Active hexose uptake in Lemna gibba G1

Growth of autotrophically growing duck-weeds (Lemna gibba L., G1) was stimulated by sucrose. The rate of respiration increased when plants had been grown on sucrose (8.7 mol O₂ g^-1 fresh weight (FW) h^-1) and was reduced after growth without sucrose in the dark or under longday conditions (2.5 mol O₂ g^-1 FW h^-1). Photosynthesis was induced already by low light intensities (0.1 klx).Short-time application of glucose or sucrose stimulated respiration in proportion to the hexose uptake rate. Sucrose is probably not taken up as the disaccharide. The transported sugar species after addition of sucrose are its hexose moieties produced by the high activity of the cell wall invertase. Fructose stimulated to a lesser extent; mannitol induced no enhancement; 2-deoxyglucose slightly inhibited O₂ uptake. After mild carbon starvation of the plants the uptake of glucose and 3-O-methylglucose proceeded without any lag phase, with similar saturation kinetics in both cases. The initial uptake rate at substrate saturation was 2.6 mol glucose g^-1 FW h^-1 in the dark. Light stimulated hexose uptake by 2 to 3 times. The results show that Lemna gibba has an energy-dependent constitutive system for hexose uptake.Abbreviation FW fresh weight - LD long day - SD short day     

Electron transport across the plasmalemma of Lemna gibba G1

Lemna gibba L., grown in the presence or absence of Fe, reduced extracellular ferricyanide with a V _max of 3.09 mol · g^-1 fresh weight · h^-1 and a K _m of 115 M. However, Fe³⁺-ethylenediaminetetraacetic acid (EDTA) was reduced only after Fe-starvation. External electron acceptors such as ferricyanide, Fe³⁺-EDTA, 2,6-dichlorophenol indophenol or methylene blue induced a membrane depolarization of up to 100 mV, but electron donors such as ferrocyanide or NADH had no effect. Light or glucose enhanced ferricyanide reduction while the concomitant membrane depolarization was much smaller. Under anaerobic conditions, ferricyanide had no effect on electrical membrane potential difference (E_m). Ferricyanide reduction induced H⁺ and K⁺ release in a ratio of 1.16 H⁺+1 K⁺/2 e^- (in +Fe plants) and 1.28 H⁺+0.8 K⁺/2 e^- (in -Fe plants). Anion uptake was inhibited by ferricyanide reduction. It is concluded that the steady-state transfer of electrons and protons proceeds by separate mechanisms, by a redox system and by a H⁺-ATPase.Abbreviations E _m electrical membrane potential difference - EDTA ethylenediaminetetraacetic acid - DCPIP dichlorophenol indophenol - +Fe control plant - -Fe iron-deficient plant - FW fresh weight - H⁺ electrochemical proton gradient     

Membrane potential changes during transport of hexoses in Lemna gibba G1

The membrane potential (pd) of duck weed (Lemna gibba G1) proved to be energy dependent. At high internal ATP levels of 74 to 105 nmol ATP g^-1 FW, pd was between -175 and -265 mV. At low ATP levels of 23 to 46 nmol ATP g^-1 FW, pd was low, about -90 to -120 mV at pH 5.7, but -180 mV at pH 8. Upon addition of glucose in the dark or by light energy the low pd recovered to the high values. The active component of the pd was depolarized by the addition of hexoses in the dark and in the light. Hexose-dependent depolarization of the pd (= pd) followed a saturation curve similar to active hexose influx kinetics. Depolarization of the pd recovered in the dark even in the presence of the hexoses and with a 10fold enhancement in the light. Depolarization and recovery could be repeated several times with the same cell. Glucose uptake caused a maximum depolarization of 133 mV, fructose uptake half that amount, sucrose had the same effect as glucose. During 3-O-methylglucose and 2-deoxyglucose uptake the depolarizing effect was only slightly lower. The pd remained unchanged in the presence of mannitol. The glucose dependent pd and especially the rate of pd recovery proved to be pH-dependent between pH 4 and pH 8. It was independent of the presence of 1 mM KCl. Although no pH could be measured in the incubation medium, these results can be best explained by a H⁺-hexose cotransport mechanism powered by active H⁺ extrusion at the plasmalemma.Abbreviations LD longday - SD shortday - pd membrane electropotential difference - pd maximum membrane potential depolarization - L light - D dark - FW fresh weight - d days of culture of Lemna gibba - 1X perfusing solution without sugar, see methods     

Effect of abscisic acid on membrane potential and transport of glucose and glycine in Lemna gibba G1

