Interaction of metal ions with polynucleotides and related compounds. XXII. Effect of divalent metal ions on the conformational changes of polyribonucleotides

Changes in the conformation of poly(G), poly(C), poly(U), and poly(I) in the presence of divalent metal ions Mg²⁺, Ca²⁺, Mn²⁺, Co²⁺, Ni²⁺, Cu²⁺, Cd²⁺, and Zn²⁺ have been measured by means of ORD and u.v. spectra. Mg²⁺ and Ca²⁺ ions stabilize helical structures of all the polynucleotides very effectively at concentrations several orders of magnitude lower than the effective concentration of Na⁺ion. Cu²⁺ and Cd²⁺ destabilize the helical structure of polynucleotides to form random coils. Zn²⁺, Ni²⁺, Co²⁺, and Mn²⁺ions do not behave in such a clear-cut manner: they selectively stabilize some ordered structures, while destabilizing others, depending on the ligand strength of the nucleotide base as well as the preferred conformation of that polynucleotide.     

Divalent Metal Cations upon Coordination to the N7 of Purines Differentially Stabilize the PyPuPu DNA Triplex due to Unequal Hoogsteen-type Hydrogen Bond Enhancement

Abstract

The PyPuPu triplexes consisting of CG*G triads are stabilized by alkaline earth cations (Ca²⁺, Mg²⁺) and transition metal cations (Mn²⁺, Co²⁺, Ni²⁺, Zn²⁺, Cd²⁺), while similar triplexes including TA*A triads are stabilized only by transition metal cations. We hypothesize that such a differential triplex stabilization by divalent metal cations can be the consequence of their coordination to the N7 of the third strand purines with concomitant polarization effects on the bases resulting in unequal Hoogsteen-type hydrogen bond enhancement.     

Heavy metal uptake by intact cells and protoplasts of Aureobasidium pullulans

Protoplasts prepared from yeast-like cells, hyphae and chlamydospores of Aureobasidium pullulans can take up heavy metals such as Zn²⁺, Co²⁺, Cd²⁺ and Cu²⁺. In relation to intact cells, the sensitivity of protoplasts to Cu²⁺ and Cd²⁺ was increased although chlamydospore protoplasts were more tolerant than yeast-like cell protoplasts. Surface binding of metals was reduced in protoplasts as compared with intact cells and this reduction was particularly evident for chlamydospore protoplasts. At the highest concentrations used, uptake of Zn²⁺, Co²⁺ and Cd²⁺ by yeast-like cell protoplasts was greater than that observed in intact cells which may have been due to toxicity, especially for Cd²⁺, resulting in increased membrane permeability, though for Zn²⁺ and Co²⁺ some barrier effect of the cell wall could not be completely discounted. Chlamydospore protoplasts were capable of intracellular metal uptake, unlike intact chlamydospores, and for Zn²⁺, uptake appeared to be via a different system less specific than that of the other cell types. For chlamydospores, the use of protoplasts confirmed the importance of the cell wall in preventing entry of metal ions into the cell.     

Toxicity of chromium and tin toAnabaena doliolum Interaction with bivalent cations

Summary The toxicity of chromium and tin on growth, photosynthetic carbon-fixation, oxygen evolution, heterocyst differentiation and nitrogenase activity ofAnabaena doliolum and its interaction with bivalent cations has been studied. Some interacting cations, viz. Ca²⁺, Mg²⁺ and Mn²⁺, substantially antagonised the toxic effects of chromium and tin with reference to growth, heterocyst differentiation and nitrogenase activity in the following hierarchal sequence: Ca²⁺ > Mg²⁺ > Mn²⁺. However, the sequence of hierarchy was Mg²⁺ > Ca²⁺ > Mn²⁺ for carbon fixation and Mn²⁺ > Mg²⁺ > Ca²⁺ for photosynthetic oxygen evolution. Synergistically inhibitory patterns were noticed for all the parameters, viz. growth,¹⁴CO₂ uptake, oxygen evolution, heterocyst differentiation and nitrogenase activity ofA. doliolum when Ni²⁺, Co²⁺ and Zn²⁺ were combined with the test metals in the growth medium. These cations followed the following sequence of synergistic inhibition: Ni²⁺ > Co²⁺ > Zn²⁺. Among all the interacting cations, Ca²⁺, Mg²⁺ and Mn²⁺ exhibited antagonistic effects which relieved the test cyanobacterium from metal toxicity. In contrast to this, Ni²⁺, CO²⁺ and Zn²⁺ showed synergistic inhibition which potentiating the toxicity of test metals in the N₂-fixing cyanobacteriumA. doliolum. It is evident from the present study that bivalent cations, viz. Ca²⁺, Mg²⁺, Mn²⁺, Ni²⁺, Co²⁺ and Zn²⁺, may appreciably regulate the toxicity of heavy metals in N₂-fixing cyanobacteria if present in aquatic media.     

