多花野牡丹红色素的提取及其稳定性研究

本文对多花野牡丹（Melastoma affine）果实胎座的色素进行了理化性质的探讨,对光、温度、氧化还原介质、7种金属离子、葡萄糖、蔗糖、苯甲酸钠对色素稳定性的影响进行了研究。结果表明,该色素为水溶性花青苷类色素,它在60 ℃以下稳定性较好,对光的耐受性较差。色素在酸性条件下稳定性好,耐还原性也较好,但耐氧化性较差。7种金属离子中,Fe3+、Cu2+、Mn2+、Zn2+对色素有明显降解作用,而Na+、Mg2+、Al3+对色素稳定性较好并有不同程度的护色作用。葡萄糖、蔗糖、苯甲酸钠对色素无不良影响。     

Comparison of Different Cations (Mn2+, Mg2+, Ca2+) on the Hydrolytic Activity of Chloroplast ATPase

The influences of Mn2+, Mg2+, and Ca2+ on the enzymic activity of chloroplast ATPase have been compared, using an HPLC method for the separation of ADP. The dissociation constants of the divalent ion-ATP complexes have been determined by a spectrophotometric method, with the dye antipyrylazo III, and enzymic constants (dissociation constant of the ion-enzyme complexes, Michaelis constants, maximum rates) have been calculated. The comparison between the rates obtained, respectively, with Mn2+ and Ca2+ alone with that given by the mixture of these two ions, allows us to conclude that, as for Mg2+, Mn2+ is also an activator of chloroplast ATPase and that metal-free ATP is the true substrate.     

Metal ion requirements for sequence-specific endoribonuclease activity of the Tetrahymena ribozyme

A shortened form of the self-splicing intervening sequence RNA of Tetrahymena thermophila acts as an enzyme, catalyzing sequence-specific cleavage of RNA substrates. We have now examined the metal ion requirements of this reaction. Mg2+ and Mn2+ are the only metal ions that by themselves give RNA enzyme activity. Atomic absorption spectroscopy indicates that Zn, Cu, Co, and Fe are not present in amounts equimolar to the RNA enzyme and when added to reaction mixtures do not facilitate cleavage. Thus, these ions can be eliminated as cofactors for the reaction. While Ca2+ has no activity by itself, it alleviates a portion of the Mg2+ requirement; 1 mM Ca2+ reduces the Mg2+ optimum from 2 to 1 mM. These results, combined with studies of the reactivity of mixtures of metal ions, lead us to postulate that two classes of metal ion binding sites are required for catalysis. Class 1 sites have more activity with Mn2+ than with Mg2+, with the other divalent ions and Na+ and K+ having no activity. It is not known if ions located at class 1 sites have specific structural roles or are directly involved in active-site chemistry. Class 2 sites, which are presumably structural, have an order of preference Mg2+ greater than or equal to Ca2+ greater than Mn2+ and Ca2+ greater than Sr2+ greater than Ba2+, with Zn2+, Cu2+, Co2+, Na+, and K+ giving no detectable activity over the concentration range tested.     

A study of the molecular mechanism of DNA interaction with divalent metal ions

The interaction of DNA with divalent metal ions: Ba2+, Mg2+, Mn2+, Ni2+, Cu2+ in solutions at different ionic strengths mu was investigated. The combination of following methods: flow birefringence, viscometry, UV-spectroscopy and circular dichroism made possible to follow the state of the secondary and tertiary structure of the DNA molecule during its interaction with ions. The presence of divalent ions in solution affects the hydrodynamic properties of DNA only at low mu. At high mu the difference in the action of mono- and divalent ions disappears. The persistence length of DNA does not change during the experiment. It is shown that the Mg2+ and Ba2+ ions interact only with phosphate groups of DNA but Mn2+, Ni2+, Cu2+ ions interact also with the nitrogen bases of the macromolecule.     

