Transport of leucine in the cyanobacterium Anabaena variabilis

The amino acid leucine was transported by the cyanobacterium Anabaena variabilis. The K _m for transport was 10.8 M; the V _max was 8.7 nmoles min^–1 mg^–1 chlorophyll a. Transport of leucine was energy dependent: uptake of leucine was inhibited in the dark, and by DCMU and cyanide. Transport was neither dependent on nor enhanced by Na⁺. Prior growth of cells with leucine did not repress transport of [¹⁴C]-leucine. Alanine, glycine, valine, and methionine were strong competitive inhibitors of leucine uptake; serine, threonine, isoleucine, norleucine, and d-alanine competitively inhibited to a lesser degree. Other amino acids or amino acid analogues, including d-leucine, -aminoisobutyrate, and d-serine did not inhibit the transport of leucine.Abbreviations Chl a chlorophyll a - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - TES N-tris(hydroxymethyl)-2-aminoethane-sulfonic acid - TCA trichloroacetic acid - Tris N-tris(hydroxymethyl)aminoethane     

Applicability of the chemiosmotic polar diffusion theory to the transport of indol-3yl-acetic acid in the intact pea (Pisum sativum L.)

The transport of exogenous indol-3yl-acetic acid (IAA) from the apical tissues of intact, light-grown pea (Pisum sativum L. cv. Alderman) shoots exhibited properties identical to those associated with polar transport in isolated shoot segments. Transport in the stem of apically applied [1-¹⁴C]-or [5-³H]IAA occurred at velocities (approx. 8–15 mm·h^-1) characteristic of polar transport. Following pulse-labelling, IAA drained from distal tissues after passage of a pulse and the rate characteristics of a pulse were not affected by chases of unlabelled IAA. However, transport of [1-¹⁴C]IAA was inhibited through a localised region of the stem pretreated with a high concentration of unlabelled IAA or with the synthetic auxins 1-napthaleneacetic acid and 2,4-dichlorophenoxyacetic acid, and label accumulated in more distal tissues. Transport of [1-¹⁴C]IAA was also completely prevented through regions of the intact stem treated with N-1-naphthylphthalamic acid (NPA) and 2,3,5-triiodobenzoic acid.Export of IAA from the apical bud into the stem increased with total concentration of IAA applied (labelled+unlabelled) but approached saturation at high concentrations (834 mmol·m^-3). Transport velocity increased with concentration up to 83 mmol·m^-3 IAA but fell again with further increase in concentration.Stem segments (2 mm) cut from intact plants transporting apically applied [1-¹⁴C]IAA effluxed 93% of their initial radioactivity into buffer (pH 7.0) in 90 min. The half-time for efflux increased from 32.5 to 103.9 min when 3 mmol·m^-3 NPA was included in the efflux medium. Long (30 mm) stem sections cut from immediately below an apical bud 3.0 h after the apical application of [1-¹⁴C]IAA effluxed IAA when their basal ends, but not their apical ends, were immersed in buffer (pH 7.0). Addition of 3 mmol·m^-3 NPA to the external medium completely prevented this basal efflux.These results support the view that the slow long-distance transport of IAA from the intact shoot apex occurs by polar cell-to-cell transport and that it is mediated by the components of IAA transmembrane transport predicted by the chemiosmotic polar diffusion theory.Abbreviations IAA indol-3yl-acetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA 1-naphthaleneacetic acid - NPA N-1-naphthylphthalamic acid - TIBA 2,3,5-triiodobenzoic acid     

STUDIES OF NITRATE TRANSPORT BY MARINE PHYTOPLANKTON USING 36Cl-ClO3− AS A TRANSPORT ANALOGUE. I. PHYSIOLOGICAL FINDINGS1

