The membrane potential in whole cells of Methanobacterium thermoautotrophicum

of whole cells of Methanobacterium thermoautotrophicum was estimated under varying conditions using an electrode sensitive to the lipophilic cation tetraphenylphosphonium chloride (TPP⁺). Since was found to be extremely sensitive to air, a special reaction vessel was developed to maintain strict anaerobiosis. The cells took up TPP⁺ under energization by H₂ and CO₂ thus allowing to calculate the from the distribution of TPP⁺ inside and outside the cells. The unspecific uptake of deenergized cells was around 10% of the total uptake of energized cells. TPP⁺ itself slightly diminished the , but had no effect on the formation of methane. Typical values of were in the range of-150 to-200 mV. showed a quantitative dependence on both the electron donor H₂ and the electron acceptor CO₂. NaCl stimulated the extent of the , whereas KCl slightly diminished it. Valinomycin resulted in a linear decline of , whereas the methane production rate was only slightly affected. In contrast, monensin reduced both methanogenesis and .Abbreviations pmf proton motive force - membrane potential - TPP⁺ tetraphenylphosphonium (chloride salt) - TPMP⁺ triphenylmethylphosphonium (chloride salt, if not otherwise indicated) - d.w. dry weight - t _d doubling time - PVC polyvinylchloride     

Uptake of organic matter by the roots of sweet potato: Analysis of the δ14C value of plants

Summary To determine whether sweet potato (Ipomoea batatas (L.) Poir.) takes up organic matter through the roots from the medium, the concentrations of natural¹⁴C (¹⁴C) in plant organic matter, atmospheric CO₂ and compost applied to media were examined under soil and sand culture conditions. In these experiments, three kinds of composts of different ¹⁴C were used. CO₂ derived from the mineralization of compost was continuously pumped out from the pots and its direct uptake by the leaves was prevented.¹⁴C of plant parts harvested after the 43 days experimental period were affected by the ¹⁴C of the compost in the treatments where the compost of rice straw was applied, and which suggested that a significant amount of plant carbon was derived from the compost.     

Growth yields and saturation constant of Desulfovibrio vulgaris in chemostat culture

Desulfovibrio vulgaris (strain Marburg) was grown on H₂ and sulfate as sole energy source in a chemostat limited by the sulfate supply. The biomass concentration and the sulfate concentration in the culture were determined as a function of the dilution rate. From the data a K _S (saturation constant) for sulfate of 10 M, a _max of 0.23 h^–1, and a of 13 g/mol were calculated. The organism was also grown in chemostat culture on H₂ and sulfite, H₂ and thiosulfate, and pyruvate (without sulfate). was found to be 35 g/mol, 36 g/mol, and Y _pyr ^max 10 g/mol. The growth yields are discussed with respect to ATP gains in dissimilatory sulfate reduction.     

Lysosomal sulfate efflux following glycosaminoglycan degradation: measurements in enzyme-supplemented Maroteaux-Lamy syndrome fibroblasts and isolated lysosomes

