Differential expression of thermophilic phosphatases in the wild type and auxotrophic mutant strains of <Emphasis Type="Italic">Thermoactinomyces vulgaris</Emphasis>

In the wild type strain (stock no. 1227) of Thermoactinomyces vulgaris, as reported earlier [Sinha and Singh (1980) Biochem. J. 190, 457–460], all phosphatase isoenzymes (three alkaline — AlpI, AlpII and AlpIII, and one acidic — Acp) are present. However, the auxotrophic mutants, the strains 1286 (thi ⁻), 1279 (nic ⁻, ura ⁻) and 1278 (thi ⁻, ura ⁻) exhibited two alkaline phosphatase isoenzymes (AlpII and AlpIII), but AlpI was lacking. In the strain 1261 (nic ⁻, thi ⁻), only AlpIII was expressed, and AlpI and AlpII isoenzymes were missing. The results suggest that the strains, which require either thiamine (1286 and 1278) or nicotinamide (1279) for their growth, were AlpI ⁻ mutants; and the strain (1261), which requires both thiamine and nicotinamide for its growth, was AlpI ⁻/AlpII ⁻ double mutant. There was no direct correlation between uracil auxotrophy and the expression of phosphatases. The uniform expression of AlpIII and Acp in all the strains, irrespective of their nutrient requirements, suggest that these constitutive phosphatases are species-specific. The specific activities of the thermophilic acid and alkaline phosphatases were maximum in the wild type strain (1227) of T. vulgaris. The next in phosphatase activity was the strain 1279 (an AlpI ⁻ mutant), followed by their decrease, in order, in the strains 1286 and 1278 (which were also AlpI ⁻ mutants); while least activity of these enzymes was observed in the obligate thermophile strain 1261 (AlpI ⁻/AlpII ⁻ double mutant).     

Mechanism of luminal Ca<Superscript>2+</Superscript> and Mg<Superscript>2+</Superscript> action on the vacuolar slowly activating channels

The non-selective slow vacuolar (SV) channel can dominate tonoplast conductance, making it necessary to tightly control its activity. Applying the patch-clamp technique to vacuoles from sugar beet (Beta vulgaris L.) taproots we studied the effect of divalent cations on the vacuolar side of the SV channel. Our results show that the SV channel has two independent binding sites for vacuolar divalent cations, (i) a less selective one, inside the channel pore, binding to which impedes channel conductance, and (ii) a Ca²⁺-selective one outside the membrane-spanning part of the channel protein, binding to which stabilizes the channels closed conformations. Vacuolar Ca²⁺ and Mg²⁺ almost indiscriminately blocked ion fluxes through the open channel pore, decreasing measured single-channel current amplitudes. This low-affinity block displays marked voltage dependence, characteristic of a permeable blocker. Vacuolar Ca²⁺—with a much higher affinity than Mg²⁺—slows down SV channel activation and shifts the voltage dependence to more (cytosol) positive potentials. A quantitative analysis results in a model that exactly describes the Ca²⁺-specific effects on the SV channel activation kinetics and voltage gating. According to this model, multiple (approximately three) divalent cations bind with a high affinity at the luminal interface of the membrane to the channel protein, favoring the occupancy of one of the SV channels closed states (C2). Transition to another closed state (C1) diminishes the effective number of bound cations, probably due to mutual repulsion, and channel opening is accompanied by a decrease of binding affinity. Hence, the open state (O) is destabilized with respect to the two closed states, C1 and C2, in the presence of Ca²⁺ at the vacuolar side. The specificity for Ca²⁺ compared to Mg²⁺ is explained in terms of different binding affinities for these cations. In this study we demonstrate that vacuolar Ca²⁺ is a crucial regulator to restrict SV channel activity to a physiologically meaningful range, which is less than 0.1% of maximum SV channel activity.Abbreviation SV Slow vacuolar     

Evidence for the Cd<Superscript>2+</Superscript> Activation of the Aryl Sulfatase from <Emphasis Type="Italic">Helix pomatia</Emphasis>

