Purified dextransucrase from Pediococcus pentosaceus CRAG3 as food additive.

The extracellular crude dextransucrase (0.67 U/mg) from P. pentosaceus CRAG3 (GenBank accession number JX679020) after PEG-1500 fractionation gave specific activity, 20.0 U/mg which by gel filtration resulted in 46.0 U/mg. The purified dextransucrase displayed molecular size of approximately, 224 kDa. The optimum assay conditions for dextransucrase activity were 5% sucrose in 20 mM sodium acetate buffer (pH 5.4) and 30 oC. The dextransucrase was stable up to 40 oC and at pH range of 5.4-7.0. The metal ions such as Co2+, Ca2+, Mg2+ and Zn2+ stimulated the dextransucrase activity by 56, 44, 14 and 12%, respectively. It was most stable at -20 oC with half-life of 307 days. Amongst various additives used, glycerol and Tween 80 provided significant stability to the enzyme with half-life 15.5 and 85.5 h, respectively as compared to control (6.9 h). The solidification of sucrose supplemented milk by purified dextransucrase due to dextran synthesis displayed its application as additive for improving the texture of dairy products.

Efeito de íons Cu2+ e Zn2+ em atividade Ca-ATPásica isolada de larvas de Pachymerus nucleorum (Fabricius) (Coleoptera: Chrysomelidae, Bruchinae) / Effect of Cu2+ and Zn2+ ions in Ca-ATPase activity isolated from Pachymerus nucleorum (Fabricius) (Coleoptera: Chrysomelidae, Bruchinae) larvae

As ATPases, um importante alvo de inseticidas, são enzimas que hidrolisam o ATP e utilizam a energia liberada no processo para realizar algum tipo de trabalho celular. A larva de Pachymerus nucleorum (Fabricius) possui uma ATPase que apresenta alta atividade Ca-ATPásica, mas não expressa atividade Mg-ATPásica. Nesse trabalho, foi testado o efeito de íons zinco e cobre na atividade Ca-ATPásica dessa enzima. Mais de 90 por cento da atividade Ca-ATPásica foi inibida em 0,5 mM de íons cobre ou 0,25 mM de íons zinco. Na presença de EDTA, mas não na sua ausência, a inibição por zinco foi revertida pelo aumento da concentração de cálcio. A inibição por íons cobre, não foi revertida nem na presença e nem na ausência de EDTA. O tratamento da fração ATPase com cobre, previamente ao ensaio de atividade ATPásica, não inibiu a atividade Ca-ATPásica sugerindo que o íon cobre não liga diretamente a enzima. Os resultados sugerem que íons zinco e cobre formam complexo com o ATP e se ligam à enzima inibindo sua atividade Ca-ATPásica.

ATPases, an important target of insecticides, are enzymes that hydrolyze ATP and use the energy released in that process to accomplish some type of cellular work. Pachymerus nucleorum (Fabricius) larvae possess an ATPase, that presents high Ca-ATPase activity, but no Mg-ATPase activity. In the present study, the effect of zinc and copper ions in the activity Ca-ATPase of that enzyme was tested. More than 90 percent of the Ca-ATPase activity was inhibited in 0.5 mM of copper ions or 0.25 mM of zinc ions. In the presence of EDTA, but not in the absence, the inhibition by zinc was reverted with the increase of calcium concentration. The inhibition by copper ions was not reverted in the presence or absence of EDTA. The Ca-ATPase was not inhibited by treatment of the ATPase fraction with copper, suggesting that the copper ion does not bind directly to the enzyme. The results suggest that zinc and copper ions form a complex with ATP and bind to the enzyme inhibiting its Ca-ATPase activity.

Divalent cations as modulators of neuronal excitability: Emphasis on copper and zinc

Based on indirect evidence, a role for synaptically released copper and zinc as modulators of neuronal activity has been proposed. To test this proposal directly, we studied the effect of copper, zinc, and other divalent cations on voltage-dependent currents in dissociated toad olfactory neurons and on their firing rate induced by small depolarizing currents. Divalent cations in the nanomolar range sped up the activation kinetics and increased the amplitude of the inward sodium current. In the micromolar range, they caused a dose dependent inhibition of the inward Na+ and Ca2+ currents (INa and ICa) and reduced de amplitude of the Ca2+-dependent K+ outward current (ICa-K). On the other hand, the firing rate of olfactory neurons increased when exposed to nanomolar concentration of divalent cations and decreased when exposed to micromolar concentrations. This biphasic effect of divalent cations on neuronal excitability may be explained by the interaction of these ions with high and low affinity sites in voltage-gated channels. Our results support the idea that these ions are normal modulators of neuronal excitability.

