Transport of ascorbate into plasma membrane vesicles ofPhaseolus vulgaris L.

Summary Incubation of bean hook plasma membrane vesicles in the presence of L-[¹⁴C]ascorbate (ASC) resulted in a specific recovery of significant levels of the ligand with the vesicles. The strong decrease in radioactive ASC detected after hypotonic disruption of the vesicles or after an assay at 4 °C indicated that ASC was probably transported from the medium into the lumen of the membrane vesicles. The concentration kinetics of this presumptive transport process revealed a saturation curve which best fitted a biphasic model. Each phase in this model showed Michaelis-Menten type kinetics. The kinetic parameters for the different phases were calculated to be 14 and 79 M (K _m1 andK _m2) and 26 and 53 pmol/min · mg protein (V _max1 andV _max2). High concentrations of iso-ascorbate, dehydroascorbate (DHA) or non-labelled ASC significantly reduced the uptake of the radioactive vitamin. It was demonstrated that sugar or amino acid carriers are not involved in the ASC transport reaction. Generation of transmembrane cation gradients (H⁺, K⁺, Ca²⁺, Na⁺) or addition of sulfhydryl reagents (pCMBS or NEM) did not affect the ASC uptake in any way. It is suggested that ASC is taken up by a facilitated diffusion mechanism.Abbreviations ASC ascorbate - DHA dehydroascorbate - FCCP carbonyl cyanidep-trifluoromethoxyphenylhydrazone - NEM N-ethylmaleimide - pCMBS p-chloromercuribenzenesulfonic acid     

Uptake of [14C]sucrose in isolated minor-vein networks of Commelina benghalensis L

Maceration with pectinase (4.5h) of Commelina benghalensis L. leaves stripped at either side yielded isolated vein networks consisting of four to five secondary veins and tertiary cross veins (=minor veins). Examination with Evans Blue and injection of Fluorescein F showed that 80% of the veins were viable. Proof of normal functioning of isolated minor veins was that [¹⁴C]sucrose fed to an apical vein network attached to the remaining intact part of the leaf was absorbed and finally arrived in the petiole. Sucrose uptake by veins obeyed Michaelis-Menten kinetics (K _m 5·10^-4 mol l^-1; V _max (light) 3.2 mol h^-1 g^-1 fresh weight, V _max (dark) 1.5 mol h^-1 g^-1 fresh weight). A linear component, not inhibited by carbonylcyanide m-chlorophenylhydrazone and p-chloromercuribenzenesulfonic acid, was present. Maximal uptake took place at 5 mmol l^-1 K⁺; concentrations of K⁺ higher than 10 mmol l^-1 decreased the rate of uptake. The uptake rates by isolated veins and veins in situ (in disks) were in the same order of magnitude. Altogether, isolated veins promise to be a useful system for the study of loading.Abbreviations CCCP carbonylcyanide m-chlorophenylhydrazone - EDTA ethylenediamine tetraacetic acid - PCMBS p-chloromercuribenzenesulfonic acid     

Evidence for the involvement of plasma-membrane-bound nitrate reductase in signal transduction during blue-light stimulation of nitrate uptake in Chlorella saccharophila

Nitrate uptake in Chlorella saccharophila (Krüger) Nadson was found to be stimulated by blue light, leading to a doubling of the rate. In the presence of background red light (300 mol photons · m^-2 · s^-1), only 15–20 mol photons · m^-2 · s^-1 of blue light was sufficient to saturate this increased uptake rate. Incubation of Chlorella cells with anti-nitrate-reductase immunoglobulin-G fragments inhibited blue-light stimulation. However, ferricyanide (10 M) doubled and dithiothreitol (100 M) inhibited the stimulatory effect of blue light. Among the protein-kinase inhibitors used, only staurosporine (10 M) prevented the blue-light stimulation. Phosphatase inhibitors were without effect and sodium vanadate totally inhibited nitrate uptake, pointing to an involvement of the plasma-membrane ATPase. Preincubation of the cells with calmodulin antagonists or calcium ionophores did not significantly reduce blue-light stimulation of nitrate uptake. The data are discussed with regard to transduction of the signal for blue-light stimulation of nitrate uptake and the possibility that the plasma-membrane-bound nitrate reductase is the blue-light receptor.Abbreviations Chl chlorophyll - DMSO dimethylsulfoxide - 1,2-DHG 1,2-dihexanoylglycerol - ML-9 1-(5-chloronaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine - NR nitrate reductase - H-7 1-(5-isoquinolinyl-sulfonyl)-2-methylpiperazine - IgG immunoglobulin G - PFD photon flux density - PM plasma membrane - W-7 N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide This work was supported by a grant from the Deutsche Forschungs-gemeinschaft to R.T.     