Transport of a nitrate analogue, ³⁶Cl-ClO₃⁻, was examined in two diatoms, Skeletonema costatum (Greve.) Cleve and Nitzschia closterium (Ehrenb) W. Sm. A dinoflagellate, Gonyaulax polyedra did not transport ClO₃⁻. Transport of ³⁶Cl-ClO₃⁻, by diatoms appeared to be active and showed saturation kinetics. The data were fitted by Michaelis-Menten equation at all but the lowest chlorate concentrations (where plots of S vs. v showed a slight concave bend). Affinity of cells for nitrate was considerably higher than for chlorate. The K_i for nitrate inhibition of chlorate transport was calculated assuming competitive inhibition. Light had little or no effect on chlorate transport. Pulse-chase experiments demonstrated that (1) ClO₃⁻ (hence NO₃⁻) was stored in two intracellular compartments of equal size, (2) internal ClO₃⁻ was exchangeable with external ClO₃⁻ (rates of efflux and influx were measured), and (3) efflux of intracellular ClO₃⁻ showed transient states following a chase of ClO₃⁻ or NO₃⁻ which stabilized after 10–20 min. Transport of chlorate was a function of growth phase.     

Determination of diffusion coefficients of octadecanoic acid in isolated cuticular waxes and their relationship to cuticular water permeabilities

Transport properties of cuticular waxes from 40 different plant species were investigated by measuring desorption rates of ¹⁴C-labelled octadecanoic acid from isolated and subsequently reconstituted wax. Diffusion coefficients (D) of octadecanoic acid in reconstituted waxes, calculated from the slopes of the regression lines fitted to the linearized portions of desorption kinetics, ranged from 1.2 × 10^?19 m² s^?1 (Senecio kleinia leaf) to 2.9 × 10^?17 m² s^?1 (Malus cf. domestica fruit). Cuticular water permeabilities (cuticular transpiration) measured with intact cuticular membranes isolated from 24 different species varied from 1.7 × 10^?11 m s^?1 (Vanilla planifolia leaf) up to 2.1 × 10^?9 m s^?1 (Malus cf. domestica fruit), thus covering a range of more than 2 orders of magnitude. Cuticular water permeabilities were highly correlated with diffusion coefficients of octadecanoic acid in isolated cuticular wax of the same species. It is therefore possible to estimate cuticular barrier properties of stomatous leaf surfaces or of leaves where isolation of the cuticle is impossible by measuring D of octadecanoic acid in isolated waxes of these leaves.     

The transport of radiolabeled indoleacetic acid and its conjugates in nodal stem segments of Phaseolus vulgaris L.

The transport of radiolabeled indoleacetic acid (IAA), and some of its conjugates, was investigated in nodal stem segments of Phaseolus vulgaris L. Donor agar blocks containing either [2-acetyl-¹⁴C]-IAA; [2-acetyl-¹⁴C]-indole-3-acetyl-L-aspartate (IAAsp); [2-acetyl-¹⁴C]-indole-3-acetyl-L-glycine (IAGly); or [2-acetyl-¹⁴C]-indole-3-acetyl-L-alanine (IAAla) were placed on either the apical or basal cut surface of stem segments each bearing an axillary bud at the midline. In some experiments, a receiver block was placed on the end opposite to the donor. After transport was terminated, the segments were divided into five equal sections plus the bud, and the radioactivity of donors, receivers and each part of the stem segment was counted.For all four substances tested, the amount of ¹⁴C transported to the axillary bud from the base was the same or greater than that from the apical end. After basipetal transport, the distribution of ¹⁴C in the segment declined sharply from apex to base. The inverse was true for acropetal transport. Transport for the three IAA conjugates did not differ substantially from each other.The IAA transport inhibitor, N-1-naphthylphthalamic acid (NPA), inhibited basipetal ¹⁴C-IAA transport to the base of the stem segment but did not alter substantially the amount of ¹⁴C-IAA recovered from the bud. Transport of ¹⁴C-IAA from the apical end to all parts of the stem segment declined when the base of the section was treated with nonradioactive IAA. Taken together with data presented in the accompanying article [Tamas et al. (1989) Plant Growth Regul 8: 165–183], these results suggest that the transport of IAA plays a role in axillary bud growth regulation, but its effect does not depend on the accumulation of IAA in the axillary bud itself.     