Often used to remove sulfate groups from carbohydrates, the regulatory properties of the aryl sulfatase from Helix pomatia remain little characterized. As many hydrolytic enzymes utilize exogenous metal ions in catalysis, the effect of various divalent metal ions on the sulfatase was investigated. Evidence for metal ion activation was collected, with Cd²⁺ being notable for effective activation. The enzyme was inhibited by Cu²⁺. The response of other common hydrolases to divalent metal ions was characterized. Activation by Cd²⁺ was not observed for chymotrypsin, rabbit liver esterase, or β-galactosidase. Instead, Cd was found to inhibit both the esterase and the galactosidase. Inhibition by Cu²⁺ and Zn²⁺ was also observed for some of these hydrolases.     

Ca<Superscript>2+</Superscript>-Dependence and Inhibition of Transformation by Trifluoperazine and Chlorpromazine in <Emphasis Type="Italic">Thermoactinomyces vulgaris</Emphasis>

Ca²⁺ enhanced the transformation frequency of Thermoactinomyces vulgaris (stock no. 1278) of an auxotrophic strain by the chromosomal DNA isolated from a prototrophic strain (stock no. 1227). The number of transformants showed a marked increase with increasing concentration of CaCl₂ upto 0.05 mM; and above this concentration, the transformation frequency decreased significantly. Antipsychotic drugs that are potent calmodulin inhibitors, like trifluoperazine and chlorpromazine, when applied in the concentration range of 0.01–0.04 mM along with optimal CaCl₂ concentration to the cultures of the recipient cells, resulted in a significant inhibition in the frequency of Ca²⁺-stimulated transformation. The results of present investigation suggest the involvement of a Ca²⁺-dependent protein activator in the development of Ca²⁺-mediated competence, which could have played an important role in the enhancement of genetic transformation in this aerobic spore forming thermophilic actinomycete. Received: 21 May 2002 / Accepted: 21 June 2002     

Characterization of a monomeric heat-labile classical alkaline phosphatase from <Emphasis Type="Italic">Anabaena</Emphasis> sp. PCC7120

Alkaline phosphatases (APs), known inducible enzymes of the Pho regulon and poorly characterized in cyanobacteria, hydrolyze phosphomonoesters to produce inorganic phosphate (P_i) during P_i starvation. In this study, two predicted alkaline phosphatase genes in the genome of Anabaena sp. PCC 7120, all2843 and alr5291, were apparently induced during P_i starvation. Sequence analysis showed that alr5291 encodes a protein that is an atypical alkaline phosphatase like other cyanobacteria PhoAs, but the protein encoded by all2843 is very similar to the classical PhoAs, such as Escherichia coli alkaline phosphatase (EAP). To date, there have been no reports about classical phoA in cyanobacterial genomes. The alkaline phosphatase AP^A, coded by all2843, is characterized as a metalloenzyme containing Mg²⁺ and Zn²⁺ with molar ratio of 1: 2. Site-directed mutagenesis analysis indicated that, though the active center of AP^A is highly conserved in comparison with EAP, differences do exist between AP^A and EAP in metal ion coordination. Besides, biochemical analysis revealed that AP^A is a monomeric protein and inactivated rapidly at 50°C. These results suggest that AP^A is the first monomeric heat-labile classical PhoA found in cyanobacteria.     

