Values Of Mg Research Articles

Groundwater Quality Assessment of Abandoned Dumpsite in Osogbo Suburb, Nigeria: Hydrogeological and Geophysical Condition Six Years after Initial Study


 
 
 In this paper, we describe the result of a recent hydrogeological and geophysical investigation carried out within and around Aduramigba - Onibu-Eja Estate dumpsite with the aim of detecting evidence of contamination as well as depths to contamination of the groundwater after a previous study six years ago. Twenty water samples from shallow water wells and boreholes were analyzed for their physico-chemical constituents while five vertical electrical soundings and five electromagnetic ground penetrating radar surveys were conducted. The result shows high concentrations of anions of nitrates (NO3-), bicarbonates (HCO3-), chloride (Cl-) and sulphate (SO42-) with values of 73.97 mg/l, 91.5 mg/l, 331.2 mg/l, 222.4 mg/l respectively, cations values of Mg2+, Fe2+, Na+, and K+ having value of 25.44 mg/l, 6.23 mg/l, 37.31 mg/l and 35.41 mg/l, respectively, which were far above the World Health Organization (WHO) and Nigeria Drinking Water Quality Standard (NDWS). Heavy metal components such as Lead, Zinc, Copper and Nickel were also observed to be on the high side, having been contributed from effluents from rust metallic materials cum chemicals dumped within the dumpsite. Detailed geophysical study reveal the depth to contamination at between 4.1 meters in VES 3 to 5.9 meters in VES 2 with resistivity as low as 5.12 Ωm in VES 2 to 7.12 Ωm in VES 3 while ground penetrating radar (GPR) revealed that the leachate has permeated to a depth of 3.5 – 5.25 meters in traverse 4 and 4.0 – 6.0 meters in traverse 3 which correspond to the depth to unconfined aquifer within the dumpsite. The study conclude that this would pose a greater threat to residents living in this vicinity and as such, this might have contributed to the closure of the dumpsite.
 
 

Bridging the 12-6-4 Model and the Fluctuating Charge Model.

Metal ions play important roles in various biological systems. Molecular dynamics (MD) using classical force field has become a popular research tool to study biological systems at the atomic level. However, meaningful MD simulations require reliable models and parameters. Previously we showed that the 12-6 Lennard-Jones nonbonded model for ions could not reproduce the experimental hydration free energy (HFE) and ion-oxygen distance (IOD) values simultaneously when ion has a charge of +2 or higher. We discussed that this deficiency arises from the overlook of the ion-induced dipole interaction in the 12-6 model, and this term is proportional to 1/r 4 based on theory. Hence, we developed the 12-6-4 model and showed it could solve this deficiency in a physically meaningful way. However, our previous research also found that the 12-6-4 model overestimated the coordination numbers (CNs) for some highly charged metal ions. And we attributed this artifact to that the current 12-6-4 scheme lacks a correction for the interactions among the first solvation shell water molecules. In the present study, we considered the ion-included dipole interaction by using the 12-6 model with adjusting the atomic charges of the first solvation shell water molecules. This strategy not only considers the ion-induced dipole interaction between ion and the first solvation shell water molecules but also well accounts for the increased repulsion among these water molecules compared to the bulk water molecules. We showed this strategy could well reproduce the experimental HFE and IOD values for Mg2+, Zn2+, Al3+, Fe3+, and In3+ and solve the CN overestimation issue of the 12-6-4 model for Fe3+ and In3+. Moreover, our simulation results showed good agreement with previous ab initio MD simulations. In addition, we derived the physical relationship between the C 4 parameter and induced dipole moment, which agreed well with our simulation results. Finally, we discussed the implications of the present work for simulating metalloproteins. Due to the fluctuating charge model uses a similar concept to the 12-6 model with adjusting atomic charges, we believe the present study builds a bridge between the 12-6-4 model and the fluctuating charge model.