Effect of metal ions on binding of bilirubin to erythrocyte membranes.

Binding of bilirubin to different erythrocyte membranes, namely, human, buffalo, sheep and goat, pre-incubated with different concentrations of metal ions was studied. The results showed that among the different metal ions used, Ca2+ had the highest potential in increasing the amount of bound bilirubin followed by Sr2+ and Mg2+, whereas Ba2+ had the lowest potential. Treatment of these membranes with Ca2+ led to an increase in the amount of bound bilirubin in all membranes. However, human erythrocyte membranes pretreated with Ca2+, bound the highest amount of bilirubin compared to other erythrocyte membranes. Increase in bilirubin binding upon Ca2+-treatment can be ascribed to shielding effect, redistribution of phospholipids as well as increase in surface hydrophobicity induced by Ca2+.

Effects of cations on ceramide-activated protein phosphatase 2A

Characterization of ceramide-effector(s), which includes protein phosphatase 2A (PP2A) is an important prelude to understanding the molecular basis of sphingolipid-mediated biological effects such as cell growth, differentiation and apoptosis. Recently, the existence of a metal-dependent form of PP2A has been reported (Cai et al., 1995). In this study, we investigated the effects of metal ions and chelators on ceramide-activated PP2A (CAPP). Our study demonstrates that at 0.5 mM concentration, Mg2+ appears to have no significant effect on either basal or ceramide-stimulated phosphatase activities, whereas Ca2+ stimulated the basal phosphatase activity, but was inhibitory towards CAPP. Moreover, the divalent cations Cr2+, Mn2+, Fe2+, Ni2+, Cu2+ and Zn2+ were tested and all were found to be inhibitory towards both CAPP and basal phosphatase activities. By contrast, Cs+ and Li+ had almost no effect on CAPP, although both stimulated basal phosphatase activity. The effects of EDTA and EGTA were tested and it was observed that EDTA decreased CAPP activity in a dose-dependent fashion, but had no effect upon basal phosphatase activity. These results suggest that CAPP is a metal-dependent protein, but, because Ca2+ inhibitied CAPP and EGTA was much less potent than EDTA in inhibiting CAPP, Ca2+ is unlikely to be its metal cofactor.

Effects of cations on ceramide-activated protein phosphatase 2A

Characterization of ceramide-effector(s), which includes protein phosphatase 2A (PP2A) is an important prelude to understanding the molecular basis of sphingolipid-mediated biological effects such as cell growth, differentiation and apoptosis. Recently, the existence of a metal-dependent form of PP2A has been reported (Cai et al., 1995). In this study, we investigated the effects of metal ions and chelators on ceramide-activated PP2A (CAPP). Our study demonstrates that at 0.5 mM concentration, Mg2+ appears to have no significant effect on either basal or ceramide-stimulated phosphatase activities, whereas Ca2+ stimulated the basal phosphatase activity, but was inhibitory towards CAPP. Moreover, the divalent cations Cr2+, Mn2+, Fe2+, Ni2+, Cu2+ and Zn2+ were tested and all were found to be inhibitory towards both CAPP and basal phosphatase activities. By contrast, Cs+ and Li+ had almost no effect on CAPP, although both stimulated basal phosphatase activity. The effects of EDTA and EGTA were tested and it was observed that EDTA decreased CAPP activity in a dose-dependent fashion, but had no effect upon basal phosphatase activity. These results suggest that CAPP is a metal-dependent protein, but, because Ca2+ inhibitied CAPP and EGTA was much less potent than EDTA in inhibiting CAPP, Ca2+ is unlikely to be its metal cofactor.

Optimisation of nutritional requirements and process control parameters for the production of HA-2-91, a new tetraene polyene antibiotic.

HA-2-91, a new tetraene polyene antibiotic produced during submerged fermentation of Streptomyces arenae var ukrainiana. Optimisation of nutritional requirements and process control parameters were studied for higher productivity of HA-2-91 during fermentative production in shaken flasks using complex media. Experimental findings indicate that jowar starch (Sorghum vulgare) is the best carbon source while cornsteep liquor in combination with peanut meal are the best nitrogen sources. Exogenous addition of amino acids, divalent cations and fatty acids suppressed the productivity of HA-2-91. Incorporation of glucose into the production medium above 5% (w/v) results in inhibition of productivity of HA-2-91 which may be due to catabolite regulation. The concentration of phosphate ions above 10 ppm also showed similar suppression effect on the productivity of HA-2-91. However, ferrous ions at 100 ppm showed slight stimulatory effect on the production of HA-2-91. The optimum process control parameters for the production of HA-2-91 were found to be temperature, 28 degrees C; inoculum concentration from seed to production medium, 1% (v/v); pH and volume of production medium 6.5 and 100 ml respectively; and fermentation cycle time, 120 hours.