The membrane potential of Lemna gibba G1 was measured with a microelectrode; glucose and glycine uptake were measured with ¹⁴C-labeled substances. The membrane potential was increased by 85 mV on the average, after the plants had been pretreated with 10 M abscisic acid (ABA) for more than 30 min. This effect is not linked to the endogenous level of soluble sugars. The concentration of these soluble sugars was increased to more than 200% by pretreatment of the plants with ABA, however, the respiration of the plants was not affected. ABA stimulated uptake of glucose and glycine. Glucose- and glycine-dependent depolarization and repolarization of the membrane was altered: depolarization was less and repolarization was slower; during uptake of glycine, the first typical phase of repolarization was suppressed. The data suggest that ABA interferes with the primary steps of substrate uptake.Abbreviations ABA abscisic acid - FW fresh weight - IAA indole acetic acid - pd membrane potential difference - 1× perfusing solution (see methods) - H⁺ electrochemical proton gradient - pd solute-induced maximum depolarization of the membrane     

Phosphate uptake inLemna gibba G1: energetics and kinetics

Phosphate uptake was studied by determining [³²P]phosphate influx and by measurements of the electrical membrane potential in duckweed (Lemna gibba L.). Phosphate-induced membrane depolarization (E _m ) was controlled by the intracellular phosphate content, thus maximal E _m by 1 mM H₂PO ₄ ^- was up to 133 mV after 15d of phosphate starvation. The E _m was strongly dependent on the extracellular pH, with a sharp optimum at pH 5.7. It is suggested that phosphate uptake is energized by the electrochemical proton gradient, proceeding by a 2H⁺/H₂PO ₄ ^- contransport mechanism. This is supported also by the fusicoccin stimulation of phosphate influx. Kinetics of phosphate influx and of E _m , which represent mere plasmalemma transport, are best described by two Michaelis-Menten terms without any linear components.Abbreviations E _m electrical membrane potential difference - E _m phosphate-induced, maximal membrane depolarization - FW fresh weight     

Energy coupling for membrane hyperpolarization in Lemna: Evidence against an ATP-fueled electrogenic pump as the exclusive mechanism

The vacuolar electrical potential of Lemna paucicostata 6746 has an active component of about-130 mV. This hyperpolarization above the diffusion potential was maintained when dicyclohexyl carbodiimide (DCCD) or arsenate (0.1 mM or 5 mM final concentrations, respectively) were added in the light or after the plants had been kept in darkness for 1 h. The ATP level was reduced to 11±3% by DCCD and to 56±6% by arsenate under conditions identical to those during the potential measurements. In this report, it is discussed whether these results could be interpreted in terms of a putative electrogenic ATPase in the plasma membrane of Lemna. Rb⁺-influx in illuminated plants was 12.5% or 52% of the control when ATP generation was inhibited by DCCD or arsenate. This finding is regarded as justifying the assumption that the availability of ATP at plasmalemma-located transport sites is drastically decreased by these inhibitors.A passive proton-permeability in the cell membrane was induced with different concentrations of carbonyl cyanide m-chlorophenyl hydrazone (CCCP). The potential decrease, caused by the current through this shunt, was not affected by DCCD. It therefore seems less conceivable that the cell membrane remains hyperpolarized because of an increase of membrane resistance concomitant to the inhibition of the pump.The significance of respiratory processes for membrane hyperpolarization is displayed by the depolarizing action of anoxia or KCN. As ATP was found to be non-limiting under these conditions, the inhibition of the electrogenic pump is regarded as being in discord with the concept of an electrogenic ATPase, which is solely responsible for membrane hyperpolarization.Abbreviations CCCP carbonyl cyanide m-chlorophenyl hydrazone - DCCD N, N-dicyclohexyl carbodiimide - DES diethylstilbestro - DNP 2,4-dinitrophenol - POPOP 1,4-bis (2-(5-phenyloxazolyl))-benzene - PPO 2,5-diphenyloxazole     

Evidence for electrogenic proton extrusion by subepidermal cells of Lemna paucicostata 6746

