Intestinal transport of manganese from human milk, bovine milk and infant formula in rats

The transport of manganese from extrinsically labeled human milk, bovine milk and infant formula was studied by the everted intestinal sac method. Tissue/mucosal flux data indicated that transport of manganese into the intestinal tissue was significantly greater with bovine milk and formula than from human milk. Similarly, the total flux of manganese from the mucosal to serosal surface was less when human milk was used. Smaller molecular weight manganese binding ligands isolated from the milk samples enhanced the mucosal to tissue movement of manganese as contrasted to the higher molecular weight manganese binding ligands. Most significantly the data suggest that the transport and uptake of manganese is less in the presence of human milk and its isolated manganese fractions than it is in bovine milk or infant formula.     

Regulation of Escherichia coli glutamine synthetase. Evidence for the action of some feedback modifiers at the active site of the unadenylylated enzyme.

The interaction of unadenylylated form of Escherichia coli glutamine synthetase with several substrates and effectors has been examined by magnetic resonance techniques. These studies show that two manganese ions bind per enzyme subunit. From the dramatic line broadening observed in the alanine spectra in the presence of manganese and enzyme, it is concluded that the binding of alanine occurs at a site nearer one of the two manganese sites. Electron spin resonance (ESR) titration experiments suggest apparent dissociation constants of 20 and 120 muM for manganese to these sites in the presence of 1.0 mM magnesium ion. The manganese concentration dependence of the broadening of alanine suggests an affinity of 30 muM for the manganese closest to the alanine binding site. This suggests that alanine binds closer to the more tightly bound manganese ion. Glutamate appears to displace the alanine and also appears to bind close to the strongly bound manganese ion. It is proposed that alanine and glutamine bind competitively and in the same site. The binding of alanine and ATP is shown to thermodynamically interact such that the presence of one ligand increases the affinity of the enzyme for the other ligand. The presence of ATP dramatically sharpens the alanine line width when manganese and glutamine synthetase are present. Addition of ADP or phosphate alone has little effect on the alanine line width but the addition of both ADP and phosphate shows the same dramatic sharpening as the addition of ATP alone, suggesting an induced fit conformational change in the enzyme induced by ATP or by both ADP and phosphate. A binding scheme is proposed in which all feedback inhibitors of the enzyme bind in a competitive fashion with substrates.     

The adsorption of bovine blood proteins onto the surface of O-(carboxymethyl)chitin

Chitin was found to interact with bovine blood proteins and the affinities of these proteins for chitin tended to be decreased by the introduction of O-carboxymethyl (CM) groups onto the chitin surface, especially with fibrinogen. As the adsorption of blood proteins to the CM-chitin (d.s. 0.35) was assumed to follow an isothermal adsorption-curve, the adsorption coefficients were estimated by applying the Langmuir equation. Bovine serum albumin showed the highest affinity among the proteins applied in this experiment [KBSA (bovine serum albumin); 20.0, KB gamma G (bovine gamma globulin); 1.96, KBF (bovine fibrinogen); 1.20]. The binding site of BSA for CM-chitin was assumed to be regulated not only by the cationic groups of BSA but also by other factors such as the recognition capacity of BSA to bind to GlcNAc residues in CM-chitin.     

A spectral probe near the subunit catalytic site of glutamine synthetase from Escherichia coli. Reduced pyridoxal 5'-phosphate.enzyme complexes.

In order to label phosphate binding sites, unadenylylated glutamine synthetase from Escherichia coli has been pyridoxylated by reacting the enzyme with pyridoxal 5'-phosphate followed by reduction of the Schiff base with NaBH4. A complete loss in Mg2+-supported activity is associated with the incorporation of 3 eq of pyridoxal-P/subunit of the dodecamer. At this extent of modification, however, the pyridoxylated enzyme exhibits substantial Mn2+-supported activity (with increased Km values for ATP and ADP). The sites of pyridoxylation appear to have equal affinities for pyridoxal-P and to be at the enzyme surface, freely accessible to solvent. At least one of the three covalently bound pyridoxamine 5'-phosphate groups is near the subunit catalytic site and acts as a spectral probe for the interactions of the manganese.enzyme with substrates. A spectral perturbation of covalently attached pyridoxamine-P groups is caused also by specific divalent cations (Mn2+, Mg2+ or Ca2+) binding at the subunit catalytic site (but not while binding to the subunit high affinity, activating Me2+ site). In addition, the feedback inhibitors, AMP, CTP, L-tryptophan, L-alanine, and carbamyl phosphate, perturb protein-bound pyridoxamine-P groups. The spectral perturbations produced by substrate and inhibitor binding are pH-dependent and different in magnitude and maximum wavelength. Adenylylation sites are not major sites of pyridoxylation.     

