Transport of potassium inChara australis: I. A symport with sodium

Summary Voltage-clamp and tracer techniques, applied simultaneously or separately to individual cells, have been used to show that K⁺-starved internodal cells ofChara australis can develop an electrogenic transport system, which requires and transports K⁺ with high affinity (K _1/2 about 30 m) and Na⁺ with lower affinity (K _1/2 about 500 m). The most likely mechanism is symport of K⁺ with Na⁺, with a stoichiometric ratio of 11. In simultaneous measurements of quantities of charge and of ions entering individual cells, the quantity of K⁺ was consistently half the quantity of electric charge, while that of Na⁺ was consistently somewhat lower than that. Possible reasons for this discrepancy are discussed. The electrogenic symport of K⁺ with Na⁺ has not previously been reported for any cell. Its functional significance inChara is apparently the active uptake of K⁺ at the expense of the electrochemical potential difference for Na⁺. This new symport reveals the unexpected presence inChara of a Na⁺-linked chemiosmotic circuit alongside the known H⁺-linked circuit.     

Amine ion porter inChara australis: Effects of alkyl substitution and external pH

Summary The rate of transport of amine ions intoChara australis internodes is studied by measuring changes in membrane current when amine solutions are presented to voltage-clamped cells. The dependence of this rate on ion concentration is investigated for a series of alkyl-amine ions: methyl-, ethyl-, isopropyl-, dimethyl-, trimethyl- and tetramethylammonium. A Michaelis-Menten relationship is displayed by all except tri- and tetramethylammonium, where currents are irregular and difficult to reproduce. Evidence suggests that the different ions cross the plasmalemma via a common uniport.K _M values for this porter increase as the amine ion becomes more highly substituted. TheV _m values are similar for all amines and lie within the range 10 to 100 mA m^–2 (for cell potential at –200 mV). The changes inK _M indicate that hydrogen bonding may be involved in the binding interaction.V _m varies with external pH in a way which suggests that an ionizable group on the transport protein with pK_a5.8 directly affects the transport rate.K _M is independent of external pH over the range 4.5 to 10.5     

Potassium-proton symport inNeurospora: kinetic control by pH and membrane potential

Summary Active transport of potassium in K⁺-starvedNeurospora was previously shown to resemble closely potassium uptake in yeast,Chlorella, and higher plants, for which K⁺ pumps or K⁺/H⁺-ATPases had been proposed. ForNeurospora, however, potassium-proton cotransport was demonstrated to operate, with a coupling ratio of 1 H⁺ to 1 K⁺ taken inward so that K⁺, but not H⁺, moves against its electrochemical gradient (Rodriguez-Navarro et al.,J. Gen. Physiol. 87:649–674).In the present experiments, the current-voltage (I–V) characteristic of K⁺–H⁺ cotransport in spherical cells ofNeurospora has been studied with a voltage-clamp technique, using difference-current methods to dissect it from other ion-transport processes in theNeurospora plasma membrane. Addition of 5-200 M K⁺ to the bathing medium causes 10–150 mV depolarization of the unclamped membrane, and yields a sigmoidI–V curve with a steep slope (maximal conductance of 10–30 S/cm²) for voltages of –300 to –100 mV, i.e., in the normal physiologic range. Outside that range the apparentI–V curve of the K⁺-H⁺ symport saturates for both hyperpolarization and depolarization. It fails to cross the voltage axis at its predicted reversal potential, however, an effect which can be attributed to failure of theI–V difference method under reversing conditions.In the absence of voltage clamping, inhibitors—such as cyanide or vanadate—which block the primary proton pump inNeurospora also promptly inhibit K⁺ transport and K⁺-H⁺ currents. But when voltage clamping is used to offset the depolarizing effects of pump blockade, the inhibitors have no immediate effect on K⁺-H⁺ currents. Thus, the inhibition of K⁺ transport usually observed with these agents reflects the kinetic effect of membrane depolarization rather than any direct chemical action on the cotransport system itself.Detailed study of the effects of [K⁺]_o and pH_o on theI–V curve for K⁺-H⁺ symport has revealed that increasing membrane potential systematicallydecreases the apparent affinity of the transporter for K⁺, butincreases affinity for protons (K _m range: for [K⁺]_o, 15–45 M; for [H⁺]_o, 10–35 nM). This behavior is consistent with two distinct reaction-kinetic models, in which (i) a neutral carrier binds K⁺ first and H⁺ last in the forward direction of transport, or (ii) a negatively charged carrier (–2) binds H⁺ first and K⁺ last.     