The cell potential of Lemna paucicostata 6746 was measured between the vacuole and the external solution. The potential in the dark (-202 mV) could be depolarized with 0.1 mM dicyclohexyl carbodiimide (DCCD) or 1 mM arsenate to-81 mV. The hyperpolarization above the latter value is therefore attributed to an ATP-dependent process. The cell potential showed a significant dependence upon the pH of the external solution. The change in the potential induced by a jump in pH between two certain values, was reversible and independent of the mode of performing the pH change (stepwise or at once). The DCCD-or arsenate-depolarized potential did not respond to external pH changes. A 0.1 mM ammonium chloride solution depolarized the cell potential reversibly to-83 mV. This potential-change could be greatly reduced by simultaneous addition of 5 mM Na isobutyrate. The pH sensitivity of the cell potential is ascribed to changes in the rate of proton extrusion upon altering the proton gradient across the plasmalemma. The effects of ammonium and isobutyrate are interpreted as being the consequence of pH shifts at the inner face of the plasmalemma, caused by the permeation of the undissociated form of the weak acid or base. A critical discussion of an alternative interpretation for the ammonium effect is presented.Abbreviation DCCD N,N-dicyclohexyl carbodiimide     

pH and membrane-potential changes during glucose uptake inLemna gibba G1 and their response to light

Extracellular pH was measured with a microelectrode positioned over the lower surface of singleLemna gibba plants. Upon addition of glucose, a transient extracellular alkalinization occurred. Saturated extracellular pH changes were observed with 5 mM glucose. Simultaneously, the membrane potential difference of –250 mV in the dark measured with intracellular glass micropipettes, trnasiently decreased by 105 mV. Uptake of [¹⁴C]glucose and extracellular alkalinization was enhanced by light whereas glucose-induced membrane-potential changes were reduced in the light and became even smaller with increasing the preillumination time. Glucose uptake was optimal at pH 6. The results are taken as further evidence in favor of H⁺-glucose cotransport inLemna.Dedicated to Professor W. Simonis on the occasion of his 70th birthdayUniversity of Missouri Agricultural Experiment Station Journal Series, paper No. 8266     

Amino acid uptake in plasma membrane vesicles isolated from proliferating tumor cells and tissues

Summary The transport of L-alanine, a natural substrate of system A, across plasma membrane vesicle preparations has been studied in the early stages of rat DENA-PH hepato-carcinogenesis and in a very undifferentiated rat ascites hepatoma cell line (Yoshida AH-130) in the exponential and stationary phase of growth.Kinetic analyses indicated an increase of the V_max value in DENA-PH-treated rats 30 h after partial hepatectomy as well as in exponential growing Yoshida ascites cells. In DENA-PH-treated rats the Km value was drastically reduced 7 and 60 days after surgery, when enzyme-altered hyperplastic and preneoplastic lesions were present in rat liver. Drastically reduced Km values were also found in Yoshida ascites cells.The results suggest that an altered alanine transporter might take place in liver plasma membranes from carcinogen-treated rats. This appears to occur also in an established tumor cell line, grown in vivo.Abbreviations AAF 2-acetylaminofluorene - DENA diethylnitrosamine - PH partial hepatectomy - PMSF phenylmethanesulfonyl fluoride     

Amino acid uptake by Saccharomyces cerevisiae plasma membrane vesicles

A procedure is described which allows for the efficient separation of Saccharomyces cerevisiae plasma membranes from other cellular membranes by discontinuous sucrose density gradient centrifugation. After vesiculization in an osmotic stabilization buffer the plasma membrane vesicles retain the ability to transport amino acids. Amino acid uptake was affected by the proton gradient dissipator $\text{m-}\text{chlorocarbonylcyanide}$ phenylhydrazone and was dependent, in some cases, on the presence of sodium ion.     

A biosensor for all D-amino acids using evolved D-amino acid oxidase

Determination of the D-amino acid content in foods and in biological samples is a very important task. In order to achieve this goal we developed a biosensor employing the flavoenzyme D-amino acid oxidase from the yeast Rhodotorula gracilis. To produce a device in which the D-amino acid composition does not alter the results, both the wild-type and a number of mutants obtained by rational design and directed evolution approaches were used. An analysis of D-amino acid oxidase mutants activity on D-amino acid mixtures containing various ratios of neutral, acidic, and basic substrates identified the Amberzyme-immobilized T60A/Q144R/K152E and M213G mutants as the best choice: their response shows an only limited dependence on the solution composition when at least 20% of the D-amino acid is made up of D-alanine (standard error is approximately 5-9%). This is the first report, to our knowledge, demonstrating that the entire D-amino acid content can be determined by using a screen-printed electrode amperometric biosensor, with a detection limit of 0.25 mM and a mean response time of 10-15 min. The D-amino acid assay based on R. gracilis DAAO-biosensor is inexpensive, simple to perform, and rapid: the D-amino acid concentration of a variety of biological samples can be investigated using this assay.     

