Phenotypic Characterization of Polysaccharidases Produced by Four Prevotella Type Strains

Four ruminal Prevotella type strains, P. ruminicola JCM8958^T, P. bryantii B₁4^T, P. albensis M384^T, and P. brevis ATCC19188^T, were characterized for polysaccharide-degrading activities with the reducing sugar release assay and zymogram analyses. Carboxymethylcellulase, xylanase, and polygalacturonate (PG)-degrading enzyme activities were determined in cultures grown on oat spelt xylan, xylose, arabinose, cellobiose, and glucose as sole growth substrates. P. ruminicola and P. albensis showed carboxymethylcellulase induction patterns. When xylan was supplied as a sole growth substrate, xylanase activities produced by P. bryantii and P. albensis were at least 18- and 11-fold higher, respectively, than during growth on other carbohydrates, suggesting that the regulation of the xylanases was highly specific to xylan. All strains constitutively produced PG-degrading enzymes. The corresponding activity of P. bryantii was more than 40-fold higher than in other strains. Zymogram analyses routinely detected the presence of high-molecular-weight (100–170 kDa) polysaccharide-degrading enzymes in ruminal Prevotella. Characteristics of the polysaccharide-degrading activities showed diversity of ruminal Prevotella species. Received: 29 November 1999 / Accepted: 1 February 2000     

Diversity of Extracellular Proteolytic Activities Among Prevotella Species from the Rumen

The current research was aimed at comparing proteolytic activities among ruminal Prevotella spp. Growth rates of Prevotella sp. 2202, Prevotella ruminicola D31d, P. brevis GA33, P. albensis M384, and P. bryantii B₁4 varied with N source, and no one N source produced the fastest growth in all species. Proteolytic activity was greatest with casein compared with peptides, AA, and NH₄Cl in all species. Proteolytic activity of Prevotella sp. 2202, P. brevis GA33, and P. bryantii B₁4 was modulated by N source. With gelatin co-polymerized SDS-PAGE, the extracellular activities of the Prevotella spp. showed wide variation in number, size, and type of proteases. Prevotella sp. 2202 and P. albensis M384 produced metalloproteases of low molecular weight (40 kDa). P. ruminicola D31d produced one cysteine protease (100–200 kDa) and two metalloproteases (90–100 kDa). P. brevis GA33 generated a diffuse clearing zone (95–160 kDa) containing serine, cysteine, and metalloproteases. P. bryantii B₁4 produced a metalloprotease greater than 200 kDa in size. The molecular sizes provided are estimations and served only to differentiate among the bacterial species in this study. Large variations in proteolytic activities among species and the known genetic diversity of the Prevotella taxon suggested that targeting this bacterial assemblage for genetic manipulation in order to alter the bacterial impact on ruminal protein degradation would be difficult. Received: 12 January 1999 / Accepted: 19 May 1999     

The Ability of “Low G + C Gram-Positive” Ruminal Bacteria to Resist Monensin and Counteract Potassium Depletion

Gram-negative ruminal bacteria with an outer membrane are generally more resistant to the feed additive, monensin, than Gram-positive species, but some bacteria can adapt and increase their resistance. 16S rRNA sequencing indicates that a variety of ruminal bacteria are found in the “low G + C Gram-positive group,” but some of these bacteria are monensin resistant and were previously described as Gram-negative species (e.g., Selenomonas ruminantium and Megasphaera elsdenii). The activity of monensin can be assayed by its ability to cause potassium loss, and results indicated that the amount of monensin needed to catalyze half maximal potassium depletion (K_d) from low G + C gram-positive ruminal bacteria varied by as much as 130-fold. The K_d values for Butyrivibrio fibrisolvens 49, Streptococcus bovis JB1, Clostridium aminophilum F, S. ruminantium HD4, and M. elsdenii B159 were 10, 65, 100, 1020, and 1330 nM monensin, respectively. B. fibrisolvens was very sensitive to monensin, and it did not adapt. S. bovis and C. aminophilum cultures that were transferred repeatedly with sub-lethal doses of monensin had higher K_d values than unadapted cultures, but the K_d was always less than 800 nM. S. ruminantium and M. elsdenii cells were highly resistant (K_d > 1000 nM), and this resistance could be explained by the ability of these low G + C Gram-positive bacteria to synthesize outer membranes. Received: 14 May 1999 / Accepted: 24 June 1999     

Selection of a Highly Monensin-Resistant Prevotella bryantii Subpopulation with Altered Outer Membrane Characteristics