Studies using lysosomal membrane vesicles have suggested that efflux of the sulfate that results from lysosomal glycosaminoglycan degradation is carrier-mediated. In this study, glycosaminoglycan degradation and sulfate efflux were examined using cultured skin fibroblasts and lysosomes deficient in the lysosomal enzymeN-acetylgalactosamine-4-sulfatase. Such fibroblasts store dermatan sulfate lysosomally, which could be labelled biosynthetically with Na ₂ ³⁵ SO₄. The addition of recombinantN-acetylgalactosamine-4-sulfatase to the media of³⁵S labelled fibroblasts degraded up to 82% of the stored dermatan [³⁵S] sulfate over a subsequent 96 h chase and released inorganic [³⁵S] sulfate into the medium. In the presence of 4-acetamido-4-isothiocyanatostilbene-2,2-disulfonic acid (SITS), sulfate was reused to a minor extent in newly synthesized proteoglycan. Isolated granules from recombinant enzyme supplemented fibroblasts degraded stored dermatan [³⁵S]sulfate to sulfate which was rapidly released into the medium at a rate that was reduced by the extra-lysosomal presence of the lysosomal sulfate transport inhibitors SITS, Na₂SO₄ and Na₂MoO₄. SITS also inhibited dermatan sulfate turnover, although it had no effect on the action of purified recombinant enzymein vitro. These data imply that sulfate clearance occurred concomitantly with dermatan sulfate turnover in the lysosome even at high substrate loading, and that lysosome-derived sulfate, while available, is reutilized minimally in synthetic pathways.Abbreviations SITS 4-acetamido-4-isothiocyanatostilbene-2,-2-disulfonic acid - GAG glycosaminoglycan - 4S N-acetylgalactosamine-4-sulfatase - r4S recombinant humanN-acetylgalactosamine-4-sulfatase - PBS phosphate buffered saline - BME basal modified Eagle's medium - FBS fetal bovine serum - GalNAc4S-GlcA-GalitolNAc4S -(N-acetyl-d-galactosamine-4-sulfate)-(1–4)--d-glucuronic acid)-(1–3)-N-acetyl-d-[1-³H]galactosaminitol-4-sulfate - DS dermatan sulfate - MPS mucopolysaccharidosis     

Relationship between the octanol-water partition coefficient of tertiary amines and their effect of ‘selective’ uncoupling of photophosphorylation

A series of tertiary amines was investigated for effects on the transmembrane proton potential difference ( H), on photophosphorylation and on electron-flux control related to the intrathylakoid proton potential ( H_I), using isolated chloroplasts ofSpinacia oleracea L. As indicated by 9-aminoacridine fluorescence and [14C]methylamine uptake, all amines studied inhibited a build-up of H and, in parallel, ATP synthesis. Even when H was low, strong H₁-dependent electron-flux control was observed under the influence of tertiary amines. The strength of flux control in the presence of low H and the effectiveness of inhibition of ATP synthesis linearly increased with the lipophilicity of the amines. The most effective of the amines tested caused 50% inhibition of ATP synthesis at a concentration of 6 M, which is about 1000-fold lower than the concentration required for inhibition by methylamine. The data presented indicate the existence of two proton domains in the thylakoid vesicles, one of them feeding the ATP-synthase, the other the sites of pH-dependent electron-flux control. It is concluded that tertiary amines develop their action in a lipophilic domain of the thylakoid membrane, in the vicinity of the ATP-synthase complex. A mechanism for selective uncoupling and for the maintenance of H_I-dependent electron flux control in the presence of low H is discussed.Abbreviations and symbols coefficient for pH-dependent electron flux control - 9-AA 9-aminoacridine - Chl chlorophyll - I₅₀ amine concentration producing 50% inhibition of ATP-synthesis - J_e flux of photosynthetic electron transport - k _H apparent rate constant for proton efflux - H₁ proton potential in the thylakoid lumen - H₁ transthylakoid proton potential difference - p partition coefficient - q _AA coefficient for 9-aminoacridine fluorescence quenching - PS photosystem - Q quantum flux of photosynthetically active light Dedicated to Professor Wilhelm Simonis, on the occasion of his 80th birthday     

Monitoring of the mitochondrial and plasma membrane potentials in human fibroblasts by tetraphenylphosphonium ion distribution

The lipophilic cation tetraphenylphosphonium (TPP⁺) is accumulated by human skin fibroblasts across both the plasma and mitochondrial membranes. We show here that TPP⁺ uptake is indeed greatly decreased under conditions leading to de-energization of mitochondria. The TPP⁺ accumulation in the presence of the proton ionophore FCCP has been used for determination of the plasma membrane potential across the plasma membrane, after correction for potential-independent binding of TPP⁺ to cellular components. Following this procedure, a value of 75 mV has been obtained. Through the amount of TPP⁺ released by FCCP treatment, an estimate of thein situ mitochondrial membrane potential has been made. Furthermore, we report that the mitochondrial component of TPP⁺ accumulation decreases with aging of fibroblast cultures.Abbreviations _m membrane potential across thein situ mitochondria - _p membrane potential across the plasma membrane - TPP⁺ tetraphenylphosphonium - HEPES N-2-hydroxyethylpiperazineN-2-ethanesulfonic acid - FCCP carbonyl cyanidep-trifluoromethoxyphenylhydrazone     