Inhibition of glycogen synthase (casein) kinase-1 by divalent metal ions

The specificity of glycogen synthase (casein) kinase-1 (CK-1) for different divalent metal ions was explored in this study. Of nine metal ions (Mg2+, Mn2+, Zn2+, Cu2+, Ca2+, Ba2+, Ni2+, Co2+, Fe2+) tested, only Mg2+ supported significant kinase activity. Several of the other metals, however, inhibited the Mg2+-stimulated kinase activity. Half-maximal inhibitions by Mn2+, Zn2+, Co2+, Fe2+, and Ni2+ were observed at 55, 65, 110, 125, and 284 microM, respectively. Kinetic analyses indicate that the metal ions are acting as competitive inhibitors of CK-1 with respect to the protein substrate (casein) and as noncompetitive inhibitors with respect to the nucleotide substrate (ATP). The inhibition of CK-1 by the different metal ions can be reversed by EGTA.     

Role of the capsule produced by Bacillus megaterium ATCC 19213 in the accumulation of metallic cations

A study was undertaken to determine if the capsule produced by Bacillus megaterium ATCC 19213 was capable of binding metallic ions. For non-toxic metallic ions, this was accomplished by determining the relative concentrations of Fe2+, Ca2+, Zn2+, Mg2+, and Mn2+ removed from a chemically defined medium by the normally capsulated parent strain and two mutants with much smaller capsules. For toxic metals, the rates of respiration of the parent strain and a small capsule mutant in the presence of Cu2+, Hg2+, and Ag1+ were compared. It was found that the parent strain accumulated more Ca2+, Mg2+, and Mn2+. Accumulation of Fe2+ and Zn2+ was similar for the parent strain and the small capsule mutants. Respiration of the parent strain was less inhibited by Cu2+, Hg2+, and Ag1+, indicating that these metals are also bound to the capsule.     

Effect of metal ions on the activity of the catalytic domain of calcineurin

Calcineurin (CN) is a heterodimer, composed of a catalytic subunit (CNA) and a regulatory subunit (CNB). There are four functional domains present in CNA, which are catalytic domain (CNa), CNB-binding domain (BBH), CaM-binding domain (CBH) and autoinhibitory domain (AI). It has been shown previously that the in vitro activity of calcineurin is relied primarily on the binding of metal ions. Mn2+ and Ni2+ are the most crucial cation-activators for this enzyme. In order to determine which domain(s) in CN is functionally regulated by metal ions, the rat CNA alpha subunit and its catalytic domain (CNa) were cloned and expressed in E. coli. The effects of Mn2+, Ni2+ and Mg2+ on the catalytic activity of these purified proteins were examined. Our results demonstrate that all the metal ions tested in this study activated either CNA or CNa. However, the activation degree of CNa by the metal ions was much higher than that of CNA. In term of different metal ions, the activating extents to CNA and CNa were different. To CNA, the activating order from high to low was Mg2+ > > Ni2+ > Mn2+, but Mn2+ > Ni2+ > > Mg2+ to CNa. No effect of CaM/Ca2+ and CNB/Ca2+ on the activity of CNa was observed in our experiments. Moreover, a weak interaction (or untight coordination binding) between metal ions and the enzyme molecule was also identified. These results suggest that the activation of these enzymes by the exogenous metal ions might be via both regulating fragment of CNA (including BBH, CBH and AI) and catalytic domain (CNa), and mainly via regulating fragment to CNA and mainly via catalytic domain to CNa. The activating extents of metal ions via catalytic domain were higher than that via regulating fragment. The results obtained in this study should be very useful for understanding the molecular mechanism underlying the interaction between calcineurin and metal ions, especially Mn2+, Ni2+ and Mg2+.     

Characteristics of the ionic composition and ultrastructure of Bordetella pertussis in controlled regulation of the mineral element content in a growth medium

The content of trace elements necessary for the normal growth of bacteria was found to have no effect on the intracellular concentration of Ca2+ and Al3+. The content of Cu2+, Fe3+, Mn2+, Mg2+ was considerably reduced. The addition of Mg2+ at different concentrations to this culture medium stimulated the capacity of cells for accumulating not only Mg2+, but also some other ions. Their maximum intracellular concentration was observed when the concentration of Mg2+ in the culture medium was 41 mM. The growth of microbial cells in the standard culture medium containing Mg2+ at a concentration of 4 mM was accompanied by the increased consumption of elements actively participating in redox reactions (Cu2+, Fe3+, Mn2+). Shifts in the ionic composition of microbial cells were manifested by the morphological features of B. pertussis, linked with the increased synthesis of crystalloid structures. The influence of Mn2+, Al3+, Zn2+ at different concentrations on the ionic composition and morphology of B. pertussis was studied.     

