Nickel uptake in Rhodopseudomonas capsulata

Nickel uptake system was investigated with a wild-type cell of Rhodopseudomonas capsulata and two mutants lacking uptake hydrogenase (Hup^-). Wild type cells grown photoheterotrophically incorporated ⁶³Ni²⁺ by a high affinity system. The uptake system had a pH of 7.0 and a temperature optimum of 28°C. Both Mg²⁺ and Co²⁺ ions severely repressed the uptake of Ni²⁺. Nickel transport was also inhibited by metabolic inhibitors including cyanide, azide, 2,4-dinitrophenol and m-chlorophenyl carbonylcyanidehydrazone. These data imply that Ni²⁺ uptake system occurs by the energy-linked system for Mg²⁺ transport. The intracellular distribution of ⁶³Ni²⁺ in Hup^- cells showed the same pattern as that of wild-type cells, indicate that the Hup^- strains have no deficiency in Ni²⁺ transport.Abbreviations CCCP m-chlorophenyl carbonylcyanidehydrazone - HEPES N-2-hydroxylethylpiperazine-N-2-ethane-sulfuric acid - HOQNO 2-n-nonly-4-hydroxyquinoline-N-oxide - TMA tetramethylammonium hydroxide     

Cobalt transport in nickel-resistant strains ofNeurospora crassa

Uptake of Co²⁺ by three nickel-resistant strains (Ni^R1, Ni^R2, and Ni^R3) ofNeurospora crassa that differed in resistance to Co²⁺ has been studied. Uptake was linear with Co²⁺ concentration (up to 1 mM), with time (up to 6 h), and with pH between 3 and 6. Uptake rates were in the order Ni^R2>Ni^R1>Ni^R3. In all strains, there was gradual increase in Co²⁺ uptake between 10° and 28°C, with a much sharper increase between 28° and 40°C. Metabolic inhibitors decreased Co²⁺ uptake partially in all strains, except for KF in Ni^R3. About 50–80 g Co²⁺/100 mg dry weight was surface bound. Ni²⁺, Zn²⁺, and Mn²⁺ competed with Co²⁺, the effects being strain specific. Mg²⁺ inhibited Co²⁺ uptake in all strains with preformed mycelia. In Ni^R1 and Ni^R2 only with young mycelia (40 h old) was Mg²⁺ inhibitory to Co²⁺ uptake,during growth in the presence of Co²⁺. The results suggested the presence of two transport systems for Co²⁺ in Ni^R1 and Ni^R2, only one of which was sensitive to Mg²⁺; in contrast, Ni^R3 had a single system, which was sensitive to Mg²⁺.     

Magnesium Transport Across the Basolateral Plasma Membrane of the Fish Enterocyte

In tilapia (Oreochromis mossambicus) intestine, Mg²⁺ transport across the epithelium involves a transcellular, Na⁺- and Na⁺/K⁺-ATPase dependent pathway. In our search for the Mg²⁺ extrusion mechanism of the basolateral compartment of the enterocyte, we could exclude Na⁺/Mg²⁺ antiport or ATP-driven transport. Evidence is provided, however, that Mg²⁺ movement across the membrane is coupled to anion transport. In basolateral plasma membrane vesicles, an inwardly directed Cl⁻ gradient stimulated Mg²⁺ uptake (as followed with the radionuclide ²⁷Mg) twofold. As Cl⁻-stimulated uptake was inhibited by the detergent saponin and by the ionophore A23187, Mg²⁺ may be accumulated intravesicularly above chemical equilibrium. Valinomycin did not affect uptake, suggesting that electroneutral symport activity occurred. The involvement of anion coupled transport was further indicated by the inhibition of Mg²⁺ uptake by the stilbene derivative, 4,4′-diisothiocyanato-stilbene-2,2′-disulfonic acid. Kinetic analyses of the Cl⁻-stimulated Mg²⁺ uptake yielded a K _m (Mg²⁺) of 6.08 ± 1.29 mmol · l⁻¹ and a K _m (Cl⁻) of 26.5 ± 6.5 mmol · l⁻¹, compatible with transport activity at intracellular Mg²⁺- and Cl⁻-levels. We propose that Mg²⁺ absorption in the tilapia intestine involves an electrically neutral anion symport mechanism. Received: 19 January 1996/Revised: 1 August 1996     