A new four-step hierarchy method for combined assessment of groundwater quality and pollution

A new four-step hierarchy method was constructed and applied to evaluate the groundwater quality and pollution of the Dagujia River Basin. The assessment index system is divided into four types: field test indices, common inorganic chemical indices, inorganic toxicology indices, and trace organic indices. Background values of common inorganic chemical indices and inorganic toxicology indices were estimated with the cumulative-probability curve method, and the results showed that the background values of Mg2+ (51.1mgL-1), total hardness (TH) (509.4mgL-1), and NO3- (182.4mgL-1) are all higher than the corresponding grade III values of Quality Standard for Groundwater, indicating that they were poor indicators and therefore were not included in the groundwater quality assessment. The quality assessment results displayed that the field test indices were mainly classified as grade II, accounting for 60.87% of wells sampled. The indices of common inorganic chemical and inorganic toxicology were both mostly in the range of grade III, whereas the trace organic indices were predominantly classified as grade I. The variabilities and excess ratios of the indices were also calculated and evaluated. Spatial distributions showed that the groundwater with poor quality indices was mainly located in the northeast of the basin, which was well-connected with seawater intrusion. Additionally, the pollution assessment revealed that groundwater in well 44 was classified as "moderately polluted," wells 5 and 8 were "lightly polluted," and other wells were classified as "unpolluted."

English

Wood charcoals were mainly used for smoking of fish, meat and frying of corns and many times dump off as wastes in southeast, Nigeria. Their nutrient recycles and release when used as soil amendment has not been considered and evaluated by farmers in the southeast, Nigeria. This study was therefore carried out to assess the efficiency of wood charcoal technology in improving soil productivity. Influence of wood charcoal from Chlorophera excelsa on soil properties and yield components of maize were evaluated. Four different rates of wood charcoal (6, 4, 2 and 0 tha-1 that received no treatment application) were used. The treatments were laid out in randomised complete block design (RCBD) and treatment means were compared using least significant difference (LSD). The findings from the study showed non-significant (P<0.05) differences in most of the growth and yield components of maize tested. Significant differences were recorded in all the soil properties assessed except for percentage nitrogen (N). Higher values were observed in 4 tha-1 rates except for the values of pH, available phosphorus and percentage base saturation, where higher values were observed in 6 tha-1 rates of wood charcoal than the other rates of wood charcoal. The percentage increase in calcium content of the soil relative to the values obtained in 0 tha-1 rate was 13.04% (2 tha-1), 11.76% (4 tha-1) and 7.69% (6 tha-1). While the percentage increase in the value of Mg2+ obtained in 4 tha-1 plots relative to control (0 tha-1) plots was 28.5%. The data generated from both growth and yield component and soil properties showed that the values of parameters tested increased as the rate of wood charcoal applied increased, though this was not consistent in the growth and yield parameters and few cases in soil parameters. In comparison of the four different rates of the wood charcoal applied, 4 tha-1 rate seems to be an ideal than the other rates as it performed competitively better and were able to liberate plant nutrients more than the other rates of wood charcoal though these liberated nutrients were not reflected in most of the growth parameters assessed.  Key words: Chlorophera excelsa , crop growth, soil properties, wood charcoal.

Structure of Hydrogenase Maturation Protein HypF with Reaction Intermediates Shows Two Active Sites

[NiFe]-hydrogenases are multimeric proteins. Thelarge subunit contains the NiFe(CN)(2)CO bimetallic active center and the small subunit contains Fe-S clusters. Biosynthesis and assembly of the NiFe(CN)(2)CO active center requires six Hyp accessory proteins. The synthesis of the CN(-) ligands iscatalyzed by the combined actions of HypF andHypE using carbamoylphosphate as a substrate.We report the structure of Escherichia coli HypF(92-750) lacking the N-terminal acylphosphatase domain. HypF(92-750) comprises the novel Zn-finger domain, the nucleotide-binding YrdC-like domain, and the Kae1-like universal domain, also binding a nucleotide and a Zn(2+) ion. The two nucleotide-binding sites are sequestered in an internal cavity, facing each other and separated by ∼14Å. The YrdC-like domain converts carbamoyl moiety to a carbamoyl adenylate intermediate, which is channeled to the Kae1-like domain. Mutations within either nucleotide-binding site compromise hydrogenase maturation but do not affect the carbamoylphosphate phosphatase activity.