The effect of amphotericin B on the permeability of lipid bilayers to divalent trace metals

In this study amphotericin B released the divalent trace metals Zn²⁺, Co²⁺, Cu²⁺, Ni²⁺, Mn²⁺, Fe²⁺, Cd²⁺ and Pb²⁺ from multilamellar liposomes containing cholesterol. This observation is consistent with amphotericin B channels being permeable to these metals, and it is proposed, therefore, that the antibiotic may be useful in investigating the metabolism of these elements.     

Characterization of the metal ion binding site in the anti-terminator protein, HutP, of Bacillus subtilis

HutP is an RNA-binding protein that regulates the expression of the histidine utilization (hut) operon in Bacillus subtilis, by binding to cis-acting regulatory sequences on hut mRNA. It requires L-histidine and an Mg²⁺ ion for binding to the specific sequence within the hut mRNA. In the present study, we show that several divalent cations can mediate the HutP–RNA interactions. The best divalent cations were Mn²⁺, Zn²⁺ and Cd²⁺, followed by Mg²⁺, Co²⁺ and Ni²⁺, while Cu²⁺, Yb²⁺ and Hg²⁺ were ineffective. In the HutP–RNA interactions, divalent cations cannot be replaced by monovalent cations, suggesting that a divalent metal ion is required for mediating the protein–RNA interactions. To clarify their importance, we have crystallized HutP in the presence of three different metal ions (Mg²⁺, Mn²⁺ and Ba²⁺), which revealed the importance of the metal ion binding site. Furthermore, these analyses clearly demonstrated how the metal ions cause the structural rearrangements that are required for the hut mRNA recognition.     

Role of Divalent Metal Ions on Activity and Stability of Thermostable Dipeptidase from Bacillus stearothermophilus

Thermostable dipeptidase from Bacillus stearothermophilus, a typical metalloenzyme containing 1.0g atom of Zn per mole of subunit of the dimeric enzyme was markedly activated by exogenous divalent metal ions such as Mn²⁺, Co²⁺, and Cd²⁺ . In contrast, several others including Ba²⁺, Hg²⁺, and Cu²⁺ considerably inhibited the enzyme, even the inherent metal, Zn²⁺, being slightly inhibitory. To study the metal-binding properties of this dipeptidase, the enzyme was completely resolved to the inactive, Zn-free apoenzyme by treatment with EDTA in the presence of guanidine hydrochloride in a weakly acidic buffer. The apoenzyme was readily reconstituted by incubation with either Zn²⁺, Mn²⁺, or Co²⁺, restoring the catalytic activity. The Mn-reconstituted enzyme had nearly twice the activity of the original Zn-enzyme. Combined with kinetic analyses of reconstitution of the apoenzyme with metal ions, these results show that the enzyme has two non-identical metal-binding sites, each with a different property. Furthermore, substitution of Mn²⁺ or Co²⁺ for Zn²⁺ considerably lowered the thermostability of the enzyme without affecting the overall conformation of the enzyme protein, suggesting that the prosthetic Zn is playing dual roles in conformational stability and catalysis of the thermostable dipeptidase.     

A new principle for activity measurement of ADP or ATP dependent enzymes: fluorescence quenching of epsilon-ADP and epsilon-ATP by divalent metal ions.

The divalent metal ions Cu²⁺, Co²⁺, Mn²⁺, and Zn²⁺ form complexes with the fluorescent etheno analogs of the adenine nucleotides. The fluorescence intensity is thereby diminished. The binding strength of the metals to etheno-adenosine triphosphate is higher than to etheno-adenosine di- and monophosphate. The quenching effect of the divalent metal ions can be exploited as a simple routine activity measurement for various kinases and phosphohydrolases.     

