Rb+ uptake by isolated pea mesophyll protoplasts in light and darkness

Rb⁺ uptake into protoplasts isolated from the mesophyll of Pisum sativum L. cv. Dan has been followed at intervals of a few minutes in the light and in the dark. The progress curve for uptake in the dark decreased in slope after about 7 min; in the light, by contrast, the slope increased. This effect was more pronounced at pH 7 than at pH 5.5. The pH profile for uptake in the dark rose with increasing pH: in the light the profile flattened, or even fell somewhat, between pH 5.5 and pH 6.5, then rose again. In the dark the proton uncoupler carbonyl cyanide m-chlorphenylhydrazone (CCCP) had little or no effect, either at pH 5.5 or at pH 7.4; in the light CCCP was strongly inhibitory, particularly at pH 7.4. Increasing concentrations of CCCP produced progressively more and more severe inhibition in the light, but in the dark produced a slight rise in uptake. The ATPase inhibitors quercetin, rutin and diethyl-stilbestrol, as well as arsenate, all depressed uptake in the light, particularly at higher pH Dark uptake was sensitive only at pH 5.5, not at pH 7.4. In marked contrast to the case of methyl-3 glucose, where protoplasts which were switched from light to dark took up sugar at the accelerated light rate for the first 7 min in the dark, a switch to darkness produced a Rb⁺ uptake rate below that for protoplasts held continuously in the dark. It is inferred that the mechanism of Rb⁺ uptake does not involve proton cotransport. Information regarding the membrane potential was obtained by following the distribution of tetraphenyl phosphonium (TPP⁺) between protoplasts and medium. The potential was more negative in the light than in the dark. It was also more negative at pH 7 than at pH 5 both in the light and in the dark. Treatment with CCCP produced no appreciable depolarization within the first 20 min, indicating thet the CCCP inhibition of Rb⁺ uptake in the light cannot be ascribed to a reduction in potential. An ATP-fueled K⁺ porter, or K⁺-H⁺ antiporter, seems the most likely explanation. The maintenance of the rising pH profile in the dark, despite the presence of a CCCP concentration which drastically inhibits light uptake, suggests that the profile does not depend on the operation of the proton pump.     

Protonation and light synergistically convert plasmalemma sugar carrier system in mesophyll protoplasts to its fully activated form

The course of sugar fluxes into and out of protoplasts isolated from the mesophyll of Pisum sativum L. has been followed over brief time intervals (minutes). Light strongly stimulated net sugar influx at pH 8 as well as at pH 5.5. The proton conductor carbonyl cyanide m-chlorophenylhydrazone (CCCP) inhibited initial influx in the light, both at pH 8.0 and at pH 5.5. CCCP was without effect in the dark at either pH. All these results applied both to sucrose and to the nonmetabolizable glucose analog 3-O-methyl-d-glucose.When protoplasts at pH 5.5 were transferred from light to darkness, "stored" light driving force maintained uptake in the dark at the full light rate for the first 7 minutes. At pH 8, however, even 4 minutes after transfer to dark, uptake was well below the light rate. Initial uptake rates over a range of external concentrations were derived from progress curves obtained in the light and in the dark, both at pH 5.5 and at 7.7. When initial rate was plotted against concentration, simple Michaelis-Menten kinetics were observed only under the condition pH 5.5, light. In the dark at both pH values, and in the light at pH 7.7, complex curves with intermediate plateaus were obtained, strongly resembling curves reported for systems where mixed negative and positive cooperativity is operating.The same "K(m) for protons" was observed in the dark and in the light (10(-7) molar). Switching protoplasts in the dark from pH 8 to 5.5 failed to drive sugar transport by imposed protonmotive force, as judged by lack of sensitivity to CCCP. Switching protoplasts which had taken up sugar in the dark at pH 5.5 to pH 7 induced net efflux of sugar. Flux analysis showed that this effect was entirely due to the prompt fall in influx.It is concluded from the kinetic experiments that protonation alone is not sufficient to convert the sugar transport system to its fully activated high affinity form. A further light-dependent factor which acts synergistically with protonation is required.     