Isolation and characterization of membrane-associated proteoglycans from normal and malignant human mammary epithelial cells

The plasma membrane-associated proteoglycans of a malignant human breast cell line (MDA-MB-231) were compared with the corresponding proteoglycans from a normal cell line (HBL-100). The labeled proteoglycans were isolated from the plasma membranes of cells grown in the presence of [³H]glucosamine and [³⁵S]Na₂SO₄ by extraction with guanidine hydrochloride and subsequently purified by DEAE-ion exchange chromatography. Their structural properties were established by treatment with nitrous acid, heparitinase and chondroitinase ABC, and by gel filtration before and after alkaline -elimination. About 18% of the proteoglycans synthesized by these cell lines were associated with the plasma membranes. The HBL plasma membranes contained 80% heparan sulfate and 20% chondroitin sulfate proteoglycans whereas MDA plasma membranes had 50% heparan sulfate and 50% chondroitin sulfate proteoglycans. The MDA plasma membrane contained two heparan sulfate proteoglycans, both having nearly the same molecular size as the two species secreted into the medium by these cells. The HBL plasma membrane also contained two hydrodynamic size heparan sulfate proteoglycans. The larger hydrodynamic size species has a slightly lower molecular size than that secreted into the medium, and the smaller hydrodynamic size species was not detectable in the medium. Even though the major chondroitin sulfate proteoglycans from MDA plasma membranes were smaller in size than those from HBL plasma membrane, a larger proportion of the glycosaminoglycan chains of the former were bigger than those from the latter.Abbreviations CHAPS 3-[(3-cholamidopropyl)dimethylammonio]propane-1-sulfonate - Di-OS 2-acetamido-2-deoxy-3-O-(-d-gluco-4-ene-pyranosyluronic acid)-d-galactose - Di-4S 2-acetamido-2-deoxy-3-O-(-d-gluco-4-ene-pyranosyluronic acid)-4-O-sulfo-d-galactose - Di-6S 2-acetamido-2-deoxy-3-O-(-d-gluco-4-ene-pyranosyluronic acid)-6-O-sulfo-d-galactose - Gdn-HCl guanidine hydrochloride - WGA wheat germ agglutinin     

The cloned β-mannanase gene from alkalophilic Bacillus sp. AM-001 produces two β-mannanases in Escherichia coli

The gene encoding -mannanase was cloned from alkalophilic Bacillus sp. AM-001 into Escherichia coli JM 101 by inserting HindIII-generated DNA fragments into the HindIII site of pUC19. A 2.0 kb XbaI-PstI fragment of the donor strain DNA was sufficient for -mannanase synthesis. The amount of -mannanase expressed in E. coli JM101 harboring pMAH3 (containing a 2.4 kb XbaI-HindIII fragment) was about 24% of the activity produced by the donor strain. E. coli JM101 harboring pMAH3 was found to produce two enzymatically active -mannanases (A and B). These two -mannanases were purified to electrophoretically homogenous states. The -mannanase A had enzymatic properties similar to those of the -mannanases M-I and M-II produced by alkalophilic Bacillus sp. AM-001, and the -mannanase B resembled its -mannanase M-III. In contrast to -mannanase production in the donor strain, that in E. coli was not inducible. The NH₂-terminal amino acid sequences from amino acid 1 (Asn) to 9 (Gln) of the three -mannanases purified from alkalophilic Bacillus sp. AM-001 coincide with those from amino acid 4 (Asn) to 12 (Gln) of the two -mannanases purified from E. coli transformant.     