Thermostable DNA polymerase from Thermus thermophilus B35: influence of divalent metal ions on the interaction with deoxynucleoside triphosphates

The interaction of DNA polymerase from Thermus thermophilus B35 (Tte-pol) with deoxynucleoside triphosphates in the presence of different divalent metal ions has been studied. DNA synthesis and competitive inhibition of the polymerase reaction by non-complementary dNTPs are described with corresponding kinetic schemes. The co-factor properties of some metals (Mg2+, Mn2+, Co2+, Ni2+, Cu2+, Ca2+, Cd2+, and Zn2+) were investigated, and their activating concentration ranges were determined. It was found that kcat values are significantly decreased and Km values slowly decrease when Mn2+ displaces Mg2+. The value of Kd for DNA template-primer is Me2+-independent, whereas Kd values for non-complementary dNTPs decrease in the presence of Mn2+. Tte-pol processivity but not DNA synthesis efficiency is Me2+-type independent.     

金属离子对地衣芽孢杆菌合成多聚γ-谷氨酸的影响

多聚γ 谷氨酸 [γ Ｐｏｌｙ(ｇｌｕｔａｍｉｃａｃｉｄ) ,γ ＰＧＡ]是由某些杆菌 (Ｂａｃｉｌｌｕｓ)合成的一种细胞外水溶性高分子氨基酸聚合物 ,是由Ｌ 谷氨酸、Ｄ 谷氨酸两种构型的单体通过γ 酰胺键聚合形成的[1 ] 。γ ＰＧＡ具有极佳的成膜性、成纤维性 ,阻氧性、可塑性、粘结性、保湿性和可生物降解等许多独特的理化和生物学特性[2 ,3] 。因此 ,γ ＰＧＡ可以被广泛用于医药制造 ,食品加工 ,蔬菜、水果、海产品防冻、保鲜 ,化妆品工业 ,烟草、皮革制造工业和植物种子保护等许多领域 ,是一种有极大开发价值和前景的多功能新型生物制…     

The Influence of Divalent Cations and Substrate Concentration on the Incorporation of Myo-inositol into Phospholipids of Isolated Bovine Oligodendrocytes

The incorporation of myo-inositol into phosphatidylinositol by two routes (CTP-independent and CTP-independent) has been investigated in homogenates prepared from isolated bovine oligodendrocyte perikarya. The CTP-dependent route has the higher maximum velocity of inositol incorporation and can utilise either Mn2+ or Mg2+ as a divalent ion cofactor. This route of inositol incorporation is also strongly inhibited by Ca2+ ions at concentrations less than 1 mM. The primary site of the inhibitory action appears to be the enzyme CDP-diglyceride inositol phosphatidyl transferase (EC 2.7.8.11) though synthesis of CDP-diacylglycerol is also inhibited by endogenous Ca2+ present in the oligodendrocyte homogenate. In contrast, CTP-independent inositol incorporation into phosphatidylinositol is only stimulated by Mn2+ (Zn2+,Cu2+, Mg2+, Ca2+ and Co2+ are ineffective) and is not inhibited by Ca2+, at least up to a concentration of 1 mM.     

DNA structural transitions induced by divalent metal ions in aqueous solutions

Using methods of IR spectroscopy, light scattering, gel-electrophoresis DNA structural transitions are studied under the action of Cu2+, Zn2+, Mn2+, Ca2+ and Mg2+ ions in aqueous solution. Cu2+, Zn2+, Mn2+ and Ca2+ ions bind both to DNA phosphate groups and bases while Mg2+ ions-only to phosphate groups of DNA. Upon interaction with divalent metal ions studied (except for Mg2+ ions) DNA undergoes structural transition into a compact form. DNA compaction is characterized by a drastic decrease in the volume occupied by DNA molecules with reversible formation of DNA dense particles of well-defined finite size and ordered morphology. The DNA secondary structure in condensed particles corresponds to the B-form family. The mechanism of DNA compaction under Mt2+ ion action is not dominated by electrostatics. The effectiveness of the divalent metal ions studied to induce DNA compaction correlates with the affinity of these ions for DNA nucleic bases: Cu2+>Zn2+>Mn2+>Ca2+>Mg2+. Mt2+ ion interaction with DNA bases (or Mt2+ chelation with a base and an oxygen of a phosphate group) may be responsible for DNA compaction. Mt2+ ion interaction with DNA bases can destabilize DNA causing bends and reducing its persistent length that will facilitate DNA compaction.     