Metabolism of14C-abscisic acid during its IAA-promoted transport in etiolated segments of pea (Pisum sativum L.) epicotyls†

The metabolism of exogenously supplied abscisic acid (ABA) during translocation attracted under the influence of indolyl-3-acetic acid (IAA) was studied in etiolated segments of pea (Pisum sativum L.). After 8 and 24 h 90% and 60% of the ABA, respectively, were found in the segments in unchanged form. Phaseic acid, dihydrophaseic acid and the glucose ester of ABA were found as ABA metabolites. Results indicated that the growth processes initiated by the application of IAA were associated neither with an increased immobilization nor increased metabolization of this growth regulator. † Part II. Influence of Auxin-like Substances upon the Transport of¹⁴C-ABA in Long Pea Epicotyl Segments.     

Effect of Plant Growth Regulators on Calcium-stimulated Serine Transport into Tobacco Cells

The transport of serine into tobacco cells (Nicotiana tabacum L.) cultured in liquid medium was examined. Transport was inhibited approximately 50% by 2,4-dichlorophenoxyacetic acid, indoleacetic acid, α-naphthalene acetic acid, and kinetin at a concentration of 10 micrograms per milliliter. Transport was not inhibited by 2,6-dichlorophenoxyacetic acid and inhibited less than 25% by p-chlorophenoxyacetic acid at this concentration. Removal of 2,4-dichlorophenoxyacetic acid from the transport medium resulted in an alleviation of inhibition. Gibberellic acid at concentrations from 2 to 20 micrograms per milliliter stimulated transport.

It was previously shown that inhibition of transport by La³⁺ was due to removal of Ca²⁺ from surface sites and inhibition of Ca²⁺ uptake by cells. None of the growth regulators tested had any significant effect on Ca²⁺ binding and/or transport.

A contributing factor to the low transport rates in the absence of Ca²⁺ is the increased rate of serine efflux. None of the growth regulators tested had any significant effect on the rate of serine efflux.

     

Simulation of Hormone Transport in Petiole Segments of Coleus

Translocation and distribution of abscisic-(1-¹⁴C) acid and of (5-³H)indol-3yl-acetic acid in agar cylinders and in boiled and fresh petiole segments of Coleus scutellarioides Bentham have been studied by liquid scintillation counting. The hormones were simultaneously applied in donor blocks of agar. Transport and distribution data of the two compounds were compared with those of (³⁶Cl^-)chloride in the same systems. From the data on movement in agar cylinders, diffusion coefficients of the transported compounds were approximated using a simulation model in which it was assumed that the movement was controlled by diffusion only. Movement in boiled tissue segments fitted the model if a correction factor was included in the equation for diffusion. Acropetal movement of indol-3yl-acetic acid in fresh petiole segments can be entirely explained in terms of passive transport and for basipetal transport it seems to be a major component. Movement of abscisic acid and of chloride in fresh petiole segments fit also a model based on diffusion. The limitations of the simulation model are discussed.     

Renal function and organic anion and cation transport during dehydration and/or food restriction in chickens

The effect of dehydration in the presence or absence of continued food intake on renal function was evaluated in chickens. In addition, renal transport of organic anions and cations under these conditions was assessed in vitro by uptake of ¹⁴C-para-aminohippuric acid and ¹⁴C-tetraethylammonium bromide by renal slices. Water restriction with continued food intake resulted in increases in serum osmolality and serum concentrations of sodium, uric acid, calcium and total protein. If food was restricted in addition to water, only serum osmolality and sodium concentration were significantly increased after 48 hours. Dehydration with continued access to food resulted in marked decreases in extracellular fluid volume, glomerular filtration rate and effective renal plasma flow. If food was restricted during dehydration, the decrease in effective renal plasma flow was attenuated despite reductions in glomerular filtration rate and extracellular fluid comparable to that seen in dehydrated birds allowed free access to food. Transport of organic anions was significantly increased after 24 and 48 hours of water restriction, regardless of whether food was withheld. Enhanced transport of organic anions in the presence of decreased glomerular filtration rate and effective renal plasma flow during dehydration may promote precipitation of urates and nephrosis in chickens.Abbreviations cpm counts per minute - dpm disintegrations per minute - ECF extracellular fluid - ERPF effective renal plasma flow - GFR glomerular filtration rate - PAH para-aminohippuric acid - SEM standard error of the mean - TEA tetraethylammonium bromide     