Stereospecificity and electrogenicity of amino acid transport in Riccia fluitans

In the aquatic liverwort Riccia fluitans, membrane depolarization (_m), change in membrane conductance (g_m), and current-voltage (I-V) characteristics in the presence of different amino acids as well as the uptake of ¹⁴C-labeled amino acids were measured. L-isomers of the tested amino acids generate larger electrical effects (_m, g_m) than D-isomers, and the I-V characteristics show that the positive electrical inward-current of 20 mA m^-2 generated by 0.5 mM D-serine is only about 50% of the current generated by adding 0.5 mM L-serine. Whereas - and -amino acids rapidly depolarize the membrane to the same extend, with -aminobutyric acid (-AB) and dipeptides no significant electrical effects have been measured. The uptake kinetics of ¹⁴C-labeled amino acids display three components: (I) A saturable high-affinity component with K_s-values of 48 M D-alanine, 12 M -aminoisobutyric acid (AIB), 9 M L-alanine, 8 M L-proline, and 6 M L-serine, respectively; (2) an apparently linear low-affinity component, and (3) an also linear but unspecific component at concentrations >20 times the given K_s-value. Uptake of ¹⁴C-labeled AIB can be inhibited competitively by all tested neutral amino acids, the L-isomers being more effective than the D-isomers, as well as by ammonium or methylamine. Vice versa, AIB competitively inhibits uptake of L-serine and L-alanine. It is concluded that an uncharged stereospecific carrier for the investigated amino acids exists in the plasmalemma of Riccia fluitans. Accumulation ratios of about 50 suggest secondary active transport driven by a transmembrane electro-chemical gradient (mainly _m) which is generated by the electrogenic proton pump. It is suggested that this carrier binds to the amino group forming either a charged binary complex with positively charged amines (Felle 1980), or an uncharged complex with -AB or dipeptides, whereas electrogenic transport of - and -amino acids is mediated by a ternary carrier complex, probably charged by a proton.Symbols and Abbreviations _m membrane potential (mV) - E_co equilibrium potential (mV) of the transport system - g_m membrane (slope) conductance (Sm^-2) - g_m change in g_m - I-V curve current-voltage curve - AIB -aminoisobutytric acid - -AB -aminobutyric acid     

Protonmotive force in freshwater sulfate-reducing bacteria,and its role in sulfate accumulation in Desulfobulbus propionicus

The protonmotive force in several sulfate-reducing bacteria has been determined by means of radiolabelled membrane-permeant probes (tetraphenyl-phosphonium cation, TPP⁺, for , and benzoate for pH). In six of ten freshwater strains tested only the pH gradient could be determine, while the membrane potential was not accessible due to nonspecific binding of TPP⁺. The protonmotive force of the other four strains was between –110 and –155 mV, composed of a membrane potential of –80 to –140 mV and a pH gradient between 0.25 and 0.8 (inside alkaline) at pH_out=7. In Desulfobulbus propionicus the pH gradient decreased with rising external pH values. This decrease, however, was compensated by an increasing membrane potential. Sulfate, which can be highly accumulated by the cells, did not affect the protonmotive force, if added in concentrations of up to 4 mM. The highest sulfate accumulation observed (2500-fold), which occurred at external sulfate concentrations below 5 M, could be explained by a symport of three protons per sulfate, if equilibrium with the protonmotive force was assumed. At higher sulfate concentrations the accumulation decreased and suggested an electroneutral symport of two protons per sulfate. At sulfate concentrations above 500 M, the cells stopped sulfate uptake before reaching an equilibrium with the protonmotive force.Abbreviations CCCP carbonyl cyanide m-chlorophenylhydrazone - MOPS morpholinopropanesulfonic acid - TPP⁺ tetraphenylphosphonium cation - EDTA ethylenediaminetetraacetic acid - pH transmembrane pH gradient (pH_in-pH_out) - transmembrane electrical potential difference     