Adsorptive characteristics of iron(III) onto hydrous manganese dioxide in model lake water under acidic conditions

The adsorption of iron(III) onto hydrous manganese dioxide was studied in the model water of Lake Tjeukemeer, The Netherlands, as a function of pH under the condition where no precipitation of iron(III) hydroxide occurs.Adsorption isotherms obtained in the pH range 3.5–5.0, fitted the Langmuir equation. Langmuir constants were strongly pH-dependent and increased with an increase in pH.Especially, the value for the conditional adsorption binding constant for iron(III), log B, was relatively high compared to that for copper in natural water. The extent of iron(III) adsorbed was more affected by the surface loading at lower pH. According to the Kurbatov plot, the average number of protons released to solution per mole of iron(III) adsorbed was estimated to be 2.2.     

Biosynthesis of the carbohydrate portions of immunoglobulin M

1. Alkaline-earth-metal cations at low concentrations form soluble complexes with bovine caseins. The relative order of binding capacities is: Mg(2+)>Ca(2+)>Ba(2+)>Sr(2+). 2. The cations interact with both free ionized carboxyl groups of aspartic acid and glutamic acid and with monoester phosphate groups covalently bound to serine and threonine; at low concentrations of the cations interactions are predominantly with the phosphate groups. 3. The order of binding capacities for purified components of the casein complex is: alpha(s1)-casein>beta-casein>kappa-casein.     

Structure-function relationships in cardiac troponin T

Regions of rabbit and bovine cardiac troponin T that are involved in binding tropomyosin, troponin C and troponin I have been identified. Two sites of contact for tropomyosin have been located, situated between residues 92-178 and 180-284 of troponin T. A cardiac-specific binding site for troponin I has been identified between residues 1-68 of cardiac troponin T, within a region of the protein that has previously been shown to be encoded by a series of exons that are expressed in a tissue-specific and developmentally regulated manner. The binding site for troponin C is located between residues 180-284 of cardiac troponin T. When isolated from fresh bovine hearts, cardiac troponin T contained 0.21 +/- 0.11 mol phosphate per mol; incubation with phosphorylase kinase increased the phosphate content to approx. 1 mol phosphate per mol. One site of phosphorylation was identified as serine-1; a second site of phosphorylation was located within peptide CB3 (residues 93-178) and has been tentatively identified as serine-176. Addition of troponin C to cardiac troponin T does not inhibit the phosphorylation of this latter protein that is catalysed by phosphorylase b kinase.     

5'-Nucleotide phosphodiesterase: features of the substrate binding site as deduced from specificity and kinetics of some novel substrates

Phosphonate monoesters and phosphate diesters with systematically varied substituents and leaving groups were synthesized and tested as substrates for homogeneous 5'-nucleotide phosphodiesterase from bovine intestine. The enzyme was shown to hydrolyze phosphorothioate and phosphonoamidate compounds but at significantly lower rates than comparable oxy compounds. The effects of bulk and structure of the ester or phosphonate substituents were also investigated. Dibenzyl phosphate, an ester of an aliphatic alcohol, was a poor substrate. The enzyme did not hydrolyze aliphatic monoesters of phosphonates, regardless of bulk. Kinetic parameters of several nitrophenyl phosphonomonoesters and phosphodiesters are presented. The results suggest that synthetic nonnucleotide substrates can bind in two different modes, only one of which is productive. Incidence of nonproductive binding, with consequent kinetic effects, is increased by increasing the symmetry of the substrates.     

Effects of adsorption to aluminum salt adjuvants on the structure and stability of model protein antigens

The effect of adsorption onto aluminum salt adjuvants on the structure and stability of three model protein antigens was studied using fluorescence and Fourier transform infrared spectroscopies, as well as isothermal titration and differential scanning calorimetric techniques. Lysozyme was preferentially adsorbed to aluminum phosphate (Adju-Phos), whereas ovalbumin and bovine serum albumin were better adsorbed to aluminum hydroxide (Alhydrogel). A linearized Langmuir adsorption isotherm was used to obtain information regarding the binding interactions between proteins and adjuvants. Binding energetics and stoichiometry data obtained from isothermal titration calorimetry measurements were complex. Based on the spectroscopic and differential scanning calorimetry studies, the structure of all three proteins, when adsorbed to the surface of an aluminum salt, was altered in such a way as to render the proteins less thermally stable. Besides the pharmaceutical significance of this destabilization, we consider the possibility that this phenomenon may facilitate the presentation of antigens and thus contribute to the adjuvant activity of the aluminum salts.     

