CO-REQUIREMENT FOR CALCIUM AND POTASSIUM IN THE GERMINATION OF SPORES OF THE FERN ONOCLEA SENSIBILIS

Spores of Onoclea sensibilis L. do not germinate on distilled H₂O if they are pretreated for sufficient time with dilute NaClO solution. However, spores will germinate, after NaClO pretreatment, on a simple mineral medium containing the major and trace elements. Complete germination after pretreatment also is obtained on a solution containing only Ca²⁺ and K⁺ as the cations, but neither ion by itself is sufficient. Rb⁺, but not Li⁺ or Na⁺, can replace K⁺. Hypochlorite-treated spores do not require the continuous presence of Ca²⁺ and K⁺ to germinate; exposure during the first 4 hr of culture, with the remainder of the time on distilled H₂O, is sufficient. Extraction of spores with ethylene glycol bis(aminoethyl ether) tetraacetic acid [EGTA] makes their germination dependent on Ca²⁺, as reported by other workers, but it does not produce a co-requirement for K⁺. Colorimetric analysis with arsenazo III confirms that Ca²⁺ is extracted from Onoclea spores by NaClO. Extractable Ca²⁺ amounts to about 78 nmol/mg spore dry wt. Of this amount, 31% is contained in the perispore. The perispore comprises 13% of the total spore dry wt.     

Calcium Dependence of Rapid Auxin Action in Maize Roots

We investigated the interaction of Ca²⁺ and auxin on root elongation in seedlings of Zea mays L. The seedlings were raised either in the presence of Ca²⁺ (high calcium; HC = imbibed and raised in 10 millimolar CaCl₂), in the absence of additional Ca²⁺ (intermediate calcium; IC = imbibed and raised in distilled H₂O, calcium supply from seed only), or without additional Ca²⁺ and subsequently depleting them of Ca²⁺ (low calcium; LC = imbibed and raised in distilled H₂O and subsequently treated with 1 millimolar ethyleneglycol-bis-[β-aminoethylether]-N,N,N′,N′ -tetraacetic acid [EGTA]). Exposure of roots of either HC or IC seedlings to auxin concentrations from 0.1 to 10 micromolar resulted in strong inhibition of elongation. In roots of LC seedlings, on the other hand, auxin concentrations as high as 10 micromolar caused only slight inhibition of elongation. Adding 0.5 millimolar Ca²⁺ to LC roots in the presence of IAA allowed normal expression of the inhibitory action of the hormone. Inhibition of elongation in IC roots by indoleacetic acid was reversible upon treatment of the roots with 1 millimolar EGTA. The inhibitory action of auxin could then be re-established by supplying 0.5 millimolar Ca²⁺. The data indicate that Ca²⁺ may be necessary to the growth-regulating action of auxin. The significance of this finding is discussed with respect to the potential role of Ca²⁺ as a second messenger of auxin action and the relevance of this model to recent evidence for gravi-induced redistribution of Ca²⁺ and its role in establishing gravitropic curvature.     

The relationship between changes in cell wall composition and the establishment of polarity in Fucus embryos

Changes in the appearance and location of fucoidin in the cell walls of Fucus embryos were related to embryo development. Fucoidin was not present in the cell wall until 10–14 hr after fertilization, when the embryos began to incorporate fucoidin preferentially into a localized area of the wall. At this time the site of rhizoid initiation was determined; that is, the embryos had undergone axis commitment. Germination of the single-celled embryo occurred between 12 and 16 hr, after fertilization, with all cell walls from germinated embryos showing fucoidin localization at the rhizoid end of the cell. The percentage of embryos with localized fucoidin at the time of axis fixation equaled the percentage of embryos that subsequently germinated. Culturing the embryos in sea water plus 0.8 M sucrose prevented the outgrowth of the rhizoid, but not the localization of fucoidin in the wall or axis commitment. Cycloheximide, nitroprusside, cytochalasin B, sulfate-free sea water, high levels of Ca²⁺, and a breakdown product of TIBA all prevented rhizoid growth and the specific localization of fucoidin. In addition, axis commitment could not be demonstrated in the presence of these inhibitors. DTNB, PCMBS, TIBA, HgCl₂, Mg²⁺ were ineffective as reversible inhibitors of rhizoid initiation. The authors propose that the fixation of axis commitment is accompanied by localized changes in the cell wall involving the incorporation of fucoidin as a structural component of the wall.     