A quantitative method for separating reaction components of CMP-sialic acid: Lactosylceramide sialyltransferase using Sep Pak C18 cartridges

A rapid procedure is described for the separation of CMP-sialic acid:lactosylceramide sialyltransferase reaction components using Sep Pak C₁₈ cartridges. The quantitative separation of the more polar nucleotide sugar, CMP-sialic acid, and its free acid from the less polar G_M3-ganglioside is simple and rapid relative to previously described methods. Recovery of G_M3 is optimized by the addition of phosphatidylcholine to the reaction mixture prior to the chromatographic step. Using rat liver Golgi membranes as a source of CMP-sialic acid: lactosylceramide sialyltransferase activity (G_M3 synthase; ST-1), the transfer of [¹⁴C] sialic acid from CMP-[¹⁴C] sialic acid to lactosylceramide can be quantified by this assay. The procedure is reliable and may be applicable to the isolation of ganglioside products in otherin vitro glycosyltransferase assays.Abbreviations G_M3 G_M3-ganglioside - II³NeuAc-LacCer NeuAc2-3Gal1-4Glc1-1Cer - G_D1a G_D1a-ganglioside, IV³NeuAc, II³NeuAc-GgOse₄Cer, NeuAc2-3Gal1-3GalNac1-4(NeuAc2-3)Gal1-4Glc1-1Cer - G_D3 G_D3-ganglioside, II³(NeuAc)₂LacCer, NeuAc2-8NeuAc2-3Gal1-4Glc1-1Cer - GgOse₄Cer asialo-G_M1 Gal1-3GalNAc1-4Gal1-4Glc1-1Cer - FucG_MI fucosyl-G_MI-ganglioside, Fuc1-2Gal1-3GalNAc1-4Gal1-4 Glc1-1Cer - ST-1 G_M3 synthase, CMP-sialic acid:lactosylceramide sialyltransferase - LacCer lactosylceramide, Gal1-4Glc1-1Cer - CMP-NeuAc cytidine 5-monophospho-N-acetylneuraminic acid - PC phosphatidylcholine - PMSF phenylmethylsulfonyl fluoride     

Genetic linkage between endosperm storage protein genes on each of the short arms of chromosomes 1A and 1B in wheat

Summary The storage proteins of the endosperm of wheat grain which are known to be controlled by genes on the short arms of the homoeologous group 1 chromosomes are (1) the -gliadins, (2) most of the -gliadins, (3) a few -gliadins and (4) the major lowmolecular-weight subunits of glutenin. Several crosses were made between varieties or genetic lines which had contrasting allelic variants for some of these proteins and which were coded by genes on chromosomes 1A or 1B. The progeny were analysed by one or more of several electrophoretic procedures. The results of all the analyses are consistent with the hypothesis that chromosomes 1A and 1B each contain just one, complex locus, named Gli-A 1 and Gli-B 1 respectively, which contain the genes for the -, - and -gliadins and the low-molecular-weight subunits of glutenin.     

Structural disorder in proteins

The refinement of X-ray structural data gives the mean square displacements, x ², at each position in the protein molecule. In order to get information on the significance of such values different refinement methods have been compared. The metmyoglobin structure was determined at 300 K and x ²-values were obtained with the restrained refinement procedure in reciprocal space of Konnert and Hendrickson. A comparison with the results of Frauenfelder et al. was used for an error estimation. The inclusion of surface bound water increases the accuracy of the results but does not change the general picture. For erythrocruorin (CTT3) a refinement was performed in reciprocal space and compared with a refinement in real space performed earlier. The x ²-values obtained from both procedures are similar although the reciprocal space refinement gives results which are physically more reasonable.A comparison of the disorder in myoglobin and erythrocruorin showed that the structural similarity results in a similarity in the disorder. Contacts of molecules in the crystal do not dominate the disorder although they locally influence x ²-values. CTT3 shows large disorder in the heme region in contrast to myoglobin. The differences in the rigidity of the F-helix can be correlated with the oxygen affinities supporting models for O₂ binding developed by Frauenfelder et al.     