Electrogenic properties of the sodium-alanine cotransporter in pancreatic acinar cells: II. Comparison with transport models

Summary In this paper, the results of the preceding electrophysiological study of sodium-alanine cotransport in pancreatic acinar cells are compared with kinetic models. Two different types of transport mechanisms are considered. In the simultaneous mechanism the cotransporterC forms a ternary complexNCS with Na⁺ and the substrateS; coupled transport of Na⁺ andS involves a conformational transition between statesNCS andNCS with inward- and outward-facing binding sites. In the consecutive (or ping-pong) mechanism, formation of a ternary complex is not required; coupled transport occurs by an alternating sequence of association-dissociation steps and conformational transitions. It is shown that the experimentally observed alanine- and sodium-concentration dependence of transport rates is consistent with the predictions of the simultaneous model, but incompatible with the consecutive mechanism. Assuming that the association-dissociation reactions are not rate-limiting, a number of kinetic parameters of the simultaneous model can be estimated from the experimental results. The equilibrium dissociation constants of Na⁺ and alanine at the extracellular side are determined to beK _N <-64mm andK _S <-18mm. Furthermore, the ratioK _N /K _N ^S of the dissociation constants of Na⁺ from the binary (NC) and the ternary complex (NCS) at the extracellular side is estimated to be <-6. This indicates that the binding sequence of Na⁺ andS to the transporter is not ordered. The current-voltage behavior of the transporter is analyzed in terms of charge translocations associated with the single-reaction steps. The observed voltage-dependence of the half-saturation concentration of sodium is consistent with the assumption that a Na⁺ ion that migrates from the extracellular medium to the binding site has to traverse part of the transmembrane voltage.     

Ion permeation in a K+ channel inChara australis: Direct evidence for diffusion limitation of ion flow in a maxi-K channel

Summary We report a study of a potassium-selective channel in the membrane delineating cytoplasmic drops fromChara australis. The relatively large conductance (170 pS in 150 mol/m³ (mm) KCl), high ion selectivity (P _Cl/P _K=0.015±0.01) and voltagedependent kinetics of this channel indicate that it is a type of maxi-K channel commonly found in animal cells but not previously detected in any plant cell.The current-voltage (I/V) characteristic of these channels was examined in drop-attached and in excised outside-out patches using the patch-clamp technique, over the unusually large voltage range of –250 to 200 mV. TheI/V characteristic is nonlinear and shows saturation at extreme voltages; the current also saturates at high [K⁺]. In solutions with symmetrical KCl concentrations the saturation behavior of the current is asymmetrical. The permeability of the channel depends on whether it is observed in excised or in drop-attached membrane patches.Here we investigate the main factors affecting the permeation of K⁺ ions through this maxi-K channel. We present the first direct evidence for the importance of diffusion external to the pore in limiting ion flow through maxi-K channels. The data are consistent with an ion translocation mechanism whose current is limited (i) at high voltages by ion diffusion external to the pore and (ii) at high [K⁺] by the maximum transport rate of the channel. We fit the data to a diffusion-limited pore model in which the pore exhibits saturation described by Michaelis-Menten kinetics with aK _m=50±25 mol/m³ andG _max=300±20 pS.     