Identification of brain ecto-apyrase as a phosphoprotein

The divalent cation requirements of NOS activity in bovine retina homogenate supernatant were investigated. Supernatants were assayed under standard conditions (in mM: EDTA 0.45, Ca²⁺ 0.25, Mg²⁺ 4.0). In order to investigate the enzyme's dependence on divalent cations, the tissue homogenate was depleted of di- and trivalent cations by passing it over a cation-exchange column (Chelex 100). Surprisingly, NOS activity was 50-100% higher in this preparation. However, addition of either EDTA (33 M) or EGTA (1 mM) almost fully inhibited NOS activity, suggesting a requirement for residual divalent metal cation(s). Phenanthroline or iminodiacetic acid at low concentrations had little effect on activity, suggesting no requirement for Fe²⁺, Zn²⁺ or Cu²⁺. Ca²⁺ had a moderate stimulatory effect, with an optimum activity around 0.01 mM. Mg²⁺ or Mn²⁺ had little effect at concentrations < 0.25 mM. However, in the presence of EDTA, Mn²⁺ or Ca²⁺ markedly stimulated NOS activity with the optimum at 0.1 mM. At high concentrations (> 0.1-0.2 mM), all divalent cations tested (Ba²⁺, Zn²⁺, Co²⁺, Mn²⁺, Mg²⁺, Ca²⁺), as well as La³⁺, dose-dependently inhibited NOS activity. We propose that retinal NOS requires low concentrations of naturally occurring divalent metal ions, most probably Ca²⁺, for optimal activity and is inhibited by high di- and trivalent metal concentrations, probably by competition with Ca²⁺.     

The phosphate-pyrophosphate exchange and hydrolytic reactions of the membrane-bound pyrophosphatase ofRhodospirillum rubrum: Effects of pH and divalent cations

The relation that exist between the Pi-PPi exchange reaction and pyrophosphate hydrolysis by the membrane-bound pyrophosphatase of chromatophores ofRhodospirillum rubrum was studied. The two reactions have a markedly different requirement for pH. The optimal pH for hydrolysis was 6.5 while the Pi-PPi exchange reaction was at 7.5; the pH affects mainly theK _m of Mg²⁺ or Pi for the enzyme; Mn²⁺ and Co²⁺ support the Pi-PPi exchange reaction partially (50%), but the reaction is slower than with Mg²⁺; other divalent cations like Zn²⁺ or Ca²⁺ do not support the exchange reaction. In the hydrolytic reaction, Zn²⁺, at low concentration, substitutes for Mg²⁺ as substrate, and Co²⁺ also substitutes in limited amount (50%). Other cations (Ca²⁺, Cu²⁺, Fe²⁺, etc.) do not act as substrates in complex with PPi. The Zn²⁺ at high concentrations inhibited the hydrolytic reaction, probably due to uncomplexed free Zn²⁺. In the presence of high concentration of substrate for the hydrolysis (Mg-PPi) the divalent cations are inhibitory in the following order: Zn²⁺>Mn²⁺>Ca²⁺Co²⁺>Fe²⁺>Cu²⁺>Mg²⁺. The data in this work suggest that H⁺ and divalent cations in their free form induced changes in the kinetic properties of the enzyme.     

Divalent cation effects on calcineurin phosphatase: differential involvement of hydrophobic and metal binding domains in the regulation of the enzyme activity