DFT and docking studies of rhodostreptomycins A and B and their interactions with solvated/nonsolvated Mg2+ and Ca2+ ions

The interactions of L-aminoglucosidic stereoisomers such as rhodostreptomycins A (Rho A) and B (Rho B) with cations (Mg²⁺, Ca²⁺, and H⁺) were studied by a quantum mechanical method that utilized DFT with B3LYP/6-311G**. Docking studies were also carried out in order to explore the surface recognition properties of L-aminoglucoside with respect to Mg²⁺ and Ca²⁺ ions under solvated and nonsolvated conditions. Although both of the stereoisomers possess similar physicochemical/antibiotic properties against Helicobacter pylori, the thermochemical values for these complexes showed that its high affinity for Mg²⁺ cations caused the hydration of Rho B. According to the results of the calculations, for Rho A–Ca²⁺(H₂O)₆, ΔH = ?72.21 kcal?mol^?1; for Rho B–Ca²⁺(H₂O)₆, ΔH = ?72.53 kcal?mol^?1; for Rho A–Mg²⁺(H₂O)₆, ΔH = ?72.99  kcal?mol^?1 and for Rho B–Mg²⁺(H₂O)₆, ΔH = ?95.00  kcal?mol^?1, confirming that Rho B binds most strongly with hydrated Mg²⁺, considering the energy associated with this binding process. This result suggests that Rho B forms a more stable complex than its isomer does with magnesium ion. Docking results show that both of these rhodostreptomycin molecules bind to solvated Ca²⁺ or Mg²⁺ through hydrogen bonding. Finally, Rho B is more stable than Rho A when protonation occurs.

Tertiary structure and energy coupling in Ca2+-pump system

Europium luminescence from europium bound to sarcoplasmic reticulum (Ca²⁺ Mg²⁺)-ATPase indicates that there are two high affinity calcium binding sites. Furthermore, the two calcium ions at the binding sites are highly coordinated by the protein as the number of H₂O molecules surrounding the Ca²⁺ ions are 3 and 0.5. In the presence of ATP, calcium ions are occluded even further down to 2 and zero H2O molecules, respectively. The Ca²⁺ - Ca²⁺ intersite distance is estimated to be 8–9 Å and the average distance from the Ca²⁺ sites to CrATP is about 18 Å.Digestion of the (Ca²⁺ + Mg²⁺)-ATPase at the T₂ site (Arg 198) causes uncoupling of Ca²⁺-transport from ATPase activity while calcium occlusion due to E₁-P formation remains unchanged. Further tryptic digestion beyond T₂ and in the presence of ATP diminishes Ca²⁺ occlusion to zero while 50% of the ATPase hydrolytic activity remains. Tryptic digestion beyond T₂ and in the absence of ATP diminishes ATPase hydrolytic activity to 50% of normal while Ca²⁺ occlusion remains intact. These data are consistent with a mechanism in which the functional enzyme must be in the dimeric form for occlusion and calcium uptake to occur, but each monomer can hydrolyze ATP.     

CALCIUM DELAYS THE ONSET OF CELL DIVISION AND RHIZOID ELONGATION IN GEMMAE OF THE CALCIFUGE FERN,VITTARIA GRAMINIFOLIA

Vittaria graminifolia gametophytes reproduce asexually by linear six-celled propagules known as gemmae. It has been shown previously that asymmetric cell division and rhizoid elongation in gemmae are inhibited by millimolar concentrations of Ca^{+ +}. The present paper shows that millimolar Ca^{+ +} delays the onset of cell division at a point prior to mitosis, without affecting the maximal rate of cell division. Ca^{+ +} is most effective in delaying cell division when it is present during the first 24 hours of culture, 2 or 3 days before the initiation of cell division. Millimolar Ca^{+ +} inhibits rhizoid elongation by delaying its onset, while also reducing its maximal rate. Ca^{+ +} is also most effective in delaying rhizoid elongation during the first 24 hours of culture. Culture of gemmae on Ethylglycol-bis-(aminoethyl ether)-N,N,N‘,N‘-tetraacetic acid-buffered media shows that the maximum frequency of cell division occurs at pCa 7, while for the initiation of rhizoid elongation, it occurs at pCa 5.     