Evidence for A Ca2+ gradient across the plasma membrane of wheat protoplasts

Fluxes of Ca²⁺ across the plasma membrane of isolated wheat protoplasts have been measured both as net accumulation and as uptake under steady-state conditions. The ATPase inhibitors, orthovanadate and diethylstibesterol, and the divalent cation ionophore, A23187, were all found to enhance net Ca²⁺ accumulation by protoplasts. The uptake of Ca²⁺ under steady-state conditions was also stimulated by A23187 but relatively unaffected by a range of plant hormones or by red or far red light. Light treatments were compared to dark controls with protoplasts isolated from etiolated wheat.The results suggest that plant cells maintain a Ca²⁺ gradient across their plasma membrane but it appears not to be under phytochrome control.     

Light-dependent rubidium uptake into isolated mesophyll protoplasts from leaves of Pisum sativum

A method is described for the isolation of large amounts of physiologically active protoplasts from leaves of Pisum sativum L. Rubidium uptake was determined after separation of the intact protoplasts from the loading medium by rapid centrifugation through a phthalate step gradient. In freshly isolated mesophyll protoplasts of Pisum sativum , rubidium uptake was carbonylcyanide- p -trifluoromethoxyphenylhydrazone reduced by metabolic inhibitors such as 5 μ M , 0.1 mW cyanide, 2 μ M DCMU and 5 m M arsenate and by dark incubation. Reduction of rubidium uptake by inhibition of aerobic respiration or the photosynthetic electron transport system demonstrates that both processes play a role in the energy supply for membrane transport in these protoplasts.     

Light-Stimulated Changes in the Acidity of Suspensions of Oat Protoplasts: Dependence upon Photosynthesis

Light induced an alkalinization and stimulated a subsequent acidification of the medium surrounding oat (Avena sativa L. cv Garry) leaf protoplasts. Blue light was less effective than would be predicted from photosynthetic action spectra. Nonetheless, 3-(3,4-dichlorophenyl)-1,1-dimethylurea prevented alkalinization and reduced acidification to the dark rate for protoplast suspensions exposed to all light regimes tested.

Alkalinization increased in parallel with initial rates of O₂ evolution as the quantum flux density of white light was raised to 75 microeinsteins per square meter per second. Alkalinization was accompanied by a decrease in the CO₂ content of the medium; therefore, it was attributed to photosynthetically induced CO₂ uptake. The effect of CO₂ depletion on the acidity of the medium appeared to be mainly restricted to the first 15 minutes of exposure to light. Consequently, subsequent pH changes primarily reflected a constant net proton efflux. Acidification occurred in the dark, but rates of acidification increased in response to increased light approximately in parallel with changes in a concomitant net O₂ efflux. The results indicated that protoplasts could acidify the medium in response to nonphotosynthetic activity, but that photosynthesis mediated light stimulation of acidification.

     

Energy coupling in the active transport of proline and glutamate by the photosynthetic halophile Ectothiorhodospira halophila.

When illuminated, washed cell suspensions of Ectothiorhodospira halophila carry out a concentrative uptake of glutamate or proline. Dark-exposed cells accumulate glutamate but not proline. Proline transport was strongly inhibited by carbonylcyanide-m-chlorophenylhydrazone (CCCP), a proton permeant that uncouples photophosphorylation, and by 2-heptyl-4-hydroxyquinoline-n-oxide (HQNO), an inhibitor of photosynthetic electron transport. A stimulation of proline uptake was effected by N,N'-dicyclohexylcarbodiimide (DCCD), an inhibitor of membrane adenosine triphosphatase (ATPase) which catalyzes the phosphorylation. These findings suggest that the driving force for proline transport is the proton-motive force established during photosynthetic electron transport. Glutamate uptake in the light was inhibited by CCCP and HQNO, but to a lesser extent than was the proline system. DCCD caused a mild inhibition of glutamate uptake in the light, but strongly inhibited the uptake by dark-exposed cells. CCCP strongly inhibited glutamate uptake in the dark. The light-dependent transport of glutamate is apparently driven by the proton-motive force established during photosynthetic electron transport. Hydrolysis of adenosine triphosphate (ATP) by membrane ATPase apparently establishes the proton-motive force to drive the light-independent transport. These conclusions were supported by demonstrating that light- or dark-exposed cells accumulate [3H]triphenylmethylphosphonium, a lipid-soluble cation. Several lines of indirect evidence indicated that the proline system required higher levels of energy than did the glutamate system(s). This could explain why ATP hydrolysis does not drive proline transport in the dark. Membrane vesicles were prepared by the sonic treatment of E. halophila spheroplasts. The vesicles contained active systems for the uptake of proline and glutamate.     