Prevotella bryantii cultures treated with monensin grew more slowly than untreated cultures, but only if the monensin concentration was greater than 1 μM. Cultures that were repeatedly transferred (eight transfers or 25 doublings) with monensin always grew rapidly, even at a 10 μM concentration. The amount of monensin needed to facilitate half-maximal potassium depletion (K_d) from monensin-selected cells was 16-fold greater than “unadapted” wild-type cultures (3,200 versus 200 nM). Cells taken from continuous culture had a K_d of 100 nM, and these inocula could not grow in batch culture when the monensin concentration was greater than 300 nM. Continuous cultures treated with monensin nearly washed out, but the surviving cells had a K_d of 1,300 nM. When wild-type cells were transferred in batch culture with 10 μM monensin, the K_d did not reach its maximum value (3,200 nM) until after eight transfers (25 doublings). K_d declined when monensin was removed, and it took eight transfers to reach the control value (200 nM). The most probable number of wild-type cells was 1,000-fold lower than of the monensin-selected cells, but calculations based on relative growth advantage and K_d indicated that the wild-type culture had 1 to 10% highly monensin-resistant cells. Cell pellets of wild-type cultures were more difficult to disperse than were monensin-selected cells, and water-soluble phenol extracts of monensin-selected cells had 1.8-fold more anthrone-reactive material than did the wild type. Wild-type cultures that were washed in Tris buffer (pH 8.0) released little alkaline phosphatase and were agglutinated by lysozyme. Monensin-selected cultures leaked ninefold more alkaline phosphatase and were not agglutinated by lysozyme. Wild-type colonies taken from high-dilution agar roll tubes retained the lysozyme agglutination phenotype even if transferred with monensin, and monensin-selected colonies were never agglutinated. These observations indicated that wild-type P. bryantii cultures had a subpopulation with different outer membrane characteristics and increased monensin resistance.     

Spermine Inhibits [3H]Glycine Binding at the NMDA Receptors from Plexiform Layers of Chick Retina

Saturable specific binding of glycine to synaptosomal membranes from plexiform layers of the retina has been described, which seems to correspond to the modulatory site on NMDA-receptors (26). Spermine inhibited specific [³H]glycine binding to membranes from synaptosomal fractions from the outer (P₁) and the inner (P₂) plexiform layers of 1–3 day-old chick retinas in a dose-dependent manner with an IC₅₀ = 35 M for the P₁ fraction and 32 M for the P₂ fraction. Kinetic experiments and non-linear regression analysis of [³H]glycine-specific binding showed a K_d ~ 100–150 nM in both fractions, and a higher B_max (4.11 ± 0.47 pmol/mg protein) for the inner plexiform layer compared to the outer plexiform layer (B_max = 2.76 ± 0.25 pmol/mg protein). Strychnine-insensitive [³H]glycine binding was inhibited by 100 M spermine, due to a reduction in B_max (P₁ = 0.84 ± 0.16 pmol/mg protein; P₂ = 0.81 ± 0.16 pmol/mg protein) without affecting the K_d. Association and dissociation constants in the absence and presence of 50 M spermine remained unchanged. Results demonstrate the presence of a single modulatory site for spermine on NMDA receptors, in both synaptic layers of the chick retina.     

Non-specific DNAases from the rumen bacteriumPrevotella bryantii

Extracellular non-specific nucleases were observed in some strains belonging to the ruminal species of the genusPrevotella, mostlyP. brevis andP. bryantii. The nuclease fromP. bryantii appeared to be extracellular; it mediates the degradation of the supercoiled plasmid DNAvia an open circle intermediate. The cleavage is not site specific although a preference for certain cleavage sites does seem to exist. Our attempts to clone the wild-typeP. bryantii B₁4 nuclease inE. coli strain ER1992 that reports on the DNA damage sustained, were unsuccessful probably due to excessive intracellular nuclease activity that killed the cells bearing the gene for the nuclease. On the other hand, the nuclease from a related strain TC1-1, which has a less active enzyme of the same type, was successfully cloned.     

Characterization of Na+-Coupled Glutamate/Aspartate Transport by a Rat Brain Astrocyte Line Expressing GLAST and EAAC1

d-Aspartate (d-Asp) uptake by suspensions of cerebral rat brain astrocytes (RBA) maintained in long-term culture was studied as a means of characterizing function and regulation of Glutamate/Aspartate (Glu/Asp) transporter isoforms in the cells. d-Asp influx is Na⁺-dependent with K _m = 5 μm and V _max= 0.7 nmoles · min⁻¹· mg protein⁻¹. Influx is sigmoidal as f[Na⁺] with _Na+ K _m ∼ 12 μm and Hill coefficient of 1.9. The cells establish steady-state d-Asp gradients >3,000-fold. Phorbol ester (PMA) enhances uptake, and gradients near 6,000-fold are achieved due to a 2-fold increase in V _max, with no change in K _m . At initial [d-Asp] = 10 μm, RBA take up more than 90% of total d-Asp, and extracellular levels are reduced to levels below 1 μm. Ionophores that dissipate the Δμ_Na+ inhibit gradient formation. Genistein (GEN, 100 μm), a PTK inhibitor, causes a 40% decrease in d-Asp. Inactive analogs of PMA (4α-PMA) and GEN (daidzein) have no detectable effect, although the stimulatory PMA response still occurs when GEN is present. Further specificity of action is indicated by the fact that PMA has no effect on Na⁺-coupled ALA uptake, but GEN is stimulatory. d-Asp uptake is strongly inhibited by serine-O-sulfate (S-O-S), threohydroxy-aspartate (THA), l-Asp, and l-Glu, but not by d-Glu, kainic acid (KA), or dihydrokainate (DHK), an inhibition pattern characteristic of GLAST and EAAC1 transporter isoforms. mRNA for both isoforms was detected by RT-PCR, and Western blotting with appropriate antibodies shows that both proteins are expressed in these cells. Received: 11 January 2001/Revised: 26 March 2001     