Inducement of a Heat-Shock Requirement for Germination and Production of Increased Heat Resistance in Bacillus fastidiosus Spores by Manganous Ions

Bacillus fastidiosus, which requires uric acid or allantoin, grows and sporulates on a simple medium containing 59.5 mM uric acid, 5.7 mM K₂HPO₄, and 2% agar in distilled water. Seventy to ninety percent sporulation was achieved in 96 h. Spores obtained on this medium do not need a heat shock prior to germination. The necessary germination conditions for this organism are 30 C, phosphate or this(hydroxymethyl)aminomethane buffer at pH 7.0, and 5.95 mM uric acid. Sporulation occurred earlier (48 h) and with higher frequency (greater than 99%) when Mn²⁺ was added to the growth medium. However, these spores germinated only after heat activation (70 C, 30 min). The effectiveness of heat activation was directly dependent upon the concentration of Mn²⁺ in the growth medium; 10⁻⁵ M Mn²⁺ was the minimal concentration for the effect. This phenomenon was not found upon addition of Ca²⁺, Mg²⁺, Fe²⁺, Zn²⁺, or Cu²⁺ to the medium. The Mn²⁺ content of the spores depended upon the concentration of Mn²⁺ in the sporulation medium. There was a significant difference in heat resistance between spores harvested from unsupplemented medium and those harvested from medium supplemented with 5 × 10⁻⁵ M Mn²⁺. A D_{85 C} value of 6.5 min was determined with the former, whereas the latter had a value of 17.0 min. Very little change in either Ca²⁺ or dipicolinic acid content was detected in spores harvested from various Mn²⁺-supplemented media. Thus Mn²⁺ may play a role in the inducement of the heat-shock requirement and the formation of spores with increased heat resistance.     

The absorption of calcium ions from the ovine reticulo-rumen

Net Ca²⁺ and Mg²⁺ absorption rates were measured in vivo from buffer solutions placed in the washed reticulo-rumen, isolated in situ in 30 conscious, trained sheep. An increase in concentration of short chain fatty acids (SCFA) in the buffer, over the range 0–50 mM, was shown to stimulate the net rates of absorption of Ca²⁺ and Mg²⁺ ions from the rumen. Similarly, the results of in vitro experiments, carried out with ovine rumen epithelium mounted in short-circuited Ussing chambers, showed that the absence of SCFA from the chamber fluid resulted in a reduction in J_net Ca²⁺ caused by reduced flux of Ca²⁺ ions in the mucosal to serosal direction (J_ms Ca²⁺). The addition of 1 mM acetazolamide, an inhibitor of carbonic anhydrase, to the ruminal buffer used in the in vivo experiments led to significant reductions in the net absorption rates of Ca²⁺and Mg²⁺ ions in the presence of SCFA (50 mmol l⁻¹) but not in the absence of SCFA. However, in the in vitro experiments, the addition of 60 μM ethoxyzolamide had no significant effect on J_net Ca²⁺. A reduction in pH of the intraruminal buffer in vivo from 6.8 to 5.4 led to significant increases in the net absorption rates of Ca²⁺and Mg²⁺ ions, an effect which was duplicated for Ca²⁺ in preliminary in vitro experiments in which the pH of the mucosal buffer was reduced from 7.4 to 5.4. This stimulatory effect was confined to J_ms Ca²⁺ and J_net Ca²⁺. Ussing chambers were also used to demonstrate that J_net Ca²⁺ was reduced by a high transmural potential difference (PD), caused by voltage clamping, independently of the mucosal K⁺ concentration. Both unidirectional Ca²⁺ fluxes consisted of a PD-dependent and a K⁺-insensitive PD-independent component. The latter may be represented by a Ca²⁺/2H⁺ antiporter. It is postulated that SCFA, and to a lesser extent H₂CO₃, can stimulate J_ms Ca²⁺ by activation of an apical Ca²⁺/2H⁺ antiporter through the provision of protons within the ruminal epithelial cell. A mild reduction in ruminal pH may also lead to a similar stimulation of this putative electroneutral exchange. Accepted: 26 July 2000     

