Conserved motif CMLD in silicic acid transport proteins of diatoms

Sequenced fragments of genes coding for silicon transporters (SITs) were analyzed for diatoms of evolutionarily distant classes (centric Chaetoceros muelleri Lemmermann, pennate araphid Synedra acus Kützing, pennate raphid Phaeodactylum tricornutum Bohlin, and pennate Cylindrotheca fusiformis Reimann et Lewin with a keeled raphe system). SITs were found to contain a conserved motif, CMLD. Hydropathy profiles showed that the motif CMLD is between two transmembrane domains lacking Lys and Arg, and the domains were consequently assumed to play a role in the formation of a channel mediating silicic acid transport. The motif CMLD proved to be rare. Since Zn²⁺ is necessary for silica incorporation into diatom cells, a hypothesis was advanced that the motif CMLD acts as a Zn-binding site. Diatom growth suppression was observed in the presence of the alkylating agent N-iodoacetylamidoethyl-1-aminonaphthalene-5-sulfonic acid (AEDANS), which does not penetrate into the cell. Cys of the motif CMLD was assumed to act as a target for AEDANS. Zinc ions inhibited Cys alkylation in the synthetic peptide NCMLDY, testifying to the above hypothesis.__________Translated from Molekulyarnaya Biologiya, Vol. 39, No. 2, 2005, pp. 303–316.Original Russian Text Copyright © 2005 by Sherbakova, Masyukova, Safonova, Petrova, Vereshagin, Minaeva, Adelshin, Triboy, Stonik, Aizdaitcher, Kozlov, Likhoshway, Grachev.     

A glycine betaine transport system in Aphanothece halophytica and other glycine betaine-synthesising cyanobacteria

Uptake of exogenous ¹⁴C-glycine betaine has been followed in the cyanobacterium Aphanothece halophytica and other species able to synthesise glycine betaine in response to osmotic stress. At 1 mmol dm^–3 uptake was rapid (flux rate=29.50 nmol m^–2 s^–1), equilibrating at an internal concentration of 120 mmol dm^–3 within 30 min. This rapid uptake, coupled with high internal accumulation, was characteristic of glycine betaine-synthesising cyanobacteria only. The ¹⁴C-glycine betaine transported was not catabolised. Kinetic studies indicated a Michaelis-Menten type relationship (K _m=2.0 mol dm^–3, V _max=45 nmol min^–1 mm^–3 cell volume), with a pH optimum of 8.0–8.5. Darkness dramatically decreased the flux rate. Higher ¹⁴C-glycine betaine levels occurred in cells growth in medium of elevated osmotic strength, and glycine betaine uptake was sensitive to changes in external salinity. A relationship between Na⁺ availability and glycine betaine uptake was observed, with >80 mmol dm^–3 Na⁺ required for optimal stimulation of uptake in seawater-grown cells. Severe hyperosmotic stress (1000 mmol dm^–3 NaCl) reduced the rate of glycine betaine uptake but increased internal glycine betaine concentration at equilibrium. Hypo-osmotic stress caused a decline in the internal glycine betaine concentration due to an increased rate of loss, indicating that the efflux system was also sensitive to ambient salinity changes. It is envisaged that this active transport system may be an adaptive mechanism in halophilic glycine betaine-synthesising cyanobacteria.     

Specific inhibitors of intracellular Ca2+ transport ATPases

Support from the National Institutes of Health and the American Heart Association is gratefully acknowledged.     