Density functional theoretical study on attachment sites of Mg2+ and Ca2+ and metal ion affinity to Crenulatin molecule

Crenulatin (C25H20O10) is a flavonol derivative and has been isolated from the roots of Rhodiola crenulata (Hook. F. et Thoms.), a widely used medicinal herb. Magnesium and calcium cations play an important physiological role in biological systems. In this work, interactions of magnesium and calcium divalent cations with Crenulatin molecule were studied. Density functional theory (DFT) was used to determine coordination geometries and absolute metal ion affinities (MIA) for all possible stable complexes. The results show that calcium and magnesium cations are able to interact with the Crenulatin molecule through mono-, bi-, and tricoordination. B3LYP/6-31G(d) bond energies for all complexes reveal that magnesium cation has a greater affinity to Crenulatin molecule than calcium cation. The calculated value of Mg2+ cation affinity, including the zero-point vibrational energy (ZPE) and basis set superposition error (BSSE), is 231.8 kcal mol−1 for the most stable complex. Entropy (ΔS) and free energy (ΔG) variations for the metalation processes considered here have also been reported.

Seasonal variations in properties of healthy and diabetic rat heart mitochondria: Mg2+-ATPase activity, content of conjugated dienes and membrane fluidity

Our previous preliminary results pointed to possible seasonal variations in Mg2+-ATPase activity of rat heart mitochondria (MIT). It is not too surprising since seasonal differences were already reported in myocardial function, metabolism and ultrastructure of the intact as well as hemodynamically overloaded rabbit hearts and also in other tissues. The present study is aimed to elucidate whether seasonal differences observed in rat heart MIT Mg2+-ATPase activity will be accompanied with changes in membrane fluidity and in the content of conjugated dienes (CD) in the lipid bilayers of MIT membranes as well as whether the above seasonal differences will also be present in the diabetic heart. Our results revealed that values of Mg2+-ATPase activity in the winter/spring-period (W/S-P) exceeded significantly (p<0.05-0.001) those in the summer/autumn-period (S/A-P). Similar trend was also observed in hearts of animals with acute (8 days) streptozotocin diabetes. With the exception of values of CD in the S/A-P, all values of Mg2+-ATPase activities, membrane fluidity and CD concentrations in diabetic hearts exceeded those observed in the healthy hearts. Our results indicate that seasonal differences may play a decisive role in the evaluation of properties and function of rat heart MIT.

Anthranilate Synthase without an LLES Motif from a Hyperthermophilic Archaeon Is Inhibited by Tryptophan

Tk-trpE and Tk-trpG, the genes that encode the two subunits of anthranilate synthase from the hyperthermophilic archaeon Thermococcus kodakaraensisKOD1, have been expressed independently in Escherichia coli. The anthranilate synthase complex (Tk–AS complex) was obtained by heat-treatment of the mixture of cell-free extracts containing each recombinant protein, Tk-TrpE (α subunit) and Tk-TrpG (β subunit), at 85°C for 10 min. Further purification of Tk–AS complex was carried out by anion-exchange chromatography followed by gel-filtration. Molecular mass estimations from gel-filtration chromatography indicated that Tk–AS complex was a heterodimer (αβ). The complex displayed both ammonia- and glutamine-dependent anthranilate synthase activities, and could not utilize asparagine as an ammonia donor. The optimal pH was pH 10.0 and the optimal temperature was 85°C in both cases. Mg2+ was necessary for the anthranilate synthase activity. At 75°C, the Km values of chorismate for ammonia- and glutamine-dependent activities were 13.8 and 3.4 μM, respectively. The Km value of Mg2+ was 20.5 μM. The Km values of glutamine and NH4Cl were 88 μM and 5.6 mM, respectively. Although Tk-TrpE displayed 47.6% similarity with TrpE of Salmonella typhimurium, conserved amino acid residues proven to be essential for inhibition of enzyme activity by l-tryptophan were not present in Tk-TrpE. Namely, residues corresponding to Glu39, Met293, and Cys465 in the enzyme from S. typhimurium were replaced by Arg28, Thr221, and Ala384 in Tk-TrpE. Nevertheless, significant inhibition by l-tryptophan was observed, with Ki values of 5.25 and 74 μM for ammonia and glutamine-dependent activities, respectively. The inhibition was competitive with respect to chorismate. The results suggest that the amino acid residues involved in the feedback inhibition by l-tryptophan in the case of Tk–AS complex are distinct from previously reported anthranilate synthases.

