Tight Junction Dynamics in the Frog Urinary Bladder

In a previous study in frog skin (Castro et al., J. Memb. Biol. 134:15–29, 1993), it was shown that TJs experimentally disrupted by a selective deposition of BaSO₄ could be re-sealed upon addition of Ca²⁺to the apical solution; in the absence of apical Ca²⁺, the normal Ca²⁺ activity of the Na₂SO₄-Ringer's bathing the basolateral side was not able to induce TJ resealing. We now show that apical Ca²⁺also activates the TJ sealing mechanism in frog urinary bladders. Three known procedures were utilized to increase TJ permeability, all in the absence of apical Ca²⁺: (i) exposure to high positive transepithelial clamping potentials; (ii) exposure of the apical surface to hypertonic solutions; and (iii) selective deposition of BaSO₄ in the TJs. The resealing of the TJs was promoted by raising the concentration of Ca²⁺ in the apical solution. This effect of Ca²⁺ is not impaired by the presence of Ca²⁺ channel blockers (nifedipine, verapamil, Mn²⁺ or Cd²⁺) in the apical solution, indicating that junction resealing does not depend on Ca²⁺ entering the cells through the apical membrane. TJ resealing that occurs in response to raised apical Ca²⁺ most likely results from a direct effect of Ca²⁺, entering the disrupted TJs from the apical solution and reaching the zonula adhaerens Ca²⁺ receptors (E-cadherins). Protein kinase C (PKC) must play a significant role in the control of TJ assembly in this tight epithelia since the PKC inhibitor (H7) and the activator (diC8) markedly affect TJ recovery after disruption by apical hypertonicity. H7 treated tissues show marked recuperation of conductance even in the absence of apical Ca²⁺. In contrast, diC8 prevents tissue recuperation which normally occurs after addition of Ca²⁺ to the apical solution.     

Effects of divalent metal ions on the fluorescence and glucose-quenching of yeast hexokinase isozymes

Titrations of the quenching of the tryptophan fluorescence of yeast hexokinase isozymes P-I and P-II by Mg²⁺, Mn²⁺, Ca²⁺, Cd²⁺, and Zn²⁺ ions and by glucose in the presence of each of these ions (10mM) were performed at pH 5.5 and 6.5 at 20°C. At the higher pH there was a reversal of the type of glucose-binding cooperativity for P-II from negative to positive when either Mn²⁺ or Ca²⁺ was present in the buffered isozyme solution before the glucose titration, whereas Mg²⁺ caused the glucose binding to become noncooperative. Zn²⁺ and Cd²⁺ decreased the glucose quenching of P-II fluorescence drastically at pH 5.5, from a value of 15% in buffer to only 4%. Thus, only these two ions, of the five studied, cause the conformation change that results in quenching of the glucose-quenchable cleft tryptophan of P-II. Glucose binding to the P-I isozyme exhibited positive cooperativity in the presence of either Ca²⁺, Mg²⁺, or Mn²⁺, as well as in buffer alone, at both pH's. At the lower pH, Ca²⁺ enhanced the efficiency of glucose quenching of P-I fluorescence several-fold, while Mn²⁺ increased it only about 40% and Mg²⁺ not at all. Further, Ca²⁺ raised the degree of cooperativity (Hill coefficient) of glucose binding to P-I at this pH from the value of 1.42 in buffer and in the presence of Mg²⁺ and Mn²⁺ to 1.94, i.e., almost up to the highest possible value, 2, for dimeric hexokinase. However, at pH 6.5 the Ca²⁺ effect on the cooperativity was negligible, while Mg²⁺ and Mn²⁺ decreased the coefficient from 1.6 in buffer to about 1.4. The biological implications of these diverse metal ion effects are discussed.     

Metal-ion-dependent hydrophobic-interaction chromatography of α-lactalbumins

α-Lactalbumins from bovine, human, goat, sheep, and horse milk bind to phenyl-Sepharose in the presence of EDTA and can be eluted by addition of Ca²⁺ (0.001–100 m ). This property has been utilized to purify these proteins in a one-step purification from milk whey. α-Lactalbumin purified in this manner has the same ultraviolet and proton nuclear magnetic resonance spectra as that purified by other methods. Using binding to phenyl-Sepharose as an assay, the conformation of bovine α-lactalbumin upon the addition of several metal ions that are known to interact with this protein was investigated. Lanthanides, Mn²⁺, Mg²⁺, and Cd²⁺ can substitute for Ca²⁺, whereas Zn²⁺, Al³⁺, and Co²⁺ cannot. Surprisingly, whereas lower concentrations of La³⁺, Mn²⁺, and Cd²⁺ (1 m and less) caused elution from the hydrophobic support, higher concentrations (10 m ) were ineffective. These observations can be rationalized assuming the presence of two distinct metal-ion binding sites with different specificities.     