Effects of polyamines on mitochondrial Ca transport

Mammalian mitochondria are able to enhance Ca²⁺ accumulation in the presence of polyamines by activating the saturable systems of Ca²⁺ inward transport and buffering extramitochondrial Ca²⁺ concentrations to levels similar to those in the cytosol of resting cells. This effect renders them responsive to regulate free Ca²⁺ concentrations in the physioloical range. The mechanism involved is due to a rise in the affinity of the Ca²⁺ transport system, induced by polyamines, most probably exhibiting allosteric behaviour. The regulatory site of this mechanism is the so-called S₁ binding site of polyamines, which operates in physiological conditions and is located in the energy well between the two peaks present in the energy profile of mitochondrial spermine transport. Spermine is bidirectionally transported across teh inner membrane by cycling, in which influx and efflux are driven by electrical and pH gradients, respectively. Most probably, polyamine affects the Ca²⁺ transport system when it acts from the outside-that is, in the direction of its uniporter channel, in order to reach the S₁ site. Important physiological functions are related to activation of Ca²⁺ transport systems by polyamines and their interactions with the S₁ site. These functions include a rise in the metabolic rate for energy supply and modulation of mitochondrial permeability transition induction, with consequent effects on the triggering of the apoptotic pathway.     

Membrane transport in stomatal guard cells: The importance of voltage control

Potassium uptake and export in the resting conditions and in response to the phytohormone abscisic acid (ABA) were examined under voltage clamp in guard cells of Vicia faba L. In 0.1 mM external K+ (with 5 mM Ca2(+)-HEPES, pH 7.4) two distinct transport states could be identified based on the distribution of the free-running membrane voltage (VM) data in conjunction with the respective I-V and G-V relations. One state was dominated by passive diffusion (mean VM = -143 +/- 4 mV), the other (mean VM = -237 +/- 10 mV) exhibited an appreciable background of primary H+ transport activity. In the presence of pump activity the free-running membrane voltage was negative of the respective K+ equilibrium potential (EK+), in 3 and 10 mM external K+. In these cases VM was also negative of the activation voltage for the inward rectifying K+ current, thus creating a strong bias for passive K+ uptake through inward-rectifying K+ channels. In contrast, when pump activity was absent VM was situated positive of EK+ and cells revealed a bias for K+ efflux. Occasionally spontaneous voltage transitions were observed during which cells switched between the two states. Rapid depolarizations were induced in cells with significant pump activity upon adding 10 microM ABA to the medium. These depolarizations activated current through outward-rectifying K+ channels which was further amplified in ABA by a rise in the ensemble channel conductance. Current-voltage characteristics recorded before and during ABA treatments revealed concerted modulations in current passage through at least four distinct transport processes, results directly comparable to one previous study (Blatt, M.R., 1990, Planta 180:445) carried out with guard cells lacking detectable primary pump activity. Comparative analyses of guard cells in each case are consistent with depolarizations resulting from the activation of an inward-going, as yet unidentified current, rather than an ABA-induced fall in H(+)-ATPase output. Also observed in a number of cells was an inward-directed current which activated in ABA over a narrow range of voltages positive of -150 mV; this and additional features of the current suggest that it may reflect the ABA-dependent activation of an anion channel previously characterized in Vicia guard cell protoplasts, but rule out its function as the primary mechanism for initial depolarization.(ABSTRACT TRUNCATED AT 400 WORDS)     

Calcium transport by pea root membranes

Calcium transport has been studied using purified endomembrane vesicles from dark-grown roots of Pisum sativum L. Membranes from a mixed microsomal (non-mitochondrial) fraction showed ATP-dependent calcium uptake which was released by the ionophore A 23187, had a pH optimum of 7.2 and required Mg²⁺ for uptake. Membranes were further purified using a rapid sucrosedensity-gradient technique yielding vesicles suitable for transport studies, and were identified using marker enzymes. Uptake by plasma membrane, tonoplast, endoplasmic reticulum and Golgi apparatus was indicated. Uptake by membranes of low density (predominantly tonoplast) had a pH optimum of 7.2–7.4 and nucleotide specificity ATP> guanosine 5-triphosphate>inosine 5-triphosphate>ADP>, while that by high-density membranes had a pH optimum of 7.5–7.9 and less specificity for ATP. The importance of regulating sucrose concentrations in calcium transport studies was demonstrated.Abbreviations ER endoplasmic reticulum - GTP guanosine 5-triphosphate - IDPase inosine diphosphatase - IIP inosine 5-triphosphate     