Transport of ascorbate into protoplasts ofNicotiana tabacum Bright Yellow-2 cell line

Summary The uptake of ascorbate into protoplasts isolated from aNicotiana tabacum Bright Yellow-2 (BY-2) cell suspension culture was investigated. Addition of¹⁴C-labelled ascorbate to freshly isolated protoplasts resulted in a time- and substrate-dependent association of radioactive molecules with the protoplasts. The kinetic characterisation of this presumptive uptake revealed kinetics of Michaelis-Menten type with an apparent maximal uptake activity of 24 pmol/min·10⁶ protoplasts and an apparent affinity constant of 139 M. The amount of ascorbate molecules transported into_{N. tabacum} protoplasts decreased when nonlabelled dehydroascorbate or iso-ascorbate were added but was not affected by addition of 5,6-o-cyclohexylidene ascorbate or ascorbate-2-sulfate. These data indicate a carrier-mediated uptake of ascorbate into the protoplasts that shows a high structural specificity. To investigate which redox status of ascorbate is preferentially taken up by theN. tabacum protoplasts, transport was tested in the presence of various compounds that can affect the redox status of ascorbate. Testing uptake in the presence of a reductant, dithiothreitol, resulted in a significant and concentration-dependent inhibition of the amount of ascorbate molecules transported into the protoplasts. On the other hand, ascorbate uptake was significantly stimulated in the presence of the enzyme ascorbate oxidase. Ferricyanide did not affect ascorbate transport. Inhibition studies revealed that ascorbate uptake in the protoplasts is sensitive to addition of sulfhydryl reagents N-ethyl maleimide andp-chloro-mercuribenzenesulfonic acid and to a disruption of the proton gradient by the protonophore carbonylcyanide-3-chlorophenylhydrazone. The uptake of ascorbate was also inhibited by addition of cytochalasin B but not sensitive to addition of phloretin or sulfinpyrazone. Taken together these data indicate the presence of an ascorbate transport system in the plasma membrane ofN. tabacum protoplasts and suggest dehydroascorbate as the preferentially transported redox species. The putative presence of different carriers for reduced and oxidised ascorbate in the plasma membrane is discussed.Abbreviations Asc ascorbate - BY-2 Bright Yellow 2 - CCCP carbonylcyanide-3-chlorophenylhydrazone - DHA dehydroascorbate - DTT dithiothreitol - MS medium Murashige and Skoog medium - NEM N-ethylmaleimide - pCMBS p-chloromercuribenzenesulfonic acid     

Iron uptake by leaf mesophyll cells: The role of the plasma membrane-bound ferric-chelate reductase

The uptake of ⁵⁹Fe from FeCl₃, ferric (Fe³⁺) citrate (FeCitr) and Fe³⁺-EDTA (FeEDTA) was studied in leaf mesophyll of Vigna unguiculata (L.) Walp. Uptake rates decreased in the order FeCl₃>FeCitrFeEDTA, and uptake depended on an obligatory reduction step of Fe³⁺ to Fe²⁺, after which the ion could be taken up independently of the chelator, citrate. Uptake was strongly increased by photosynthetically active light (>630 nm), and kinetic analysis revealed saturation kinetics with a K _m (FeCitr) of 80–110 M. In the presence of an external Fe²⁺ scavenger, bathophenanthroline disulfonate, the mesophyll also reduced external FeCitr with a K _m of approx. 50–60 M. The reduction rates for FeCitr were five-to eightfold higher than necessary for uptake. Purified plasma membranes from leaves revealed an NADH-dependent FeCitr- and FeEDTA-reductase activity, which had a pH optimum of 6.5–6.8 and a K _m of approx. 20 M for NADH. Under anaerobic conditions, a K _m of 130–170 M for ferric chelates was obtained, while in the presence of oxygen a K _m (FeCitr) of approx. 100 M was found. It is concluded that the leaf plasma membrane provides a ferric-chelate-reductase activity, which plays a crucial role in iron uptake of leaf cells. Under in-vivo conditions, however, reactive oxygen species or strong (blue) light may also contribute to the obligatory reduction of Fe³⁺ prior to uptake.Abbreviations BPDS bathophenanthroline disulfonate - DCMU 3-(3,4 dichlorophenyl)-1,1-dimethyl urea - FCR ferricchelate reductase - FeCitr Fe³⁺-citrate - FeEDTA Fe³⁺-EDTA - PM plasma membrane This work was supported by the SCIENCE program of the European Community (contract no. SC1000344; P.R.M.). We wish to thank P. Siersma and C. Winter for their cooperation at the Central Isotope Laboratory of the Biological Centre of the University of Groningen.     