A kinetic analysis of the electrogenic pump ofChara corallina: IV. Temperature dependence of the pump activity

Summary The sigmoidal current-voltage curve (i _p -V curve) of the electrogenic H⁺-pump of theChara membrane was simulated satisfactorily with a simple reaction kinetic model which assumed consecutive changes in state of H⁺-ATPase. Four rate constants, i.e., forward and backward ones in voltage-dependent and-independent steps could be evaluated from the data. The emf of the pump (E _p ), the voltage at which the pump current changes its sign, varies only slightly with temperature. However, the pump current (i _p ) is highly temperature dependent, and there-fore the conductance (g _p ) of the pump, calculated as the chord conductance from thei _p-V curve, is also highly voltage dependent having a peak at a level somewhat less negative than the resting potential. In contrast tog _p , the conductance (i _p ) of the passive channel does not change appreciably with temperature. Arrhenius plots ofg _p and also of the rate constants showed a clear bend at about 19°C. Great temperature dependence of the kinetic parameters offers useful information on the pumping mechanism of theChara membrane.     

Transport of an anionic substrate by the H+/monosaccharide symport inRhodotorula gracilis: Only the protonated form of the carrier is catalytically active

Summary The yeastRhodotorula gracilis accumulated glucuronate by an H⁺/symport. The transport was electroneutral, driven by the chemical gradient of protons pH. The observed stoichiometry amounted to 1 proton per molecule glucuronate. At pH 4, the half-saturation constantK _T was at its lowest value (K _T =8mm), whereas the maximal velocityV _T reached a maximum (V _T =15 nmol/min×mg dry wt). Monosaccharides competitively inhibited the uptake of glucuronate and vice versa. Hence, the two substrates share the same transport system. The steady-state accumulation of glucuronate reflected the course of the pH gradient. It is concluded that glucuronate is transported as an anionic substrate by the protonated carrier, the driving force being the chemical gradient of the H⁺ (pH). The ternary carrier/H⁺/glc-COOO-complex is electroneutral and independent of the membrane potential. Simultaneous uptake of organic acids (acetic or propionic acid) which is also energized by the pH gradient led to a noncompetitive inhibition of glucuronate transport. Thus, manipulation of the driving force, pH, reducedV _T without affectingK _T . Kinetic and energetic arguments are presented which stronly suggest that only the protonated carrier is catalytically active inR. gracilis.     

Electrogenic properties of the cloned Na+/glucose cotransporter: II. A transport model under nonrapid equilibrium conditions

Summary The results of the accompanying electrophysiological study of the cloned Na⁺/glucose cotransporter from small intestine (Parent, L., Supplisson, S., Loo, D.D.F., Wright, E.M. (1992) J. Membrane Biol. 125:49–62) were evaluated in terms of a kinetic model. The steady-state and presteady-state cotransporter properties are described by a 6-state ordered kinetic model (mirror symmetry) with a Na⁺:MDG stoichiometry of 2. Carrier translocation in the membrane as well as Na⁺ and sugar binding and dissociation are treated as a function of their individual rate constants. Empty carrier translocation and Na⁺ binding/ dissociation are the only steps considered to be voltage dependent. Currents were associated with the translocation of the negatively charged carrier in the membrane. Negative membrane potential facilitates sugar transport. One numerical solution was found for the 14 rate constants that account quantitatively for our experiment observations: i.e., (i) sigmoidal shape of the sugar-specific current-voltage curves (absence of outward currents and inward current saturation at high negative potentials), (ii) Na⁺ and voltage dependence of K _0.5 ^sugar and i _max ^sugar , (iii) sugar and voltage dependence of K _0.5 ^Na and i _max ^Na , (iv) presteady-state currents and their dependence on external Na⁺, MDG and membrane potential, and (v) and carrier Na⁺ leak current. We conclude that the main voltage effect is on carrier translocation. Na⁺ ions that migrate from the extracellular medium to their binding sites sense 25 to 35% of the transmembrane voltage, whereas charges associated with the carrier translocation experiences 60 to 75% of the membrane electrical field. Internal Na⁺ ion binding is not voltage dependent. In our nonrapid equilibrium model, the rate-limiting step for sugar transport is a function of the membrane potential, [Na]₀ and [MDG]₀. At 0 mV and at saturating [Na]₀ and [MDG]₀, the rate-limiting step for sugar transport is the empty carrier translocation (5 sec^–1). As the membrane potential is made more negative, the empty carrier translocation gets faster and the internal Na⁺ dissociation becomes increasingly rate limiting. However, as [Na]₀ is decreased to less than 10 mm, the rate-limiting step is the external Na⁺ ions binding in the 0 to –150 mV potential range. At 0 mV, the external Na⁺ dissociation constant KNa is 80 mm and decreases to 24 mm at –150 mV. The external sugar dissociation constant KNaS is estimated to be 200 m and voltage independent. Finally, the internal leak pathway (CNa₂ translocation) is insignificant. While we cannot rule out a more complex kinetic model, the electrical properties of the cloned Na⁺/glucose cotransporter are found to be adequately described by this 6-state kinetic model.We are grateful to Drs. A. Berteloot, S. Ciani, and J.-Y. Lapointe for stimulating discussions and thank our colleagues for comments. L.P. was recipient of a post-doctoral fellowship from the Medical Research Council of Canada. This work was supported by a grant from the U.S. Public Health Service DK 19567.     