Model of active transport of ions in cardiac cell

A model of the active transport of ions in a cardiac muscle cell, which takes into account the active transport of Na⁺, K⁺, Ca²⁺, Mg²⁺, HCO₃⁻ and Cl⁻ ions, has been constructed. The model allows independent calculations of the resting potential at the biomembrane and concentrations of basic ions (sodium, potassium, chlorine, magnesium and calcium) in a cell. For the analysis of transport processes in cardiac cell hierarchical algorithm “one ion-one transport system” was offered. The dependence of the resting potential on concentrations of the ions outside a cell has been established. It was shown, that ions of calcium and magnesium, despite their rather small concentration, play an essential role in maintenance of resting potential in cardiac cell. The calculated internal concentrations of ions are in good agreement with the corresponding experimental values.     

Biosynthetically directed fractional 13C labeling facilitates identification of Phe and Tyr aromatic signals in proteins

Analysis of 2D [¹³C,¹H]-HSQC spectra of biosynthetic fractionally ¹³C labeled proteins is a reliable, straightforward means to obtain stereospecific assignments of Val and Leu methyl sites in proteins. Herein we show that the same fractionally labeled protein sample facilitates observation and identification of Phe and Tyr aromatic signals. This is the case, in part, because the fractional ¹³C labeling yields aromatic rings in which some of the ¹³C-¹³C J-couplings, present in uniformly labeled samples, are absent. Also, the number of homonuclear J-coupling partners differs for the -, - and -carbons. This enabled us to vary their signal intensities in distinctly different ways by appropriately setting the ¹³C constant-time period in 2D [¹³C,¹H]-HSQC spectra. We illustrate the application of this approach to an 18 kDa protein, c-VIAF, a modulator of apoptosis. In addition, we show that cancellation of the aromatic ¹³C CSA and ¹³C-¹H dipolar interactions can be fruitfully utilized in the case of the fractionally labeled sample to obtain high resolution ¹³C constant-time spectra with good sensitivity.     

Cadmium inhibits plasma membrane calcium transport

Summary The interaction of Cd²⁺ with the plasma membrane Ca²⁺-transporting ATPase of fish gills was studied. ATP-driven Ca²⁺-transport in basolateral membrane (BLM) vesicles was inhibited by Cd²⁺ with anI ₅₀ value of 3.0nm at 0.25 m free Ca²⁺ using EGTA, HEEDTA and NTA to buffer Ca²⁺ and Cd²⁺ concentrations. The inhibition was competitive in nature since theK _0.5 value for Ca²⁺ increased linearly with increasing Cd²⁺ concentrations while theV _max remained unchanged. The Ca²⁺ pump appeared to be calmodulin dependent, but we conclude that the inhibition by Cd²⁺ occurs directly on the Ca²⁺ binding site of the Ca²⁺-transporting ATPase and not via the Ca²⁺-binding sites of calmodulin. It is suggested that Cd²⁺-induced inhibition of Ca²⁺-transporting enzymes is the primary effect in the Cd²⁺ toxicity towards cells followed by several secondary effects due to a disturbed cellular Ca²⁺ metabolism. Our data illustrate that apparent stimulatory effects of low concentrations of Cd²⁺ on Ca²⁺-dependent enzymes may derive from increased free-Ca²⁺ levels when Cd²⁺ supersedes Ca²⁺ on the ligands.     

Changes in glycine and leucine transport during red cell maturation in the rat

Both glycine and leucine transport in rat red blood cells have been studied. The glycine uptake showed two different components, one sodium-dependent and another diffusion-like process. In contrast, leucine uptake was sodium independent. Both, Na⁺-dependent glycine and the overall leucine uptake in red blood cells showed a saturable pattern. Kinetic parameters in reticulocytes were: i) glycine: apparent Km 0.16 mM; V_max 100.2 nmol/ml ICW/min; ii) leucine: apparent Km 2.11 mM; V_max 3.88 mol/ml ICW/min. The erythrocytes kinetic parameters were: i) glycine: apparent Km 0.17 mM; V_max 9.47 nmol/ml ICW/min; leucine; apparent Km 4.77 mM; V_max 7.42 mol/ml ICW/min. The Kd values (sodium independent glycine uptake) were similar in both kind of cells, but the importance of this component in total glycine uptake in erythrocytes was much higher than in reticulocytes. Our results confirm that rat red blood cells have both saturable leucine and Na⁺-dependent glycine uptake, but some important changes occur during cell maturation.     