Interaction of divalent metal ions with human serum albumin

ESR study was carried out of the interaction between Zn2+, Cu2+, Ca2+, Mg2+ ions and human serum albumin (HSA) in the presence of Mn2+ ions which depends on pH. Competitive binding of these ions with "manganese-binding" sites of albumin was shown to depend on pH. An analysis of concentration dependence of binding these ions with human serum albumin confirmed earlier supposition about the nature of the binding sites of Mn2+ ions with HSA.     

Oxidoreductase activities of polyclonal IgGs from the sera of Wistar rats are better activated by combinations of different metal ions

It was shown that IgGs purified from the sera of healthy Wistar rats contain several different bound Me2+ ions and oxidize 3,3'-diaminobenzidine through a H2O2-dependent peroxidase and H2O2-independent oxidoreductase activity. IgGs have lost these activities after removing the internal metal ions by dialysis against EDTA. External Cu2+ or Fe2+ activated significantly both activities of non-dialysed IgGs containing different internal metals (Fe > or = Pb > or = Zn > or = Cu > or = Al > or = Ca > or = Ni > or = Mn > Co > or = Mg) showing pronounced biphasic dependencies corresponding to approximately 0.1-2 and approximately 2-5 mM of Me2+, while the curves for Mn2+ were nearly linear. Cu2+ alone significantly stimulated both the peroxidase and oxidoreductase activities of dialysed IgGs only at high concentration (> or = 2 mM), while Mn2+ weakly activated peroxidase activity at concentration >3 mM but was active in the oxidoreductase oxidation at a low concentration (<1 mM). Fe2+-dependent peroxidase activity of dialysed IgGs was observed at 0.1-5 mM, but Fe2+ was completely inactive in the oxidoreductase reaction. Mg2+, Ca2+, Zn2+, Al2+ and especially Co2+ and Ni2+ were not able to activate dialysed IgGs, but slightly activated non-dialysed IgGs. The use of the combinations of Cu2+ + Mn2+, Cu2+ + Zn2+, Fe2+ + Mn2+, Fe2+ + Zn2+ led to a conversion of the biphasic curves to hyperbolic ones and in parallel to a significant increase in the activity as compared with Cu2+, Fe2+ or Mn2+ ions taken separately; the rates of the oxidation reactions, catalysed by non-dialysed and dialysed IgGs, became comparable. Mg2+, Co2+ and Ni2+ markedly activated the Cu2+-dependent oxidation reactions catalysed by dialysed IgGs, while Ca2+ inhibited these reactions. A possible role of the second metal in the oxidation reactions is discussed.     

Kinetic effects of ATP, divalent metal ions and pH on chicken liver mevalonate 5-diphosphate decarboxylase

The activity of chicken liver mevalonate 5-diphosphate decarboxylase was measured over a wide range of Mg2+ and ATP concentrations. It was found that free ATP activated the enzyme, whereas free Mg2+ had no effect on the enzyme activity. Computed analyses of free species concentrations and pH studies indicated that MgATP2- is the true substrate. The relative efficiencies of Mg2+, Mn2+, Cd2+, and Zn2+ as activating metal ions were evaluated in terms of V/Km for the corresponding (metal-ATP)2- complexes, and the relative ratios were: Mn2+ 100, Cd2+ 37, Mg2+ 14, Zn2+ 1.7. Inhibitory effects were demonstrated for all free divalent cations tested, except for Mg2+, and were in the order Zn2+ greater than Cd2+ greater than Mn2+.     