Divalent cations and the phosphatase activity of the (Na + K)-dependent ATPase

Phosphatase activity of a kidney (Na + K)-ATPase preparation was optimally active with Mg²⁺ plus K⁺. Mn²⁺ was less effective and Ca²⁺ could not substitute for Mg²⁺. However, adding Ca²⁺ with Mg²⁺ or substituting Mn²⁺ for Mg²⁺ activated it appreciably in the absence of added K⁺, and all three divalent cations decreased apparent affinity for K⁺. Inhibition by Na⁺ decreased with higher Mg²⁺ concentrations, when Ca²⁺ was added, and when Mn²⁺ was substituted for Mg²⁺. Dimethyl sulfoxide, which favorsE ₂ conformations of the enzyme, increased apparent affinity for K⁺, whereas oligomycin, which favorsE ₁ conformations, decreased it. These observations are interpretable in terms of activation through two classes of cation sites. (i) At divalent cation sites, Mg²⁺ and Mn²⁺, favoring (under these conditions)E ₂ conformations, are effective, whereas Ca²⁺, favoringE ₁, is not, and monovalent cations complete. (ii) At monovalent cation sites divalent cations compete with K⁺, and although Ca²⁺ and Mn²⁺ are fairly effective, Mg²⁺ is a poor substitute for K⁺, while Na⁺ at these sites favorsE ₁ conformations. K⁺ increases theK _m for substrate, but both Ca²⁺ and Mn²⁺ decrease it, perhaps by competing with K⁺. On the other hand, phosphatase activity in the presence of Na⁺ plus K⁺ is stimulated by dimethyl sulfoxide, by higher concentrations of Mg²⁺ and Mn²⁺, but not by adding Ca²⁺; this is consistent with stimulation occurring through facilitation of an E₁ to E₂ transition, perhaps an E₁-P to E₂-P step like that in the (Na + K)-ATPase reaction sequence. However, oligomycin stimulates phosphatase activity with Mg²⁺ plus Na⁺ alone or Mg²⁺ plus Na⁺ plus low K⁺: this effect of oligomycin may reflect acceleration, in the absence of adequate K⁺, of an alternative E₂-P to E₁ pathway bypassing the monovalent cation-activated steps in the hydrolytic sequence.     

Purification and biochemical characterization of thermostable alkaline phosphatases produced by <Emphasis Type="Italic">Rhizopus microsporus</Emphasis> var. <Emphasis Type="Italic">rhizopodiformis</Emphasis>

The biochemical properties of the alkaline phosphatases (AlPs) produced by Rhizopus microsporus are described. High enzymic levels were produced within 1–2 d in agitated cultures with 1 % wheat bran. Intra- and extracellular AlPs were purified 5.0 and 9.3×, respectively, by DEAE-cellulose and ConA-sepharose chromatography. Molar mass of 118 and 120 kDa was estimated by gel filtration for both forms of phosphatases. SDS-PAGE indicated dimeric structures of 57 kDa for both forms. Mn²⁺, Na⁺ and Mg²⁺ stimulated the activity, while Al³⁺ and Zn²⁺ activated only the extracellular form. Optimum temperature and pH for both phosphatases were 65 °C and pH 8.0, respectively. The enzymes were stable at 50 °C for at least 15 min. Hydrolysis of 4-nitrophenyl phosphate exhibited a K _m 0.28 and 0.22 mmol/L, with υ _lim 5.89 and 4.84 U/mg, for intra- and extracellular phosphatases, respectively. The properties of the reported AlPs may be suitable for biotechnological application.     

Ca<Superscript>2+</Superscript>-independent effects of spermine on pyruvate dehydrogenase complex activity in energized rat liver mitochondria incubated in the absence of exogenous Ca<Superscript>2+</Superscript> and Mg<Superscript>2</Superscript><Superscript>+</Superscript>

In the absence of exogenous Ca²⁺ and Mg²⁺ and in the presence of EGTA, which favours the release of endogenous Ca²⁺, the polyamine spermine is able to stimulate the activity of pyruvate dehydrogenase complex (PDC) of energized rat liver mitochondria (RLM). This stimulation exhibits a gradual concentration-dependent trend, which is maximum, about 140%, at 0.5 mM concentration, after 30 min of incubation. At concentrations higher than 0.5 mM, spermine still stimulates PDC, when compared with the control, but shows a slight dose-dependent decrease. Changes in PDC stimulation are very close to the phosphorylation level of the E_1α subunit of PDC, which regulates the activity of the complex, but it is also the target of spermine. In other words, progressive dephosphorylation gradually enhances the stimulation of RLM and progressive phosphorylation slightly decreases it. These results provide the first evidence that, when transported in RLM, spermine can interact in various ways with PDC, showing dose-dependent behaviour. The interaction most probably takes place directly on a specific site for spermine on one of the regulatory enzymes of PDC, i.e. pyruvate dehydrogenase phosphatase (PDP). The interaction of spermine with PDC may also involve activation of another regulatory enzyme, pyruvate dehydrogenase kinase (PDK), resulting in an increase in E_1α phosphorylation and consequently reduced stimulation of PDC at high polyamine concentrations. The different effects of spermine in RLM are discussed, considering the different activities of PDP and PDK isoenzymes. It is suggested that the polyamine at low concentrations stimulates the isoenzyme PDP₂ and at high concentrations it stimulates PDK₂.     