Nickel resistance in fission yeast associated with the magnesium transport system

We isolated and characterized a nickel (Ni²⁺)-resistant mutant (GA1) of Schizosaccharomyces pombe. This mutant strain displayed resistance to both Ni²⁺ and Zn²⁺, but not to Cd²⁺, Co²⁺, and Cu²⁺. The growth rate of GA1 increased proportionally with increasing Mg²⁺ concentrations until 50 mM Mg²⁺. The GA1 mutation phenotype suggests a defect in Mg²⁺ uptake. Sequence analysis of the GA1 open reading frame (ORF) O13779, which is homologous to the prokaryotic and eukaryotic CorA Mg²⁺ transport systems, revealed a point mutation at codon 153 (ccc to acc) resulting in a Pro 153Thr substitution in the N-terminus of the CorA domain. Our results provide novel genetic information about Ni²⁺ resistance in fission yeast. Specifically, that reducing Mg²⁺ influx through the CorA Mg²⁺ transport membrane protein confers Ni²⁺ resistance in S. pombe. We also report that Ni²⁺ ion detoxification of the fission yeast is related to histidine metabolism and pH.     

Gastrointestinal transport of Ca<Superscript>2+</Superscript> and Mg<Superscript>2+</Superscript> during the digestion of a single meal in the freshwater rainbow trout

A diet containing an inert marker (ballotini beads, quantified by X-radiography) was used to quantify the transport of two essential minerals, Ca²⁺ and Mg²⁺ from the diet during the digestion and absorption of a single meal of commercial trout food (3% ration). Initially, net uptake of Ca²⁺ was observed in the stomach followed by subsequent Ca²⁺ fluxes along the intestine which were variable, but for the most part secretory. This indicated a net secretion of Ca²⁺ along the intestinal tract resulting in a net assimilation of dietary Ca²⁺ of 28%. Similar handling of Ca²⁺ and Mg²⁺ was observed along the gastrointestinal tract (GI), although net assimilation differed substantially between the cations, with Mg²⁺ assimilation being close to 60%, mostly a result of greater uptake by the stomach. The stomach displayed the highest net uptake rates for both cations (1.5 and 1.3 mmol kg⁻¹ fish body mass for Ca²⁺ and Mg²⁺, respectively), occurring within 2 h following ingestion of the meal. Substantial secretions of both Ca²⁺ and Mg²⁺ were observed in the anterior intestine, which were attributed to bile and other intestinal secretions, while fluxes in the mid and posterior intestine were small and variable. The overall patterns of Ca²⁺ and Mg²⁺ handling in the GI tract were similar to those observed for Na⁺ and K⁺ (but not Cl⁻) in a previous study. Overall, these results emphasize the importance of dietary electrolytes in ionoregulatory homeostasis.     

Nickel Uptake by Pseudomonas aeruginosa: Role of Modifying Factors

Pseudomonas aeruginosa cells growing in minimal medium were 40-fold more sensitive to Ni²⁺ than cells growing in enriched medium, suggesting a possible protective role of medium ingredients. Likewise, cells pre-grown in enriched medium showed a high K _m (6.15 mM) and increased Ni²⁺ uptake (950 nmol mg⁻¹ protein, 1h) over cells pre-sown in minimal medium (K _m , 0.48 mM; 146 nmol mg⁻¹ protein, 1 h). The overall pattern indicates that cells pre-grown in enriched medium were characterized by having lowered affinity towards Ni²⁺ than those with minimal medium background. The enhanced Ni²⁺ uptake by enriched medium-grown cells can be correlated with the improved metabolic state of the cells. Ni²⁺ uptake was optimum at neutrality (pH 7.0). A major Ni²⁺ transport system was competitively inhibited by Mg²⁺, Zn²⁺, Cd²⁺, or Co²⁺ (400 μM each). Noticeably, a minor Ni²⁺ transport pathway was still operative even in the higher concentration range of Mg²⁺ (4 mM and 40 mM). The stimulation of Ni²⁺ uptake monitored in the presence of different carbon sources (0.5% wt/vol, each) showed the sequence: glucose (1.6-fold) > phenol = gallic acid (1.5-fold). Succinate, in comparison, reduced Ni²⁺ uptake (0.5-fold) possibly because of its acting as a metal chelator as well. Sensitivity of Ni²⁺ transport towards methyl viologen, azide, 2-4 DNP, and DCCD suggested that transport was energy-linked. Received: 13 January 1998 / Accepted: 21 May 1998     