Effects of Ca2+ on ethanolaminephosphotransferase and cholinephosphotransferase in rabbit platelets

The effects of Ca2+ on ethanolaminephosphotransferase [EC 2.7.8.1] and cholinephosphotransferase [EC 2.7.8.2] activities in rabbit platelet membranes were studied using endogenous diglyceride and CDP-[3H]ethanolamine or CDP-[14C]choline as substrates. Both transferases required Mn2+, Co2+, or Mg2+ as a metal cofactor and the optimal concentrations of the metals for both activities were about 5, 10, and 5 mM, respectively. When 5 mM Mg2+ was used as a cofactor, both transferase activities were inhibited by a low concentration of Ca2+ (half maximal inhibition at approx. 15 microM). In the presence of 5 mM Mn2+, however, approx. 5 mM Ca2+ was required to produce half maximal inhibition. The Ca2+-induced inhibition was reversible and the rate of the inhibition was not affected either by the concentrations of the CDP-compound or by exogenously added diacylglycerol. The relationship between Ca2+ and both Mg2+ and Mn2+ on the transferase activities was competitive. 45Ca2+ binding (and/or uptake) to the platelet membranes was inhibited by Mn2+, Mg2+, and Co2+, in a concentration-dependent manner. However, the inhibitory effects of the three metal ions on the total Ca2+ binding (and/or uptake) did not correlate with the activation of both transferase activities by the three metal ions in the presence of Ca2+. These results suggest that both transferase activities are regulated by low concentrations of Ca2+ in the presence of optimal concentrations of Mg2+, and that the inhibition is mediated directly by Ca2+, which interacts with a specific metal cofactor binding site(s) of the transferases.     

Effects of bivalent cations on prostaglandin biosynthesis and phospholipase A2 activation in rabbit kidney medulla slices.

The bivalent cations Ca2+, Mg2+, Co2+, Mn2+, Sr2+ and Ba2+ were compared for their stimulatory or inhibitory effect on prostaglandin formation in rabbit kidney medulla slices. Ca2+, Mn2+ and Sr2+ ions stimulated prostaglandin generation up to 3--5-fold in a time- and dose-dependent manner (Ca2+ greater than Mn2+ congruent to Sr2+). The stimulation by Mn2+ (but not by Sr2+) was also observed in incubations of medulla slices in the presence of Ca2+. Mg2+ and Co2+ ions were without significant effects on either basal or Ca2+-stimulated prostaglandin synthesis. The stimulatory effects of Ca2+, Mn2+ and Sr2+ on medullary generation of prostaglandin E2 were found to correlate with their stimulatory effects on the release of arachidonic acid and linoleic acid from tissue lipids. The release of other fatty acids was unaffected, except for a small increase in oleic acid release. As both arachidonic acid and linoleic acid are predominantly found in the 2-position of the glycerol moiety of phospholipids, the stimulation by these cations of prostaglandin E2 formation appears to be mediated via stimulation of phospholipase A2 activity.     

Metal requirements of a diadenosine pyrophosphatase from Bartonella bacilliformis: magnetic resonance and kinetic studies of the role of Mn2+