Intracellular Mg<Superscript><Emphasis Type="Bold">2+</Emphasis></Superscript> Influences Both Open and Closed Times of a Native Ca<Superscript><Emphasis Type="Bold">2+</Emphasis></Superscript>-activated BK Channel in Cultured Human Renal Proximal Tubule Cells

Effects of intracellular Mg²⁺ on a native Ca²⁺-and voltage-sensitive large-conductance K⁺ channel in cultured human renal proximal tubule cells were examined with the patch-clamp technique in the inside-out mode. At an intracellular concentration of Ca²⁺ ([Ca²⁺]_i) of 10⁻⁵–10⁻⁴ M, addition of 1–10 mM Mg²⁺ increased the open probability (P_o) of the channel, which shifted the P_o –membrane potential (V_m) relationship to the negative voltage direction without causing an appreciable change in the gating charge (Boltzmann constant). However, the Mg²⁺-induced increase in P_o was suppressed at a relatively low [Ca²⁺]_i (10^−5.5–10⁻⁶ M). Dwell-time histograms have revealed that addition of Mg²⁺ mainly increased P_o by extending open times at 10⁻⁵ M Ca²⁺ and extending both open and closed times simultaneously at 10^−5.5 M Ca²⁺. Since our data showed that raising the [Ca²⁺]_i from 10⁻⁵ to 10⁻⁴ M increased P_o mainly by shortening the closed time, extension of the closed time at 10^−5.5 M Ca²⁺ would result from the Mg²⁺-inhibited Ca²⁺-dependent activation. At a constant V_m, adding Mg²⁺ enhanced the sigmoidicity of the P_o–[Ca²⁺]_i relationship with an increase in the Hill coefficient. These results suggest that the major action of Mg²⁺ on this channel is to elevate P_o by lengthening the open time, while extension of the closed time at a relatively low [Ca²⁺]_i results from a lowering of the sensitivity to Ca²⁺ of the channel by Mg²⁺, which causes the increase in the Hill coefficient. M. Kubokawa and Y. Sohma contributed equally to this work.     

ATPase and acid phosphatase activities associated with vacuoles isolated from storage roots of red beet (Beta vulgaris L.)

Phosphatase activities were measured in preparations of vacuoles isolated from storage roots of red beet (Beta vulgaris L.). The vacuoles possessed both acid phosphatase and ATPase activities which could be distinguished by their susceptibility to inhibition by low concentrations of ammonium molybdate [(NH₄)₆Mo₇O₂₄·4H₂O]. The acid phosphatase was completely inhibited by 100 M ammonium molybdate but the ATPase was unaffected. The acid phosphatase was a soluble enzyme which hydrolysed a large number of phosphate esters and had a pH optimum of 5.5. In contrast, the ATPase was partially membrane-bound, had a pH optimum of 8.0 and hydrolysed ATP preferentially, although it was also active agianst PP_i, GTP and GDP. At pH 8.0 both the ATPase and PPase activities were Mg²⁺-dependent and were further stimulated by KCl. The ATPase and PPase activities at pH 8.0 may be different enzymes. The recovery and purification of the ATPase during vacuole isolation were determined. The results indicate that the Mg²⁺-dependent, KCl-stimulated ATPase activity is not exclusively associated with vacuoles.Abbreviations BSA bovine serum albumen - MES 2-(N-Morpholino)ethanesulphonic acid - MOPS 3-(N-Morpholino)propanesulphonic acid - Na₂EDTA ethylenediaminetetra-acetic acid, disodium salt - P_i inorganic phosphate - PP_i inorganic pyrophosphate - PPase inorganic pyrophosphatase - TCA trichloroacetic acid - TES N-tris(hydroxymethyl)methyl-2-amino-ethanesulphonic acid - Tris tris(hydroxymethyl)methylamine     