Response of the carotenoidless mutant Rhodobacter sphaeroides growing cells to cobalt and nickel exposure

The interaction of cobalt (Co²⁺) and nickel (Ni²⁺) ions with whole cells of the photosynthetic purple bacterium Rhodobacter sphaeroides strain R26 was investigated. Active and passive uptakes were examined in cells grown in the presence of increasing amounts of Co²⁺ and Ni²⁺. Inductively coupled plasma atomic emission spectroscopy (ICP-AES), pH titration, and attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy were used to assess the role of cell envelope and metabolism in accumulating the two heavy metals. The chosen microorganism was able to uptake cobalt and nickel up to 2.2 and 0.25 mg per gram of dried cells respectively, with the largest part found bound to the cell surface. Carboxylate groups lying on the cell wall of this Gram-negative bacterium proved to be the major candidates for binding protons and metal cations. Co²⁺ was found to interfere with Mg²⁺ extracellular immobilization and transport across the membrane, indicating that these ions share binding sites on the cell envelope and ion transport systems. According to the presence of a competition mechanism, bacterial growth experiments showed that high Mg²⁺ concentrations are able to rescue R. sphaeroides from Co²⁺ toxicity.     

The homologous Arabidopsis MRS2/MGT/CorA-type Mg2+ channels,AtMRS2-10 and AtMRS2-1 exhibit different aluminum transport activity

Magnesium (Mg²⁺) plays a critical role in many physiological processes. The AtMRS2/MGT family, which consists of nine Arabidopsis genes (and two pseudo-genes) belongs to a eukaryotic subset of the CorA superfamily of divalent cation transporters. AtMRS2-10 and AtMRS2-1 possess the signature GlyMetAsn sequence conserved in the CorA superfamily; however, they have low sequence conservation with CorA. Direct measurement using the fluorescent dye mag-fura-2 revealed that reconstituted AtMRS2-10 and AtMRS2-1 mediated rapid Mg²⁺ uptake into proteoliposomes. The rapid Mg²⁺ uptake through AtMRS2-10 was inhibited by aluminum. An assay using the Al-sensitive dye morin indicated Al uptake into the proteoliposomes through AtMRS2-10. AtMRS2-10 also exhibited Ni²⁺ transport activity but almost no Co²⁺ transport activity. The rapid Mg²⁺ uptake through AtMRS2-1 was not inhibited by aluminum. Al uptake into the proteoliposomes through AtMRS2-1 was not observed. The functional complementation assay in Escherichia coli strain TM2 showed that AtMRS2-1 was capable of mediating Mg²⁺ uptake. Heterologous expression using the E. coli mutant cells also showed that the E. coli cells expressing AtMRS2-1 was more resistant to aluminum than the E. coli cells expressing AtMRS2-10. The results suggested that AtMRS2-10 transported Al into the E. coli cells, and then the transported Al inhibited the growth of E. coli. AtMRS2-1 has been localized to the Arabidopsis tonoplast, indicating that AtMRS2-1 is exposed to much higher concentration of aluminum than AtMRS2-10. Under the conditions, it may be required that the Mg²⁺ transport of AtMRS2-1 is insensitive to Al inhibition, and AtMRS2-1 is impermeable to Al.     