Uptake of sulfate,sulfite and thiosulfate by proton-anion symport in Desulfovibrio desulfuricans

pH changes and sulfide production upon addition of sulfate, sulfite or thiosulfate to non-buffered H₂-saturated cell suspensions of Desulfovibrio desulfuricans were studied by means of electrodes. The addition of these electron acceptors resulted in a rapid alkalinization of the suspension which was accompanied by sulfide production. At-2° C, alkalinization without immediate sulfide production could be obtained. After addition of ³⁵S-labelled sulfate at-2° C, the label was found to be concentrated 7,500-fold in the cells, while 2 protons per sulfate molecule had disappeared from the outer bulk phase. Alkalinization and sulfide production from micromolar electron acceptor additions depended on the transmembraneous proton gradient ( pH), and were reversibly inhibited in alkaline solution (pH>8.0) or by the protonophore carbonylcyanide m-chlorophenylhydrazone (CCCP). Protonophore-inhibited sulfide production from sulfite or thiosulfate could be restored if the cell membranes were permeabilized by the detergent cetyltrimethylammonium bromide (CTAB), or if downhill transport was made possible by the addition of electron acceptors at millimolar concentrations. Sulfate was not reduced under these conditions, presumably because the cells did not contain ATP for its activation. K⁺-and Na⁺-ionophores such as nigericin, valinomycin or monensin appeared to be of limited efficiency in D. desulfuricans. In most experiments, sulfate reduction was inhibited by the K⁺–H⁺ antiporter nigericin in the presence of K⁺, but not by the thiocyanate anion or the K⁺-transporter valinomycin. The results indicate that sulfate, sulfite and thiosulfate are taken up by proton-anion symport, presumably as undissociated acids with an electroneutral mechanism, driven by the transmembraneous pH gradient ( pH) or by a solute gradient. Kinetics of alkalinization and sulfide production in cells grown with different electron acceptors revealed that D. desulfuricans has different specific uptake systems for sulfate and thiosulfate, and obviously also for sulfite. It is proposed that the electron acceptor transport finally will not consume net energy during growth in buffered medium: The protons taken up during active electron acceptor transport leave the cell with the reduced end-product by simple passive diffusion of H₂S.Abbreviations CCCP carbonyl cyanide m-chlorophenylhydrazone - FCCP carbonyl cyanide p-trifluoromethoxy phenylhydrazone - CTAB cethyltrimethylammonium bromide     

Electrical properties of the plasma membrane of microplasmodia ofPhysarum polycephalum