Summary The effects of divalent metals, metal chelators (EDTA, EGTA) and sodium dodecyl sulfate were investigated on the phosphatase activity of isolated bovine brain calcineurin assayed in the absence (called intrinsic) and presence of calmodulin. Intrinsic phosphatase was increased by Mn²⁺, was unaffected by Mg²⁺, Ca^2–, and Ba⁺, and was markedly inhibited by Ni^2–, Fe²⁺, Zn²⁺ and Cu^2–. When assayed in the presence of calmodulin, many divalent metals (Ni^2–, Zn²⁺, Pb²⁺, Cd²⁺), besides Mn²⁺, increased modestly the phosphatase activity at low concentrations (10–100 M) and inhibited it markedly at high concentrations. Ca^2–-calmodulin stimulated phosphatase activity was antagonized by Ni²⁺, Zn²⁺, Fe²⁺, Cu²⁺, Pb²⁺, at low concentrations (50 M), and by Ba²⁺, Cd²⁺ at slightly higher concentrations (> 100 M); Mn²⁺ and Co^2– (50 M to 1 mM) in fact augmented it. EDTA and EGTA in a concentration and time dependent fashion inhibited the intrinsic phosphatase activity, particularly that of trypsinized calcineurin. SDS in low concentrations (0.005%) augmented the phosphatase activity and inhibited it at high concentrations. Mn²⁺ (± calmodulin) and Ca²⁺ only with calmodulin present increased the phosphatase activity assayed with low concentrations of SDS. The EDTA dependent inhibition of intrinsic phosphatase was almost abolished in assays containing SDS. Prior exposure of calcineurin to Mn²⁺ led to a high activity conformation state of calcineurin that was long-lived or pseudo-irreversible. Such Mn²⁺-activated state of calcineurin exhibited no discerbible change in the affinity towards myelin basic protein or its inhibition by trifluoperazine. At alkaline pH, Mg²⁺ supported the intrinsic phosphatase activity, although to a lesser degree than Mn²⁺. The latter cation, compared to Mg²⁺ and Ni²⁺, was also a more powerful stimulator of the calcineurin phosphatase assayed with histone (III-S) and myosin light chain as substrates.     

Kinetic evidence for a dual cation role for muscle pyruvate kinase

The activation of muscle pyruvate kinase by divalent cations was studied by steady-state kinetics. Under experimental conditions the enzyme exhibits activation by Mg²⁺, Co²⁺, Mn²⁺, Ni²⁺, and Zn²⁺ in descending order of maximal velocity. Combinations of cations were also studied. A synergistic activation was observed with a fixed concentration of Mg²⁺ and varying concentrations of Mn²⁺ or of Co²⁺. This synergism indicates at least two roles for the cations for enzymatic activation and a differential specificity among the cations for the separate functions. Synergistic activation was also observed with fixed Co²⁺ and varying Mn²⁺. These results are consistent with a cation specifically required to activate the enzyme and a cation which serves as a cation-nucleotide complex which is a substrate for the reaction. The response observed suggests that Mn²⁺ is a better activator of the enzyme than is Mg²⁺, however, MgADP is a better substrate than is MnADP. The lack of a synergistic effect by Ni²⁺ or Zn²⁺ with Mg²⁺ suggests that Ni²⁺ and Zn²⁺ are poor activators either because they serve one catalytic function poorly but bind to that site tightly or they serve both catalytic functions poorly in contrast to Mg²⁺. These studies yield the first simple kinetic evidence that muscle pyruvate kinase, under catalytic conditions of the overall reaction, has a dual divalent cation requirement for activity.     

Interaction of divalent metal ions with the carboxyl-terminal domain of human voltage-gated proton channel Hv1

The voltage-gated proton channel Hv1 functions as a dimer, in which the intracellular C-terminal domain of the protein is responsible for the dimeric architecture and regulates proton permeability. Although it is well known that divalent metal ions have effect on the proton channel activity, the interaction of divalent metal ions with the channel in detail is not well elucidated. Herein, we investigated the interaction of divalent metal ions with the C-terminal domain of human Hv1 by CD spectra and fluorescence spectroscopy. The divalent metal ions binding induced an obvious conformational change at pH 7 and a pH-sensitive reduction of thermostability in the C-terminal domain. The interactions were further estimated by fluorescence spectroscopy experiments. There are at least two binding sites for divalent metal ions binding to the C-terminal domain of Hv1, either of which is close to His²⁴⁴ or His²⁶⁶ residue. The binding of Zn²⁺ to the two sites both enhanced the fluorescence of the protein at pH 7, whereas the binding of other divalent metal ions to the two sites all resulted fluorescence quenching. The orders of the strength of divalent metal ions binding to the two sites from strong to weak are both Co²⁺, Ca²⁺, Ni²⁺, Mg²⁺, and Mn²⁺. The strength of Ca²⁺, Co²⁺, Mg²⁺, Mn²⁺ and Ni²⁺ binding to the site close to His²⁴⁴ is stronger than that of these divalent metal ions binding to the site close to His²⁶⁶.     