Recombinant IalA protein from Bartonella bacilliformis is a monomeric adenosine 5'-tetraphospho-5'-adenosine (Ap4A) pyrophosphatase of 170 amino acids that catalyzes the hydrolysis of Ap4A, Ap5A, and Ap6A by attack at the delta-phosphorus, with the departure of ATP as the leaving group [Cartwright et al. (1999) Biochem. Biophys. Res. Commun. 256, 474-479]. When various divalent cations were tested over a 300-fold concentration range, Mg2+, Mn2+, and Zn2+ ions were found to activate the enzyme, while Ca2+ did not. Sigmoidal activation curves were observed with Mn2+ and Mg2+ with Hill coefficients of 3.0 and 1.6 and K0.5 values of 0.9 and 5.3 mM, respectively. The substrate M2+ x Ap4A showed hyperbolic kinetics with Km values of 0.34 mM for both Mn2+ x Ap4A and Mg2+ x Ap4A. Direct Mn2+ binding studies by electron paramagnetic resonance (EPR) and by the enhancement of the longitudinal relaxation rate of water protons revealed two Mn2+ binding sites per molecule of Ap4A pyrophosphatase with dissociation constants of 1.1 mM, comparable to the kinetically determined K0.5 value of Mn2+. The enhancement factor of the longitudinal relaxation rate of water protons due to bound Mn2+ (epsilon b) decreased with increasing site occupancy from a value of 12.9 with one site occupied to 3.3 when both are occupied, indicating site-site interaction between the two enzyme-bound Mn2+ ions. Assuming the decrease in epsilon(b) to result from cross-relaxation between the two bound Mn2+ ions yields an estimated distance of 5.9 +/- 0.4 A between them. The substrate Ap4A binds one Mn2+ (Kd = 0.43 mM) with an epsilon b value of 2.6, consistent with the molecular weight of the Mn2+ x Ap4A complex. Mg2+ binding studies, in competition with Mn2+, reveal two Mg2+ binding sites on the enzyme with Kd values of 8.6 mM and one Mg2+ binding site on Ap4A with a Kd of 3.9 mM, values that are comparable to the K0.5 for Mg2+. Hence, with both Mn2+ and Mg2+, a total of three metal binding sites were found-two on the enzyme and one on the substrate-with dissociation constants comparable to the kinetically determined K0.5 values, suggesting a role in catalysis for three bound divalent cations. Ca2+ does not activate Ap4A pyrophosphatase but inhibits the Mn2+-activated enzyme competitively with a Ki = 1.9 +/- 1.3 mM. Ca2+ binding studies, in competition with Mn2+, revealed two sites on the enzyme with dissociation constants (4.3 +/- 1.3 mM) and one on Ap4A with a dissociation constant of 2.1 mM. These values are similar to its Ki suggesting that inhibition by Ca2+ results from the complete displacement of Mn2+ from the active site. Unlike the homologous MutT pyrophosphohydrolase, which requires only one enzyme-bound divalent cation in an E x M2+ x NTP x M2+ complex for catalytic activity, Ap4A pyrophosphatase requires two enzyme-bound divalent cations that function in an active E x (M2+)2 x Ap4A x M2+ complex.     

Autophosphorylation of phosphorylase kinase. Divalent metal cation and nucleotide dependency

This study reports on the divalent metal ion specificity for phosphorylase kinase autophosphorylation and, in particular, provides a comparison between the efficacy of Mg2+ and Mn2+ in this role. As well as requiring Ca2+ plus divalent metal ion-ATP2- as substrate, both phosphorylase kinase autoactivation and phosphorylase conversion are additionally modulated by divalent cations. However, these reactions are affected differently by different ions. Phosphorylase kinase-catalyzed phosphorylase conversion is maximally enhanced by a 4- to 10-fold lower concentration of Mg2+ than is autocatalysis and, whereas both reactions are stimulated by Mg2+, autophosphorylation is activated by Mn2+, Co2+, and Ni2+ while phosphorylase a formation is inhibited. This difference may be due to an effect of free Mn2+ on phosphorylase rather than the inability of phosphorylase kinase to use MnATP as a substrate when catalyzing phosphorylase conversion since Mn2+, when added at a level which minimally decreases [MgATP], greatly inhibits phosphorylase phosphorylation. The interactions of Mn2+ with phosphorylase kinase are different from those of Mg2+. Not only are the effects of these ions on phosphorylase activation opposite, but they also provoke different patterns of subunit phosphorylation during phosphorylase kinase autocatalysis. With Mn2+, the time lag of phosphorylation of both the alpha and beta subunits of phosphorylase kinase in autocatalysis is diminished in comparison to what is observed with Mg2+, and the beta subunit is only phosphorylated to a maximum of 1 mol/mol of subunit. With both Mg2+ and Mn2+ the alpha subunit is phosphorylated to a level in excess of 3 mol/mol, a level similar to that obtained for beta subunit phosphorylation in the presence of Mg2+. The support of autophosphorylation by both Co2+ and Ni2+ has characteristics similar to those observed with Mn2+. Although Mn2+ stimulation of autophosphorylation occurs at levels much higher than normal physiological levels, the possible potential of phosphorylase kinase autophosphorylation as a control mechanism is illustrated by the 80- to 100-fold activation that occurs in the presence of Mn2+, a level far in excess of the enzyme activity change normally seen with covalent modification. Autophosphorylation of phosphorylase kinase demonstrates a Km for Mg X ATP2- of 27.7 microM and a Ka for Mg2+ of 3.1 mM. The reaction mechanism of autophosphorylation is intramolecular. This latter observation may indicate that phosphorylase kinase autocatalysis could be of potential physiological relevance and could occur with equal facility in cells containing either constitutively high or low levels of this enzyme.