Summary Microplasmodia ofPhysarum polycephalum have been investigated by conventional electrophysiological techniques. In standard medium (30mm K⁺, 4mm Ca⁺⁺, 3mm Mg⁺⁺, 18mm citrate buffer, pH 4.7, 22°C), the transmembrane potential differenceV _m is around –100 mV and the membrane resistance about 0.25 m².V _m is insensitive to light and changes of the Na⁺/K⁺ ratio in the medium. Without bivalent cations in the medium and/or in presence of metabolic inhibitors (CCCP, CN^–, N ₃ ^– ),V _m drops to about 0 mV. Under normal conditions,V _m is very sensitive to external pH (pH _o ), displaying an almost Nernstian slope at pH _o =3. However, when measured during metabolic inhibition,V _m shows no sensitivity to pH _o over the range 3 to 6, only rising (about 50 mV/pH) at pH _o =6. Addition of glucose or sucrose (but not mannitol or sorbitol) causes rapid depolarization, which partially recovers over the next few minutes. Half-maximal peak depolarization (25 mV with glucose) was achieved with 1mm of the sugar. Sugar-induced depolarization was insensitive to pH _o . The results are discussed on the basis of Class-I models of charge transport across biomembranes (Hansen, Gradmann, Sanders and Slayman, 1981,J. Membrane Biol. 63:165–190). Three transport systems are characterized: 1) An electrogenic H⁺ extrusion pump with a stoichiometry of 2 H⁺ per metabolic energy equivalent. The deprotonated form of the pump seems to be negatively charged. 2) In addition to the passive K⁺ pathways, there is a passive H⁺ transport system; here the protonated form seems to be positively charged. 3) A tentative H⁺-sugar cotransport system operates far from thermodynamic equilibrium, carrying negative charge in its deprotonated states.     

Kinetics and specificity of the renal Na+/myo-inositol cotransporter expressed in Xenopus Oocytes

The two-microelectrode voltage clamp technique was used to examine the kinetics and substrate specificity of the cloned renal Na⁺/myo-inositol cotransporter (SMIT) expressed in Xenopus oocytes. The steady-state myo-inositol-induced current was measured as a function of the applied membrane potential (V _m ), the external myo-inositol concentration and the external Na⁺ concentration, yielding the kinetic parameters: K _0.5 ^MI , K _0.5 ^Na , and the Hill coefficient n. At 100 mM NaCl, K _0.5 ^MI was about 50 m and was independent of V _m . At 0.5 mm myo-inositol, K _0.5 ^Na ranged from 76 mm at V _m =–50 mV to 40 mm at V _m =–150 mV. n was voltage independent with a value of 1.9±0.2, suggesting that two Na⁺ ions are transported per molecule of myo-inositol. Phlorizin was an inhibitor with a voltage-dependent apparent K _I of 64 m at V _m =–50 mV and 130 m at V _m = –150 mV. To examine sugar specificity, sugar-induced steady-state currents (at V _m =–150 mV) were recorded for a series of sugars, each at an external concentration of 50 mm. The substrate selectivity series was myo-inositol, scyllo-inositol > l-fucose > l-xylose > l-glucose, d-glucose, -methyl-d-glucopyranoside > d-galactose, d-fucose, 3-O-methyl-d-glucose, 2-deoxy-d-glucose > d-xylose. For comparison, oocytes were injected with cRNA for the rabbit intestinal Na⁺/glucose cotransporter (SGLT1) and sugar-induced steady-state currents (at V _m =–150 mV) were measured. For oocytes expressing SGLT1, the sugar selectivity was: d-glucose, -methyl-d-glucopyranoside, d-galactose, d-fucose, 3-O-methyl-d-glucose > d-xylose, l-xylose, 2-deoxy-d-glucose > myo-inositol, l-glucose, l-fucose. The ability of SMIT to transport glucose and SGLT1 to transport myo-inositol was independently confirmed by monitoring the Na⁺-dependent uptake of ³H-d-glucose and ³H-myo-inositol, respectively. In common with SGLT1, SMIT gave a relaxation current in the presence of 100 mm Na⁺ that was abolished by phlorizin (0.5 mm). This transient current decayed with a voltage-sensitive time constant between 10 and 14 msec. The presteady-state current is apparently due to the reorientation of the cotransporter protein in the membrane in response to a change in V _m . The kinetics of SMIT is accounted for by an ordered six-state nonrapid equilibrium model. Present address: W.M. Keck Biotechnology Resource Laboratory, Boyer Center for Molecular Medicine, Rm, 305A, Yale University, 295 Congress Ave., New Haven, Connecticut 06536-0812 Present address: National Institute for Physiological Sciences, Department of Cell Physiology, Okazaka, 444, JapanContributed equally to this workWe thank John Welborn for the HPLC analysis of the sugar substrates. This work was supported by grants from the National Institutes of Health DK19567, DK42479 and NS25554.     