Effects of Metal Ions on the Conformation and Activity of Acutolysin D from Agkistrodon Acutus Venom

Acutolysin D, isolated from the venom of Agkistrodon acutus, possesses marked haemorrhagic and proteolytic activities. The molecular weight and the absorption coefficients (A ^1% ₂₈₀) of acutolyisn D have been determined to be 47,850 ± 8 amu and 9.3 by mass spectrometer and UV spectrum, respectively. The effects of metal ions on the conformation and activity of acutolysin D have been studied by following fluorescence, circular dichroism and biological activity measurements. Acutolysin D contains two Ca²⁺-binding sites and two Zn²⁺-binding sites determined by atomic absorption spectrophotometer. Zn²⁺ is essential for the enzyme activities of acutolysin D, however, the presence of 1 mM Zn²⁺ significantly decreases its caseinolytic activity and intrinsic fluorescence intensity at pH 9.0 due to Zn(OH)₂ precipitate formation. Ca²⁺ is important for the structural integrity of acutolysin D, and the presence of 1 mM Ca²⁺ markedly enhances its caseinolytic activity. Interestingly, the caseinolytic activity which is inhibited partly by Cu²⁺, Co²⁺, Mn²⁺ or Tb³⁺ and inhibited completely by Cd²⁺, is enhanced by Mg²⁺. The fluorescence intensity of the protein decreases in the presence of Cu²⁺, Co²⁺, Cd²⁺ or Mn²⁺, but neither for Ca²⁺, Mg²⁺ nor for Tb³⁺. Zn²⁺, Ca²⁺, Mg²⁺, Cu²⁺, Mn²⁺, Co²⁺ and Tb³⁺ have slight effects on its secondary structure contents. In addition, Cd²⁺ causes a marked increase of antiparallel β-sheet content from 45.5% to 60.2%.     

Bidirectional Modulation of GABA-Gated Chloride Channels by Divalent Cations: Inhibition by Ca2+ and Enhancement by Mg2+

Abstract: The effects of the divalent cations Ca²⁺, Sr²⁺, Ba²⁺, Mg²⁺, Mn²⁺, and Cd²⁺ were studied on γ-aminobutyric acid_A (GABA_A) responses in rat cerebral cortical synaptoneurosomes. The divalent cations produced bidirectional modulation of muscimol-induced ³⁶Cl^? uptake consistent with their ability to permeate and block Ca²⁺channels. The order of potency for inhibition of muscimol responses was Ca²⁺ > Sr²⁺ > Ba²⁺, similar to the order for permeation of Ca²⁺ channels in neurons. The order of potency for enhancement of muscimol responses was Cd²⁺> Mn²⁺ > Mg²⁺, similar to the order for blockade of Ca²⁺channels in neurons. Neither Ca²⁺ nor Mg²⁺ caused accumulation of GABA in the extravesicular space due to increased GABA release or decreased reuptake of GABA by the synaptoneurosomes. The inhibition of muscimol responses by Ca²⁺ was most likely via an intracellular site of action because additional inhibition could be obtained in the presence of the Ca²⁺ ionophore, A23187. This confirms electrophysiologic findings in cultured neurons from several species. In contrast, the effects of Cd²⁺, Mn²⁺, and Mg²⁺ may be mediated via blockade of Ca²⁺ channels or by intracellular sites, although the results of these studies do not distinguish between the two loci. The effects of Zn²⁺ were also studied, because this divalent cation is reported to have widely divergent effects on GABA_A responses. In contrast to other studies, we demonstrate that Zn²⁺ inhibits GABA_A responses in an adult neuronal preparation. Zn²⁺ produced a concentration-dependent inhibition (limited to 40%) of muscimol responses with an EC₅₀ of 60 μM. The inhibition of muscimol-induced ³⁸Cl^? uptake by Zn²⁺ was noncompetitive. The effect of Zn²⁺was reduced in the presence of Mg²⁺ in a competitive or allosteric manner. The portion of GABA_A receptors sensitive to Zn²⁺ may reflect a specific subunit composition in cerebral cortex as previously observed for recombinant GABA_A receptors in several expression systems. The modulation of GABA_A receptor function by Ca²⁺ and other divalent cations may play an important role in the development and/or attenuation of neuronal excitability associated with pathologic conditions such as seizure activity and cerebral ischemia.     