Binding Energies of Hexahydrated Alkaline Earth Metal Ions, M2+(H2O)6, M = Mg, Ca, Sr, Ba: Evidence of Isomeric Structures for Magnesium

Binding Energies of Hexahydrated Alkaline Earth Metal Ions, M²⁺(H₂O)₆, M = Mg, Ca, Sr, Ba: Evidence of Isomeric Structures for Magnesium

Lysine 194 Is Functional in Isocitrate Lyase from Escherichia coli

Lysine 194 in conserved stretch 1 of tetrameric isocitrate lyase from Escherichia coli has been replaced by using the restriction-enzyme-site elimination method of directed mutagenesis. Expression of subunits of each variant and of wild-type (wt) enzyme was equivalent and all variants assembled into tetrameric proteins. The variants K194R and K194H had kcat values relative to that of wt enzyme taken as 100 of 11 and 7, respectively. Km values for Mg2+-Ds-isocitrate (in mM units) were: 0.13 for wt-enzyme; 0.12 for the K194R variant; and 0.55 for the K194H variant. Substitution at position 194 of Leu or Glu resulted in zero catalytic activity. These results establish that Lys 194 is another functional residue in conserved stretch one of isocitrate lyase from E. coli besides H184, K193, C195, and H197. Because K194 can be specifically replaced by the basic residues His and Arg with resultant lowered activity and by His with an increased Km value, it may contribute to a cation center and facilitate substrate binding as well as orientation of the developing transition state.

Convenient Isolation and Kinetic Mechanism of Glutathionylspermidine Synthetase from Crithidia fasciculata

Trypanothione, the essential metabolite in the oxidant defense system of trypanosomatids, is synthesized by two distinct proteins, glutathionylspermidine synthetase and trypanothione synthetase. Glutathionylspermidine synthetase was purified to homogeneity from the trypanosomatid Crithidia fasciculata by aqueous two-phase systems and chromatography. The enzyme showed a specific activity of 38 micromol of glutathionylspermidine formed per min per mg of protein. Its molecular mass was 78 kDa in SDS-polyacrylamide gel electrophoresis, and it appeared predominantly monomeric in native polyacrylamide gel electrophoresis and gel filtration. The isoelectric point was at pH 4.6, and the pH optimum was near 7.6. Partial amino acid sequencing revealed homology with, but low similarity to, the glutathionylspermidine synthetase/amidase of Escherichia coli, and amidase activity was not detected in glutathionylspermidine synthetase of C. fasciculata. The kinetics of trypanosomatid glutathionylspermidine synthetase revealed a rapid equilibrium random mechanism with limiting Km values for Mg2+-ATP, GSH, and spermidine of 0.25 +/- 0.02, 2.51 +/- 0.33, and 0.47 +/- 0. 09 mM, respectively, and a kcat of 415 +/- 78 min-1. Partial reactions at restricted cosubstrate supply were not detected by 31P NMR, supporting the necessity of a quarternary complex formation for catalysis. ADP inhibited competitively with respect to ATP (Ki = 0. 08 mM) and trypanothione exerted a feedback inhibition competitive with GSH (Ki = 0.48 mM).

Evidence for two catalytically different magnesium-binding sites in acetohydroxy acid isomeroreductase by site-directed mutagenesis

Alignment of procaryotic and plant acetohydroxy acid isomeroreductase (EC 1.1.1.86) reveals five conserved regions designated domains I, II, III, IV, and V. Domain I has been previously proposed to correspond to the NADPH-binding site [Dumas et al. (1991) Biochem. J. 277, 469-475] and domain III to a putative magnesium-binding site [Sista & Bowman (1992) Gene 120, 115-118]. The binding and the function of this cation are of particular importance. First, Mg2+ is essential for the two-step reaction catalyzed by this enzyme: an isomerization followed by an NADPH-dependent reduction. Second, the plant acetohydroxy acid isomeroreductase exhibits Kd and Km values for Mg2+ of 5 microM and 6 microM, respectively. Such values correspond to the strongest affinity known between an enzyme and the metal ion. To determine if domain III of acetohydroxy acid isomeroreductase is effectively involved in magnesium binding, and with the goal to assign a function to the other conserved domains, site-directed mutagenesis was performed on each charged or polar conserved amino acids of domains II-V of the spinach acetohydroxy acid isomeroreductase. The results demonstrate that mutation of each of these amino acids leads to a partial or complete inactivation of enzyme activity. Steady-state kinetic analysis and equilibrium binding experiments show that both domains III and IV are directly involved in the binding of magnesium. Also, they suggest that magnesium bound to domain III plays a role in the reductive half-reaction, whereas, magnesium bound to domain IV is involved in the isomerization half-reaction.