Regulation of the basolateral potassium conductance of theNecturus proximal tubule

Summary Two methods, the measurement of the response of the basolateral membrane potential (V _bl) of proximal tubule cells ofNecturus to step changes in basolateral K⁺ concentration, and cellular cable analysis, were used to assess the changes in basolateral potassium conductance (G _K) caused by a variety of maneuvers. The effects of some of these maneuvers on intracellular K⁺ activity (a _K ⁱ ) were also evaluated using double-barreled ion-selective electrodes. Perfusion with 0mm K⁺ basolateral solution for 15 min followed by 45 min of 1mm K⁺ solution resulted in a fall in basolateral potassium (apparent) transference number (t _K),V _bl anda _K ⁱ . Results of cable analysis showed that total basolateral resistance,R _b , rose. The electrophysiological effects of additional manipulations, known to inhibit net sodium reabsorption across the proximal tubular epithelium ofNecturus, were also investigated. Ouabain caused a fall int _K accompanied by large decreases ina _K ⁱ andV _bl. Lowering luminal sodium caused a fall int _K and a small reduction inV _bl. Selective reduction of peritubular sodium, a maneuver that has been shown to block sodium transport from lumen to peritubular fluid, also resulted in a significant decrease int _K. These results suggest thatG _K varies directly with rate of transport of the sodium pump, irrespective of the mechanism of change in pump turnover.Part of this material has been presented at the 10th International Conference on Biological Membranes (Cohen & Giebisch, 1984).     

Biological role of endoreplication in the process of spermatogenesis inChara vulgaris L.

Summary During cell division in antheridial filaments ofChara vulgaris an increase in DNA content occurs in both shield cells and manubria within an antheridium, reaching 16C–64C and 8C–32C levels, respectively. Endoreplication ceases prior to the formation of spermatids and initiation of spermiogenesis, probably as a result of symplasmic isolation of the antheridium from the thallus. As the DNA content of the nuclei increases, the shield cells³H-leucine incorporation increases, and they grow intensively in the tangential plane. Translation decreases considerably after termination of shield cell growth. DNA content of mature manubria is half of that in shield cells, although their size is 10 times that of manubria. Translational activity of manubria also increases as DNA content rises and cells grow. However, during spermiogenesis, this activity remains at its maximum, which is associated with the secretory function of the manubria. Spermiogenesis is also accompanied by far-reaching ultrastructural changes within the manubrial cytoplasm.The level of endopolyploidy in both shield cells and manubria of antheridia formed in the spring is higher by one replication cycle, than in autumnal antheridia. AMO-1618, at a concentration of 10^–5M reduces the DNA content in the autumnal manubria. The higher the manubrial level of endopolyploidy in spermiogenesis, the greater their size, and the higher the translational activity and number of joined spermatids. The number of spermatozoids in the antheridium is also positively correlated with the internal volume of an antheridium, which is itself dependent on the endopolyploidy level of shield cells.The results obtained confirm the assumption that endoreplication favours the higher growth dynamics and potential translational activity, which occurs in the dynamic growth phase only in shield cells, while in manubria, i.e. cells producing substances necessary to spermatozoids development, it remains high until the end of spermiogenesis.     