Studies on a Factor in Sweet Potato Root Which Agglutinates Spores of Ceratocystis fimbriata, Black Rot Fungus

A factor which agglutinated the spores of Ceratocystis fimbriata in the presence of Ca²⁺ was purified from sweet potato (Ipomea batatas Lam cv. Norin[1]) root. Element composition of the purified factor was as follows; analysis found: C (29.8%), H (3.97%), O (65.34%), N (0.81%): calculated for C₄₃H₆₉O₇₀N₁: C (30.02%), H (4.01%), O (65.15%), N (0.81%). The factor was mainly composed of galacturonic acid (53% of dry weight) and contained arabinose, fucose, and unidentified component as minor components. The factor also agglutinated A-, B-, AB-, and O types of human erythrocytes to almost the same degree in the presence of Ca²⁺. The differential spore-agglutinating activity of the factor depended on the pH of the assay medium; it agglutinated similarly the germinated spores of sweet potato and coffee strains at pH 7.5 and 5.5, whereas it displayed a distinct differential agglutinating activity at pH 6.5. The factor was assayed for spore-agglutinating activity at pH 6.5, using the germinated and ungerminated spores of seven strains of C. fimbriata; sweet potato, coffee, prune, cacao, oak, taro, and almond strains. The factor agglutinated ungerminated spores of all seven strains similarly, although small differences were observed among strains. On the other hand, a clear differential agglutination was observed among the germinated spores of various strains; sweet potato and almond strains were highly insensitive in comparison with other strains. The growth of the agglutinated spores of C. fimbriata was inhibited. These results are discussed in relation to host-parasite specificity.     

A Ca2+-ATPase and a Mg2+/H+-antiporter are present on tonoplast membranes from roots of Zea mays L.

The primary or secondary energized transport of Ca²⁺, Mg²⁺ and H⁺ into tonoplast membrane vesicles from roots of Zea mays L. seedlings was studied photometrically by using the fluorescent Ca²⁺ indicator Indo 1 and the pH indicator neutral red. The localization of an ATP-dependent, vanadate-sensitive Ca²⁺ pump on tonoplast-type vesicles was demonstrated by the co-migration of the Ca²⁺-pumping and tonoplast H⁺-pyrophosphatase (PP_iase) activity on continuous sucrose density gradients. In ER-membrane fractions, only a low Ca²⁺-pumping activity could be detected. The ATP-dependent Ca²⁺ uptake into tonoplast vesicles (using Ca²⁺ concentrations from 0.8–1 μM) was completely inhibited by the Ca²⁺ ionophore ionomycin (1 μM) whereas the protonophore nigericin (1 μM) which eliminates ATP-dependent intravesicular H⁺ accumulation had no effect. Vanadate (IC₅₀ = 43 μM) and diethylstilbesterol (IC₅₀ = 5.2 μM) were potent inhibitors of this type of Ca²⁺ transport. The nucleotides GTP, UTP, ITP, and ADP gave 27%–50% of the ATP-dependent activity (K _m = 0.41 mM). From these results, it was suggested that this ATP-dependent high-affinity Ca²⁺ transport mechanism is the only functioning Ca²⁺ transporter of the tonoplast under in-vivo conditions i.e. under the low cytosolic Ca²⁺ concentration. In contrast, the secondary energized Ca²⁺-transport mechanism of the tonoplast, the low-affinity Ca²⁺/H⁺-antiporter, which was reported to allow the uptake of Ca²⁺ in exchange for H⁺, functions chiefly as an Mg²⁺ transporter under physiological conditions because cytosolic Mg²⁺ is several orders of magnitude higher than the Ca²⁺ concentration. This conclusion was deduced from experiments showing that Mg²⁺ ions in a concentration range of 0.01 to 1 mM triggered a fast efflux of H⁺ from acid-loaded vesicles. Furthermore, the proton-pumping activity of the tonoplast H⁺-ATPase and H⁺-PP_iase was found to be influenced by Ca²⁺ differently from and independently of the Mg²⁺ concentration. Calcium was a strong inhibitor for the H⁺-PP_iase (IC₅₀ = 18 μM, Hill coefficient nH = 1.7) but a weak one for the H⁺-ATPase (IC₅₀ = 330 μM, nH = 1). From these results it is suggested that at the tonoplast membrane a functional interaction exists between (i) the Ca²⁺-and Mg²⁺-regulated H⁺-PP_iase, (ii) the newly described high-affinity Ca²⁺-AT-Pase, (iii) the low-affinity Mg²⁺(Ca²⁺)/H⁺-antiporter and (iv) the H²⁺-ATPase.     