Inhibition of Ca2+-transport ATPase from synaptosomal vesicles by flavonoids

The inhibitory action of the flavonoid quercetin has been examined on the calcium-transport ATPase of synaptosomal vesicles and compared to that of two other flavonoids, morin and rutin. We have found that while quercetin caused a 50% inhibition of calcium transport at a concentration of 15 microM, morin and rutin had similar effects at concentrations of about 200 microM. A similar order of potency was observed also for ATP hydrolysis, though at higher concentrations. Quercetin also strongly inhibited phosphorylation of membrane proteins by ATP in synaptosomal vesicles. Rutin and morin had an almost negligible effect on membrane protein phosphorylation. The order of inhibitory potency of the flavonoids on the Ca2+-transport ATPase from synaptosomal vesicles: quercetin greater than morin greater than rutin, could be linked to their possible solubility in the membrane lipid phase since: (1) it paralleled their partitioning between a mixture of oil and water; (2) it paralleled their uptake from the reaction mixture by synaptosomal vesicles and phosphatidylcholine liposomes; (3) they had almost equal potency as inhibitors of the water soluble system of histone phosphorylation by protein kinase.     

Regulation of the cytosolic adenylate ratio as determined by rapid fractionation of mesophyll protoplasts of oat

Adenylate levels in chloroplasts, mitochondria and the cytosol of oat mesophyll protoplasts were determined under light and dark conditions, in the absence and presence of plasmalemma-permeable inhibitors of electron transfer and uncouplers of phosphorylation. This was achieved using a microgradient technique which allowed an integrated homogenization and fractionation of protoplasts within 60 s (Hampp et al. 1982, Plant Physiol. 69, 448–455), under conditions which quench bulk activities of metabolic interconversion in less than 2 s. In illuminated controls, ATP/ADP ratios were found to be 2.1 in chloroplasts, about unity in mitochondria, and 11 in the cytosol; whereas, in the dark, this ratio only showed a large drop in chloroplasts (0.4). None of the compounds used [carbonylcyanide m-chlorophenylhydrazone (CCCP), carbonylcyanide p-trifluoromethoxy-phenylhydrazone (FCCP), antimycin A, dibromothymoquinone (DBMIB), dichlorophenyldi-methylurea (DCMU), or salicylhydroxamic acid (SHAM)] affected the stroma adenylate ratio in the dark. Under illumination, however, the ATP/ADP ratios were partly reduced in the presence of antimycin (inhibitor of cyclic photophosphorylation) and of DCMU (inhibitor of linear electron flow), while in the presence of DBMIB, DCMU+ antimycin (inhibition of both cyclic and linear electron flow), and CCCP (uncoupling) the ratio obtained was the same as that occurring in the dark. In contrast, mitochondrial adenylate levels did not exhibit large variations under the various treatments. The cytosolic ATP/ADP ratio, however, showed dramatic changes: in darkened protoplasts, cytosolic values dropped to 0.2 and 0.1 in the presence of uncouplers and antimycin, respectively, while SHAM did not induce any significant alteration. In the light, a similar pronounced decrease in ATP levels was observed only after the application of uncouplers or inhibitors of both mitochondrial and photosynthetic electron transport, whereas selective inhibition of the latter was largely ineffective in reducing the cytosolic ATP/ADP ratio. Thus, the results show that the antimycin-sensitive electron transport is, potentially, equally active in light and darkness. In addition, they indicate that antimycin-insensitive electron transport in mitochondria (alternative pathway) does not significantly contribute to the cytosolic energy state.Abbreviations CCCP carbonylcyanide m-chlorophenylhydrazone - DBMIB dibromothymoquinone (2,5-dibromo-3-methyl-6-isopropy-p-benzoquinone) - DCMU dichlorophenyldimethylurea - FCCP carbonylcyanide-p-trifluoromethoxy-phenylhydrazone - SHAM sancylhydroxamic acid     