Excretion of pyruvate in nickel toxicity in wild type and Ni2+ resistant mutants ofNeurospora crassa

The parent wild strainNeurospora crassa Em 5297a and three Ni²⁺ resistantNeurospora crassa mutants have been shown to excrete pyruvate into the culture medium in Ni²⁺ and Co²⁺ toxicities. Ni²⁺ has a more pronounced effect in this regard. The excretion is progressive with growth inhibition and is abolished by Mg²⁺ in all strains and by Fe³⁺ partially in the Em strain but not inNeurospora crassa Ni^R1. Pyruvate, citrate and malate supplementation reverse growth inhibition caused by excess Ni²⁺, but with concomitant suppression of Ni²⁺ accumulation. It is suggested that one of the features of Ni²⁺ toxicity inNeurospora crassa is a derangement in carbohydrate metabolism at step(s) beyond pyruvate and that this is possibly due to decreased invivo activity of Mg²⁺ dependent processes     

Kinetics of uptake and intracellular location of cobalt,manganese and zinc in the estuarine green alga Chlorella salina

Summary The uptake kinetics and intracellular location of cobalt (⁶⁰Co), manganese (⁵⁴Mn) and zinc (⁶⁵Zn) have been characterized in Chlorella salina. Uptake of all three metals was biphasic. The initial rapid phase was independent of light, temperature or the presence of metabolic inhibitors. This first phase of metabolism-independent biosorption was followed by a slower phase of uptake that was apparently dependent on metabolism and decreased by incubation in the dark, or in the light at low temperature or in the presence of metabolic inhibitors. This latter phase of metal accumulation followed Michaelis-Menten kinetics. However, when expressed in the form of a Lineweaver-Burk plot two distinct phases were apparent for each metal with the following K_m values (M); Co²⁺, 19 and 266; Mn²⁺, 2 and 760; Zn²⁺, 4 and 635. For all three metals cellular compartmentation analysis showed that large amounts of metal were bound to intracellular components and to the cell wall. There was also a higher concentration of each metal in the vacuole than in the cytosol, indicating transport of the metals across the tonoplast which may, in part, account for the multi-phasic uptake systems detected. The influence of competing divalent ions on the active uptake of Co²⁺ and Mn²⁺ was also studied. When the concentration of divalent ion was the same as that of Co²⁺ the uptake of the latter was not affected, indicating a specific system for the uptake of Co²⁺. However, Mn²⁺ uptake inhibited by Mg²⁺, Zn²⁺ and Cd²⁺, but not by Co²⁺, which indicated that Mn²⁺, Mg²⁺ and Cd²⁺ may enter the cells via a common system with different affinities for each metal.     

Stimulation of valyl- and isoleucyl-tRNA synthetase reactions by polyamines

The aminoacylation of tRNA catalysed by valyl-tRNA synthetase (EC 6.1.1.9) and isoleucyl-tRNA synthetase (EC 6.1.1.5) fromMycobacterium smegmatis is dependent on the presence of divalent metal ions. Polyamines alone, in the absence of metal ions, do not bring about aminoacylation. In the presence of suboptimal concentrations of Mg²⁺, polyamines significantly stimulate the reaction. Of the cations tested, only Mn²⁺, Co²⁺ and Ca²⁺ can partially substitute for Mg²⁺ in aminoacylation, and spermine stimulates aminoacylation in the presence of these cations also. At neutral pH, spermine deacylates nonenzymatically aminoacyl tRNA. AMP and pyrophosphate-dependent enzymatic deacylation of aminoacyl-tRNA (reverse reaction) is also stimulated by spermine. The inhibitory effect of high concentration of KC1 on aminoacylation is counteracted, by spermine. The low level of activity between pH 8.5–9.0 at 1.2 mM Mg²⁺ is restored to normal level on the addition of spermine. The inhibitory effect of high pH on aminoacylation in the presence of low concentration of Mg²⁺ is also prevntedvby spemine.     