Protein-phosphate complexes as an inhibitor of iron-induced lipid peroxidation

Effect of phosphate buffer (pH 6.2) alone or in the presence of bovine serum albumin and other proteins on iron (II)-induced lipid peroxidation was studied. Phosphate buffer alone and in the presence of bovine serum albumin was found to inhibit lipid peroxidation. The inhibition was higher when bovine serum albumin was also present. Other proteins also inhibited lipid peroxidation in the presence of phosphate. Inhibition by proteins in the presence of phosphate seems to be due to binding of iron with phosphate and with protein-phosphate complexes. Reversal in inhibition was observed with an increase in iron concentration in reaction mixture. Equilibrium dialysis showed more binding of iron to protein in the presence of phosphate than in the presence of chloride ions.     

An RNA aptamer that discriminates bovine factor IX from human factor IX

An RNA aptamer has been selected by SELEX against bovine factor IX using an RNA pool containing 74-nucleotides randomized region. Selected RNA aptamer (Clone 5) could discriminate bovine factor IX effectively from human factor IX. Interestingly, the nucleotide regions 73-78 and 80-83 of the selected aptamer were determined to be important for bovine factor IX-binding using phosphate interference. Based on phosphate interference and binding studies the minimal motif for aptamer with discriminating ability is found with the nucleotide regions from 65 to 106. The discriminating ability of this mini aptamer is calculated as more than 1,000 fold. The equilibrium dissociation constant (K(d)) for the above complex was 10 nM as determined by surface plasmon resonance. Based on the available structural informations, probable binding site of aptamer on the target was predicted.     

Manganese adsorption by sediment in Wadi Tafna,Algeria

The removal of manganese from aqueous solution by sediment in Wadi Tafna, north-western Algeria, was investigated under batch conditions in 2003. Kinetic data equilibrium removal isotherms were measured at 20 °C. The influence of different experimental parameters, such as contact time, manganese concentration and sediment mass on the removal kinetics of manganese was studied. The content of carbonate in sediment increased the adsorption rate, indicating the active phase towards manganese cations. The process followed pseudo-second-order kinetics. Manganese uptake by sediment was quantitatively evaluated using sorption isotherms. In order to describe the adsorption isotherm mathematically, the experimental data of the removal equilibrium were analysed with Langmuir and Freundlich models revealing that the equilibrium data were perfectly represented using both isotherms.     

Methyl acetyl phosphate as a covalent probe for anion-binding sites in human and bovine hemoglobins

An allosteric modulator of oxygen release in human erythrocytes is 2,3-diphosphoglycerate, but bovine erythrocytes apparently utilize chloride for this purpose since they contain little, if any, 2,3-diphosphoglycerate. In order to identify the sites to which these anions bind, the site-specific acetylating agent, methyl acetyl phosphate, has been employed to compete with these allosteric modulators and to mimic their effects on hemoglobin function. With human hemoglobin A, methyl acetyl phosphate competes with 2,3-diphosphoglycerate and acetylates only Val-1(beta), Lys-82(beta), and Lys-144(beta) within or near the cleft that binds this organic phosphate (Ueno, H., Pospischil, M. A., Manning, J. M., and Kluger, R. (1986) Arch Biochem. Biophys. 244, 795). With bovine hemoglobin, the acetylation is competitive with chloride ion. The sites of acetylation in oxy bovine hemoglobin are Met-1(beta) and Lys-81(beta) and for deoxy bovine hemoglobin, they are Val-1(alpha) and Lys-81(beta). Thus, these sites are expected to be involved in the binding of chloride to bovine hemoglobin. Treatment of either human or bovine hemoglobins with methyl acetyl phosphate under anaerobic conditions leads to a lowering of their oxygen affinity and hence the covalent modifier has the same effect on hemoglobin function as the non-covalent regulators, 2,3-diphosphoglycerate and chloride. The Hill's coefficient of hemoglobin is unaffected by treatment with methyl acetyl phosphate. Under aerobic conditions, specifically acetylated bovine hemoglobin also has a lowered oxygen affinity, and human hemoglobin A shows a slight change in its oxygen affinity. In general, bovine hemoglobin is more responsive than human hemoglobin to both chloride and methyl acetyl phosphate; the latter agent results in a permanent covalent labeling of the protein. Therefore, the results support the idea that methyl acetyl phosphate may be a useful probe for deciphering the sites of binding of anions to proteins.     