Transition from octahedral to tetrahedral geometry causes the activation or inhibition by Zn<Superscript>2+</Superscript> of <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> phosphorylcholine phosphatase

Pseudomonas aeruginosa phosphorylcholine phosphatase (PchP) catalyzes the hydrolysis of phosphorylcholine, which is produced by the action of hemolytic phospholipase C on phosphatidylcholine or sphyngomielin, to generate choline and inorganic phosphate. Among divalent cations, its activity is dependent on Mg²⁺ or Zn²⁺. Mg²⁺ produced identical activation at pH 5.0 and 7.4, but Zn²⁺ was an activator at pH 5.0 and became an inhibitor at pH 7.4. At this higher pH, very low concentrations of Zn²⁺ inhibited enzymatic activity even in the presence of saturating Mg²⁺ concentrations. Considering experimental and theoretical physicochemical calculations performed by different authors, we conclude that at pH 5.0, Mg²⁺ and Zn²⁺ are hexacoordinated in an octahedral arrangement in the PchP active site. At pH 7.4, Mg²⁺ conserves the octahedral coordination maintaining enzymatic activity. The inhibition produced by Zn²⁺ at 7.4 is interpreted as a change from octahedral to tetrahedral coordination geometry which is produced by hydrolysis of the [ \textZn ²⁺ \textL ₂ ^{- 1} \textL ₂⁰ ( \textH ₂ \textO ) ₂ ] \left[ {{\text{Zn}}^{ 2+ } {\text{L}}_{ 2}^{ - 1} {\text{L}}_{ 2}^{0} \left( {{\text{H}}_{ 2} {\text{O}}} \right)_{ 2} } \right] complex.     

Distinct Ca<Superscript>2+</Superscript>-permeable Cation Currents Are Activated by Internal Ca<Superscript>2+</Superscript>-Store Depletion in RBL-2H3 Cells and Human Salivary Gland Cells,HSG and HSY

Store-operated Ca²⁺ influx, suggested to be mediated via store-operated cation channel (SOC), is present in all cells. The molecular basis of SOC, and possible heterogeneity of these channels, are still a matter of controversy. Here we have compared the properties of SOC currents (I _SOC) in human submandibular glands cells (HSG) and human parotid gland cells (HSY) with I _CRAC (Ca²⁺ release-activated Ca²⁺ current) in RBL cells. Internal Ca²⁺ store-depletion with IP₃ or thapsigargin activated cation channels in all three cell types. 1 μM Gd³⁺ blocked channel activity in all cells. Washout of Gd³⁺ induced partial recovery in HSY and HSG but not RBL cells. 2-APB reversibly inhibited the channels in all cells. I _CRAC in RBL cells displayed strong inward rectification with E _rev(Ca) = >+90 mV and E _rev (Na) = +60 mV. I _SOC in HSG cells showed weaker rectification with E _rev(Ca) = +25 mV and E _rev(Na) = +10 mV. HSY cells displayed a linear current with E _rev = +5 mV, which was similar in Ca²⁺- or Na⁺-containing medium. pCa/pNa was >500, 40, and 4.6 while pCs /pNa was 0.1,1, and 1.3, for RBL, HSG, and HSY cells, respectively. Evidence for anomalous mole fraction behavior of Ca²⁺/Na⁺ permeation was obtained with RBL and HSG cells but not HSY cells. Additionally, channel inactivation with Ca²⁺ + Na⁺ or Na⁺ in the bath was different in the three cell types. In aggregate, these data demonstrate that distinct store-dependent cation currents are stimulated in RBL, HSG, and HSY cells. Importantly, these data suggest a molecular heterogeneity, and possibly cell-specific differences in the function, of these channels.This revised version was published online in June 2005 with a corrected cover date.     