Selective suppression of rod signal transmission by cobalt ions of low levels in carp retina

Selective suppression of rod signal transmission by cobalt ions was reported in carp retina. Using 10 μnol/L Co²⁺, rod-driven horizontal cells were hyperpolarized and light responses were completely suppressed in superfused, isolated retina, while cone-driven horizontal cells were almost unaffected. Similarly, scotopic electroretinographic bwave was suppressed by 10 μnol/L Co²⁺, while the photopic b-wave remained unaffected. Furthermore, the glutamate-isolated receptor potential (PIII) was not altered by low Co²⁺ under dark-adapted conditions. Other divalent ions with high affinity to calcium channels, such as cadmium and manganese ions, did not show similar suppressive effect on the rod horizontal cells. When rod horizontal cells were hyperpolarized by 10 μnol/L Co²⁺, the use of 3 mmol/L glutamate caused a significant depolarization of the cells, indicating that Co²⁺ application did not impair the ability of these cells to respond to glutamate. On the other hand, application of 200 μnol/L β-hydroxyaspartate, a glutamate transport blocker, mimicked the effect of low Co²⁺, suggesting a possibility that the low Co²⁺ effect might be related to a blockade of glutamate uptake by rods. Project supported by the State Commission of Science and Technology of China, the National Natural Science Foundation of China, the National Eye Institute, the Human Frontier Science Program.     

Kinetic studies on sodium-dependent calcium uptake by myocardial cells and neuroblastoma cells in culture

Kinetic analyses were made on intracellular Na⁺-dependent Ca²⁺ uptake by myocardial cells and neuroblastoma cells (N-18 strain) in culture. Cells loaded with various concentrations of Na⁺ could be prepared by incubating them in Ca²⁺-free medium containing various concentrations of Na⁺. Cells pre-loaded with various concentrations of Na⁺ were incubated in medium containing Ca²⁺ and ⁴⁵Ca. The resulting ⁴⁵Ca uptake by the two types of cell depended greatly on the initial intracellular concentrations of Na⁺. Lineweaver-Burk plots of the initial rate of Ca²⁺ uptake against the external concentration of Ca²⁺ fitted well to straight lines obtained by linear regression (r > 0.95). This result shows that Ca²⁺ uptake by the two types of cell was achieved by a carrier-mediated transport system. This Na⁺-dependent Ca²⁺ uptake was accompanied by Na⁺ release and the ratio of Na⁺ release to Ca²⁺ uptake was close to 3 : 1. A comparison of the kinetic data between myocardial cells and N-18 cells suggested that N-18 cells possess a carrier showing the same properties as that of myocardial cells, i.e.: (1) a similar dependency on the intracellular concentration of Na⁺; (2) the coincidence of the apparent Michaelis constants for Ca²⁺ (0.1 mM); (3) the similarities of the K_i values for Co²⁺, Sr²⁺ and Mg²⁺ (Co²⁺ < Sr²⁺ < Mg²⁺) and (4) a similar dependency on pH. However, the maximal initial rate, V, of N-18 cells was about $\text{1}\text{100}$ that of myocardial cells. The rate of Na⁺-dependent Ca²⁺ uptake by non-excitable cells was much lower than that by myocardial cells.     

The effect of glucagon on the kinetics of hepatic mitochondrial calcium uptake

Summary Previous work by this and other laboratories has shown that glucagon administration stimulates calcium uptake by subsequently isolated hepatic mitochondria. This stimulation of hepatic mitochondrial Ca²⁺ uptake byin vivo administration of glucagon was further characterized in the present report. Maximal stimulation of mitochondrial Ca²⁺ accumulation was achieved between 6–10 min after the intravenous injection of glucagon into intact rats. Under control conditions, Ca²⁺ uptake was inhibited by the presence of Mg²⁺ in the incubation medium. Glucagon treatment, however, appeared to obliterate the observed inhibition by Mg²⁺ of mitochondrial Ca²⁺ uptake. Kinetic experiments revealed the usual sigmoidicity associated with initial velocity curves for mitochondrial calcium uptake. Glucagon treatment did not alter this sigmoidal relationship. Glucagon treatment significantly increased the V_max for Ca²⁺ uptake from 292±22 to 377±34 nmoles Ca²⁺ /min per mg protein (n=8) but did not affect the K_0.5, (6.5–8.6 μM). Since the major kinetic change in mitochondrial Ca²⁺ uptake evoked by glucagon is an increase in V_max, the enhancement mechanism is likely to be an increase either in the number of active transport sites available to Ca²⁺ or in the rate of Ca²⁺ carrier movement across the mitochondrial membranes.     