Amino acid uptake and protein synthesis during early meiosis in Saccharomyces cerevisiae

Uptake and incorporation into proteins of an externally supplied amino acid were followed during early meiosis in yeast. Under conditions optimal for development, an insufficient permeability of the cell leads to an incorporation pattern which reflects the changes in the activity of the amino acid transporting system rather than those in protein synthesis. A more correct picture of protein synthesis during early meiosis is obtained by the use of a mutant with an enhanced level of amino acid uptake.Abbreviation SPM Sporulation medium     

Chloroquine,a novel inhibitor of amino acid transport by rat renal brush border membrane vesicles

Summary Chloroquine is an antimalarial and antirheumatic lysosomotropic drug which inhibits taurine uptake into and increases efflux from cultured human lymphoblastoid cells. It inhibits taurine uptake by rat lung slices and affects the uptake and release of cystine from cystinotic fibroblasts. Speculations on its mode of action include a proton gradient effect, a non-specific alteration in membrane integrity, and membrane stabilization. In this study, the effect of chloroquine on the uptake of several amino acids by rat renal brush border membrane vesicles (BBMV) was examined. Chloroquine significantly inhibited the secondary active, NaCl-dependent component of 10µM taurine uptake at all concentrations tested, but did not change equilibrium values. Analysis of these data indicated that the inhibition was non-competitive. Taurine uptake was reduced at all osmolarities tested, but inhibition was greatest at the lowest osmolarity. Taurine efflux was not affected by chloroquine, nor was the NaCl-independent diffusional component of taurine transport. Chloroquine (1 mM) inhibited uptake of the imino acids L-proline and glycine, and the dibasic amino acid L-lysine. It inhibited the uptake of D-glucose, but not the neutral-amino acids L-alanine or L-methionine. Uptake of the dicarboxylic amino acids, L-glutamic acid and L-aspartic acid, was slightly enhanced. With regard to amino acid uptake by BBMV, these findings may support some of the currently proposed mechanisms of the action of chloroquine but further studies are indicated to determine why it affects the initial rate of active amino acid transport.     

Secondary transport of amino acids by membrane vesicles derived from lactic acid bacteria

Lactococci are fastidious bacteria which require an external source of amino acids and many other nutrients. These compounds have to pass the membrane. However, detailed analysis of transport processes in membrane vesicles has been hampered by the lack of a suitable protonmotive force (pmf)-generating system in these model systems. A membrane-fusion procedure has been developed by which pmf-generating systems can be functionally incorporated into the bacterial membrane. This improved model system has been used to analyze the properties of amino acid transport systems in lactococci. Detailed studies have been made of the specificity and kinetics of amino acid transport and also of the interaction of the transport systems with their lipid environment. The properties of a pmf-independent, arginine-catabolism specific transport system in lactococci will be discussed.Abbreviations pmf protonmotive force - transmembrane electrical potential - pH transmembrane pH gradient - PE phosphatidylethanolamine - PC phosphatidylcholine Paper adapted from a treatise Secondary Transport of Amino Acids by Membrane Vesicles Derived from Lactic Acid Bacteria and awarded the Kluyver Prize 1988 by the Netherlands Society of Microbiology.     

Amino acids,polyamines and proteins during seed germination of two species of Dipterocarpaceae

Summary Amino acid, polyamine and protein concentrations in seeds and their evolution during seed germination of two dipterocarp species, Hopea odorata and Dipterocarpus alatus, were determined with the help of a multianalytical system. Glutamic acid and glutamine were the major amino compounds present. Hopea seeds also contain high levels of aspartic acid/asparagine, serine, threonine, arginine and alanine, while those of Dipterocarpus contain high levels of alanine, arginine and threonine. These species were quite different in their germination behavior and thus in their protein and amine metabolism rates. In Hopea, polyamines increased during the first 3 days of germination and reached a maximum by the 3rd day, 1 day before maximum germination rate. In Dipterocarpus polyamines reached their maximum at the 6th day while maximum germination rate is observed by the 7th day. This suggests that polyamine compounds could play a role in the early part of the germination process in Hopea and Dipterocarpus seeds. The possibility that control of polyamine biosynthesis could be used for the establishment of biochemical methods to improve seed storage and to control germination of these recalcitrant seeds is discussed.     