Quantification of Muscle Tissue Magnesium and Potassium Using Atomic Absorption Spectrometry

Abstract Flame atomic absorption spectrometry (AAS) has been used to measure magnesium (Mg2+) and potassium (K+) contents of wistar rat myocardium and skeletal muscle. Ten to twenty mg of tissue was homogenized in 10 mM hydrochloric acid, centrifuged, and the clear supernatant was diluted three-fold in deionized distilled water before determination of Mg2+ and K+ concentrations. The calibration curves of both ions were linear from 0.25 to 2.0 mg/L for Mg2+ and from 1.5 to 15.0 mg/L for K+. The mean values (±SD) of Mg2+ and K+ contents for the analyzed cardiac muscle were 371.97±12.16 mg/Kg of wet tissue with a coefficient of variation (CV) of 3.3%, and 4571.02±359.74 mg/Kg with CV of 7.9%, respectively. Those for various skeletal muscles varied between 323.35-420.60 mg/Kg Mg2+, with CV ranging from 3.2% to 8.7%, and 4883.84-8156.68 mg/Kg K+, with CV ranging from 4.4% to 10.1%. The within-day CV were 2.8% to 3.8% for Mg2+ and 3.2% to 5.9% for K+, whereas day-to-day CV were 6.2% to 10.2% for Mg2+ and 8.7% to 10.5% for K+. Recoveries were 92.7% to 104.9% for Mg2+, and 93.2% to 106.1% for K+. Values for Mg2+ obtained by the proposed method were significantly different from those obtained by a published tissue digestion method, those for K+ were not significantly different.

Solubilization and Reconstitution of the Mg2+/2H+ Antiporter of the Lutoid Tonoplast from Hevea brasiliensis Latex.

The Mg2+/2H+ antiporter recently described on lutoid membrane (Z. Amalou, R. Gibrat, C. Brugidou, P. Trouslot, J.d'Auzac [1992] Plant Physiol 100: 255-260) was solubilized by octylglucoside and reconstituted into soybean liposomes using the detergent dilution method. Magnesium efflux or influx experiments were used to generate a H+ influx or efflux, respectively, monitored with the fluorescent probe 9-amino-6-chloro-2-methoxyacridine. Both experiments gave saturable H+ fluxes as a function of internal or external Mg2+ concentrations with similar kinetic parameters Km and Vmax. The Km value for Mg2+ (about 2 mM) was identical to that previously found in lyophilized-resuspended lutoid (reference therein), whereas the Vmax value was 14-fold higher. Since only 10% of the initial proteins were recovered in proteoliposomes, and electrophoretic patterns of the two kinds of vesicles differed significantly, it was inferred that the increase in Vmax was due essentially to an enrichment of the protein antiporter in the reconstituted fraction, owing to a selective effect of octylglucoside at both solubilization and reconstitution steps. None of the various divalent cations used could dissipate the pH gradient of control liposomes of soybean lipids, unless the divalent/H+ exchanger A23187 was added, whereas a rapid dissipation of the pH gradient was observed with reconstituted proteoliposomes from lutoid proteins, with the cation selectivity sequence Zn2+ > Cd2+ > Mg2+ in the millimolar concentration range. The divalent ions Ca2+, Ba2+, and Mn2+ were incapable of generating a H+ efflux in reconstituted proteoliposomes, whereas both Mg2+/H+ and Ca2+/H+ exchanges were observed in lyophilized-resuspended lutoids. Therefore, the lutoid membrane seems to contain separate Mg2+/H+ and Ca2+/H transport systems, the latter being eliminated during the solubilization/reconstitution of lutoid membrane proteins.

An improved assay method for UDP-glucuronyltransferase activity towards 5-hydroxytryptamine and the characteristic properties of the enzyme.