Effect of the chelation of metal cation on the antioxidant activity of chondroitin sulfates

The antioxidant potencies of chondroitin sulfates (CSs) from shark cartilage, salmon cartilage, bovine trachea, and porcine intestinal mucosa were compared by three representative methods for the measurement of the antioxidant activity; DPPH radical scavenging activity, superoxide radical scavenging activity, and hydroxyl radical scavenging activity. CSs from salmon cartilage and bovine trachea showed higher potency in comparison with CSs from shark cartilage and porcine intestinal mucosa. Next, CS from salmon cartilage chelating with Ca²⁺, Mg²⁺, Mn²⁺, or Zn²⁺ were prepared, and their antioxidant potencies were compared. CS chelating with Ca²⁺ or Mg²⁺ ions showed rather decreased DPPH radical scavenging activity in comparison with CS of H⁺ form. In contrast, CS chelating with Ca²⁺ or Mg²⁺ ion showed remarkably enhanced superoxide radical scavenging activity than CS of H⁺ or Na⁺ form. Moreover, CS chelating with divalent metal ions, Ca²⁺, Mg²⁺, Mn²⁺, or Zn²⁺, showed noticeably higher hydroxyl radical scavenging activity than CS of H⁺ or Na⁺ form. The present results revealed that the scavenging activities of, at least, superoxide radical and hydroxyl radical were enhanced by the chelation with divalent metal ions.     

Intrinsic fluorescence studies on troponin C.

Evidence for a proton transfer mechanism in the Ca²⁺-induced enhancement of the Tyr fluorescence of troponin C was obtained by studying the effects, in D₂O and H₂O, of Ca²⁺, Mg²⁺, and H⁺ on the fluorescence of both the protein and a model system containing L-Tyr in the presence of citrate. In the model system, it is shown that citrate quenches the fluorescence of L-Tyr, that there is a large deuterium isotope effect on the quenching, and that binding of Ca², Mg²⁺, or H⁺ by citrate results in a fluorescence enhancement. These results can be explained by the transfer of the phenolic proton of the excited Tyr to the carboxylates of citrate. Similar effects on the fluorescence of troponin C suggest that, in the apoprotein, the fluorescence of Tyr is quenched by a similar mechanism. Thus, the Ca²⁺, Mg²⁺, and H⁺-induced Tyr fluorescence enhancement in troponin C is due to “dequenching” resulting from coordination or protonation of vicinal carboxylates. Studies of troponin C fluorescence and fluorescence depolarization as a function of urea concentration enabled an estimate of the separate fluorescence contributions of its two Tyr residues (Nos. 10 and 109) in its various conformational states. Further evidence was also obtained to support the earlier proposal that the Ca²⁺ enhancement is primarily due to the direct loss of quenching by nearby carboxylates, by showing that the pH-induced fluorescence enhancement did not occur in parallel with the ellipticity increase at 222 nm.     