D-amino acid N-acetyltransferase of Saccharomyces cerevisiae: a close homologue of histone acetyltransferase Hpa2p acting exclusively on free D-amino acids

d-Amino acid N-acetyltransferase is a unique enzyme of Saccharomyces cerevisiae acting specifically on d-amino acids. The enzyme was found to be encoded by HPA3, a putative histone/protein acetyltransferase gene, and we purified its gene product, Hpa3p, from recombinant Escherichia coli cells. Hpa3p shares 49% sequence identity and 81% sequence similarity with a histone acetyltransferase, Hpa2p, of S. cerevisiae. Hpa3p acts on a wide range of d-amino acids but shows extremely low activity toward histone. However, Hpa2p does not act on any of the free amino acids except l-lysine and d-lysine. Kinetic analyses suggest that Hpa3p catalyzes the N-acetylation of d-amino acids through an ordered bi-bi mechanism, in which acetyl-CoA is the first substrate to be bound and CoA is the last product to be liberated.     

Influence of permeant acids and bases on net potassium uptake by Chlorella

Salts of membrane-permeant acids and bases strongly influence net K uptake by Chlorella fusca. Na phenylacetate, acetate, isobutyrate, propionate, and butyrate added to buffered algal suspensions containing 0.1–0.2 mM KCl increasingly stimulated net K uptake. In contrast, K release was induced by the chlorides of imidazole, ammonia and methylamine. All these effects were found in the light and, less pronounced, in the dark. The dependence of the net K movements on the concentrations of the salts added and on the pH of the medium suggests that the free acids or bases are the effective agents. Between net uptake of K and uptake of labeled propionate a molar ratio close to 1 was found. It is concluded that the internal pH of the cell is changed by the permeants. Acidification of the cytoplasm stimulates extrusion of protons coupled to uptake of K. Alcalization brings about proton uptake and K extrusion. Apparently K/H exchange serves as a pH-stat of the cell.Abbreviations DA dark/air - DMO 5,5-dimethyloxazolidine-2,4-dione - LA light/air - PIPES piperazine-N-N-bis-2-ethane sulfonic acid     

Bioassays with a floating aquatic plant (Lemna minor) for effects of sprayed and dissolved glyphosate

Macrophytes in forested areas and in prairie wetlands furnish critical habitat for aquatic communities and for several species of birds and mammals. North American agriculture relies heavily on herbicides and these compounds are detected routinely in surface waters of Western Canada. The question is whether these residues have biological meaning. There is surprisingly little literature on the responses of macrophytes to herbicides, or indeed to other chemicals. Previously we have used common duckweed in efforts to detect effects of herbicides and other chemicals. Duckweed clones were developed from local collections and grown axenically. In this study the plants were exposed to glyphosate herbicide either by dissolving formulated Roundup^® (Monsanto Canada Inc.) in the culture media or by spraying of the cultures in a laboratory spray chamber. Plant growth was monitored by counting the fronds present on several occasions over a 2-week period following treatment and by taking wet and dry weights of plants after the final counting period. Plant growth, as measured by increased numbers of fronds or increased wet or dry weights was relatively insensitive to glyphosate dissolved in the culture medium. However, the plants were killed by application of glyphosate as a spray.     

Glutamine uptake by a sodium-dependent secondary transport system inCorynebacterium glutamicum

Corynebacterium glutamicum took up glutamine by a sodium-dependent secondary transport system. Both the membrane potential and the sodium gradient were driving forces. Glutamine uptake showed Michaelis-Menten kinetics, with aK _m of 36 μM and aV _max of 12.5 nmol min⁻¹ (mg dry weight)⁻¹ at pH 7. Despite a pH optimum in the alkaline range around pH 9, it was shown that uncharged glutamine is the transported species. The affinity for the cotransported sodium was relatively low; an apparentK _m of 1.4 mM was determined. Among various substrates tested, only asparagine, when added in 50-fold excess, led to an inhibition of glutamine transport. It was concluded that glutamine uptake occurs via a specific transport system in symport with at least one sodium ion.     

Production of amino acids by free-living heterotrophic nitrogen-fixing bacteria

Summary Interest of microbial production of amino acids has been increased greatly since development of biotechnological methods. These methods represent a perspective way applied in a future large-scale manufacture of inexpensive amino acids. In this context, the isolation of producing organisms that may be exploited in the desing of alternative methods for the production of amino acids could be of primary importance.In this review we will describe the liberation of amino acids (methionine, lysine, arginine, tryptophane and glutamic acid) byAzotobacter andAzospirillum during growth in culture media with different carbon sources under diazotrophic and adiazotrophic conditions. These organisms may be useful in developing new methods for the industrial production of amino acids.