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.     

Processes Maintaining Calcium Homeostasis in Acinar Cells of the Rat Submandibular Salivary Gland

Using a Ca²⁺-sensitive fluorescent indicator, fura-2/AM, we recorded calcium transients in secretory cells of isolated acini of the rat submandibular salivary gland; these transients were induced by hyperpotassium-induced depolarization (after an increase in [K⁺] _e up to 50 mM) of the plasma membrane of the above cells. Calcium transients were significantly suppressed by 50 M nifedipine. Addition of 10 M carbonyl cyanide m-chlorophenylhydrazone to the normal extracellular solution was accompanied by a rise in [Ca²⁺] _i , whereas when hyperpotassium solution is used the effect was less expressed. Blockers of CA²⁺-ATPase in the cellular membrane and in the endoplasmic reticulum, eosin Y (5 M) and cyclopiazonic acid (CPA, 5 M), respectively, evoked a significant increase in [Ca²⁺] _i and a decrease in the K⁺-depolarization-induced calcium transient. Extracellular application of caffeine (2, 10, or 30 mM) was accompanied by a concentration-dependent rise in [Ca²⁺] _i . Therefore, potassium depolarization of the plasma membrane of acinar cells of the rat submandibular salivary gland activates both the voltage-dependent Ca²⁺ influx and Ca²⁺-induced Ca²⁺ release from the endoplasmic reticulum; the initial level of [Ca²⁺] _i was restored at the joint involvement of Ca²⁺-ATPases in the plasma membrane and the membranes of the endoplasmic reticulum and mitochondria.     

A nystatin-resistant mutant of Rhodotorula gracilis. Transport properties and sterol content

A nystatin-resistant mutant of Rhodotorula gracilis was obtained by treatment of the wild strain cells with N-methyl-N-nitro-N-nitrosoguanidine and selected on agar plates containing 150 g nystatin/ml. Three important transport functions of the plasma membrane of mutant cells: the accumulation of monosaccharides, the generation and maintenance of the pH-gradient and of the membrane potential, as well as the cell respiration were insensitive to at least 10^-5 M nystatin. This concentration of nystatin inhibited completely all these processes in wild strain cells. Analysis of cellular sterols revealed a defect of ergosterol biosynthesis in the mutant, which was localized at the last oxidative step between 5,6-dihydroergosterol and ergosterol.     

Shifts in the intracellular ATP pools of immobilisedNostoc cells (Cyanobacteria) induced by water stress

Summary Immobilised, desiccated cells ofNostoc commune UTEX 584 have the capacity to increase the size of their extractable intracellular ATP pool upon rewetting. The time taken to recover the pool size depends on the conditions of storage at a particular water potential and the duration of storage. Under the conditions employed, the rewetting of cells induced an increase in ATP pool size at the expense of photophosphorylation or electron transport (oxidative) phosphorylation. The rise in the ATP pool size was instantaneous and was shown to be due to ATP synthesis. This increase did not occur when cells were rewetted in the presence of sodium azide (10 mmol/l), while a partial inhibition was observed with CCCP (carbonyl cyanidem-chlorophenylhydrazone; 2 mol/l). For cells dried at more extreme water potentials, the lag ofc 48 h observed before the ATP pool reached control values is of similar duration to that observed in the recovery of nitrogenase upon rewetting. Chloramphenicol (10 mol/l) stimulated significantly the upshift in the size of the ATP pool ofNostoc cells upon rewetting, yet inhibited completely the rise in nitrogenase activity.     