A simple and reproducible assay method for UDP-glucuronyltransferase (GT) towards 5-hydroxytryptamine (5-HT) was developed. It consists of the removal of unconjugated 5-HT by 0.6 N NH4OH-saturated n-amyl alcohol and the colorimetric estimation of 5-HT glucuronide. Using this assay method, some properties of the enzyme activity in rat liver microsomes were studied. Simple Michaelis-Menten kinetics was followed with respect to 5-HT and the apparent Km value for 5-HT was 0.1 mM. However, the deviation from this kinetics was observed with respect to UDP-glucuronic acid (UDPGA). The apparent Km values for UDPGA were 0.6 mM and 5 mM. The enzyme activity was stimulated by divalent cations. For Mg2+, the enzyme did not obey this kinetics, and the apparent Km values for Mg2+ were 1 mM and 10 mM. In the presence of Mg2+, the apparent Km value for 5-HT did not change but the Vmax value increased. On the other hand, the addition of a low concentration of Mg2+ decreased the apparent Km value for UDPGA and increased the Vmax value. The addition of a high concentration of Mg2+ did not change the apparent Km value for UDPGA but increased the Vmax value. These results indicate that the enzyme activity is stimulated by the formation of Mg(2+)-UDPGA complex which showed higher affinity for the enzyme than UDPGA at the low concentration of Mg2+ and further stimulated by the formation of Mg(2+)-enzyme complex at the higher concentration of Mg2+.

Interactions of metal ions with mu-monothiopyrophosphate.

mu-Monothiopyrophosphate (MTP) binds monovalent and divalent metal ions with dissociation constants (Kd) similar to those for pyrophosphate (PPi). The values of Kd for metal-MTP complexes are the following, as measured kinetically in the hydrolysis of MTP (microM): Mg2+, 32 +/- 4; Mn2+, 5.4 +/- 1.4; and Co2+, 27 +/- 15. The thermodynamically measured (EPR) values for Mg2+ and Co2+ are 28 +/- 13 microns and 11 +/- 4 microM, respectively; and the Kd for the complex MnPPi is 3.4 +/- 0.5 microM. The metal-MTP complexes undergo hydrolysis at rates modestly faster or slower than the rate at which MTP itself reacts. The complexes MgMTP2-, CoMTP2-, and MnMTP2- undergo hydrolytic cleavage with release of thiophosphate with observed first-order rate constants of 1.6 x 10(-2) min-1, 2.3 x 10(-2) min-1, and 0.6 x 10(-2) min-1, respectively, at 35 degrees C, compared with 1.1 x 10(-2) min-1 for MTP4- under the same conditions. Alkali metal cations also stimulate or retard the hydrolysis of MTP. At 25 degrees C and pH 12.2, the observed rate constant for tetramethylammonium MTP4- is 2.1 x 10(-3) min-1, and the estimated rate constants (min-1) for saturating alkali metals under the same conditions are as follows: Li+, 0.25 x 10(-3); Na+, 3.9 x 10(-3), K+, 6.7 x 10(-3); and Cs+, 6.7 x 10(-3). Divalent metal ions markedly retard the hydrolysis of MTP at pH 7 and 8 because complexation shifts the pH rate profile more than 2 pH units toward the acid side.(ABSTRACT TRUNCATED AT 250 WORDS)

Purification and properties of a kinase from Escherichia coli K-12 that phosphorylates two periplasmic transport proteins.

The phosphorylation in vivo and in vitro of the arginine-ornithine and the lysine-arginine-ornithine (LAO) periplasmic transport proteins of Escherichia coli K-12 was previously reported (Celis, R. T. F. (1984) Eur. J. Biochem. 145, 403-411). The phosphorylative reaction required ATP (as a direct energy donor), Mg2+, and a kinase that can be released by osmotic shock treatment of the cells. The enzyme was purified to electrophoretic homogeneity. The enzyme exhibited an ATPase activity and a kinase activity. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate gave an apparent molecular weight of 43,000 for the enzyme. The native protein showed the same molecular weight, suggesting that the protein is a monomer. The protein showed an apparent isoelectric point of 4.8 on isoelectric focusing. The two enzymatic reactions required a divalent cation and the apparent Km value for Mg2+ for the kinase activity was 0.5 mM. Mn2+ and Co2+ served as well as Mg2+, whereas Zn2+ and Ca2+ did not support activity. The ATPase activity of the enzyme yielded an apparent Km value for ATP of 50 microM. A similar value, Km of 100 microM, was calculated for the kinase activity with different concentrations of ATP. The enzyme showed a pH optimum of 7.3.