Some characteristics of anion transport at the tonoplast of oat roots,determined from the effects of anions on pyrophosphatedependent proton transport

The effects of anions on inorganicpyrophosphate-dependent H⁺-transport in isolated tonoplast vesicles from oat (Avena sativa L.) roots were determined. Both fluorescent and radioactive probes were used to measure formation of pH gradients and membrane potential in the vesicles. Pyrophosphate hydrolysis by the H⁺-translocating pyrophosphatase was unaffected by anions. Nonetheless, some anions (Cl^-, Br^- and NO₃-) stimulated H⁺-transport while others (malate, and iminodiacetate) did not. These differential effects were abolished when the membrane potential was clamped at zero mV using potassium and valinomycin. Stimulation of H⁺-transport by Cl^- showed saturation kinetics whereas that by NO₃- consisted of both a saturable component and a linear phase. For Cl^- and NO₃-, the saturable phase had a K _m of about 2 mol·m^-3. The anions that stimulated H⁺-transport also dissipated the membrane potential (.) generated by the pyrophosphatase. It is suggested that the stimulatory anions cross the tonoplast in response to the positive generated by the pyrophosphatase, causing dissipation of and stimulation of pH, as expected by the chemiosmotic hypothesis. The work is discussed in relation to recent studies of the effects of anions on ATP-dependent H⁺-transport at the tonoplast, and its relevance to anion accumulation in the vacuole in vivo is considered.Abbreviations and symools BTP 1,3-bis[tris(hydroxymethyl)-methylamino]-propane - EGTA ethylene glycol-bis(-aminoethyl ether)-N,N,N,N-tetraacetic acid - Hepes 4-(2-hydroxyethyl)-1-piperazine ethanesulphonic acid - IDA iminodiacetate - membrane potential - pH pH gradient - PPase inorganic pyrophosphatase - PPi morganic pyrophosphate     

Transport of H+, K+, Na+ and Ca++ in Streptococcus

Summary The streptococci differ from other bacteria in that cation translocations (with the possible exception of one of the K⁺ uptake systems) occur by primary transport systems, i.e., by cation pumps which use directly the free energy released during hydrolysis of chemical bonds to power transport. Transport systems in other bacteria, especially for Na⁺ and Ca⁺⁺, are often secondary, using the free energy of another ion gradient to drive cation transport. In streptococci H⁺ efflux occurs via the F₁F₀-ATPase. This enzyme is composed of eight distinct subunits. Three of the subunits are embedded in the membrane and form a H⁺ channel; this is called the F₀ portion of the enzyme. The other five subunits form the catalytic part of the enzyme, called F₁, which faces the cytoplasm and can easily be stripped from the membrane. Physiologically, this enzyme functions as a H⁺-ATPase, pumping protons out of the cell to form an electrochemical proton gradient, . The F₁F₀-ATPase, however, is fully reversible and if supplied with P_i, ADP and a + of sufficient magnitude (ca –200 mv) catalyzes the synthesis of ATP. Streptococcus faecalis can accumulate K⁺ and establish a gradient of 50 000:1 (in>out) under some conditions. Uptake occurs by two transport systems. The dominant, constitutive system requires both an electrochemical proton gradient and ATP to operate. The minor, inducible K⁺ transport system, which has many similarities to the K⁺-ATPase of the Kdp transport system found in Escherichia coli, requires only ATP to power K⁺ uptake.Sodium extrusion occurs by a Na⁺/H⁺-ATPase. Exchange is electroneutral and there is no requirement for a . The possibility that the Na⁺/H⁺-ATPase may consist of two parts, a catalytic subunit and a Na⁺/H⁺ antiport subunit, is suggested by the finding that damage to the Na⁺ transport system either through mutation or protease action leads to the appearance of -requiring Na⁺/H⁺ antiporter activity.Ca⁺⁺ like Na⁺ is extruded from metabolizing, intact cells. Transport requires no but does require ATP. Reconstitution of Ca⁺⁺ transport activity with accompanying Ca⁺⁺-stimulated ATPase activity into proteoliposomes suggests that Ca⁺⁺ is transported by a Ca⁺⁺-translocating ATPase.Where respiring organelles and bacteria use secondary transport systems the streptococci have developed cation pumps. The streptococci, which are predominantly glycolyzing bacteria, generate a much inferior to that of respiring organisms and organelles. The cation pumps may have developed simply in response to an inadequate .Abbreviations electrochemical potential of protons - membrane potential - pH pH gradient - p proton-motive force - DCCD N,Na¹-dicyclohexlcarbodiimide - TCS tetrachlorosalicylanilide - FCCP carbonylcyanide-p-trifluoromethylphenylhydrazone - CCCP carbonylcyanie-m-chlorophenylhydrazone - TPMP⁺ triphenylmethyl phosphonium ion - DDA⁺ dibenzyldimethylammonium ion - Hepes 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid - EGTA ethyleneglycol-bis (amino-ethyl-ether)-N,N-tetraacetic acid     