Noncyclic electron transport in chromatophores from photolithotrophically grown Rhodobacter sulfidophilus

Chromatophores isolated from the marine phototrophic bacterium Rhodobacter sulfidophilus were found to photoreduce NAD with sulfide as the electron donor. The apparent K _m for sulfide was 370 M and the optimal pH was 7.0. The rate of NAD photoreduction in chromatophore suspensions with sulfide as the electron donor (about 7–12 M/h·mol Bchl) was approximately onetenth the rate of sulfide oxidation in whole cell suspensions. NAD photoreduction was inhibited by rotenone, carbonyl cyanide-m-chlorophenylhydrazone, and antimycin A. Sulfide reduced ubiquinone in the dark when added to anaerobic chromatophore suspensions. These results suggest that electron transport from sulfide to NAD involves an initial dark reduction of ubiquinone followed by reverse electron transport from ubiquinol to NAD mediated by NADH dehydrogenase.Abbreviations Bchl bacteriochlorophyll - CCCP carbonyl cyanide-m-chlorophenylhydrazone - MOPS 3(N-morpholino)-propane sulfonate - Uq ubiquinone     

Sucrose-dependent H+ transport in plasma-membrane vesicles isolated from sugarbeet leaves (Beta vulgaris L.)

The mechanism of sucrose transport across the plasma membrane (PM) was investigated in membrane vesicles isolated from sugarbeet (Beta vulgaris L.) leaves. In the presence of a membrane potential () generated as a K⁺-diffusion potential, negative inside, sucrose induced a rapid and transient alkalization of the medium. Alkalization was inhibited by carbonyl cyanide m-chlorophenylhydrazone, was specific for the sucrose sugar and was dependent on the sucrose concentration with a K_m of approx. 1 mM. Sucrose-induced alkalization and sucrose transport were inhibited by the sulfhydryl-reactive reagent, p-chloromercuribenzene sulfonic acid, and by the histidine-reactive reagent, diethyl pyrocarbonate. Parallel analysis of sucrose uptake and alkalization indicated that the stoichiometry of sucrose uptake to proton consumed was 11. These results provide clear evidence that the saturable mechanism of sucrose transport across the PM in plants is a coupled H⁺-sucrose symport.Abbreviations and Symbols CCCP carbonyl cyanide m-chlorophenylhydrazone - DEPC diethyl pyrocarbonate - PCMBS p-chloromercuribenzene sulfonic acid - pH pH gradient - membrane potential difference - PM plasma membrane The financial support for a portion of thus study was provided by the Deutsche Forschungsgemeinschaft. We thank Kimberly A. Mitchell for her excellent technical assistance and dedicate this report to the memory of Mr. William A. Dungey.     

Alkaloid N-oxides as transport and vacuolar storage compounds of pyrrolizidine alkaloids in Senecio vulgaris L

Cell-suspension cultures of pyrrolizidinealkaloid-producing species selectively take up and accumulate senecionine (sen) and its N-oxide (sen-Nox). Cultures established from non-alkaloid-producing species are unable to accumulate the alkaloids. The uptake and accumulation of ¹⁴C-labelled alkaloids was studied using a Senecio vulgaris cell-suspension culture as well as protoplasts and vacuoles derived from it. The alkaloid uptake exhibits all characteristics of a carrier-mediated transport. The uptake of sen-Nox follows a multiphasic saturation kinetics. The K_m-values for sen Nox of 53 M and 310 M are evaluated. Senecionine competitively inhibits sen-Nox uptake, indicating that the tertiary alkaloid and its N-oxide share the same membrane carrier. The N-oxide of sen shows a pH optimum below 5.5, whereas sen is taken up over a range from pH 4 to 8. Activation energies of 90 and 53 kJ·mol^-1 are calculated for sen-Nox and sen transport, respectively. At concentrations of 10 to 100 M, sen-Nox is rapidly taken up by cells and protoplasts; within 2 h >90% of total N-oxide is within the cells. By contrast the uptake of sen is less efficient. Vacuoles isolated from protoplasts preloaded with sen-Nox totally retained the alkaloid N-oxide, whereas sen is rapidly lost during the procedure of vacuole preparation. N-oxidation converts the weak lipophilic tertiary base into a charged polar molecule which is excellently adapted to serve as the cellular transport and storage form of pyrrolizidine alkaloids.Abbreviations CCCP carbonylcyanide m-chlorophenylhydrazone - DCCD N,N-dicyclohexylcarbodiimide - DIDS 4,4-diisothiocyanatostilbene-2,2-disulfonic acid - DNP 2,4-dinitrophenol - sen senecionine - sen-Nox senecionine N-oxide     