Effect of calcium on synthesis of dipicolinic acid inPenicillium citreoviride and its feedback resistant mutant

Dipicolinic acid synthesis inPenicillium citreoviride strain 3114 was inhibited by Ca²⁺ ions, but not by Ba²⁺, Cu²⁺or Fe²⁺. Among the metals tested, only Zn²⁺ inhibited the synthesis of dipicolinic acid and promoted sporulation. None of these metals reversed the inhibition by Ca²⁺ or Zn²⁺. A mutant 27133-dpa-ca selected for resistance to feedback inhibition by dipicolinic acid: Ca²⁺ complex showed cross-resistance to inhibition by dipicolinic acid: Zn²⁺. Both 3114 and271 33-dpa-ca excreted a number of aliphatic and amino acids during secondary metabolism of dipicolinic acid. In the presence of 1000 ppm of Ca²⁺, accumulation of citric acid and α-aminoadipic acid was completely inhibited under conditions of inhibition of dipicolinic acid in parent strain 3114 but not in the mutant. Citric acid with or without Ca²⁺ did not inhibit thede novo synthesis of dipicolinic acid in the strain 3114. In fact, citric acid in the presence of Ca²⁺ improved significantly rate of dipicolinic acid synthesis. Apart from resistance to feed back inhibition by dipicolinic acid: Ca²⁺ complex, mutant differed from the parent in three other aspectsviz. (i) dipicolinic acid synthesis was not subject to catabolite repression by glucose, (ii) sporulation as well as dipicolinic acid synthesis was dependent on the presence of Ca²⁺ ions in the medium and (iii) Mg²⁺ requirement for the mutant increased three fold. Higher requirement of the Mg²⁺ could be partially relieved by Ca²⁺ during secondary metabolism. The results support the inference thatde novo synthesis of dipicolinic acid is regulated through feedback inhibition by dipicolinic acid: Ca²⁺complex.     

Mg2+ release coupled to Ca2+ uptake: A novel Ca2+ accumulation mechanism in rat liver

Isolated hepatocytes release 2–3 nmol Mg²⁺/mg protein or ~10% of the total cellular Mg²⁺ content within 2 minutes from the addition of agonists that increase cellular cAMP, for example, isoproterenol (ISO). During Mg²⁺ release, a quantitatively similar amount of Ca²⁺ enters the hepatocyte, thus suggesting a stoichiometric exchange ratio of 1 Mg²⁺:1Ca²⁺. Calcium induced Mg²⁺ extrusion is also observed in apical liver plasma membranes (aLPM), in which the process presents the same 1 Mg²⁺:1Ca²⁺ exchange ratio. The uptake of Ca²⁺ for the release of Mg²⁺ occurs in the absence of significant changes in Δψ as evidenced by electroneutral exchange measurements with a tetraphenylphosphonium (TPP⁺) electrode or ³H-TPP⁺. Collapsing the Δψ by high concentrations of TPP⁺ or protonophore carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) does not inhibit the Ca²⁺-induced Mg²⁺ extrusion in cells or aLPM. Further, the process is strictly unidirectional, serving only in Ca²⁺ uptake and Mg²⁺ release. These data demonstrate the operation of an electroneutral Ca²⁺/Mg²⁺ exchanger which represents a novel pathway for Ca²⁺ accumulation in liver cells following adrenergic receptor stimulation. This work was supported by National Institutes of Health Grant HL 18708.     