Regulation of Maize (Zea mays L.) Phosphoenolpyruvate Carboxylase Isoenzymes by pH

Summary The activity of phosphoenolpyruvate carboxylase (PEPC) isoenzymes from maize leaves is regulated by pH. The S0.5 values for phosphoenolpyruvate (PEP) and Mg2+ ions decrease for both isoenzymes at pH below 7.5 and above 8.5. The Hill coefficient values for PEP are about 1.0 at pH above 8.0 for both isoenzymes, however, they increase to values higher than 1.0 with the decrease of pH below 7.5 (mainly with the PEPC I isoenzyme). The Hill coefficient values for Mg 2+ are pH-independent. The pK values of the amino acid groups present in the active site of the protein molecule of both PEPC isoenzymes are determined from the dependence of the enzyme's activity on the concentration of hydrogen ions. The pK values of about 7.3, 8.5 and 9.0 indicate that histidine, cysteine and lysine residues, respectively, are present in the active centre of both isoenzymes.

Properties of phosphofructokinase from carp, eel and rainbow trout liver.

Phosphofructokinase (EC 2.7.1.11) from rainbow trout liver, eelliver, and carp hepatopancreas was purified by ammonium sulfate fractionation, followed by ethanol fractionation, DEAF-cel-lulose chromatography, and Sephadex G-200 gel filtration. The rainbow trout and eel enzymes were obtained as a homogeneous peak by gel filtration. In the carp liver, there was a considerable loss of enzyme activity during dialysis. The molecular weight of the enzyme was estimated to be about 340, 000 by gel filtration, and the pH optimum of the activity was found to be around 8. The Km values for F6P of the rainbow trout, eel and carp enzymes were 19, 24, and 71μM, respectively, and that for ATP were 15, 19, and 22μM, respectively. The reaction mechanism by which the carp enzyme differed from that of the other two fish enzymes. Fish phosphofructokinase required Mg2+ for activation. The Km values for Mg2+ of the three fish enzymes ranged from O.83 to 1.1mM. The enzyme showed maximum activity around i mM ATP in the presence of 2mM F6P beyond which the activity decreased with increasing ATP concentration. ADP inhibited the enzyme activity, depending of F6P and ATP concentrations. AMP was found to activate the enzyme in the presence of ATP at inhibitory concentrations. The enzyme also utilized ITP, UTP, and CTP as a phosphoryl donor.

Stimulation of guanylate cyclase activity and reduction of adenylate cyclase activity by granulocyte-macrophage colony-stimulating factor in human blood neutrophils.

Human neutrophils were incubated with granulocyte-macrophage (GM)-CSF and examined for changes in second messenger systems. Twofold increases in cGMP but not cAMP were measured after 5 to 20 min with 100 U/ml GM-CSF. Guanylate cyclase activities in membrane and cytosol fractions were increased to the same extent whether measured in the presence of Mg2+ or Mn2+, or in the cytosol with Mg2+ + N-methyl-N'-nitro-N-nitroso-guanidine. Kinetic studies of the cytosol enzyme showed no changes in the Km values for Mg2+ and Mn2+dependent guanylate cyclase activities (0.91 and 0.022 mM, respectively), whereas Vm values were increased after treating intact cells with GM-CSF. Two peaks of guanylate cyclase activity were observed, one at 10 and another at 60 min after adding 100 U/ml GM-CSF, whereas only one peak at 5 min occurred with 1 U/ml. Adenylate cyclase activity was reduced by nearly 50% after adding 100 U/ml GM-CSF for 10 to 30 min. These effects were also seen in the presence of several hormonal and nonhormonal adenylate cyclase stimulators. In contrast, small increases in adenylate cyclase activity occurred after adding 1 U/ml GM-CSF. In experiments to examine the pathway of guanylate cyclase activation by GM-CSF, we observed no changes in inositol phosphates, intracellular calcium ion, or cytosolic protein kinase C. The augmentation of chemotactic peptide-induced superoxide production by GM-CSF concentrations, may be related to the effects of the higher levels of GM-CSF to stimulate late increases in guanylate cyclase or decreases in adenylate cyclase.