The specific transport system for lysine is fully inhibited by ammonium in Penicillium chrysogenum: An ammonium-insensitive system allows uptake in carbon-starved cells

The regulation exerted by ammonium and other nitrogen sources on amino acid utilization was studied in swollen spores of Penicillium chrysogenum. Ammonium prevented the L-lysine, L-arginine and L-ornithine utilization by P. chrysogenum swollen spores seeded in complete media, but not in carbon-deficient media. Transport of L-[¹⁴C]lysine into spores incubated in presence of carbon and nitrogen sources was fully inhibited by ammonium ions (35 mM). However, in carbon-derepressed conditions (growth in absence of sugars, with amino acids as the sole carbon source) L-[¹⁴C]lysine transport was only partially inhibited. Competition experiments showed that L-lysine (1 mM) inhibits the utilization of L-arginine, and vice versa, L-arginine inhibits the L-lysine uptake. High concentrations of L-ornithine (100 mM) prevented the L-lysine and L-arginine utilization in P. chrysogenum swollen spores. In summary, ammonium seems to prevent the utilization of basic amino acids in P. chrysogenum spores by inhibiting the transport of these amino acids through their specific transport system(s), but not through the general amino acid transport system that is operative under carbon-derepression conditions.     

Dicarboxylic acid transport in Rhizobium meliloti: isolation of mutants and cloning of dicarboxylic acid transport genes

The role of the dicarboxylic acid transport (dct) system in the Rhizobium meliloti-Alfalfa symbiosis was investigated. Mutants of R. meliloti CM2 unable to grow on medium containing succinate as the sole carbon source were isolated following chemical and transposon mutagenesis. These mutants were also unable to utilize malate or fumarate as the sole source of carbon. Transport studies with ¹⁴C-labelled succinate showed that the mutants were specifically defective in succinate transport. Revertants of both chemical and transposon mutants were obtained at a frequency of 10^-5–10^-6. The R. meliloti dct mutants were able to nodulate Alfalfa plants but the nodules formed were unable to fix nitrogen. Revertants of the mutants were fully effective on plants. The mutants unable to transport succinate were used to isolate dct genes from a R. meliloti gene bank. Two plasmids containing a common 26.5 Mdal insert were found to complement some of the mutants. The presence of this DNA insert in the complementing mutant strains restored their effectivenss of plants. This DNA fragment encoding succinate transport function(s) was used to produce genetically engineered R. meliloti strains with an increased rate of succinate uptake.Abbreviation dct dicarboxylic acid transport     

Effect of ethylene treatment on transport and metabolism of indole-3-butyric acid in citrus leaf midribs

Transport and metabolism of radiolabeled indole-3-butyric acid (IBA) were studied in midrib sections of Cleopatra mandarin (Citrus reticulata Blanco) and compared to that of indole-3-acetic acid (IAA). Exogenous IBA was metabolized by the midribs to a polar compound, probably an ester conjugate. Ethylene pretreatment of the midribs reduced their capacity to metabolize IBA by ca. 70% as compared to air pretreatment. IBA transport capacity in the leaf midribs was ca. two times greater in the basipetal direction than the acropetal. The basipetal transport capacity of ³H-IBA was lower than that of ¹⁴C-IAA (ca. 24% and 39% of the uptake, respectively). While ethylene treatment reduced basipetal transport of IAA by ca. 70% it did not affect the transport of IBA. Most of the transported label was found as free IBA, but the reduction of IBA conjugation by ethylene treatment did not affect the transport capacity.     