Metal ion inhibition of corn root plasma membrane atpase

In the presence of K⁺, the hydrolysis of ATP catalysed by the ATPase of corn plasma membrane showed negative cooperative kinetics. When the complexes of ATP and Mg²⁺, Mn²⁺, Ca²⁺ or Cd²⁺ were used as substrates, the catalysed hydrolysis changed to follow simple Michaelis-Menten kinetics. However, this change was not observed with Zn²⁺-ATP as the substrate. A substantial enhancement of the hydrolysis was observed only when the complexes of Mg²⁺ and Mn²⁺ were used. Kinetic parameter determination indicated that the enzyme exhibited a similar binding property but a different catalytic efficiency to Mg²⁺, Mn²⁺ and Ca²⁺-ATP. The enzyme formed a more stable but less reactive complex with Cd²⁺-ATP. The presence of aluminium ions competitively inhibited the membrane-catalysed hydrolysis of Mg²⁺-ATP, but showed no effect when free ATP was the substrate. This finding suggested that aluminium might bind in the vicinity of the Mg²⁺ of Mg²⁺-ATP in the active site of the enzyme. On the basis of these observed inhibitory effects, possible origins of metal ion toxicity to root plasma membrane ATPase activity are discussed.     

Metal Selectivity of Exocytosis in α-Toxin-Permeabilized Bovine Chromaffin Cells

Abstract: Metal selectivity of exocytosis was analyzed by comparing the effects of polyvalent metal cations Ca²⁺, Ba²⁺, Sr²⁺, Pb²⁺, La³⁺, Cd²⁺, Co²⁺, Tb³⁺, Mn²⁺, and Zn²⁺ on the release of norepinephrine (NE) from staphylococcal α-toxin-permeabilized bovine chromaffin cells. Pb²⁺, La³⁺, Cd²⁺, Sr²⁺, and Ba²⁺ activated NE secretion accompanied by the release of intragranular dopamine β-hydroxylase but not cytosolic lactate dehydrogenase, indicating the activation of the mechanism of exocytosis. The release triggered by saturating concentrations of Pb²⁺, La³⁺, Cd²⁺, and Sr²⁺ was nonadditive with Ca²⁺, indicating a common site of action. In contrast, the Ba²⁺-evoked NE release was additive with Ca²⁺ and the Ca²⁺ agonists Pb²⁺, La³⁺, Cd²⁺, and Sr²⁺, suggesting that Ba²⁺ activates secretion at a site distinct from the Ca²⁺ receptor. In distinction to the NE release evoked by Pb²⁺, La³⁺, Cd²⁺, and Ba²⁺, the Sr²⁺-evoked NE release was associated with a significant elevation of Ca²⁺ concentration in the medium and abolished by Ca²⁺ chelation. This indicates that the secretagogue effect of Sr²⁺ was indirect and secondary to the displacement of bound Ca²⁺. Co²⁺ and Mn²⁺ inhibited the NE release evoked by Ca²⁺, Sr²⁺, Pb²⁺, La³⁺, and Cd²⁺ but had no effect on the Ba²⁺-dependent secretion. Tb³⁺ and Zn²⁺ were without effect on exocytosis.     

Small Synthetic Peptides Homologous to Segments of the First External Loop of Occludin Impair Tight Junction Resealing

This study shows that resealing of opened tight junctions (TJs) is impaired by interaction with oligopeptides homologous to the external domain of chick occludin. The experiments were carried out with confluent A6 cell monolayers grown on collagen supports under stable transepithelial electrical resistance (TER). The monolayers were bathed on the apical side with a 75 mm KCl solution and on the basolateral side by NaCl-Ringer's solution. TJ opening was induced by basolateral Ca²⁺ removal and was characterized by a marked drop of TER. The reintroduction of Ca²⁺ triggered junction resealing as indicated by an elevation of TER to control values. Custom-made peptides SNYYGSGLSY (corresponding to the residues 100 to 109) and SNYYGSGLS (residues 100 to 108), homologous to segments of the first external loop of chick occludin molecule, impaired junction resealing when the peptides were included in the apical bathing fluid (concentrations in the range of 0.5 to 1.5 mg/ml). Peptide removal from the apical solution usually triggered a slow recovery of TER, indicating a slow recovery of the TJ seal. Changes in localization of ZO-1, a cytoplasmic protein that underlies the membrane at the TJs, were evaluated immunocytochemically following Ca²⁺ removal and reintroduction. The presence or absence of the oligopeptides showed no influence on the pattern of change of ZO-1 localization. These observations support the hypothesis that the TJ seal results from the interaction of specific homologous segments of occludin on the surface of adjacent cells. Additionally, our results show that small peptides homologous to segments of the occludin first external loop can be used as specific reagents to manipulate the permeability of tight junctions. Received: 4 December 1998/Revised: 22 January 1999     