Purification, characterisation and steady state kinetic properties of cytosolic pyruvate kinase free of phosphoenol pyruvate phosphatase activity from germinating mung beans (Vigna radiata L.)

Mung bean pyruvate kinase (PK) practically free from PEP-phosphatase has been purified about 36 fold. The enzyme is irreversibly inactivated on desalting by gel filtration or dialysis (without EDTA). The inactivation is also observed in the presence of ATP, Mg2+ or thiols but is prevented by a non-proteinous, heat stable, small molecular mass factor present in the mung bean extract. Mung bean PK has a molecular mass of 210 kDa. It shows single exponential decay of activity at various temperatures (-4 to 60 degrees C). The Km of PEP and ADP are found to be 0.12 and 0.24 mM, respectively at pH 6.5, when the enzyme is saturated with the second substrate. The Km values for PEP and ADP are 0.05 and 0.16 mM, at pH 8.5 and 0.09 and 0.17 mM, respectively at pH 7.5. The optimum pH is 7.5. The enzyme shows an absolute requirement for Mg2+ (Km 0.43 mM) or Mn2+ ions (Km 0.125 mM). Potassium ions are not essential but activate the enzyme in the presence of Mg2+ or Mn2+ ions. ATP shows competitive inhibition with ADP and non-competitive with PEP. Kinetic studies at different pHs and effects of ATP suggest the formation of a ternary complex (E.ADP.PEP) by a combination of random and compulsory ordered pathways depending on the experimental conditions.

Effect of trace metals on synthesis of exoproteins and exotoxins by Staphylococcus aureus.

Staphylococcus aureus did not grow in chemically defined medium completely free of trace metals. At low concentrations all the trace metals showed stimulating effect on growth and exoprotein production. Increasing concentrations of Ca2+ and Mg2+ were growth stimulatory whereas Co2+, Cu2+, Fe3+, Mn2+, Ni2+ and Zn2+ were inhibitory. The amount of exoprotein and haemolytic exotoxins produced in relation to final viable count increased with increasing concentrations of growth inhibitory trace metals whereas it decreased with increasing concentrations of Ca2+ and Mg2+. Trace metals did not show significant influence on amount of haemolytic exotoxins produced in relation to total exoproteins. The results indicate presence of a common regulatory mechanism for all types of exoproteins produced by S. aureus.

Divalent cations and radiation induced lipid peroxidation.

Response of Co2+, Cu2+, Fe2+, Ni2+ and Mn2+ against radiation induced lipid peroxidation has been examined. Liposomes prepared from L-alpha-lecithin were irradiated with or without divalent cations, Co2+ and Cu2+ inhibited lipid peroxidation in concentration dependent manner. On the other hand Fe2+, Ni2+ and Mn2+ enhanced the peroxidation. The enhancement of peroxidation due to Ni2+ was biphasic in nature. Higher concentration of Mn2+ decreased the peroxidation. Except Cu2+ no other cation studied could change the non-linear pattern of radiation induced lipid peroxidation. The pro- or antioxidant effect of divalent cations may be due to their reactivity towards oxygen and oxygen free radicals, and at least in part to their hard/soft acid character.

Purification and characterization of aminopeptidase activity from rat urine

The aminopeptidase activity of rats urine was tested using L-aminoacy1-2-naphtylamides and L-Leu-p-nitroanilide. The enzyme preparation was purified by ion-exchange and gel filtration chromatography and showed only a single active protein active protein band on 7.5% polyacrylamide gel electrophoresis. The enzyme was characterized by studying the effects of ions (divalent cations and chloride), chelating agents and -SH and -S-S- group reagents and by determining kinetic parameters (Km and Vmax for different substrates and K1 for amino acids, antibiotics and anti-inflammatory drugs)

Influence of divalent cations on rabbit atria and modification of catecholamine response on pacemaker cells.

Spontaneously beating isolated atria of rabbits responded to Mn++ and Co++ with a progressively increasing negative chronotropism and inotropism, which was reversible by washout and by elevating the bath concentration of Ca++. The cumulative dose response curve for adrenaline for chronotropic response was markedly shifted to the right in the presence of Mn++ or Co++. This effect was also reversible. Verapamil produced only a moderate decline in spontaneous rate and contractility and did not block the chronotropic response to adrenaline. It is concluded that Mn++ and Co++ block the action of catecholamine on the pacemaker cells and they differ at least in part from organic calcium channel antagonists in their mechanism of action.