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.     

Heart sarcolemmal Ca2+ transport in endotoxin shock: I. Impairment of ATP-dependent Ca2+ transport

Effects of endotoxin administration on the ATP-dependent Ca²⁺ transport in canine cardiac sarcolemma were investigated. The results show that the sidedness of the sarcolemmal vesicles was not affected but the ATP-dependent Ca²⁺ transport in cardiac sarcolemma was decreased by 22 to 46% (p < 0.05) at 4 h following endotoxin administration. The kinetic analysis indicates that the V_max for ATP and for Ca²⁺ were decreased by 50% (p < 0.01) and 32% (p < 0.01), respectively, while the Km values for ATP and Ca²⁺ were not significantly affected after endotoxin administration. Magnesium (1–5 mM) stimulated while vanadate (0.25–3.0 M) inhibited the ATP-dependent Ca²⁺ transport, but the Mg²⁺-stimulated and the vanadate-inhibitable activities remained significantly lower in the endotoxin-treated animals. These data demonstrate that endotoxin administration impairs the ATP-dependent Ca²⁺ transport in canine cardiac sarcolemma and that the impairment is associated with a mechanism not affecting the affinity towards ATP and Ca²⁺. Additional experiments show that the Ca²⁺ sensitivity of the Ca²⁺-ATPase activity was indifferent between the control and endotoxic groups suggesting that endotoxic injury impairs Ca²⁺ pumping without affecting Ca²⁺-ATPase activity. Since sarcolemmal ATP-dependent Ca²⁺ transport plays an important role in the regulation of cytosolic Ca²⁺ homeostasis, an impairment in the sarcolemmal ATP-dependent Ca²⁺ transport induced by endotoxin administration may have a pathophysiological significance in contributing to the development of myocardial dysfunction in endotoxin shock.     

Oxalate transport across the isolated rat colon. A re-examination

A net absorption of oxalate and chloride was observed when isolated, shortcircuited segments of rat colon were bathed by a calcium-containing buffer. Removal of calcium promoted a two-fold decrease in transmural resistance, while the net chloride flux was reduced and the net oxalate transport abolished. It was concluded that net oxalate absorption was not observed in previous studies (employing calcium-free buffers) because calcium is required to maintain the integrity of the conductive pathways across colonic epithelia.     

Differential alterations in ATP-supported calcium transport activities of sarcoplasmic reticulum and sarcolemma of aging myocardium