Inhibition of Ca-transport ATPase from synaptosomal vesicles by flavonoids

The inhibitory action of the flavonoid quercetin has been examined on the calcium-transport ATPase of synaptosomal vesicles and compared to that of two other flavonoids, morin and rutin. We have found that while quercetin caused a 50% inhibition of calcium transport at a concentration of 15 μM, morin and rutin had similar effects at concentrations of about 200 μM. A similar order of potency was observed also for ATP hydrolysis, though at higher concentrations. Quercetin also strongly inhibited phosphorylation of membrane proteins by ATP in synaptosomal vesicles. Rutin and morin had an almost negligible effect on membrane protein phosphorylation. The order of inhibitory potency of the flavonoids on the Ca²⁺-transport ATPase from synaptosomal vesicles: quercetin > morin > rutin, could be linked to their possible solubility in the membrane lipid phase since: (1) it paralleled their partitioning between a mixture of oil and water; (2) it paralleled their uptake from the reaction mixture by synaptosomal vesicles and phosphatidylcholine liposomes; (3) they had almost equal potency as inhibitors of the water soluble system of histone phosphorylation by protein kinase.     

Inhibition of Ca2+-transport ATPase from synaptosomal vesicles by flavonoids

The inhibitory action of the flavonoid quercetin has been examined on the calcium-transport ATPase of synaptosomal vesicles and compared to that of two other flavonoids, morin and rutin. We have found that while quercetin caused a 50% inhibition of calcium transport at a concentration of 15 μM, morin and rutin had similar effects at concentrations of about 200 μM. A similar order of potency was observed also for ATP hydrolysis, though at higher concentrations. Quercetin also strongly inhibited phosphorylation of membrane proteins by ATP in synaptosomal vesicles. Rutin and morin had an almost negligible effect on membrane protein phosphorylation. The order of inhibitory potency of the flavonoids on the Ca²⁺-transport ATPase from synaptosomal vesicles: quercetin > morin > rutin, could be linked to their possible solubility in the membrane lipid phase since: (1) it paralleled their partitioning between a mixture of oil and water; (2) it paralleled their uptake from the reaction mixture by synaptosomal vesicles and phosphatidylcholine liposomes; (3) they had almost equal potency as inhibitors of the water soluble system of histone phosphorylation by protein kinase.     

Regulation of the glucose phosphotransferase system in Brochothrix thermosphacta by membrane energization.

Uptake of 2-deoxyglucose, alpha-methylglucopyranoside, and glucose into intact cells of Brochothrix thermosphacta (formerly Microbacterium thermosphactum, ATCC 11509) was stimulated by KCN or CCCP. The glucose analogs were recovered almost totally as the sugar phosphates. Membrane vesicles were isolated from protoplasts and shown to be right side out by freeze fracturing and by using ATPase as a marker for the cytoplasmic membrane surface. Uptake of glucose into vesicles was dependent on the presence of phosphoenolpyruvate. NADH oxidation, K+ -diffusion gradients, and externally directed lactate gradients (pH greater than 7 initially) were used to generate transmembrane potentials across membrane vesicles. Above a threshold value of about -50 mV, uptake of glucose into membrane vesicles was reduced. Likewise, the maximum uptake of glucose and its two analogs into cells occurred when the protonmotive force was less than about -50 mV.     

Sugar uptake by the dermal transfer cells of developing cotyledons of Vicia faba L.