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.     

Ca2+ and Mg2+ ions counteract the reduction by fosetyl-Al (aluminum tris[ethyl phosphonate]) of peroxidase activity from suspension-cultured grapevine cells

Grapevine (Vitis vinifera cv. Monastrell) cell suspension cultures were treated with 1.5 mM fosetyl-Al, a frequently used systemic fungicide for grapevine diseases caused by oomycetes. These cells showed a reduction in the level of peroxidase activity secreted into the culture media when compared to non-treated cells, the effect being mainly related to a decrease in the level of the basic B₁ peroxidase isozyme. The effect of fosetyl-Al on peroxidase was analogous to that observed with the Ca²⁺-channel blockers Co²⁺, Cd²⁺ and La³⁺, and was counteracted by Ca²⁺ ions, but was not reversed when the Ca²⁺-ionophore A23187 was added to the culture media. Moreover, the effect of fosetyl-Al on peroxidase activity and peroxidase isozymes was also partially reversed by Mg²⁺ ions but not by Sr²⁺, and was accentuated by Ba²⁺ ions. These results suggested that Ca²⁺ and Mg²⁺ ions specifically overcome the inhibitory effect of fosetyl-Al on peroxidase. In this context, an apoplastic Ca²⁺/Mg²⁺-displacement hypothesis is proposed for the mechanism of action of fosetyl-Al on peroxidase from grapevine cells.     

Metal cations as synaptosomal calcium blockers in studies with Fura-2

Metal ions are often used to block calcium channels in various tissues, including synaptosomes. In the present study, Fura-2 was used to determine the effectiveness of various metal ions as calcium channel blockers in rat brain synaptosomes in vitro. In buffer solutions, La³⁺ and Cd²⁺ increased the Fura-2 fluorescence in a manner similar to Ca²⁺. Ni²⁺ and Mn²⁺ appeared to be fluorescence quenching cations, and Sr²⁺ and Co²⁺ had little effect on the fluorescence of Fura-2. In suspensions of synaptosomes under resting conditions, Cd²⁺, Ni²⁺ and Mn²⁺ were found to be not suitable for use in synaptosome studies. On the other hand, La³⁺ and Co²⁺ had little effect on the Fura-2 fluorescence of resting synaptosomes, and under depolarizing conditions, La³⁺ and Co²⁺ decreased the Fura-2 fluorescence. These resuls, therefore, suggest that La³⁺ and Co²⁺ may be suitable as calcium channel blockers in synaptosome studies.     

Redistribution and Phosphorylation of Occludin During Opening and Resealing of Tight Junctions in Cultured Epithelial Cells

We studied the expression, distribution, and phosphorylation of the tight junction (TJ) protein occludin in confluent MDCK cell monolayers following three procedures for opening and resealing of TJs. When Ca²⁺ is transiently removed from the culture medium, the TJs open and the cells separate from each other, but the occludin band around each cell is retained. When Ca²⁺ is reintroduced, the TJs reseal. When the monolayers are exposed to prolonged Ca²⁺ starvation the cells maintain contact, but occludin disappears from the cell borders and can be detected only in a cytoplasmic compartment. When Ca²⁺ is reintroduced, new TJs are assembled and the transepithelial electrical resistance (TER) is reestablished in about 20 hr. Monolayers treated with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) show a different pattern of TJ opening: the cell-cell contact is maintained but the TJ strand network, as seen in freeze-fracture replicas, becomes discontinuous. Occludin is still localized at the cell periphery, but in a pattern of distribution that matches the discontinuous TJ. These TJs do not reseal even 24 hr after removal of the TPA. Western blot analysis showed that the 62–65 kD double band of occludin did not change with these treatments. However, in vivo phosphorylation analysis showed that the TPA treatment reduced the phosphorylation levels of occludin, while the prolonged Ca²⁺ starvation completely dephosphorylated the two occludin bands. In addition, a highly phosphorylated 71 kD band that immunoprecipitates with occludin is not present when TJ is opened by the Ca²⁺ removal. Phosphoaminoacid analysis showed that the 62–65 kD occludin bands are phosphorylated on serine and threonine, while the 71 kD band was phosphorylated exclusively on serine. Our results provide further evidence that phosphorylation of occludin is an important step in regulating TJ formation and permeability. Received: 28 December 1998/Revised: 8 April 1999     