Two membrane fractions, one enriched in sarcoplasmic reticulum and the other enriched in sarcolemma, were isolated from the myocardium of young (3–4-months-old) and aged (24–25-months old) rats. ATP-supported Ca²⁺ binding and accumulating activities as well as (Mg²⁺ + Ca²⁺)-ATPase activities of these membrane fractions were studied in an effort to determine the influence of age on the Ca²⁺ pump function of the two myocardial membrane systems. Sarcoplasmic reticulum from aged hearts showed significantly reduced (approx. 50%) rates of ATP-supported (oxalate-facilitated) Ca²⁺ accumulation compared to sarcoplasmic reticulum from young hearts; the amount of Ca²⁺ accumulated by this membrane of aged heart at steady state was also lower. On the other hand, sarcolemma from aged hearts displayed 2-fold higher rates of ATP-supported Ca²⁺ accumulation compared to sarcolemma from young hearts; at steady state, sarcolemma from aged hearts accumulated significantly higher amounts of Ca²⁺ than did sarcolemma from young hearts. Similar age-related differences were also observed in the ATP-dependent Ca²⁺ binding activities of the two membranes, determined in the absence of oxalate. The divergent age-associated changes in Ca²⁺ binding and accumulating activities of sarcoplasmic reticulum and sarcolemma were seen at varying Ca²⁺ concentrations (0.24–39.1 μM).With either membrane, kinetic analysis showed 2-fold age-related differences in the V values for ATP-supported Ca²⁺ accumulation (V (nmol Ca²⁺/mg protein per min): sarcoplasmic reticulum — young, 119 ± 8; aged, 59 ± 5; sarcolemma — young, 11 ± 2; aged, 21 ± 3); the concentrations of Ca²⁺ required for half-maximal velocities did not differ significantly with age (K_0.5 for Ca²⁺ (μM): sarcoplasmic reticulum — young, 2.5 ± 0.20; aged, 2.9 ± 0.25; sarcolemma — yount, 2.7 ± 0.25; aged, 3.2 ± 0.30). Kinetic parameters of ATP-dependent Ca²⁺ binding also indicated that the velocity of Ca²⁺ binding but not the concentration of Ca²⁺ required for half-maximal binding was altered due to aging. At identical Ca²⁺ concentrations, the combined Ca²⁺ accumulating activity of sarcoplasmic reticulum and sarcolemma from aged hearts was significantly lower (38–47%) than the combined Ca²⁺ accumulating activity of the two membranes from young hearts. No significant age-related differences were observed in the ATP-independent (passive) Ca²⁺ binding (or accumulation) by sarcoplasmic reticulum and sarcolemma, the (Mg²⁺ + Ca²⁺)-ATPase activities of these membranes, their polypeptide composition or relative purity. These results indicate that differential alterations occur in the ATP-supported Ca²⁺ pump activities of sarcoplasmic reticulum and sarcolemma in aging myocardium and such alterations may be due to age-associated changes in the efficacy of coupling ATP hydrolysis to Ca²⁺ transport. Further, the age-related increment in the Ca²⁺ pump activity of sarcolemma is inadequate to fully compensate for the diminished Ca²⁺ pump activity of sarcoplasmic reticulum. It is, therefore, suggested that deterioration of the Ca²⁺ pump function of sarcoplasmic reticulum may contribute to the increased relaxation time observed in aging heart.     

Transport of glutamine inXenopus laevis oocytes: Relationship with transport of other amino acids

Summary We have investigated transport of the amino acid glutamine across the surface membranes of prophase-arrestedXenopus laevis oocytes. Glutamine accumulation was linear with time for 30 min; it was stereospecific with aK _m of 0.12±0.02mm andV _max of 0.92±0.17 pmol/oocyte · min forl-glutamine. Transport ofl-glutamine was Na⁺-dependent, the cation not being replaceable with Li⁺, K⁺, choline, tris(hydroxymethyl)-aminomethane (Tris), tetramethylammonium (TMA) or N-methyld-glucamine NMDG); external Cl^– appeared to be necessary for full activation of Na⁺-dependent glutamine transport. Two external Na⁺ may be required for the transport of one glutamine molecule.l-glutamine transport (at 50 m glutamine) was inhibited by the presence of other amino acids:l-alanine,d-alanine,l-leucine,l-asparagine andl-arginine (about 60% inhibition at 1mm);l-histidine,l-valine and glycine (25 to 40% inhibition at 1mm);l-serine,l-lysine,l-phenylalanine andl-glutamate (45 to 55% inhibition at 10mm). N-methylaminoisobutyric acid (meAIB) had no effect at 10mm, but 2-aminobicyclo[2,2,1]heptane-2-carboxylic acid (BCH) inhibited Na⁺/glutamine transport by about 50% at 10mm.l-glutamine was a competitive inhibitor of the Na⁺-dependent transport ofl-alanine,d-alanine andl-arginine; this evidence is consistent with the existence of a single system transporting all four amino acids. Glutamine uptake in oocytes appears to be catalyzed by a transport system distinct from the cotransport Systems A, ASC, N and Gly, although it resembles System B^0,+.     