Conformation and reactivity of DNA. IV. Base binding ability of transition metal ions to native DNA and effect on helix conformation with special reference to DNA-Zn(II) complex

The effects of metal ions of the first-row transition and of alkaline earth metals on the DNA helix conformation have been studied by uv difference spectra, circular dichroism, and sedimentation measurements. At low ionic strength (10^?3 M NaClO₄) DNA shows a maximum in the difference absorption spectra in the presence of Zn²⁺, Mn²⁺, Co²⁺, Cd²⁺, and Ni²⁺ but not with Mg²⁺ or Ca²⁺. The amplitude of this maximum is dependent on GC content as revealed by detailed studies of the DNA-Zn²⁺ complex of eight different DNA's. Pronounced changes also occur in the CD spectra of DNA transition metal complexes. A transition appears up to a total ratio of approximately 1 Zn²⁺ per DNA phosphate at 10^?3 M NaClO₄; then no further change was observed up to high concentrations. The characteristic CD changes are strongly dependent on the double-helical structure of DNA and on the GC content of DNA. Differences were also observed in hydrodynamic properties of DNA metal complexes as revealed by the greater increase of the sedimentation coefficient of native DNA in the presence of transition metal ions. Spectrophotometric acid titration experiments and CD measurements at acidic pH clearly indicate the suppression of protonation of GC base-pair regions on the addition of transition metal ions to DNA. Similar effects were not observed with DNA complexes with alkaline earth metal ions such as Mg²⁺ or Ca²⁺. The data are interpreted in terms of a preferential interaction of Zn²⁺ and of other transition metal ions with GC sites by chelation to the N-7 of guanine and to the phosphate residue. The binding of Zn²⁺ to DNA disappears between 0.5 M and 1 M NaClO₄, but complex formation with DNA is observable again in the presence of highly concentrated solutions of NaClO₄ (3?7.2 M NaClO₄) or at 0.5 to 2 M Mn²⁺. At relatively high cation concentration Mg²⁺ is also effective in changing the DNA comformation. These structural alterations probably result from both the shielding of negatively charged phosphate groups and the breakdown of the water structure along the DNA helix. Differential effects in CD are also observed between Mn²⁺, Zn²⁺ on one hand and Mg²⁺ on the other hand under these conditions. The greater sensitivity of the double-helical conformation of DNA to the action of transition metal ions is due to the affinity of the latter to electron donating sites of the bases resulting from the d electronic configuration of the metal ions. An order of the relative phosphate binding ability to base-site binding ability in native DNA is obtained as follows: Mg²⁺, Ba²⁺, < Ca²⁺ < Fe²⁺, Ni²⁺, Co²⁺ < Mn²⁺, Zn²⁺ < Cd²⁺ < Cu²⁺. The metal-ion induced conformational changes of the DNA are explained by alternation of the winding angle between base pairs as occurs in the transition from B to C conformation. These findings are used for a tentative molecular interpretation of some effects of Zn²⁺ and Mn²⁺ in DNA synthesis reported in the literature.     

Uptake of Lead and Cadmium by Maize Seedlings and the Effect of Heavy Metals on the Activity of Phosphoenolpyruvate Carboxylase Isolated from Maize