The mechanism of carrier-mediated sucrose uptake by the dermal transfer cells of developing Vicia faba L. cotyledons was studied using excised cotyledons and isolated transfer cell protoplasts. Addition of sucrose resulted in a transitory alkalinization of the bathing solution whereas additions of glucose, fructose or raffinose had no effect. Dissipating the proton motive force by exposing cotyledons and isolated transfer cell protoplasts to an alkaline pH, carbonylcyanide m-chlorophenylhydrazone, weak acids (propionic acid and 5,5-dimethyl-oxazolidine-2,4-dione) or tetraphenylphos-phonium ion resulted in a significant reduction of sucrose uptake. The ATPase inhibitors, erythrosin B (EB), diethylstilbestrol (DES) and N,N-dicyclohexylcarbodiimide (DCCD) were found to abolish the sucrose-induced medium alkanization as well as reduce sucrose uptake. Cytochemical localization of the ATPase, based on lead precipitation, demonstrated that the highest activity was present in the plasma membranes located in wall ingrowth regions of the dermal transfer cells. The presence of a transplasma-membrane redox system was detected by the extracellular reduction of the electron acceptor, hexacyanoferrate III. The reduction of the ferric ion was coupled to a release of protons. The redox-induced proton extrusion was abolished by the ATPase inhibitors EB, DES and DCCD suggesting that proton extrusion was solely through the H⁺-ATPase. Based on these findings, it is postulated that cotyledonary dermal transfer cells take up sucrose by a proton symport mechanism with the proton motive force being generated by a H ⁺ -ATPase. Sucrose uptake by the storage parenchyma and inner epidermal cells of the cotyledons did not exhibit characteristics consistent with sucrose-proton symport.Abbreviations CCCP carbonylcyanide m-chlorophenylhydrazone - DCCD N,N-dicyclohexylcarbodiimide - DES diethylstilbestrol - EB erythrosin B - Em membrane potential - FC fusicoccin - HCF II hexacyanoferrate II - HCF III hexacyanoferrate III - Mes 2-(N-morpholino)ethanesulfonic acid - pmf proton motive force - TPP⁺ tetraphenylphosphonium ion The investigation was supported by funds from the Research Management Committee, The University of Newcastle and the Australian Research Council. One of us, R. McDonald, gratefully acknowledges the support of an Australian Postgraduate Research Award. We are indebted to Stella Savory for preparing the ultrathin sections for electron microscopy.     

A comparative study of metabolite levels in plant leaf material in the dark

Metabolite levels have been compared in the dark and during photosynthesis in leaves and protoplasts from spinach, pea, wheat and barley. In protoplasts the subcellular distribution was also studied. The levels of triose phosphates and sugar bisphosphates were high in the light and low in the dark. The hexose phosphates and 3-phosphoglycerate levels in the dark were very variable depending on the plant material. In most conditions, hexose phosphates and triose phosphates were mainly in the extrachloroplast compartment, while 3-phosphoglycerate and the sugar bisphosphates were mainly in the chloroplast compartment. Leaves always had a very low triose phosphate: 3-phosphoglycerate ratio in the dark, but in protoplasts this ratio was higher. Detailed studies with spinach showed that metabolite levels were very dependent on the availability of carbohydrate in the leaf, particularly starch. Starch mobilisation is not controlled just by the availability of inorganic phosphate and accumulation of phosphorylated intermediates. Hydrolysis of starch may provide precursors for sucrose synthesis while phosphorolysis leads to provision of substrates for respiration. Starch breakdown generates high enough levels of hexose phosphate to support substantial rates of sucrose synthesis in the dark. Respiration is not greatly increased when metabolite levels are high during starch mobilisation. Higher levels of metabolites shorten the length of the induction phase of photosynthesis.Abbreviations Chl chlorophyll - DHAP dihydroxyacetone phosphate - Fru2,6bisP fructose-2,6-bisphosphate - NMR nuclear magnetic resonance - PGA 3-phosphoglyceric acid - Pi inorganic phosphate - RuBP ribulose-1,5-bisphosphate - UDPGlc uridine-5-diphosphate glucose     

Evidence that H+ efflux stimulated by redox activity is independent of plasma membrane ATPase activity

Proton efflux from mesophyll cells of Asparagus sprengeri Regel was inhibited completely by diethylstilbestrol (DES) and NN'-dicyclohexylcarbodiimide (DCCD), known inhibitors of the plasma membrane ATPase. At the concentrations of inhibitors employed, fusicoccin did not reactivate proton efflux. Subsequent addition of ferricyanide however resulted in significant rates of acidification of the medium and reduction of ferricyanide. Similar results were obtained in the light and in the dark. Thus, medium acidification in response to redox activity appears to be independent of the ATP-dependent acidification process.     