TRANSPORT OF IAA AND ACC IN FLORAL ORGANS OF IPOMOEA NIL (CONVOLVULACEAE)

The involvement of the stamens as transporters of plant growth regulators in flowers was examined by measuring the movement of ¹⁴C-indole-3-acetic acid (IAA) and ^l4C-l-aminocyclopropane-1-carboxylic acid (ACC) through floral organs of Ipomoea nil. During the transport of ¹⁴C-IAA through isolated filament segments, the polar accumulation of ¹⁴C-IAA in receiver blocks increased with time during filament development, which correlated with polar efflux rates at older stages of filament development. An inhibitor of polar IAA transport, 2,3,5-triiodobenzoic acid, disrupted the polarity of auxin transport by reducing the movement of ¹⁴C- IAA from filaments into receiver blocks. Transport of both ¹⁴C-IAA and ^l4C-ACC through filaments into other floral organs also was monitored in isolated flower buds in the laboratory and intact buds in the greenhouse. In isolated and intact buds 21 hr before anthesis, substantially higher levels of isotope were recovered in corolla tissue when ¹⁴C-ACC was transported through the filaments than when ¹⁴C-IAA was transported from the filaments. In isolated buds, substantial levels of both isotopes accumulated in the pistil (69 hr and 45 hr before anthesis), but minimal amounts were observed in receptacle and calyx tissues (69 hr to 21 hr before anthesis). In intact buds, high levels of both isotopes were recovered in receptacle, calyx, and pistil tissues (69 hr to 21 hr before anthesis). The results from this study support the hypothesis that Ipomoea stamens are transporters for ACC and IAA to regulate ethylene production in the corolla and other floral tissues.     

Auxin-directed transport of radiophosphorus in stems

Summary The role of indolylacetic acid (IAA) in influencing the movement of radiophosphorus from a remote locus has been investigated in the decapitated stems of Pisum sativum (var. Meteor) and disbudded shoots of Populus robusta. It has been shown after only a few hours that the radioisotope accumulates at the region where the auxin was applied. By steam-girdling experiments with pea, it was established that auxin-induced transport occurs via the phloem and other obserbations suggest that this effect may be independent of growth. When tri-iodobenzoic acid was applied together with IAA at the stump or to the surface of the stem between the IAA source and the injection point of the tracer, the transport of ³²P along the stem was greatly reduced. Of a number of auxin-type substances tested, only naphthoxyacetic acid gave a comparable response to that obtained with IAA, whilst other growth regulators such as kinetin or gibberellic acid did not stimulate ³²P transport.Studies with isolated poplar cuttings showed that auxin-induced transport could only be demonstrated by completely disbudded shoots. Again, phloem transport seemed to be involved and the movement of ³²P could be more readily induced in an acropetal direction.The results are discussed in relation to some recently proposed theories concerning the mechanism of translocation in the phloem.

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Zusammenfassung Der Einfluß der Indolylessigsäure (IES) auf die Wanderung von radioaktivem Phosphor wurde an dekapitierten Sprossen von Populus robusta nach Entfernung ihrer Knospen und an dekapitierten Stengeln von Pisum sativum (var. Meteor) untersucht. Bereits nach wenigen Stunden war eine Anhäufung von ³²P am Ort der Auxinzufuhr zu erkennen. Durch Dampf-Ringelungsversuche ließ sich bei Pisum zeigen, daß der Auxin-induzierte Transport über das Phloem erfolgt; weitere Beobachtungen lassen darauf schließen, daß dieser Effekt unabhängig vom Wachstum sein dürfte. Wenn man gleichzeitig mit IES Trijodbenzoesäure (TIBA) auf die apikale Schnittfläche aufbringt oder in einer Zone zwischen Auxinquelle und Injektionsstelle des ³²P als Paste auf die Oberfläche des Sprosses aufträgt, so wird der Längstransport des markierten Phosphors erheblich gehemmt.Es wurden auch noch einige andere Verbindungen von Wuchsstoffcharakter geprüft. Von ihnen führte nur Naphthyloxyessigsäure zu einem ähnlichen Effekt wie IES; andere Wachstumsregulatoren wie Kinetin oder Gibberellinsäure förderten den ³²P-Transport nicht.Untersuchungen an isolierten Pappelsprossen zeigten, daß der auxininduzierte Stofftransport nur in solchen Sprossen zu beobachten ist, deren Knospen vollständig entfernt worden waren. Auch hier scheint es sich um einen Transport im Phloem zu handeln. Die Wanderung von ³²P ließ sich stets leichter in akropetaler Richtung induzieren.Die geschilderten Ergebnisse werden in Beziehung zu einigen neuerdings vorgeschlagenen Theorien des Transportmechanismus im Phloem diskutiert.