Calcium moderation of cadmium stress explored using a stress-inducible transgenic strain of Caenorhabditis elegans

In a transgenic strain of Caenorhabditis elegans carrying a stress-inducible lacZ reporter gene, short-term sublethal exposure to heavy metals activates transgene expression. The transgene response to Cd²⁺ is strongly inhibited by Ca²⁺ ions; furthermore, Ca²⁺ reduces the net accumulation of Cd²⁺ by worms. Both Ca²⁺ and a variety of calcium uptake inhibitors (nifedipine, La³⁺, verapamil) depress the dose response of the transgene to Cd²⁺. Calcium ionophore (A23187) slightly increases transgene activity in control and Cd²⁺ treated worms, but has a much larger effect in the case of Mn²⁺, reflecting its much greater affinity for this ion.     

Differential effects of heavy metal ions on Ca2+-dependent K+ channels

Summary 1. The ability of various divalent metal ions to substitute for Ca²⁺ in activating distinct types of Ca²⁺-dependent K⁺ [K⁺(Ca²⁺] channels has been investigated in excised, inside-out membrane patches of human erthrocytes and of clonal N1E-115 mouse neuroblastoma cells using the patch clamp technique. The effects of the various metal ions have been compared and related to the effects of Ca²⁺.2. At concentrations between 1 and 100 µM Pb²⁺, Cd²⁺ and Co²⁺ activate intermediate conductance K⁺(Ca²⁺) channels in erythrocytes and large conductance K⁺(Ca²⁺) channels in neuroblastoma cells. Pb²⁺ and Co²⁺, but not Cd²⁺, activate small conductance K⁺(Ca²⁺) channels in neuroblastoma cells. Mg²⁺ and Fe²⁺ do not activate any of the K⁺(Ca²⁺) channels.3. Rank orders of the potencies for K⁺(Ca²⁺) activation are Pb²⁺, Cd²⁺>Ca²⁺, Co²⁺>>Mg²⁺, Fe²⁺ for the intermediate erythrocyte K⁺(Ca²⁺) channel, and Pb²⁺, Cd²⁺>Ca²⁺>Co²⁺>>Mg²⁺, Fe²⁺ for the small, and Pb²⁺>Ca²⁺>Co²⁺>>Cd²⁺, Mg²⁺, Fe²⁺ for the large K⁺(Ca²⁺) channel in neuroblastoma cells.4. At high concentrations Pb²⁺, Cd²⁺, and Co²⁺ block K⁺(Ca²⁺) channels in erythrocytes by reducing the opening frequency of the channels and by reducing the single channel amplitude. The potency orders of the two blocking effects are Pb²⁺>Cd²⁺, Co²⁺>>Ca²⁺, and Cd²⁺>Pb²⁺, Co²⁺>>Ca²⁺, respectively, and are distinct from the potency orders for activation.5. It is concluded that the different subtypes of K⁺(Ca²⁺) channels contain distinct regulatory sites involved in metal ion binding and channel opening. The K⁺(Ca²⁺) channel in erythrocytes appears to contain additional metal ion interaction sites involved in channel block.     

Evidence of metal binding activities of pentadecapeptide from Panax ginseng

A tetradecapeptide from ginseng (Panax ginseng) root showing anti-lipolytic activity in an isolated rat fat cell assay was chemically synthesized for analysis of metal binding activities in vitro. Binding activities against several metal ions were analysed by measuring mobility shifts during capillary zone electrophoresis experiments. The ginseng polypeptide (GPP) showed the greatest increase in effective molecular electrophoretic mobility in the presence of Mg²⁺. Mobility was also affected in the presence of La³⁺, Mn²⁺, Ca²⁺ and Zn²⁺ ions. Analysis with the dye Stains-all revealed GPP to possess a cation binding site similar to those in Ca²⁺-binding proteins. GPP thus appears to be a metal binding peptide. The results of this analysis suggested that GPP may perform its anti-lipolytic activities through an ability to modulate the level of free cellular Mg²⁺ and Mn²⁺ ions.     