Ca uptake and plasma membrane depolarization associated with blue light-sensitive exogenous NADH oxidation by Cuscuta protoplasts

Protoplasts isolated from the apical segments of Cuscuta reflexa exhibited blue light-sensitive PM-linked NADH oxidase activity and increased rate of Ca²⁺-uptake in presence of NADH in dark, which was also stimulated by blue light. Contrary to marginal inhibition by Con A treatment, the ATPase inhibitors significantly inhibited the Ca²⁺ uptake by the protoplasts both in dark and under blue light. The Ca²⁺-calmodulin antagonists, W-7 and calmidazolium, also inhibited Ca²⁺-uptake by protoplasts under similar conditions. The state of PM polarization was monitored by the fluorescent dye 9-amino acridine. It was observed that PM-linked NADH oxidation caused hyperpolarization of the membrane, the exposure of which to blue light resulted in membrane depolarization. The presence of Ca²⁺-calmodulin antagonists or Con A treatment completely abolished the blue light-induced membrane depolarization. It is argued that these actities at the PM, having some glycoproteic components, are functionally closely involved in blue light-induced signal transduction in Cuscuta     

Involvement of the Plasma Membrane ATPase in the Osmoregulatory Mechanism of the Alga Dunaliella salina

The unicellular halotolerant alga Dunaliella salina recovers normally from a hypertonic shock even when suspended in NaCl and buffer only. Furthermore, addition of Cu²⁺, valinomycin and KCl, or permeable ions such as methyltriphenylphosphonium or thiocyanate, do not affect the recovery. However, treatment with two specific inhibitors of the plasma membrane adenosine triphosphatase (ATPase), diethylstilbestrol, or vanadate, fully inhibit the recovery. The inhibition is manifested by the inability of the cells to both synthesize glycerol and return to their original volume. The inhibitions are nonlethal, reversible and equally effective in the dark or the light. Since the plasma membrane ATPase is the only enzyme known to be inhibited by both diethylstilbestrol and vanadate, it is concluded that its activity is essential for the recovery of Dunaliella from a hypertonic shock. Mechanisms by which the plasma membrane ATPase may participate in the activation of glycerol production in the algae are discussed.     

Uptake of sodium in quince, sugar beet, and wheat protoplasts determined by the fluorescent sodium-binding dye benzofuran isophthalate

The uptake of sodium into protoplasts of quince (Cydonia oblonga Mill, clone BA29), sugar beet (Beta vulgaris L. cv. Monohill), and wheat (Triticum aestivum L. cv. Kadett) was determined by use of the acetoxy methyl ester of the fluorescent sodium-binding benzofuran isopthalate (SBFI-AM). In the presence of 1 mM CaCl2, little sodium was taken up in the cytosol of quince mesophyll cells compared to cytosols of sugar beet and wheat. Upon addition of 40 mM NaCl, approximately the same amount of sodium was taken up in leaf and root protoplasts of wheat, but no sodium was taken up in quince. However, in calcium-free medium, obtained by addition of ethylene glycol tetra acetic acid (EGTA), quince protoplasts transiently took up sodium in the cytosol when 200-400 mM NaCl was added to the protoplast medium. Moreover, after cultivation of quince in the presence of 200 mM sodium for 4 weeks, the cytosol of isolated protoplasts did not take up any sodium at all from a calcium-free medium. The results show that protoplasts from salt tolerant quince only temporarily take up sodium in the cytosol and that they have a mechanism for fast extrusion of sodium from that compartment. These mechanisms are probably important for the high salt tolerance of quince. Calcium blocks the sodium uptake into the cytosol of both quince and wheat protoplasts.     