The role of phosphate groups in the VS ribozyme-substrate interaction

The VS ribozyme trans-cleavage substrate interacts with the catalytic RNA via tertiary interactions. To study the role of phosphate groups in the ribozyme–substrate interaction, 18 modified substrates were synthesized, where an epimeric phosphorothioate replaces one of the phosphate diester linkages. Sites in the stem–loop substrate where phosphorothioate substitution impaired reaction cluster in two regions. The first site is the scissile phosphate diester linkage and nucleotides downstream of this and the second site is within the loop region. The addition of manganese ions caused recovery of the rate of reaction for phosphorothioate substitutions between A621 and A622 and U631 and C632, suggesting that these two phosphate groups may serve as ligands for two metal ions. In contrast, significant manganese rescue was not observed for the scissile phosphate diester linkage implying that electrophilic catalysis by metal ions is unlikely to contribute to VS ribozyme catalysis. In addition, an increase in the reaction rate of the unmodified VS ribozyme was observed when a mixture of magnesium and manganese ions acted as the cofactor. One possible explanation for this effect is that the cleavage reaction of the VS ribozyme is rate limited by a metal dependent docking of the substrate on the ribozyme.     

Airborne manganese exposure differentially affects end points of oxidative stress in an age-and sex-dependent manner

Juvenile female and male (young) and 16-mo-old male (old) rats inhaled manganese in the form of manganese sulfate (MnSO₄) at 0, 0.01, 0.1, and 0.5 mg Mn/m³ or manganese phosphate at 0.1 mg Mn/m³ in exposures of 6h/d, 5d/wk for 13 wk. We assessed biochemical end points indicative of oxidative stress in five brain regions: cerebellum, hippocampus, hypothalamus, olfactory bulb, and striatum. Glutamine synthetase (GS) protein levels, metallothionein (MT) and GS mRNA levels, and total glutathione (GSH) levels were determined for all five regions. Although most brain regions in the three groups of animals were unaffected by manganese exposure in terms of GS protein levels, there was significantly increased protein (p<0.05) in the hippocampus and decreased protein in the hypothalamus of young male rats exposed to manganese phosphate as well as in the aged rats exposed to 0.1 mg/m³ MnSO₄. Conversely, GS protein was elevated in the olfactory bulb of females exposed to the high dose of MnSO₄. Statistically significant decreases (p<0.05) in MT and GS mRNA as a result, of manganese exposure were observed in the cerebellum, olfactory bulb, and hippocampus in the young male rats, in the hypothalamus in the young female rats, and in the hippocampus in the senescent males. Total GSH levels significantly (p<0.05) decreased in the olfactory bulb of manganese exposed young male rats and increased in the olfactory bulb of female rats exposed to manganese. Both the aged and young female rats had significantly decreased (p<0.05) GSH in the striatum resulting from manganese inhalation. The old male rats also had depleted GSH levels in the cerebellum and hypothalamus as a result, of the 0.1-mg/m³ manganese phosphate exposure. These results demonstrate that age and sex are variables that must be considered whenassessing the neurotoxicity of manganese.     

Effect of phosphate ion on the activity of bovine plasma amine oxidase.

The system bovine plasma amine oxidase-polyamine-phosphate ion was investigated by activity measurements and 31P NMR spectroscopy. Lineweaver-Burk plots showed that phosphate ion, under physiological conditions, is an apparent competitive inhibitor of bovine plasma amine oxidase. While NMR measurements of the T1 of 31P do not suggest the binding of phosphate to/or near the paramagnetic Cu(II) sites of bovine plasma amine oxidase, the chemical shift dependence of 31P on spermidine concentration indicates the formation of a spermidine-phosphate complex. The value of the dissociation constant of this complex was found 18.5 +/- 1.4 mM, at pH 7.2, by NMR, in good agreement with the value 17.0 +/- 0.8 mM calculated from activity measurements, assuming the enzyme activity is proportional to the free amine concentration, under second order conditions. Our data suggest that the decrease of the free spermidine, due to the binding of phosphate ion, is responsible of the observed inhibition of bovine plasma amine oxidase.     