The behaviour of inositol 1,3,4,5,6-pentakisphosphate in the presence of the major biological metal cations

The inositol phosphates are ubiquitous metabolites in eukaryotes, of which the most abundant are inositol hexakisphosphate (InsP ₆) and inositol 1,3,4,5,6-pentakisphosphate [Ins(1,3,4,5,6)P ₅)]. These two compounds, poorly understood functionally, have complicated complexation and solid formation behaviours with multivalent cations. For InsP ₆, we have previously described this chemistry and its biological implications (Veiga et al. in J Inorg Biochem 100:1800, 2006; Torres et al. in J Inorg Biochem 99:828, 2005). We now cover similar ground for Ins(1,3,4,5,6)P ₅, describing its interactions in solution with Na⁺, K⁺, Mg²⁺, Ca²⁺, Cu²⁺, Fe²⁺ and Fe³⁺, and its solid-formation equilibria with Ca²⁺ and Mg²⁺. Ins(1,3,4,5,6)P ₅ forms soluble complexes of 1:1 stoichiometry with all multivalent cations studied. The affinity for Fe³⁺ is similar to that of InsP ₆ and inositol 1,2,3-trisphosphate, indicating that the 1,2,3-trisphosphate motif, which Ins(1,3,4,5,6)P ₅ lacks, is not absolutely necessary for high-affinity Fe³⁺ complexation by inositol phosphates, even if it is necessary for their prevention of the Fenton reaction. With excess Ca²⁺ and Mg²⁺, Ins(1,3,4,5,6)P ₅ also forms the polymetallic complexes [M₄(H₂L)] [where L is fully deprotonated Ins(1,3,4,5,6)P ₅]. However, unlike InsP ₆, Ins(1,3,4,5,6)P ₅ is predicted not to be fully associated with Mg²⁺ under simulated cytosolic/nuclear conditions. The neutral Mg²⁺ and Ca²⁺ complexes have significant windows of solubility, but they precipitate as [Mg₄(H₂L)]·23H₂O or [Ca₄(H₂L)]·16H₂O whenever they exceed 135 and 56 μM in concentration, respectively. Nonetheless, the low stability of the [M₄(H₂L)] complexes means that the 1:1 species contribute to the overall solubility of Ins(1,3,4,5,6)P ₅ even under significant Mg²⁺ or Ca²⁺ excesses. We summarize the solubility behaviour of Ins(1,3,4,5,6)P ₅ in straightforward plots.     

The pH Dependence and the Binding Equilibria of the Calcium Indicator — Arsenazo III

The underlying principles of binding equilibria of arsenazo III with Ca²⁺ and Mg²⁺ are presented. Ca²⁺ and Mg²⁺ can bind arsenazo III in several different protonated forms depending on pH. The binding affinities of these different protonated forms of arsenazo III with Ca²⁺ increase in the order of H₄A_4- <H₃A^5- >H₂A^6- and with Mg²⁺, H₄A^4- > H₃A^5- > H₂A^6-. Arsenazo III is not membrane bound. The sensitivity ratio of arsenazo III with Ca²⁺ to arsenazo III with Mg²⁺ is close to two orders of magnitude. Arsenazo III and its complexes are extremely sensitive to pH changes. With 5 μM arsenazo III, the minimum detectable amount of Ca²⁺ can be as low as 0.08 μM. Contrary to current belief, we found that Mg²⁺ can bind to arsenazo III in a slightly acidic medium. Potential applications of arsenazo III to the study of membrane Ca²⁺ transport are also discussed.     