The sodium cycle. I. Na+-dependent motility and modes of membrane energization in the marine alkalotolerant vibrio Alginolyticus

Respiration, membrane potential generation and motility of the marine alkalotolerant Vibrio alginolyticus were studied. Subbacterial vesicles competent in NADH oxidation and delta psi generation were obtained. The rate of NADH oxidation by the vesicles was stimulated by Na+ in a fashion specifically sensitive to submicromolar HQNO (2-heptyl-4-hydroxyquinoline N-oxide) concentrations. The same amounts of HQNO completely suppressed the delta psi generation. Delta psi was also inhibited by cyanide, gramicidin D and by CCCP + monensin. CCCP (carbonyl cyanide m-chlorophenylhydrazone) added without monensin exerted a much weaker effect on delta psi. Na+ was required to couple NADH oxidation with delta psi generation. These findings are in agreement with the data of Tokuda and Unemoto on Na+-motive NADH oxidase in V. alginolyticus. Motility of V. alginolyticus cells was shown to be (i) Na+-dependent, (ii) sensitive to CCCP + monensin combination, whereas CCCP and monensin, added separately, failed to paralyze the cells, (iii) sensitive to combined treatment by HQNO, cyanide or anaerobiosis and arsenate, whereas inhibition of respiration without arsenate resulted only in a partial suppression of motility. Artificially imposed delta pNa, i.e., addition of NaCl to the K+ -loaded cells paralyzed by HQNO + arsenate, was shown to initiate motility which persisted for several minutes. Monensin completely abolished the NaCl effect. Under the same conditions, respiration-supported motility was only slightly lowered by monensin. The artificially-imposed delta pH, i.e., acidification of the medium from pH 8.6 to 6.5 failed to activate motility. It is concluded that delta mu Na+ produced by (i) the respiratory chain and (ii) an arsenate-sensitive anaerobic mechanism (presumably by glycolysis + Na+ ATPase) can be consumed by an Na+ -motor responsible for motility of V. alginolyticus.     

Uptake of Lucifer Yellow CH into plant-cell protoplasts: a quantitative assessment of fluid-phase endocytosis

The highly fluorescent dye Lucifer Yellow CH (LYCH), now in common use in microinjection studies, has been shown to enter the vacuole of a range of plant-cell protoplasts from the external medium. Uptake was quantified by lysing the protoplasts following incubation and determining the amount of LYCH incorporated by spectrofluorimetry. Uptake was biphasic with respect to both time and substrate concentration, enhanced at low pH and inhibited by low temperature and metabolic inhibitors. The kinetics of uptake showed several similarities with those reported for the fluid-phase endocytosis of LYCH in animal cells and yeast cells. A calculated membrane permeability coefficient for LYCH, based on the observed rates of uptake, was too high to be consistent with simple diffusion of the undissociated form of the molecule and inconsistent with the membrane-impermeant properties of the dye. The data are discussed in the light of the possibility of fluid-phase endocytosis versus active transmembrane transport.Abbreviations CCCP carbonyl cyanide M-chlorophenyl hydrazone - LYCH Lucifer Yellow CH     

Uptake of phosphate by symbiotic and free-living dinoflagellates

Uptake of phosphate in the light by Amphidinium carterae, Amphidinium klebsii, cultured and symbiotic Gymnodinium microadriaticum conformed to Michaelis-Menten type saturation kinetics with all organisms showing similar K _m values, namely 0.005 to 0.016 M phosphorus. V _max values were 0.009–0.32 nmol phosphorus · 10⁵ cells^-1 · 10 min^-1. Phosphate uptake by all the dinoflagellates was greater in the dark than in the light. The metabolic inhibitor 3-(3,4-dichlorophenyl) 1,1-dimethylurea stimulated phosphate uptake in the light by A. carterae and A. klebsii, but inhibited uptake by cultured and symbiotic G. microadriaticum. Carbonylcyanide 3-chlorophenylhydrazone (CCCP) inhibited phosphate uptake by A. carterae and A. klebsii under both light and dark conditions. Uptake of phosphate by cultured and symbiotic G. microadriaticum in the light, but not in the dark, was inhibited by CCCP. Low concentrations of arsenate (5 g As · l^-1) stimulated phosphate by A. carterae and A. klebsii, but inhibited uptake by cultured and symbiotic G. microadriaticum. High concentrations of arsenate (100 g As · l^-1) did not affect uptake of phosphate by A. carterae and A. klebsii.