Auxin carriers in Cucurbita vesicles

Carrier-mediated uptake of indole-3-acetic acid (IAA) by microsomal vesicles from Cucurbita pepo L. hypocotyls was strongly inhibited by 2,4-dichlorophenoxyacetic acid (2,4-D; i ₅₀= 0.3 M) but only weakly by 1-naphthylacetic acid (NAA). The fully ionised auxin indol-3-yl methanesulphonic acid also inhibited (i ₅₀=3 M). The same affinity ranking of these auxins for the uptake carrier, an electroimpelled auxin anion-H⁺ symport, is demonstrable in hypocotyl segments. The specificity of the auxin-anion eflux carrier was tested by the ability of different nonradioactive auxins to compete with [³H]IAA and reduce the stimulation of net radioactive uptake by N-1-naphthylphthalamic acid (NPA), a noncompetitive inhibitor of this carrier. By this criterion, NAA and IAA had comparable affinities, with 2,4-D interaction more weakly. Stimulation of [³H]IAA uptake by NAA, as a result of competition for the efflux carrier, could also be demonstrated when a suitable concentration of 2,4-D was used selectively to inhibit the uptake carrier. However, when [³H]NAA was used, no stimulation of its association with vesicles by NPA, 2,3,5-triiodobenzoic acid, or nonradioactive NAA was found. In hypocotyl segments, [³H]NAA net uptake was much less sensitive to NPA stimulation than was [¹⁴C]IAA uptake. The apparent contradictions concerning NAA could be explained by carrier-mediated auxin efflux making a smaller relative contribution to the overall transport of NAA than of IAA. The relationship between carrier specificity as manifested in vitro and the specificity of polar auxin transport is discussed.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - ION3 mixture of 4 M carbonylcyanide m-chlorophenylhydrazone, nigericin and valinomycin - IMS indol-3-yl methanesulphonic acid - NAA 1-naphthylacetic aci - NPA N-1-naphthylphthalamic acid     

Transport of branched-chain amino acids in Corynebacterium glutamicum

The transport of branched-chain amino acids was characterized in intact cells of Corynebacterium glutamicum ATCC 13032. Uptake and accumulation of these amino acids occur via a common specific carrier with slightly different affiniteis for each substrate (K _m[Ile]=5.4 M, K _m[Leu]=9.0 M, K _m[Val]=9.5 M). The maximal uptake rates for all three substrates were very similar (0.94–1.30 nmol/mg dw · min). The optimum of amino acid uptake was at pH 8.5 and the activation energy was determined to be 80 kJ/mol. The transport activity showed a marked dependence on the presence of Na⁺ ions and on the membrane potential, but was independent of an existing proton gradient. It is concluded, that uptake of branched-chain amino acid transport proceeds via a secondary active Na⁺-coupled symport mechanism.Abbreviations CCCP Carboxyl cyanide m-chlorophenylhydrazone - dw dry weight - MES 2[N-morpholino]ethanesulfonic acid - mon monensin - nig nigericin - TPP tetraphenylphosphonium bromide - Tris tris[hydroxymethyl]aminomethane - val valinomycin     

Accumulation of alkaloids in plant vacuoles does not involve an ion-trap mechanism

Alkaloid uptake into vacuoles isolated from a Fumaria capreolata L. cell suspension culture was investigated. The uptake is carrier-mediated as shown by its substrate saturation, its sensitivity to metabolic inhibitors and especially by its exclusive preference for the (S)-forms of reticuline and scoulerine while the (R)-enantiomers which do not occur in this plant species were strictly discriminated. The carrier has a high affinity for (S)-reticuline with a K _m=0.3 M. The rate of alkaloid uptake was 6 pmol·h^-1·l^-1 vacuole, and 0.03 mg alkaloid·mg^-1 vacuolar protein were taken up. Transport was stimulated five-to seven-fold by ATP and was inhibited by the ATPase inhibitors N,N-dicyclohexylcarbodiimide and 4-4-diisothiocyanatostilbene-2,2 disulfonic acid, as well as by the protonophore carbonyl cyanide m-chlorophenylhydrazone. A number of alkaloids did not compete with labelled (S)-reticuline for uptake into vacuoles. The uptake system is absolutely specific for alkaloids indigenous to the plant from which the vacuoles were isolated. Slight modifications of the topography of an alkaloid molecule even with full retention of its electrical charge results in its exclusion. Alkaloid efflux was also shown to be mediated by a highly specific energy-dependent carrier. These results contradict the previously proposed ion-trap mechanism for alkaloid accumulation in vacuoles. A highly specific carrier-mediated and energy-dependent proton antiport system for alkaloid uptake and release is postulated.Abbreviations CCCP carbonylcyanide m-chlorophenylhydrazone - DCCD N,N-dicyclohexylcarbodiimide - DIDS 4-4-diisothiocyanatostilbene-2,2 disulfonic acid Dedicated to Professor Harry Beevers, Santa Cruz, on the occasion of his 60^th birthday     

