion, chelating all of the four calcium ions, is close to a water molecule bridging two of the bound calcium ions. Fluoride ion, which is expected to replace this water molecule, is an uncompetitive inhibitor of the enzyme. The enzyme is able to hydrolyze any of the six phosphate groups of phytate. CONCLUSIONS: The enzyme reaction is likely to proceed through a direct attack of the metal-bridging water molecule on the phosphorous atom of a substrate and the subsequent stabilization of the pentavalent transition state by the bound calcium ions. The enzyme has two phosphate binding sites, the "cleavage site", which is responsible for the hydrolysis of a substrate, and the "affinity site", which increases the binding affinity for substrates containing adjacent phosphate groups. The existence of the two nonequivalent phosphate binding sites explains the puzzling formation of the alternately dephosphorylated myo-inositol triphosphates from phytate and the hydrolysis of myo-inositol monophosphates.     

Phosphorylation from inorganic phosphate and ATP synthesis of sarcoplasmic membranes

The incorporation of inorganic phosphate in the fragmented sarcoplasmic membranes induced by the removal of calcium ions bound to high affinity binding sites at the cytoplasmic surface of the membranes gives rise to the formation of two species of phosphoenzyme. The properties of the phosphoproteins formed depend on the absence or the presence of a gradient of calcium ions across the membranes. The phosphoenzymes differ by the affinity of the protein for phosphate, the enthalpy of formation, the kinetics of phosphate incorporation, and by the sensitivity to ionophores and ADP. In the absence of a calcium gradient less than 0.5 nmol phosphoenzyme per mg protein are formed in media containing less than 5 mM phosphate at pH7 and 10 degrees C. Under the same conditions approximately 2 nmol of phosphoenzyme per mg protein are formed with an initial rate of 0.5 nmol mg-1-s-1 when a calcium gradient exists. When the gradient is abolished by the addition of the ionophore X537A, the level of phosphoprotein drops to the same value as observed in the absence of a gradient. On addition of ADP at concentrations increasing from 0.3 to 10 muM continuous ATP formation is activated to its maximum rate, and simultaneously, the level of phosphoprotein declines. These concentrations of ADP scarcely affect phosphoprotein formed in the absence of a gradient, the phosphoryl residue of which is displaced when the concentration of ADP exceeds 10 micrometer without the formation of an equivalent amount of ATP. Minimum mechanisms for the formation of gradient-independent and gradient-dependent phosphoprotein are discussed.     

Risk Assessment and Class-Based Evaluation of Three Phosphate Esters

A category approach was used in the evaluation of a group of chemicals (triethyl phosphate, tripropyl phosphate, and tributyl phosphate) in drinking water, in support of determination of safe-harbor level for use by a standard-setting organization. Both non-cancer and cancer endpoints were considered, and risk assessment methodology developed by the U.S. Environmental Protection Agency (USEPA) was used to determine a class-based evaluation level (CBEL) and a total allowable concentration (TAC) in drinking water considered adequately protective of human health. The critical study was a chronic oncogenicity study on tributyl phosphate, with a no-observed-adverse-effect-level (NOAEL) of 9 mg/kg-day. The critical effect, combined incidence of bladder papilloma/transitional cell carcinoma, was likely non-genetic in nature, as supported by the lack of genetic toxicity observed in tributyl phosphate. The effect thus demonstrated a threshold, and this study used an oral reference dose approach and a total uncertainty factor of 100. This conservative analysis demonstrated the TAC should be twenty times greater than the default value of 0.01 mg/L.     

Three metal ions participate in the reaction catalyzed by T5 flap endonuclease

Protein nucleases and RNA enzymes depend on divalent metal ions to catalyze the rapid hydrolysis of phosphate diester linkages of nucleic acids during DNA replication, DNA repair, RNA processing, and RNA degradation. These enzymes are widely proposed to catalyze phosphate diester hydrolysis using a "two-metal-ion mechanism." Yet, analyses of flap endonuclease (FEN) family members, which occur in all domains of life and act in DNA replication and repair, exemplify controversies regarding the classical two-metal-ion mechanism for phosphate diester hydrolysis. Whereas substrate-free structures of FENs identify two active site metal ions, their typical separation of > 4 A appears incompatible with this mechanism. To clarify the roles played by FEN metal ions, we report here a detailed evaluation of the magnesium ion response of T5FEN. Kinetic investigations reveal that overall the T5FEN-catalyzed reaction requires at least three magnesium ions, implying that an additional metal ion is bound. The presence of at least two ions bound with differing affinity is required to catalyze phosphate diester hydrolysis. Analysis of the inhibition of reactions by calcium ions is consistent with a requirement for two viable cofactors (Mg2+ or Mn2+). The apparent substrate association constant is maximized by binding two magnesium ions. This may reflect a metal-dependent unpairing of duplex substrate required to position the scissile phosphate in contact with metal ion(s). The combined results suggest that T5FEN primarily uses a two-metal-ion mechanism for chemical catalysis, but that its overall metallobiochemistry is more complex and requires three ions.     

Stabilisation of tributyl phosphate-biodegradative ability of naturally-occurring pseudomonads using ampicillin

The biodegradation of tributyl phosphate (TBP) by a mixed microbial culture was demonstrated but growth on TBP and its catabolism were erratic. Pseudomonads isolated from the culture initially degraded TBP but were unstable and irreversibly lost this ability. Maintenance of cultures in the presence of ampicillin or growth in the presence of the antibiotic stabilised, and in some cases enhanced, TBP degradation, suggesting a central role for plasmid-borne activity.     

Adenosine triphosphate-lead histochemical reactions in ependymal epithelia of murine brains do not represent calcium transport adenosine triphosphatase

Summary The strong enzyme histochemical reactions for adenosine triphosphatase (ATPase) seen in ependymal tanycytes after incubation in calcium-containing media have previously been reported as calcium transport ATPase. Investigation of these reactions showed that: (1) any nucleoside triphosphate can serve as a substrate; (2) diphosphates and monophosphates cannot replace triphosphates; this includes p-nitrophenyl phosphate which is readily hydrolysed by plasma membrane transport ATPases; (3) strong localization occurs in the presence of millimolar concentrations of either calcium or magnesium ions; there is no absolute requirement for calcium ions; (4) they are not inhibited by sulphydryl inhibitors or calmodulin antagonists; (5) lead phosphate precipitates are localized almost entirely on the external face of tanycyte plasma membranes. In addition, the technique gives strong localization to vessels in the choroid plexus but not to the choroidal epithelium. Immunohistochemistry with a primary antibody raised against Ca²⁺,Mg²⁺-ATPase stains the choroidal epithelium but not the vessels or the ependymal tanycytes. These results are inconsistent with identification of the reaction as calcium transport ATPase but support characterization as an ecto-ATPase.