ROLE OF ASYMMETRIC CELL DIVISION IN PTERIDOPHYTE DIFFERENTIATION. II. EFFECT OF CA2+ ON ASYMMETRIC CELL DIVISION,RHIZOID ELONGATION,AND ANTHERIDIUM DIFFERENTIATION IN VITTARIA GEMMAE

Gametophytes of Vittaria graminifolia reproduce vegetatively by means of gemmae. Each gemma consists of a linear array of six cells: four body cells and a knob-shaped terminal cell at each end. When gemmae are shed from the gametophyte onto Knop's mineral medium, the two terminal cells do not divide, but elongate to form primary rhizoids. The body cells undergo asymmetric cell division, and the smaller daughter cells differentiate into either secondary rhizoids or prothalli. When gibberellic acid is included in the medium, antheridia are formed as a result of asymmetric cell division instead of vegetative structures. We studied the effect of Ca²⁺ on asymmetric cell division, rhizoid elongation, and antheridium formation in gemmae cultured on Knop's mineral medium and variations of Knop's medium. Ca²⁺ inhibited the onset of cell division and rhizoid elongation, but was required for differentiation of antheridia. Treatments which lowered the Ca²⁺ content of gemmae (EGTA and dilute HCl extraction, culture on verapamil-containing and Ca²⁺-deficient medium) caused an early onset of cell division and rhizoid elongation. The stimulation of growth was most pronounced when gemmae were deprived of Ca²⁺ during the first 24 hr of culture. The proportion of cell divisions which differentiated into antheridia in response to GA was greatly reduced when the Ca²⁺ status of gemmae was lowered with verapamil and Ca²⁺-EGTA buffers.     

Osmolality and electrolyte concentration of hemolymph and the problem of ion and volume regulation of cells in higher insects

• 1.1. The study was carried out on 22 species of insects from 5 orders. The osmolality of their hemolymph varied from 319 to 421 mOsm/kg H₂O, concentration of Na⁺ 4.6 to 118 mM/l, K⁺ 6.3 to 73mM/l, Ca²⁺ 3.6 to 12.9 mM/l, Mg²⁺ 2.3 to 76 mM/l. The most abundant cation in the hemolymph of insects from higher orders is either K⁺ or Mg²⁺.
• 2.2. In the muscles of lower and higher insects K⁺ is usually within 80–120 mM/kg wet wt.
• 3.3. Most Ca²⁺ and Mg²⁺ in hemolymph is bound with protein and low molecular anions, concentration of free Ca²⁺ is 0.9-2.1mM/l Mg²⁺ 3.7–8.0 mM/l.
• 4.4. It is concluded that, in insects, potassium hemolymph, cell volume regulation and accumulation of ions in the cell, are ensured by an increased osmolality of hemolymph due to a high percentage contribution of low molecular organic substances which are retained in the hemolymph due to the absence of filtration apparatus in the Malpighian tubules.

     

Reversible sporicidal action of cuprous ions

At 10 mM, Cu⁺ was highly protective against killing of spores of Bacillus megaterium ATCC 19213 by H₂O₂, while at higher concentrations, from 15–100 mM, killing was augmented. In contrast, Cu²⁺, Fe²⁺, Fe³⁺, Co²⁺ or Co³⁺ ions acted only protectively. Cu⁺ itself was sporicidal in the absence of H₂O₂ or ascorbate, and its sporicidal action did not depend on generation of highly reactive oxygen species. It appeared that killing involved either inhibition of germination or copper toxicity to germinated cells in that Cu⁺-inactivated spores did not germinate readily but chemical decoating of the cells prior to plating on a solid medium resulted in reversal of the sporicidal effect. Received 12 July 1996/ Accepted in revised form 03 November 1996     

Kinetics of the Ca, H, and Mg Interaction with the Ion-Binding Sites of the SR Ca-ATPase