Towards extracellular Ca<Superscript>2+</Superscript> sensing by MRI: synthesis and calcium-dependent <Superscript>1</Superscript>H and <Superscript>17</Superscript>O relaxation studies of two novel bismacrocyclic Gd<Superscript>3+</Superscript> complexes

Two new bismacrocyclic Gd³⁺ chelates containing a specific Ca²⁺ binding site were synthesized as potential MRI contrast agents for the detection of Ca²⁺ concentration changes at the millimolar level in the extracellular space. In the ligands, the Ca²⁺-sensitive BAPTA-bisamide central part is separated from the DO3A macrocycles either by an ethylene (L¹) or by a propylene (L²) unit [H₄BAPTA is 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid; H₃DO₃A is 1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid]. The sensitivity of the Gd³⁺ complexes towards Ca²⁺ and Mg²⁺ was studied by ¹H relaxometric titrations. A maximum relaxivity increase of 15 and 10% was observed upon Ca²⁺ binding to Gd₂L¹ and Gd₂L², respectively, with a distinct selectivity of Gd₂L¹ towards Ca²⁺ compared with Mg²⁺. For Ca²⁺ binding, association constants of log K = 1.9 (Gd₂L¹) and log K = 2.7 (Gd₂L²) were determined by relaxometry. Luminescence lifetime measurements and UV–vis spectrophotometry on the corresponding Eu³⁺ analogues proved that the complexes exist in the form of monohydrated and nonhydrated species; Ca²⁺ binding in the central part of the ligand induces the formation of the monohydrated state. The increasing hydration number accounts for the relaxivity increase observed on Ca²⁺ addition. A ¹H nuclear magnetic relaxation dispersion and ¹⁷O NMR study on Gd₂L¹ in the absence and in the presence of Ca²⁺ was performed to assess the microscopic parameters influencing relaxivity. On Ca²⁺ binding, the water exchange is slightly accelerated, which is likely related to the increased steric demand of the central part leading to a destabilization of the Ln–water binding interaction. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Designing a Highly Efficient Refolding System for Alkaline Phosphatase using Combination of Cyclodextrin and Mg<Superscript>2+</Superscript> Ion

Two different folding aids including α-cyclodextrin and Mg²⁺ ions were applied to alkaline phosphatase refolding. The refolding yield depending on concentration of each of the refolding agents reached to 55 and 52% in the presence of 100 mM α-cyclodextrin and/or 5 mM Mg²⁺ ions, respectively. However, the refolding yield, mediated by combination of α-cyclodextrin and Mg²⁺ ions, was more than 96%. Replacement of Mg²⁺ ion with other type of ions which interact with α-cyclodextrin interfere with the function of cyclodextrin resulting in low refolding yields.     

Functional analysis of the <Emphasis Type="Italic">Burkholderia cenocepacia</Emphasis> J2315 BceA<Subscript>J</Subscript> protein with phosphomannose isomerase and GDP-<Emphasis Type="SmallCaps">d</Emphasis>-mannose pyrophosphorylase activities

The bceA _J gene from the cystic fibrosis isolate Burkholderia cenocepacia J2315 encodes a 56-kDa bifunctional protein, with phosphomannose isomerase (PMI) and guanosine diphosphate (GDP)-mannose pyrophosphorylase (GMP) activities, a new member of the poorly characterised type II PMI class of proteins. Due to the lack of homology between the type II PMIs and the human PMI, this class of proteins are being regarded as interesting potential targets to develop new antimicrobials. The BceA_J protein conserves the four typical motifs of type II PMIs: the pyrophosphorylase signature, the GMP active site, the PMI active site and the zinc-binding motif. After overproduction of BceA_J by Escherichia coli as a histidine tag derivative, the protein was purified to homogeneity by affinity chromatography. The GMP activity is dependent on the presence of Mg²⁺ or Ca²⁺ as cofactors, while the PMI activity uses a broader range of divalent ions, in the order of activation Mg²⁺ > Ca²⁺ > Mn²⁺ > Co²⁺ > Ni²⁺. The kinetic parameters K _m, V _max and K _cat/K _m for the PMI and GMP activities were determined. Results suggest that the enzyme favours the formation of GDP-mannose instead of mannose catabolism, thus channelling precursors to the formation of glycoconjugates.     