Transport of basic amino acids in Riccia fluitans: Evidence for a second binding site

The transport of several amino acids with different side-chain characteristics has been investigated in the aquatic liverwort Riccia fluitans. i) The saturation of system I (neutral amino acids) by addition of excess -aminoisobutyric acid to the external medium completely eliminated the electrical effects which are usually set off by neutral amino acids. Under these conditions arginine and lysine significantly depolarized the plasmalemma. ii) L- and D-lysine/arginine were discriminated against in favour of the L-isomers. iii) Increasing the external proton concentration in the interval pH 9 to 4.5 stimulated plasmalemma depolarization, electrical net current, and uptake of [¹⁴C]-basic amino acids. iv) Uptake of [¹⁴C]-glutamic acid took place only at acidic pHs. v) [¹⁴C]-histidine uptake had an optimum between pH 6 and 5.5. vi) Overlapping of the transport of basic, neutral, and acidic amino acids was common. It is suggested that besides system I, a second system (II), specific for basic amino acids, exists in the plasmalemma of Riccia fluitans. It is concluded that the amino-acid molecule with an uncharged side chain is the substrate for system I, which also binds and transports the neutral species of acidic amino acids, whereas system II is specific for amino acids with a positively charged side chain. The possibility of system II being a proton cotransport is discussed.Abbreviation AiB -aminoisobutyric acid     

Host specificity testing ofRhodometra sacraria [Lep.: Geometridae], a possible biological control candidate forEmex australis in Australia

The foliage feeding geometridRhodometra sacraria L. was collected onEmex australis Steinheil in South Africa, cultured, then tested as a possible biological control agent forE. australis, in Australia. Tests on its host specificity were carried out in South Africa and Australia.R. sacraria was specific to plants of the familyPolygonaceae rather than toE. australis. Request for the reintroduction and release ofR. sacraria into Australia has not been made.      

Sodium-alanine cotransport in oocytes ofXenopus laevis: Correlation of alanine and sodium fluxes with potential and current changes

Summary The sodium-dependentl-alanine transport across the plasma membrane of oocytes ofXenopus laevis was studied by means of [¹⁴C]-l-alanine,²²Na⁺ and electrophysiological measurements. At fixed sodium concentrations, the dependence of alanine transport on alanine concentration follows Michaelis-Menten kinetics; at fixed alanine concentrations, the transport varies with sodium concentration with a Hill coefficient of 2. In the presence of sodium the uptake of alanine is accompanied by a depolarization of the membrane. Under voltage-clamp conditions this depolarization can be compensated by an inward-directed current. Assuming that this current is carried by sodium we arrive at a 21 stoichiometry for the sodium-alanine cotransport. The assumption was confirmed by direct measurements of both sodium and alanine fluxes at saturating concentrations of the two substrates, which also yielded a stoichiometry close to 21. The sodium-l-alanine cotransport is neither inhibited by furosemide (0.5 mmol/liter) nor by N-methyl amino isobutyric acid (5 mmol/liter). A 20-fold excess ofd-alanine overl-alanine caused about 60% inhibition.     

Phosphorylation of a putative myosin light chain inChara by calcium-dependent protein kinase

Summary Ca²⁺-dependent protein kinase (CDPK) has been proposed to mediate inhibition by Ca²⁺ of cytoplasmic streaming in the green algaChara. We have identified the in vivo substrate(s) of CDPK inChara by using vacuolar perfusion of individual internodal cells with [-³²P]ATP. Phosphorylation of several polypeptides is enhanced when perfusions are performed at 10^–4M free Ca²⁺ compared to <10^–9M free Ca²⁺. The Ca²⁺-stimulated phosphorylation of these proteins is inhibited by the presence of a monoclonal antibody to soybean CDPK. One of these proteins is 16 to 18kDa and is recognized by an antibody against gizzard myosin light chains. These results demonstrate that inChara, several polypeptides are phophorylated by CDPK and one of these proteins has been tentatively identified as a myosin light chain. These observations support the hypothesis that Ca²⁺-regulated phosphorylation of myosin is involved in the regulation of cytoplasmic streaming.Abbreviations CDPK calcium-dependent protein kinase - mAb monoclonal antibody     