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%.     

Pulses of Cell Ca<Superscript>2+</Superscript> and the Dynamics of Tight Junction Opening and Closing

A mathematical modeling of tight junction (TJ) dynamics was elaborated in a previous study (Kassab, F., Marques, R.P., Lacaz-Vieira, F. 2002. Modeling tight junction dynamics and oscillations. J. Gen. Physiol. 120:237–247) to better understand the dynamics of TJ opening and closing, as well as oscillations of TJ permeability that are observed in response to changes of extracellular Ca²⁺ levels. In this model, TJs were assumed to be specifically controlled by the Ca²⁺ concentration levels at the extracellular Ca²⁺ binding sites of zonula adhaerens. Despite the fact that the model predicts all aspects of TJ dynamics, we cannot rule out the likelihood that changes of intracellular Ca²⁺ concentration (Ca²⁺ _cell), which might result from changes \ of extracellular Ca²⁺ concentration (Ca²⁺ _extl), contribute to the observed results. In order to address this aspect of TJ regulation, fast Ca²⁺-switch experiments were performed in which changes of Ca²⁺ _cell were induced using the Ca²⁺ ionophore A23187 or thapsigargin, a specific inhibitor of the sarco-endoplasmic reticulum Ca²⁺-ATPase. The results indicate that the ionophore or thapsigargin per se do not affect basal tissue electrical conductance (G), showing that the sealing of TJs is not affected by a rise in Ca²⁺ _cell. When TJs were kept in a dynamic state, as partially open structures or in oscillation, conditions in which the junctions are very sensitive to disturbances that affect their regulation, a rise of Ca²⁺ _cell never led to a decline of G, indicating that a rise of Ca²⁺ _cell does not trigger per se TJ closure. On the contrary, always the first response to a rise of Ca²⁺ _cell is an increase of G that, in most cases, is a transient response. Despite these observations we cannot assure that a rise of Ca²⁺ _cell is without effect on the TJs, since an increase of Ca²⁺ _cell not only causes a transient increase of G but, in addition, during oscillations a rise of Ca²⁺ _cell induced by the Ca²⁺ ionophore transiently halted the oscillatory pattern of TJs. The main conclusion of this study is that TJ closure that is observed when basolateral Ca²⁺ concentration (Ca²⁺ _bl) is increased after TJs were opened by Ca²⁺ _bl removal cannot be ascribed to a rise of Ca²⁺ _cell and might be a consequence of Ca²⁺ binding to extracellular Ca²⁺ sites.     

Tight Junction Dynamics: Oscillations and the Role of Protein Kinase C

The present study aimed to characterize the role of protein kinase C (PKC) on the dynamics of tight junction (TJ) opening and closing in the frog urinary bladder. The early events of TJ dynamics were evaluated by the fast Ca⁺⁺ switch assay (FCSA), which consisted in opening the TJs by removing basolateral Ca⁺⁺ ([Ca⁺⁺] _bl ), and closing them by returning [Ca⁺⁺] _bl to normal values. Changes in TJ permeability can be reliably gauged through changes of transepithelial electrical conductance (G) determined in the absence of apical Na⁺. The FCSA allows the appraisal of drugs and procedures acting upon the mechanism controlling the TJs. The time courses of TJ opening and closing in an FCSA were shown to follow single exponential time courses. PKC inhibition by H7 (100 μm) caused a reduction of the rate of junction opening in response to removing [Ca⁺⁺] _bl , without affecting junction closing, indicating that PKC is a key element in the control of TJ opening dynamics in this preparation. H7 at 250 μm almost completely inhibits TJ opening in response to basolateral Ca⁺⁺ withdrawal. Subsequent H7 removal caused a prompt inhibition release characterized by a sharp G increase which, however, once started cannot be stopped by H7 reintroduction, Ca⁺⁺ being necessary to allow TJ recovery. A step rise of apical Ca⁺⁺ concentration ([Ca⁺⁺] _ap ) causes a reduction of the rate of TJ opening in a FCSA, an effect that is believed to be mediated by apical Ca⁺⁺ entering the open TJs. The specific condition of having Ca⁺⁺ only in the apical solution and the TJs located midway between the Ca⁺⁺ source (apical solution) and the Ca⁺⁺-binding sites presumably located at the zonula adhaerens, might configure a situation in which a control feedback loop is set up. A rise of [Ca⁺⁺] _ap during the phase of G increase in an FCSA causes a transient recovery of G followed by a subsequent escape phase where G increases again. Oscillations of G also appear in response to a rise of apical Ca⁺⁺. Both escape and oscillations result from the properties of the TJ regulatory feedback loop. In conclusion, the present results indicate that PKC plays a key role in TJ opening in response to extracellular Ca⁺⁺ withdrawal without major effect on the reverse process. In addition, PKC inhibition by H7 not only prevents TJ opening in response to basolateral Ca⁺⁺ removal but induces a prompt blockade of TJ oscillations induced by apical Ca⁺⁺, oscillations which reappear again when H7 is removed. Received: 9 May 2000/Revised: 30 August 2000     