Electrochromic styryl dyes were used to investigate mutually antagonistic effects of Ca²⁺ and H⁺ on binding of the other ion in the E₁ and P-E₂ states of the SR Ca-ATPase. On the cytoplasmic side of the protein in the absence of Mg²⁺ a strictly competitive binding sequence, H₂E₁?HE₁?E₁?CaE₁?Ca₂E₁, was found with two Ca²⁺ ions bound cooperatively. The apparent equilibrium dissociation constants were in the order of K_1/2(2 Ca) = 34 nM, K_1/2(H) = 1 nM and K_1/2(H₂) = 1.32 μM. Up to 2 Mg²⁺ ions were also able to enter the binding sites electrogenically and to compete with the transported substrate ions (K_1/2(Mg) = 165 μM, K_1/2(Mg₂) = 7.4 mM). In the P-E₂ state, with binding sites facing the lumen of the sarcoplasmatic reticulum, the measured concentration dependence of Ca²⁺ and H⁺ binding could be described satisfactorily only with a branched reaction scheme in which a mixed state, P-E₂CaH, exists. From numerical simulations, equilibrium dissociation constants could be determined for Ca²⁺ (0.4 mM and 25 mM) and H⁺ (2 μM and 10 μM). These simulations reproduced all observed antagonistic concentration dependences. The comparison of the dielectric ion binding in the E₁ and P-E₂ conformations indicates that the transition between both conformations is accompanied by a shift of their (dielectric) position.     

Interaction of Adenosine 5′-Triphosphate with Metal Ions X-ray Structure of Ternary Complexes Containing Mg(II), Ca(II), Mn(II), Co(II), ATP and 2,2′ -Dipyridylamine

Abstract

The X-ray structures of the isomorphous Mg²⁺, Ca²⁺, Mn²⁺ and Co²⁺ complexes of ATP have been determined. The metal ions are wrapped in hexa-coordination by the α, β and γ phosphate groups of two ATP molecules thus blocking the interaction of the metal ions with the adenine base. A second metal ion which is fully hydrated, M(H₂O)²⁺ ₆, is engaged in a strong hydrogen bond with the γ phosphate group of ATP and suggests a possible step in facilitating the cleavage between the β and γ phosphates in phosphoryl transfer reactions.     

Characterisation of a Ca2+-dependent ATP hydrolysis associated with the vacuolar membrane of wheat roots

Microsomal fractions from wheat tissues exhibit a higher level of ATP hydrolytic activity in the presence of Ca²⁺ than Mg²⁺. Here we characterise the Ca²⁺-dependent activity from roots of Triticum aestivum lev. Troy) and investigate its possible function. Ca²⁺-dependent ATP hydrolysis in the microsomal fraction occurs over a wide pH range with two slight optima at pH 5.5 and 7.5. At these pHs the activity co-migrates with the major peak of nitrate-inhibited Mg²⁺. Cl-ATPase on continuous sucrose gradients indicating that it is associated with the vacuolar membrane. Ca²⁺-dependent ATP hydrolysis can be distinguished from an inhibitory effect of Ca²⁺ on the plasma membrane K⁺, Mg²⁺-ATPase following microsomal membrane separation using aqueous polymer two phase partitioning. The Ca²⁺-dependent activity is stimulated by free Ca²⁺ with a K_m of 8.1 μM in the absence of Mg²⁺ ([CaATP] = 0.8 mM). Vacuoiar membrane vacuolar preparations contain a higher Ca²⁺-dependent than Mg²⁺-dependent ATP hydrolysis, although the two activities are not directly additive. The nucleotide specificity of the divalent ion-dependent activities in vacuolar membrane-enriched fractions was low. hydrolysis of CTP and UTP being greater than ATP hydrolysis with both Ca²⁺ and Mg²⁺ The Ca²⁺-dependent activity did discriminate against dinucleotides, and mononucleotides. and failed to hydrolyse phosphatase substrates. Despite low nucleotide specificity the Mg²⁺-dependent activity functioned as a bafilomycin sensitive H⁺-pump in vacuolar membrane vesicles. Ca²⁺-dependent ATP hydrolysis was not inhibited by the V-, P-, or F-type ATPase inhibitors bafilomycin. vanadate and azide, respectively. nor by the phosphatase inhibitor molybdate, but was inhibited 20% at pH 7.5 by K⁺. Possible functions of Ca²⁺-dependent hydrolysis as a H⁺-pump or a Ca²⁺-pump was investigated using vacuolar membrane vesicles. No H⁺ or Ca²⁺ translocating activity was observed under conditions when the Ca²⁺-dependent ATP hydrolysis was active.