Ionic relations of aeroponically-grown olive genotypes,during salt stress

Two olive (Olea europaea L.) genotypes, Frantoio and Leccino, were exposed to increasing concentrations of NaCl (0-30-60-120 mM) in an aeroponic cultivation system for 60 days. Dry weights and sodium and potassium contents of apical and basal leaves, new and old wood, and roots were measured to determine Na uptake rate, Na translocation rate and K-Na selectivity ratio (S_K,Na). Frantoio showed a higher salt resistance than Leccino. Frantoio and Leccino had a similar Na uptake rate, but largely differed for Na translocation to the shoot. Furthermore Frantoio exhibited a higher K-Na selectivity than Leccino at both whole plant level and above all at the level of shoot system. Resistance mechanism of Frantoio is probably related to Na esclusion by roots and to the ability to maintain an appropriate K/Na ratio in actively growing tissues.Research supported by National Research Council of Italy, Special project RAISA.     

Light and GTP dependence of transducin solubility in retinal rods

The physical origin and functional significance of the near infra-red light scattering changes observable upon flash illumination of diluted suspensions of magnetically oriented, permeabilised frog retinal rods has been reinvestigated with particular attention paid to the degree with which transducin remains attached to the membrane. In the absence of GTP, the so called binding signal is shown to include two components of distinctive origins, widely different kinetics, and whose relative amplitudes depend on the dilution of the suspension and resulting detachment of transducin from the disc membrane. The fast component is a consequence of the fast interaction between photoexcited rhodopsin (R^*) and the transducin remaining on the membrane. Its kinetics monitors a structural modification of the discs caused by a change in electrostatic interaction between closely packed membranes upon the formation of R^*-T complexes. The slow component monitors the slow rebinding to the membrane and possible subsequent interaction with excess R^* of T-GDP which, in spite of its low solubility, had eluted into solution given the high dilution of the permeated rods. In the presence of GTP, the so called dissociation signal includes a fast, anisotropic release component that specifically monitors the release into the interdiscal space of T -GTP formed from the membrane-bound pool, and a slower isotropic loss component monitoring the leakage from the permeated rod of the excess T -GTP which did not interact with the cGMP phosphodiesterase. The amplitudes of both components depend exclusively on the membrane bound T-GDP pool. The kinetics of the loss component is limited by the size and degree of permeation of the rod fragments, rather than by the dissociation rate of T -GTP from the membrane.Abbreviations ROS rod outer segment - R rhodopsin - R^* photoactivated rhodopsin - T, T-GDP, T -GDP, T -GTP, T transducin and its various forms - T _mb, T _sol: T bound to membrane or soluble - PDE cGMP-phosphodiesterase - GTP guanosine 5-triphosphate - GDP guanosine 5-diphosphate - GDP S guanosine 5-O-(2-thiodiphosphate) - cGMP guanosine-3-5 cyclic-monophosphate - DTT dithiothreitol - HEPES 4-(2-hydroxyethyl)-1-piperazine-ethane sulfonic acid - TRIS Tris (hydroxymethyl)aminomethane - SDS sodium dodecyl sulfate     