The effects of lime and superphosphate on manganese toxicity in steam-sterilized soil

Summary The effects of liming and superphosphate application on the manganese nutrition of lettuce were studied in factorial experiments. A fairly acid silt loam of the Hamble series, which on steam-sterilization released considerable amounts of manganese in readily available forms, was used in the investigation.Manganese toxicity in lettuce was prevented by liming to increase the pH of the soil. The amounts of water-soluble, exchangeable and total active manganese present in the soil, and the manganese content of lettuce plants, decreased with increasing soil pH; easily reducible soil manganese increased with increasing soil pH.The effect of added superphosphate depended on the pH of the untreated soil. When the soil had a high pH, application of phosphate invariably decreased the pH and increased the manganese uptake. In acid soil supplying excessive amounts of manganese, application of phosphate reduced the manganese content of the plants; the pH of the soil was either unchanged or slightly increased by the treatment.Correlations were calculated between soil pH, various fractions of soil manganese, and the manganese content and yield of lettuce.From a comparison of plants grown in soil and sand culture it was concluded that the presence of a further toxic factor in the soil was probable.The results are discussed in relation to those obtained by other investigators.     

Crystal structure of heart 6‐phosphofructo‐2‐kinase/fructose‐2,6‐bisphosphatase (PFKFB2) and the inhibitory influence of citrate on substrate binding

The heart‐specific isoform of 6‐phosphofructo‐2‐kinase/fructose‐2,6‐bisphosphatase (PFKFB2) is an important regulator of glycolytic flux in cardiac cells. Here, we present the crystal structures of two PFKFB2 orthologues, human and bovine, at resolutions of 2.0 and 1.8 Å, respectively. Citrate, a TCA cycle intermediate and well‐known inhibitor of PFKFB2, co‐crystallized in the 2‐kinase domains of both orthologues, occupying the fructose‐6‐phosphate binding‐site and extending into the γ‐phosphate binding pocket of ATP. This steric and electrostatic occlusion of the γ‐phosphate site by citrate proved highly consequential to the binding of co‐complexed ATP analogues. The bovine structure, which co‐crystallized with ADP, closely resembled the overall structure of other PFKFB isoforms, with ADP mimicking the catalytic binding mode of ATP. The human structure, on the other hand, co‐complexed with AMPPNP, which, unlike ADP, contains a γ‐phosphate. The presence of this γ‐phosphate made adoption of the catalytic ATP binding mode impossible for AMPPNP, forcing the analogue to bind atypically with concomitant conformational changes to the ATP binding‐pocket. Inhibition kinetics were used to validate the structural observations, confirming citrate's inhibition mechanism as competitive for F6P and noncompetitive for ATP. Together, these structural and kinetic data establish a molecular basis for citrate's negative feed‐back loop of the glycolytic pathway via PFKFB2. Proteins 2016; 85:117–124. © 2016 Wiley Periodicals, Inc.     

Significance and mechanism of divalent-ion binding to transfer RNA.

Phosphorus NMR shows that divalent ions (manganese) bind to tRNA phosphates as to those of DNA or isolated phosphodiesters. The time for dissociation of a phosphate-divalent ion complex is in the microsecond range. For no single phosphate is the affinity to divalent ions greater than 10 times that of the average phosphate. It is often stated that a small number of strong binding sites exist and are structurally and functionally important. This concept originates from binding curves whose properties should, instead, be traced to the polyelectrolyte nature of nucleic acids. The 31P NMR data preclude the existence of strong sites to which divalent ions would bind very selectively. The Spectroscopic and crystallographic observations of sites for divalent ions do not in fact demonstrate selective binding to these sites.     

The binding of sodium dodecyl sulphate to bovine serum albumin at high binding ratios

The extent of binding of sodium dodecyl sulphate to bovine serum albumin at high binding ratios was investigated by gel filtration. The weight ratio of bound sodium dodecyl sulphate to bovine serum albumin increases with the NaCl concentration, and, except at low salt concentrations, with the concentration of sodium dodecyl sulphate. In the presence of 1.0g of sodium dodecyl sulphate/l, the binding ratio varied from 1.0 (at 0.04m-Na(+)) to 2.2 (at 0.44m-Na(+)). In the presence of 0.24m-Na(+), the binding ratio increased with sodium dodecyl sulphate concentration, from 0.9 (0.2g of sodium dodecyl sulphate/l) to 2.0 (5g of sodium dodecyl sulphate/l), at 26 degrees C, in a dilute sodium phosphate buffer. No significant dependence of the binding ratio upon temperature in the range 26-45 degrees C was observed. These results differ from those of Reynolds & Tanford (1970a) obtained by equilibrium dialysis.