Transport and catabolism of proline betaine in salt-stressed Rhizobium meliloti

Exogenous proline betaine (N,N-dimethylproline or stachydrine) highly stimulated the growth rate of Rhizobium meliloti, in media of inhibitory concentration of NaCl whereas proline was ineffective. High levels of proline betaine uptake occurred in cells grown in media of elevated osmotic strength; on the contrary, only low activity was found in cells grown in minimal medium. The apparent K _m was 10 M with a maximal transport rate of 25 nmol min^-1 mg^-1 of protein in 0.3 M NaCl-grown cells. The concentrative transport was totally abolished by KCN (2 mM), 2,4-dinitrophenol (2 mM), and carbonyl cyanide-m-chlorophenyl hydrazone (CCCP 10 M) but was insensitive to arsenate (5 mM). Glycine betaine was a very potent inhibitor of proline betaine uptake while proline was not. Proline betaine transport was not reduced in osmotically shocked cells and no proline betaine binding activity was detected in the crude periplasmic shock fluid. In the absence of salt stress, Rhizobium meliloti actively catabolized proline betaine but this catabolism was blocked by increasing the osmotic strength of the medium. The osmolarity in the growth medium regulates the use of proline betaine either as a carbon and nitrogen source or as an osmoprotectant.Abbreviations LAS lactate-aspartate-salts - MSY mannitol-salts-yeast - CCCP carbonyl cyanide-m-chlorophenyl hydrazone - DCCD dicyclohexylcarbodiimide - KCN potassium cyanide - Hepes 4-(2-hydroxyethyl)-1-piperzine-ethanesulphonic acid     

Phosphate-limited growth of Chromatium vinosum in continuous culture

Chromatium vinosum DSM 185 was grown in continuous culture at a constant dilution rate of 0.071 h^-1 with sulfide as the only electron donor. The organism was subjected to conditions ranging from phosphate limitation (S _R-phosphate=2.7 M and S _R-sulfide=1.8 mM) to sulfide limitation (S _R-phosphate=86 M and S _R-sulfide=1.8 mM). At values of S _R-phosphate below 7.5 M the culture was washed out, whereas S _R-phosphate above this value resulted in steady states. The saturation constant (K ) for growth on phosphate was estimated to be between 2.6 and 4.1 M. The specific phosphorus content of the cells increased from 0.30 to 0.85 mol P mg^-1 protein with increasing S _R-phosphate. The specific rate of phosphate uptake increased with increasing S _R-phosphate, and displayed a non-hyperbolic saturation relationship with respect to the concentration of phosphate in the inflowing medium. Approximation of a hyperbolic saturation function yielded a maximum uptake rate (V _max) of 85 nmol P mg^-1 protein h^-1, and a saturation constant for uptake (K _t) of 0.7 M. When phosphate was supplied in excess 8.5% of the phosphate taken up by the cells was excreted as organic phosphorus at a specific rate of 8 nmol P mg^-1 protein h^-1.Non-standard abbreviations BChla bacteriochlorophyll a - D dilution rate; _max, maximum specific growth rate - maximum specific growth rate if the substrate were not inhibitory - K saturation constant for growth on phosphate - V _max maximum rate of phosphate uptake - K _i saturation constant for phosphate uptake - K _i inhibition constant for growth in the presence of sulfide - S _R concentration of substrate in the inflowing medium     

Ammonium uptake by cowpea Rhizobium strain MNF 2030 and Rhizobium trifolii MNF 1001