Effects of light on nitrate-limited Oscillatoria agardhii in chemostat cultures

The effects of the average light irradiance (I) on growth and nitrate uptake kinetics of the cyanobacterium Oscillatoria agardhii, in nitrate-limited chemostat cultures, were studied. Light was nonsaturating for I <9.4 Wm^–2, for all growth rates () studied. However, was throughout limited by the availability of nitrate. Under light-saturating conditions the kinetics of nitrate-limited growth could be adequately described by both the Monod and Droop equations. Under light-non-saturating conditions the internal nitrogen content (Q) was a function of both and I, for which new formulas were derived. The high uptake capacity (V _max) of nitrate-limited cells was independent of , but was significantly increased for cells growing at I <9.4 Wm^–2. The half-saturation constant for nitrate uptake (K _s ^u ) increased with increasing , but was independent of the prevailing light conditions. The effects of light during nitrate-limited growth were associated with the regulation in the nitrogen-containing pigments.The results reported herein have important consequences for the use of Q, K _s ^u and V _max values as indicators of nutrient-deficiency of natural populations.     

Energetics of CO formation and CO oxidation in cell suspensions of Acetobacterium woodii

Cell suspensions of Acetobacterium woodii produced CO from H₂ and CO₂. Depending on the conditions, more than 1,000 ppm CO were measured in the gas phase. This concentration was more than 10-fold higher than the thermodynamic equilibrium concentration that can be calculated to be 83.5 ppm for the experimental conditions used. This finding is taken as evidence that, besides the activation of formate, also CO production from CO₂ is an energy-dependent step in the reduction of CO₂ to acetate. Studies on the influence of ionophores and dicyclohexylcarbodiimide (DCCD) as well as that of CO and formaldehyde on acetate synthesis were undertaken in order to determine whether ATP or is the driving for CO₂ reduction to CO.Cells of A. woodii also catalyzed the conversion of CO (5% in the gas phase) to CO₂ and H₂. This process was coupled to the generation of metabolic energy, which could be used by the cells to drive the uptake of histidine into the cells; histidine uptake was almost completely inhibited by the ionophores valinomycin plus nigericin. The data were taken to indicate that in this acetogen the energy derived from CO oxidation can be converted to metabolic energy.Abbreviations DCCD dicyclohexylcarbodiimide - THF tetrahydrofolate - TCS tetrachlorosalicylanilide - TPP⁺ tetraphenylphosphonium ion - Val valinomycin; Nig, nigericin - DTT dithiothreitol - DTE dithioerythritol - DTE dithioerythritol - membrane potential - electrochemical proton potential - ppm parts per million     

Effects of root temperature on uptake of nitrate and ammonium ions by barley grown in flowing-solution culture

Summary Barley plants (Hordeum vulgare L.) grown from seed for 28 days in flowing solution culture were subjected to different root temperatures (3, 5, 7, 9, 11, 13, 17, 25°C) for 14 days with a common air temperature of 25/15°C (day/night). Uptake of NH₄ and NO₃ ions was monitored separately and continuously from solutions maintained at 10 M NH₄NO₃ and pH 6.0. Effects of root temperature on unit absorption rate , flux and inflow were compared. After 5 days , and increased with temperature over the range 3–11°C for NH₄ ions and over the range 3–13°C for NO₃ ions, with little change for either ion above these temperatures. Q₁₀ temperature coefficients for NH₄ ions (3–13°C) were 1.9, 1.7 and 1.6 for , and respectively, the corresponding values for NO₃ ions being 5.0, 4.5 and 4.6. For both ions, , and changed with time as did their temperature dependence over the range 3–25°C, suggesting that rates of ontogenetic development and the extent of adaptation to temperature may have varied among treatments.