Charge and acidity compensation during proton-sugar symport in Chlorella: The H+-ATPase does not fully compensate for the sugar-coupled proton influx

This study was undertaken in order to demonstrate the extent to which the activity of the plasmalemma H⁺-ATPase compensates for the charge and acidity flow caused by the sugar-proton symport in cells of chlorella vulgaris Beij.. Detailed analysis of H⁺ and K⁺ fluxes from and into the medium together with measurements of respiration, cytoplasmic pH, and cellular ATP-levels indicate three consecutive phases after the onset of H⁺ symport. Phase 1 occurred immediately after addition of sugar, with an uptake of H⁺ by the hexoseproton symport and charge compensation by K⁺ loss from the cells and, to a smaller degree, by loss of another ion, probably a divalent cation. This phase coincided with strong membrane depolarization. Phase 2 started approximately 5 s after addition of sugar, when the acceleration of the H⁺-ATPase caused a slow-down of the K⁺ efflux, a decrease in the cellular ATP level and an increase in respiration. The increased respiration was most probably responsible for a pronounced net acidification of the medium. This phase was inhibited in deuterium oxide. In phase 3, finally, a slow rate of net H⁺ uptake and K⁺ loss was established for several further minutes, together with a slight depolarization of the membrane. There was hardly any pH change in the cytoplasm, because the cytoplasmic buffering capacity was high enough to stabilize the pH for several minutes despite the net H⁺ fluxes. The quantitative participation of the several phases of H⁺ and K⁺ flow depended on the pH of the medium, the ambient Ca²⁺ concentration, and the metabolic fate of the transported sugar. The results indicate that the activity of the H⁺-ATPase never fully compensated for H⁺ uptake by the sugar-symport system, because at least 10% of symport-caused charge inflow was compensated for by K⁺ efflux. The restoration of pH in the cytoplasm and in the medium was probably achieved by metabolic reactions connected to increased glycolysis and respiration.Abbreviations DMO dimethyloxazolidinedione - EDTA ethylcnediaminetetraacetic acid - p.c. packed cell volume     

Effects of experimental infection with Eimeria bovis on the balance of sodium, potassium and water in calves

Balance trials were performed to investigate the effects of experimental Eimeria bovis coccidiosis on the metabolism of water, sodium and potassium in calves. Non-infected pair-fed controls and controls fed according to plan were included in the study to allow differentiation between the effects due to infection and due to changes in feed intake. Primary infection with 5 × 10⁴ (group A) or 1 × 10⁵ (group B) oocysts caused mild diarrhoea in three out of four group A calves and mild to severe haemorrhagic diarrhoea in all five group B calves. Losses of sodium and potassium via faeces tended to increase in the infected calves during patency and apparent digestibility (AD) of these minerals was comparably low. In the urine of the infected calves the Na/K-ratio decreased due to a reduced urinary excretion of sodium. The retention (RT) of sodium was particularly high in the calves that had received the higher oocyst dose. Potassium RT did not underlie significant changes during the course of coccidiosis. In the infected calves the plasma level of sodium was reduced transiently while the level of potassium remained fairly stable. Infections with the higher oocyst dose caused a distinct reduction of fluid excretion via urine which compensated for the increased faecal water losses during severe diarrhoea. Reinfection of the group A calves with 1 × 10⁵ oocysts did not cause any significant metabolic impairment. The results of this study indicate that although acute sublethal bovine coccidiosis alters electrolyte and water metabolism the overall balance of electrolytes and water is largely maintained by physiologic adaptation.