News & Notes: Sorption and Desorption of Cobalt by Oscillatoria anguistissima

Oscillatoria anguistissima rapidly adsorbs appreciable amounts of cobalt from the aqueous solutions within 15 min of initial contact with the metal solution. O. anguistissima showed a high sequestration of cobalt at low equilibrium concentrations, and it followed the Freundlich model of adsorption. The adsorption is a strongly pH-dependent and temperature-independent phenomenon. The presence of Mg²⁺ and Ca²⁺ (100–200 ppm) resulted in decline in Co²⁺ adsorption capacity of Oscillatoria biomass. Sulphate and nitrate (0.75–10 mM) drastically reduced the extent of Co²⁺ biosorption. The biosorption of cobalt is an ion-exchange process as the Co²⁺ binding was accompanied by release of a large amounts of Mg²⁺ ions. Na₂CO₃ (1.0 mM) resulted in about 76% desorption of Co²⁺ from the loaded biomass. Received: 30 January 1999 / Accepted: 3 March 1999     

Functional reconstitution and characterization of the Arabidopsis Mg2+ transporter AtMRS2-10 in proteoliposomes

Magnesium (Mg²⁺) plays critical role in many physiological processes. The mechanism of Mg²⁺ transport has been well documented in bacteria; however, less is known about Mg²⁺ transporters in eukaryotes. The AtMRS2 family, which consists of 10 Arabidopsis genes, belongs to a eukaryotic subset of the CorA superfamily proteins. Proteins in this superfamily have been identified by a universally conserved GlyMetAsn motif and have been characterized as Mg²⁺ transporters. Some members of the AtMRS2 family, including AtMRS2-10, may complement bacterial mutants or yeast mutants that lack Mg²⁺ transport capabilities. Here, we report the purification and functional reconstitution of AtMRS2-10 into liposomes. AtMRS2-10, which contains an N-terminal His-tag, was expressed in Escherichia coli and solubilized with sarcosyl. The purified AtMRS2-10 protein was reconstituted into liposomes. AtMRS2-10 was inserted into liposomes in a unidirectional orientation. Direct measurement of Mg²⁺ uptake into proteoliposomes revealed that reconstituted AtMRS2-10 transported Mg²⁺ without any accessory proteins. Mutation in the GMN motif, M400 to I, inactivated Mg²⁺ uptake. The AtMRS2-10-mediated Mg²⁺ influx was blocked by Co(III)hexamine, and was independent of the external pH from 5 to 9. The activity of AtMRS2-10 was inhibited by Co²⁺ and Ni²⁺; however, it was not inhibited by Ca²⁺, Fe²⁺, or Fe³⁺. While these results indicate that AtMRS2-10 has similar properties to the bacterial CorA proteins, unlike bacterial CorA proteins, AtMRS2-10 was potently inhibited by Al³⁺. These studies demonstrate the functional capability of the AtMRS2 proteins in proteoliposomes to study structure–function relationships.     

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%.     

Ion antagonism in phytochrome-mediated calcium-dependent germination of turions of Spirodela polyrhiza (L.) Schleiden

The light-dependent germination response of turions (resting fronds) is mediated by phytochrome and requires the presence of Ca²⁺ in the medium (K.-J. Appenroth and H. Augsten, 1990, Photochem. Photobiol. 52: 61–65). The Ca²⁺ requirement of germination is apparent only in the presence of exogenous Mg²⁺. A competitive ion antagonism was demonstrated between Ca²⁺ and Mg²⁺ in this physiological response; Mg²⁺ could also be replaced by Ba²⁺ or Sr²⁺. Without exog-enous Mg²⁺, a Ca²⁺ concentration as low as 0.9 μM fulfilled the Ca²⁺ requirement. This type of ion antagonism resembled the competitive Ca/Mg interaction reported previously for calcium-binding proteins. The physiological response was blocked by inhibitors of Ca²⁺ uptake (verapamil, La³⁺). It was concluded that uptake of Ca²⁺ from the external medium is an essential step in the phytochrome-mediated germination of turions. The results are in agreement with the assumption that the uptake of Ca²⁺ is blocked at the side of entry by other alkaline earth ions. Treatment of turions with Mg²⁺ (1 mM) for 24 h at varying times after the red light pulse in otherwise virtually Ca²⁺-free KNO₃ solution resulted in a response similar to a Ca²⁺ step-down treatment. This is in agreement with the assumption that the Ca²⁺- and the Mg²⁺-sensitive periods coincide. The ion interaction described here represents the first photophysiological example in plants of an antagonistic effect between Ca²⁺ and Mg²⁺ similar to that which occurs in vitro with calmodulin. Received: 12 June 1998 / Accepted: 28 December 1998