Cd<Superscript>2+</Superscript> extrusion by P-type Cd<Superscript>2+</Superscript>-ATPase of <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> 17810R via energy-dependent Cd<Superscript>2+</Superscript>/H<Superscript>+</Superscript> exchange mechanism

Cd²⁺ is highly toxic to Staphylococcus aureus since it blocks dithiols in cytoplasmic 2-oxoglutarate dehydrogenase complex (ODHC) participating in energy conservation process. However, S. aureus 17810R is Cd²⁺-resistant due to possession of cadA-coded Cd²⁺ efflux system, recognized here as P-type Cd²⁺-ATPase. This Cd²⁺ pump utilizing cellular energy—ATP, ?μ _H ⁺ (electrochemical proton potential) and respiratory protons, extrudes Cd²⁺ from cytoplasm to protect dithiols in ODHC, but the mechanism of Cd²⁺ extrusion remains unknown. Here we propose that two Cd²⁺ taken up by strain 17810R via Mn²⁺ uniporter down membrane potential (?ψ) generated during glutamate oxidation in 100 mM phosphate buffer (high P_iB) are trapped probably by high affinity sites in cytoplasmic domain of Cd²⁺-ATPase, forming SCdS. This stops Cd²⁺ transport towards dithiols in ODHC, allowing undisturbed NADH production, its oxidation and energy conservation, while ATP could change orientation of SCdS towards facing transmembrane channel. Now, increased number of P_i-dependent protons pumped electrogenically via respiratory chain and countertransported through the channel down ?ψ, extrude two trapped cytoplasmic Cd²⁺, which move to low affinity sites, being then extruded into extracellular space via ?ψ-dependent Cd²⁺/H⁺ exchange. In 1 mM phosphate buffer (low P_iB), external Cd²⁺ competing with decreased number of P_i-dependent protons, binds to ψ_s of Cd²⁺-ATPase channel, enters cytoplasm through the channel down ?ψ via Cd²⁺/Cd²⁺ exchange and blocks dithiols in ODHC. However, Mg²⁺ pretreatment preventing external Cd²⁺ countertransport through the channel down ?ψ, allowed undisturbed NADH production, its oxidation and extrusion of two cytoplasmic Cd²⁺ via Cd²⁺/H⁺ exchange, despite low P_iB.     

Measurement of protein backbone <Superscript>13</Superscript>CO and <Superscript>15</Superscript>N relaxation dispersion at high resolution

The ³¹P NMR pressure response of guanine nucleotides bound to proteins has been studied in the past for characterizing the pressure perturbation of conformational equilibria. The pressure response of the ³¹P NMR chemical shifts of the phosphate groups of GMP, GDP, and GTP as well as the commonly used GTP analogs GppNHp, GppCH₂p and GTPγS was measured in the absence and presence of Mg²⁺-ions within a pressure range up to 200 MPa. The pressure dependence of chemical shifts is clearly non-linear. For all nucleotides a negative first order pressure coefficient B ₁ was determined indicating an upfield shift of the resonances with pressure. With exception of the α-phosphate group of Mg²⁺·GMP and Mg²⁺·GppNHp the second order pressure coefficients are positive. To describe the data of Mg²⁺·GppCH₂p and GTPγS a Taylor expansion of 3rd order is required. For distinguishing pH effects from pressure effects a complete pH titration set is presented for GMP, as well as GDP and GTP in absence and presence of Mg²⁺ ions using indirect referencing to DSS under identical experimental conditions. By a comparison between high pressure ³¹P NMR data on free Mg²⁺-GDP and Mg²⁺-GDP in complex with the proto-oncogene Ras we demonstrate that pressure induced changes in chemical shift are clearly different between both forms.     