     

Transport of benzenesulfonic acid derivatives through the rat erythrocyte membrane

Summary Transport of benzenesulfonic acid derivatives through the rat erythrocyte membrane was studied. The transport properties, such as pH-dependence and effects of reagents reacting with amino-groups, were similar to those of anions like Cl^– through the human erythrocyte membrane. The rate of transport of anions through rat erythrocyte membranes is higher than through those of other mammals, such as guinea pig and bovine erythrocyte membranes. This relatively high rate of transport makes the rat erythrocyte membrane suitable for use in comparative studies on the transports of slowly penetrating substances, such as organic anions. The transport velocities of benzenesulfonic acid derivatives were compared with their physico-chemical properties. It was shown that the hydrophobicity has no effect on the transport, but the electronic property has a significant effect: the transport rate is mainly dependent on thee ^– donor capacities. This feature is the inverse to the well-known inhibitory effect of these derivatives on other anion transport: the inhibition is mainly dependent on thee ^– acceptor capacities. It is suggested that the transport is regulated by the binding capacity of anions to the transport site.     

Relations polyphénols — croissance: Mise en évidence d'un effet inhibiteur des composés phénoliques sur le transport polarisé de l'auxine

Polyphenols and Growth: Inhibition of Polar Auxin Transport by Phenolic Compounds. The possible effects of polyphenols on auxin transport in tomato plants (Lycopersicum esculentum Mill.) were investigated. For this purpose, the phenolic content of the material was stimulated by exogenously supplied quinic acid. After the apical bud had been excised, labelled compounds were applied to the cut surface, and the radioactivity transported to the roots was measured. Quinic acid treatment significantly delayed polar transport of labelled auxins (IAA or NAA). It did not affect the migration rate of sucrose^?14C and leucine^?3H. A number of evidences seems to demonstrate that the phenolics are responsible for these modifications, since similar results were recorded when the labelled compounds were supplied simultaneously with polyphenols from tomato. Moreover, a decreased polarity of NAA transport could be observed when the plants were submitted to treatments which lead to an increased level of phenols (boron deficiency, infection by Fusarium oxysporum). The data presented in this paper suggest that phenolic compounds could act on growth processes via the regulation of polar auxin transport.     

Mutations in Neurospora crassa which affect multiple amino acid transport systems

Characterization of a double mutant, his-6: hgu-4, which is unable to utilize l-histidyl-glycine as a source of histidine has revealed a new locus on linkage group V. The hgu-4 genotype results in a generalized reduced transport activity for amino acids, with a concomitant increased resistance to amino acid analogs. Transport rates and analog resistance for amino acids by this mutant are compared to the previously reported transport deficient mutants fpr-1, nap and un-3.Transport of l-aspartate as a function of temperature is examined in a variety of transport deficient strains in an attempt to explain the mode of action of mutation which pleiotropically affect several genetically and biochemically distinct amino acid transport systems.     

Structure and functional characterization of a bile acid 7α dehydratase BaiE in secondary bile acid synthesis

Conversion of the primary bile acids cholic acid (CA) and chenodeoxycholic acid (CDCA) to the secondary bile acids deoxycholic acid (DCA) and lithocholic acid (LCA) is performed by a few species of intestinal bacteria in the genus Clostridium through a multistep biochemical pathway that removes a 7α‐hydroxyl group. The rate‐determining enzyme in this pathway is bile acid 7α‐dehydratase (baiE). In this study, crystal structures of apo‐BaiE and its putative product‐bound [3‐oxo‐Δ^4,6‐lithocholyl‐Coenzyme A (CoA)] complex are reported. BaiE is a trimer with a twisted α + β barrel fold with similarity to the Nuclear Transport Factor 2 (NTF2) superfamily. Tyr30, Asp35, and His83 form a catalytic triad that is conserved across this family. Site‐directed mutagenesis of BaiE from Clostridium scindens VPI 12708 confirm that these residues are essential for catalysis and also the importance of other conserved residues, Tyr54 and Arg146, which are involved in substrate binding and affect catalytic turnover. Steady‐state kinetic studies reveal that the BaiE homologs are able to turn over 3‐oxo‐Δ⁴‐bile acid and CoA‐conjugated 3‐oxo‐Δ⁴‐bile acid substrates with comparable efficiency questioning the role of CoA‐conjugation in the bile acid metabolism pathway. Proteins 2016; 84:316–331. © 2016 Wiley Periodicals, Inc.     