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.     

Purification and characteristics of Ca2+,Mg2+- and Ca2+,Mn2+-dependent and acid DNases from spermatozoa of the sea urchin Strongylocentrotus intermedius

Ca²⁺,Mg²⁺- and Ca²⁺,Mn²⁺-dependent and acid DNases were isolated from spermatozoa of the sea urchin Strongylocentrotus intermedius. The enzymes have been purified by successive chromatography on DEAE-cellulose, phenyl-Sepharose, Source 15Q, and by gel filtration, and the principal physicochemical and enzymatic properties of the purified enzymes were determined. Ca²⁺,Mg²⁺-dependent DNase (Ca,Mg-DNase) is a nuclear protein with molecular mass of 63 kD as the native form and its activity optimum is at pH 7.5. The enzyme activity in the presence of bivalent metal ions decreases in the series (Ca²⁺ + Mg²⁺) > Mn²⁺ = (Ca²⁺ + Mn²⁺) > (Mg²⁺ + EGTA) > Ca²⁺. Ca,Mg-DNase retains its maximal activity in sea water and is not inhibited by G-actin and N-ethylmaleimide, whereas Zn²⁺ inhibits the enzyme. The endogenous Ca,Mg-DNase is responsible for the internucleosomal cleavage of chromosomal DNA of spermatozoa. Ca²⁺,Mn²⁺-dependent DNase (Ca,Mn-DNase) has molecular mass of 25 kD as the native form and the activity optimum at pH 8.5. The enzyme activity in the presence of bivalent metal ions decreases in the series (Ca²⁺ + Mn²⁺) > (Ca²⁺ + Mg²⁺) > Mn²⁺ > (Mg²⁺ + EGTA). In seawater the enzyme is inactive. Zinc ions inhibit Ca,Mn-DNase. Acid DNase of spermatozoa (A-DNase) is not a nuclear protein, it has molecular mass of 37 kD as a native form and the activity optimum at pH 5.5, it is not activated by bivalent metal ions, and it is inhibited by N-ethylmaleimide and iodoacetic acid. Mechanisms of the endonuclease cleavage of double-stranded DNA have been established for the three enzymes. The possible involvement of DNases from sea urchin spermatozoa in programmed cell death is discussed.     

Effects of agents affecting Ca2+ homeostasis onTrichoderma viride growth and conidiation

Agents known to influence Ca²⁺ homeostasis affected significantly the vegetative growth and starvation-induced conidiation ofTrichoderma viride. Ca²⁺ in millimolar concentrations stimulated both growth and conidiation; a Ca²⁺ deprivation of the fungus by the chelation of extracellular Ca²⁺ (not Mg²⁺ or divalent trace metals) with EGTA (ethyleneglycolbis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid) restricted both the vegetative growth rate and starvation-induced conidiation. Both processes were affected by either Ca²⁺ or EGTA with different efficiencies. Divalent cations (Sr²⁺, Ba²⁺, Co²⁺, Ni²⁺, Mg²⁺, Cd²⁺, Cu²⁺, Mn²⁺) and La³⁺ (inorganic Ca²⁺ blockers) in millimolar concentrations exerted complex (stimulatory, inhibitory, or biphasic) effects on growth and conidiation. In general, their effects on the two processes were mutually different either qualitatively, or quantitatively, or both. Organic Ca²⁺ antagonists (verapamil and dihydropyridines) inhibited the vegetative growth. The results show that Ca²⁺ is required for vegetative growth and conidiation, and that different Ca²⁺-dependent mechanisms may be involved in the two processes. Divalent cations could serve as a tool for investigating the relationship between growth and conidiation.     

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⁺.