Ca<Superscript>2+</Superscript> Dependence and Inhibitory Effects of Trifluoperazine on Plasma Membrane ATPase of <Emphasis Type="Italic">Thermoactinomyces vulgaris</Emphasis>

Ca²⁺ enhanced the plasma membrane Ca²⁺-ATPase (PMCA) specific activities in wild-type strain 1227 and mutant strains 1278, 1286, and 1261 of Thermoactinomyces vulgaris. The Ca²⁺-ATPase specific activities showed marked increase with increasing concentrations of Ca²⁺ added in the form of CaCl₂ in the culture medium and reached the optimum values at 0.6 mM in strains 1227, 1278, and 1286 and at 0.7 mM in strain 1261 of T. vulgaris. Trifluoperazine, a specific blocker of calmodulin, when added in vivo at concentrations of 2 M and 8 M along with the respective optimal concentrations of Ca²⁺, decreased the PMCA-specific activities to a low level in a dose-dependent manner. The results of the present investigation suggest the presence of a Ca²⁺-dependent protein activator (CaDPA) in the microenvironment constituting this enzyme; and such Ca²⁺-modulated protein has been assigned to play an important role in the enhancement of PMCA levels in this aerobic, spore-forming, thermophilic actinomycete.     

<Superscript>65</Superscript>Zn<Superscript>2+</Superscript> transport by lobster hepato-pancreatic baso-lateral membrane vesicles

The lobster (Homarus americanus) hepato-pancreatic epithelial baso-lateral cell membrane possesses three transport proteins that transfer calcium between the cytoplasm and hemolymph: an ATP-dependent calcium ATPase, a sodium-calcium exchanger, and a verapamil-sensitive cation channel. We used standard centrifugation methods to prepare purified hepato-pancreatic baso-lateral membrane vesicles and a rapid filtration procedure to investigate whether ⁶⁵Zn²⁺ transfer across this epithelial cell border occurs by any of these previously described transporters for calcium. Baso-lateral membrane vesicles were osmotically reactive and exhibited a time course of uptake that was linear for 10–15 s and approached equilibrium by 120 s. In the absence of sodium, ⁶⁵Zn²⁺ influx was a hyperbolic function of external zinc concentration and followed the Michaelis-Menten equation for carrier transport. This carrier transport was stimulated by the addition of 150 M ATP (increase in K_m and J_max) and inhibited by the simultaneous presence of 150 mol l^–1 ATP+250 mol l^–1 vanadate (decrease in both K_m and J_max). In the absence of ATP, ⁶⁵Zn²⁺ influx was a sigmoidal function of preloaded vesicular sodium concentration (0, 5, 10, 20, 30, 45, and 75 mmol l^–1) and exhibited a Hill Coefficient of 4.03±1.14, consistent with the exchange of 3 Na⁺/1Zn²⁺. Using Dixon analysis, calcium was shown to be a competitive inhibitor of baso-lateral membrane vesicle ⁶⁵Zn²⁺ influx by both the ATP-dependent (K_i=205 nmol l^–1 Ca²⁺) and sodium-dependent (K_i=2.47 mol l^–1 Ca²⁺) transport processes. These results suggest that zinc transport across the lobster hepato-pancreatic baso-lateral membrane largely occurred by the ATP-dependent calcium ATPase and sodium-calcium exchanger carrier proteins.Communicated by: I.D. Hume     

The spatial distribution of cations in nematocytes of Hydra vulgaris

The spatial distribution of cations was assayed qualitatively and quantitatively in tentacular nematocytes of Hydra vulgaris in a scanning transmission electron microscope by means of x-ray microanalysis performed on 100 nm thick freeze-dried cryosections. The matrix of undischarged cysts (stenoteles, desmonemes and isorhizas) was found to contain mainly K⁺. In isolated nematocysts of Hydra the intracapsular potassium can be readily substituted by practically any other mono- and divalent cation (Na⁺, NH₄ ⁺, Mn²⁺, Co²⁺, Mg²⁺, Ca²⁺, Fe²⁺) all, except Fe²⁺, without impairing the ability of the cyst to respond to the chemical triggering with dithioerythritol or proteases. Monovalent cations increase the osmotically generated intracapsular pressure compared to divalent ions.