Calcium and copper transport ATPases: analogies and diversities in transduction and signaling mechanisms

The calcium transport ATPase and the copper transport ATPase are members of the P-ATPase family and retain an analogous catalytic mechanism for ATP utilization, including intermediate phosphoryl transfer to a conserved aspartyl residue, vectorial displacement of bound cation, and final hydrolytic cleavage of Pi. Both ATPases undergo protein conformational changes concomitant with catalytic events. Yet, the two ATPases are prototypes of different features with regard to transduction and signaling mechanisms. The calcium ATPase resides stably on membranes delimiting cellular compartments, acquires free Ca²⁺ with high affinity on one side of the membrane, and releases the bound Ca²⁺ on the other side of the membrane to yield a high free Ca²⁺ gradient. These features are a basic requirement for cellular Ca²⁺ signaling mechanisms. On the other hand, the copper ATPase acquires copper through exchange with donor proteins, and undergoes intracellular trafficking to deliver copper to acceptor proteins. In addition to the cation transport site and the conserved aspartate undergoing catalytic phosphorylation, the copper ATPase has copper binding regulatory sites on a unique N-terminal protein extension, and has also serine residues undergoing kinase assisted phosphorylation. These additional features are involved in the mechanism of copper ATPase intracellular trafficking which is required to deliver copper to plasma membranes for extrusion, and to the trans-Golgi network for incorporation into metalloproteins. Isoform specific glyocosylation contributes to stabilization of ATP7A copper ATPase in plasma membranes.     

Action of mercurials on the active and passive transport properties of sarcoplasmic reticulum

The effect of Hg²⁺ and Ch₃-Hg⁺ on the passive and active transport properties of the Ca²⁺-Mg²⁺-ATPase-rich fraction of skeletal sarcoplasmic reticulum (SR) is reported. The agents abolish active transport, at 10^–5 and 10^–4M concentrations, respectively. Addition of the mercurials was also shown to release actively accumulated Ca²⁺. The mercurials increase the passive Ca²⁺ and Mg²⁺ permeability in the absence of ATP at the same concentrations at which they inhibit transport. It is proposed that both effects are the result of direct binding of the mercurials to the SH groups of the Ca²⁺-Mg²⁺-ATPase pump, altering the conformational equilibria of the pump. The agents were also shown to increase the passive KCl permeability. The SR preparation consists of two vesicle populations with respect to K⁺ permeability, one with rapid KCl equilibration faciliated by a monovalent cation channel function and one with slow KCl equilibration. The mercurials increase the rates of KCl equilibration in both fractions, but produce higher rates in the fraction containing the channel function. The results are discussed in terms of pump and channel function and are compared with results for the electrical behavior of the Ca²⁺-Mg²⁺-ATPase and other SR proteins in black lipid membranes, as presented by others.     

Characterisation and cloning of a Na-dependent broad-specificity neutral amino acid transporter from NBL-1 cells: a novel member of the ASC/B transporter family

Na⁺-dependent neutral amino acid transport into the bovine renal epithelial cell line NBL-1 is catalysed by a broad-specificity transporter originally termed System B⁰. This transporter is shown to differ in specificity from the B⁰ transporter cloned from JAR cells [J. Biol. Chem. 271 (1996) 18657] in that it interacts much more strongly with phenylalanine. Using probes designed to conserved transmembrane regions of the ASC/B⁰ transporter family we have isolated a cDNA encoding the NBL-1 cell System B⁰ transporter. When expressed in Xenopus oocytes the clone catalysed Na⁺-dependent alanine uptake which was inhibited by glutamine, leucine and phenylalanine. However, the clone did not catalyse Na⁺-dependent phenylalanine transport, again as in NBL-1 cells. The clone encoded a protein of 539 amino acids; the predicted transmembrane domains were almost identical in sequence to those of the other members of the B⁰/ASC transporter family. Comparison of the sequences of NBL-1 and JAR cell transporters showed some differences near the N-terminus, C-terminus and in the loop between helices 3 and 4. The NBL-1 B⁰ transporter is not the same as the renal brush border membrane transporter since it does not transport phenylalanine. Differences in specificity in this protein family arise from relatively small differences in amino acid sequence.