Acquisition of alanyl-alanine in an Agnathan: Characteristics of dipeptide transport across the hindgut of the Pacific hagfish Eptatretus stoutii

This study used ³H-_L-alanyl-_L-alanine to demonstrate dipeptide uptake using in vitro gut sacs prepared from the hindgut of the Pacific hagfish Eptatretus stoutii. Concentration-dependent kinetic analysis resulted in a sigmoidal distribution with a maximal (± SE) uptake rate (J_max-like) of 70 ± 3 nmol cm⁻² h⁻¹ and an affinity constant (K_m-like) of 1072 ± 81 μM. Addition of high alanine concentrations to transport assays did not change dipeptide transport rates, indicating that hydrolysis of the dipeptide in mucosal solutions and subsequent uptake via apical amino acid transporters was not occurring, which was further supported by a K_m distinct from that of amino acid transport. Transport occurred independent of mucosal pH, but uptake was reduced by 42% in low mucosal sodium. This may implicate cooperation between peptide transporters and sodium-proton exchangers, previously demonstrated in several mammalian and teleost species. Finally, apical _L-alanyl-_L-alanine uptake rates (i.e., mucosal disappearance) were significantly increased following a meal, demonstrating regulation of uptake. Overall, this examination of dipeptide acquisition in the earliest extant Agnathan suggests evolutionarily conserved mechanisms of transport between hagfish and later-diverging vertebrates such as teleosts and mammals.     

Gibberellic-acid-stimulated Ca2+ accumulation in endoplasmic reticulum of barley aleurone: Ca2+ transport and steady-state levels

The steady-state levels of Ca²⁺ within the endoplasmic reticulum (ER) and the transport of ⁴⁵Ca²⁺ into isolated ER of barley (Hordeum vulgare L. cv. Himalaya) aleurone layers were studied. The Ca²⁺-sensitive dye indo-1. Endoplasmic reticulum was isolated and purified from indo-1-loaded protoplasts, and the Ca²⁺ level in the ER was measured using the Ca²⁺-sensitive dye indo-1. Endoplasmic reticulum was isolated and purified from indo-1-loaded protoplasts, and the Ca²⁺ level in the lumen of the ER was determined by the fluorescence-ratio method to be at least 3 M. Transport of ⁴⁵Ca²⁺ into the ER was studied in microsomal fractions isolated from aleurone layers incubated in the presence and absence of gibberellic acid (GA₃) and Ca²⁺. Isopycinic sucrose density gradient centrifugation of microsomal fractions isolated from aleurone layers or protoplasts separates ER from tonoplast and plasma membranes but not from the Golgi apparatus. Transport of ⁴⁵Ca²⁺ occurs primarily in the microsomal fraction enriched in ER and Golgi. Using monensin and heat-shock treatments to discriminate between uptake into the ER and Golgi, we established that ⁴⁵Ca²⁺ transport was into the ER. The sensitivity of ⁴⁵Ca²⁺ transport to inhibitors and the K_m of ⁴⁵Ca²⁺ uptake for ATP and Ca²⁺ transport in the microsomal fraction of barley aleurone cells. The rate of ⁴⁵Ca²⁺ transport is stimulated several-fold by treatment with GA₃. This effect of GA₃ is mediated principally by an effect on the activity of the Ca²⁺ transporter rather than on the amount of ER.Abbreviations CCR cytochrome-c reductase - DCCD dicyclohexylcarbodiimide - EGTA ethylene glycol bis(-aminoethyl ether)-N,N,N,N-tetraacetic acid - ER endoplasmic reticulum - FCCP carbonylcyanide p-trifluoromethoxyphenyl hydrazone - GA₃ gibberellic acid - IDPase inosine diphosphatase - Mon monensin