Free-living Rhizobium trifolii MNF 1001 and cowpea Rhizobium MNF 2030 grown in chemostat culture under nitrogen limitation had high activities of an ammonium permease. In phosphate-limited, nitrogen-excess conditions, strains MNF 1001 and MNF 2030 retained 20% and 50%, respectively, of the ammonium uptake activity found in nitrogen-limited cells. Uptake in both strains was sensitive to azide, cyanide, carbonyl cyanide m-chlorophenyl hydrazone and 2,4-dinitrophenol. A gradient of ammonium concentration greater than 150-fold developed across the membrane within 20 min in cells of strain MNF 1001 grown under ammonia limitation. The pH optimum for ammonium uptake by N-limited cells of both MNF 1001 and MNF 2030 was around pH 7. The apparent K _m values for the ammonium permease in strains MNF 2030 and MNF 1001 were 3.9±1.6 M and 2.0±1.6 M respectively, and the V _max was 47±2.6 nmol min^-1 (mg protein)^-1 for MNF 2030 and 101±5.1 nmol min^-1 (mg protein)^-1 for MNF 1001. Isolated snake bean bacteroids of strain MNF 2030 capable of transporting succinate and l-glutamate had no detectable ammonium uptake activity. It therefore appears that the ammonium permeases in cells of these two strains are not as tightly regulated as in R. leguminosarum MNF 3841.Abbreviations CCCP Carbonyl cyanide m-chlorophenyl hydrzone - HEPES N-Hydroxyethylpiperazine-N-2-ethanesulphonic acid     

Nitrate reductase activity in spheroplasts from Rhodobacter capsulatus E1F1 requires a periplasmic protein

Spheroplasts from Rhodobacter capsulatus E1F1 cells grown in nitrate maintained nitrate uptake and nitrate reductase activity only when they were illuminated under anaerobiosis in the presence of the periplasmic fraction and nitrate. The effects on nitrate uptake and nitrate reductase activity of spheroplasts were observed at low concentrations of periplasmic protein (about 50 x ml^-1). Periplasm from nitrate-grown cells was also required for nitrate reductase activity in spheroplasts isolated from ammonia-grown or diazotrophic cells which initially lacked this enzymatic activity. Both the maintenance of nitrate reductase in spheroplasts from nitrate-grown cells and the appearance of the activity in spheroplasts from diazotrophic cells were dependent on de novo protein synthesis. A periplasmic, 45-kDa protein which maintained the activity of nitrate reductase in spheroplasts was partially purified by gel filtration chromatography of periplasm obtained from nitrate-grown cells.Abbreviations NR nitrate reductase - CCCP carbonyl cyanide m-chlorophenylhydrazone - CAM chloramphenicol     

Active transport of proline into washed cells of Rhodopseudomonas sphaeroides f. sp. denitrificans grown with nitrate

Washed cells of Rhodopseudomonas sphaeroides f. sp. denitrificans, prepared from cultures grown anaerobically in light with NO ₃ ^- as the terminal acceptor, readily incorporated [¹⁴C]-proline both in light and in the dark. The proline uptake was coupled to the reduction of either NO ₃ ^- , NO ₂ ^- , N₂O or O₂. Light stimulated the accumulation of proline in these cells. The addition of NO ₃ ^- to washed cells in light decreased the K _m for proline from 40 M to 5.7 M. Proline transport was inhibited by antimycin A, 2-n-heptyl-4-hydroxyquinoline-N-oxide both in light and in the dark with nitrate indicating that electron transfer from both denitrification and photosynthesis are involved in this uptake. Inhibition by carbonyl cyanide-m-chlorophenyl hydrazone and 2.4-dinitrophenol indicate that proline transport is energy dependent. The H⁺/proline stoichiometry increased from 1 to 2.5 when the external pH was increased from 6.0 to 8.0. Under these conditions pro increased but p decreased markedly above pH 7.0.Abbreviations TPP⁺ Tetraphenylphosphonium bromide - EDTA ethylenediamine-tetra-acetic acid - CCCP carbonyl cyanide-m-chlorophenyl hydrazone - DNP 2,4-dinitrophenol - HOQNO 2-n-heptyl-4-hydroxyquinoline-N-oxide - DBMIB dibromo-methyl-isopropyl-p-benzoquinone - DCCD N,N-dicyclohexylcarbodiimide