Maize seeds and five-day-old maize seedlings were incubated in media containing Pb²⁺ at concentrations of 50, 100, and 200 mg 1^-1 and Cd²⁺ at concentrations of 1, 5, 10 and 50 mg 1^-1. After five days of incubation, both heavy metals were determined by means of AAS following wet mineralisation of roots and shoots. The results obtained indicate that Pb²⁺ were transported to shoots from roots at a lower rate than Cd²⁺. Phosphoenolpyruvate carboxylase (PEPC) isolated from germinating maize seeds was inhibited to a comparable degree by solutions containing 0.001 mmol 1^-1 Pb²⁺, 0.01 mmol 1^-1 Cd²⁺, and 0.005 mmol 1^-1 Cu²⁺. The enzyme was protected against this inhibition by the addition of mercaptoethanol, the substrate (PEP), or the cofactor (Mg²⁺). The inhibition increased during a 20 min incubation of the enzyme with salts of the metals. Mn²⁺, Ni²⁺, and Co²⁺ ions could partially substitute for the metal cofactor Mg²⁺. K_m values for these metal ions were as follows: for Mg²⁺ 0.07 mmol 1^-1 in the range from 0 to 0.30 mmol 1^-1 Mg²⁺; 0.71 mmol 1^-1 for 0.30 to 2.50 mmol 1^-1 Mg²⁺; for Mn²⁺ 0.36 mmol 1^-1; for Ni²⁺ 0.34 mmol 1{-1}; and for Co²⁺ 0.20 mmol 1^-1. The activity of the enzyme reached with Mn²⁺ 85 %, with Ni²⁺ 65 %, and with Co²⁺ 55 % of the activity recorded with Mg²⁺.     

Characterization of the Effects of Divalent Cations on the Coupled Activities of the H-ATPase in Tonoplast Vesicles

The substrate requirement of the H⁺-ATPase in purified corn root tonoplast vesicles was investigated. The coupled activities, ATP hydrolysis and proton pumping, were simultaneously supported only by Mg²⁺ or Mn²⁺. The presence of Ca²⁺ or Ba²⁺ did not significantly affect the coupled activities. The addition of Cd²⁺, Co²⁺, Cu²⁺, and Zn²⁺ inhibited both the hydrolysis of Mg-ATP and the proton transport. However, the inhibition of proton pumping was more pronounced. Based on equilibrium analysis, both ATP-complexed and free forms of these cations were inhibitory. Inhibition of the hydrolysis of Mg-ATP could be correlated to the concentrations of the ATP-complex of Zn. On the other hand, the free Cu²⁺ and Co²⁺ were effective in inhibiting hydrolysis. For proton pumping, the ATP complexes of Co²⁺, Cu²⁺, and Zn²⁺ were effective inhibitors. However, this inhibition could be further modulated by free Co²⁺, Cu²⁺, and Zn²⁺. While the equilibrium concentrations of Cd-ATP and free Cd²⁺ were not estimated, the total concentration of this cation needed to inhibit the coupled activities of the H⁺-ATPase was found to be in the range of 10 to 100 micromolars. The presence of free divalent cations also affected the structure of the lipid phase in tonoplast membrane as demonstrated by the changes of emission intensity and polarization of incorporated 1,6-diphenyl-1,3,5-hexatriene. The differential inhibition caused by these cations could be interpreted by interactions with the protogenic domain of the membrane as previously proposed in “indirect-link” mechanism.     

Effects of heavy metals on pollen tube growth and ultrastructure

Summary The influence of different concentrations of the heavy metals cadmium (Cd²⁺), cobalt (Co²⁺), copper (Cu²⁺), iron (Fe²⁺ and Fe³⁺), mercury (Hg²⁺), manganese (Mn²⁺), and zinc (Zn²⁺), plus aluminium (Al³⁺) (a toxic metal in polluted areas), on pollen germination and tube growth ofLilium longiflorum was investigated using light microscopy. Effects could be observed with 3 M and 100 M of heavy metal, added as chloride salts to the medium. Cd²⁺, Cu²⁺, and Hg²⁺, showed the greatest toxicity, whereas germination and growth rate was less affected by Mn²⁺. Affected tubes showed swelling of the tip region. Tubes treated with Cd²⁺, Co²⁺, Fe²⁺, Fe³⁺, Hg²⁺, and Mn²⁺ were also prepared for ultrastructural studies. In all cases, the main effect was abnormal cell wall organization, mostly at the tip, where round, fibrillar aggregates, the shape and size of secretory Golgi vesicles were formed. They built up a loose network which could be up to 10 m thick compared to untreated tubes where the cell wall was composed of thin layers of long fibrils and about 100 nm thick. Cd²⁺ was the only metal which produced effects at the intracellular level: organelle distribution within the tip region appeared disorganized. A general mechanism of heavy metal action on pollen tube growth is discussed.