Changes in the kinetics of phosphate and potassium absorption in nutrient-deficient barley roots measured by a solution-depletion technique

From measurements of the rates of depletion of labelled ions from solution in the low concentration range, we described the phosphate and potassium uptake characteristics of the roots of intact barley plants in terms of the kinetic parameters, K _m and I _max (the maximum rate of uptake). In relatively young (13 d) and older (42 d) plants, cessation of phosphate supply for 4 d or more caused a marked increase in I _max (up to four times), without concomitant change in K _m, which remained between 5 and 7 M. By contrast, 1 d of potassium starvation with 14-d plants caused a decline in the K _m (i.e. an increased apparent affinity for potassium) from 53 M to 11 M, without alteration to I _max. After longer periods of potassium starvation, I _max increased (about two times) while the K _m remained at the same low value. Growth of shoots and roots were unaffected by these treatments, so that concentrations of ions in the tissues declined after 1 d or more of nutrient starvation, but we could not identify a characteristic endogenous concentration for either nutrient at which changes in kinetic parameters were invariably induced. The possible mechanisms regulating carriermediated transport, and the importance of changes induced in kinetic parameters in ion uptake from solution and soil are discussed.Symbol I _max the maximum rate of absorption at saturating concentrations     

Allosteric effects of atropine and carbachol on the activity of α<Subscript>2</Subscript>-adrenoceptors in rat brain cortex membranes

Activation and inhibition of muscarinic cholinoceptors by atropine and carbachol are shown to exert allosteric effects on the binding of specific nonselective α₂-adrenoceptor antagonist [³H]RX821002 in rat brain cortex membranes. The ligand-receptor interaction for α₂-adrenoceptors corresponded to the model suggesting the presence of one homogeneous pool of receptors with two specific binding sites. The parameters of the [³H]RX821002 binding were as follows: [³H]RX821002 -K _d = 1.94 ± 0.08 nM, B _max = 13.4 ± 1.8 fmol/mg protein, n = 2. The inhibition of muscarinic cholinoceptors by atropine induced an increase of affinity (K _d = 1.36 ± 0.12 nM) and a decrease of the α2-adrenoceptor density (B _max = 10.18 ± 0.48 fmol/mg protein). The muscarinic cholinoceptor agonist carbachol induced an increase of the affinity (K _d = 1.56 ± 0.05 nM) and quantity of binding sites (B _max = 16.61 ± 0.29 fmol/mg protein). As a result, under the influence of atropine and carbachol, the efficiency of binding (E = B _max/2K _d) increased from 3.50 ± 0.40 to 5.60 ± 0.79 and 6.86 ± 0.20 fmol/mg protein/nM, respectively. The data suggest that α₂-adrenoceptors exist in rat brain cortex as homodimers.     

Construction and one-step purification of Bacillus kaustophilus leucine aminopeptidase fused to the starch-binding domain of Bacillus sp. strain TS-23 α-amylase

The starch-binding domain of Bacillus sp. strain TS-23 α-amylase was introduced into the C-terminal end of Bacillus kaustophilus leucine aminopeptidase (BkLAP) to generate a chimeric enzyme (BkLAPsbd) with raw-starch-binding activity. BkLAPsbd, with an apparent molecular mass of approximately 65 kDa, was overexpressed in Escherichia coli M15 cells and purified to homogeneity by nickel–chelate chromatography. Native PAGE and chromatographic analyses revealed that the purified fusion protein has a hexameric structure. The half-life for BkLAPsbd was 12 min at 70°C, while less than 20% of wild-type enzyme activity retained at the same heating condition. Compared with the wild-type enzyme, the 60% decrease in the catalytic efficiency of BkLAPsbd was due to a 91% increase in K _m value. Starch-binding assays showed that the K _d and B _max values for the fusion enzyme were 2.3 μM and 0.35 μmol/g, respectively. The adsorption of the crude BkLAPsbd onto raw starch was affected by starch concentration, pH, and temperature. The adsorbed enzyme could be eluted from the adsorbent by 2% soluble starch in 20 mM Tris–HCl buffer (pH 8.0). About 49% of BkLAPsbd in the crude extract was recovered through one adsorption–elution cycle with a purification of 11.4-fold.     

Oxidation of arsenite by two β-proteobacteria isolated from soil

Two heterotrophic As(III)-oxidizing bacteria, SPB-24 and SPB-31 were isolated from garden soil. Based on 16S rRNA gene sequence analysis, strain SPB-24 was closely related to genus Bordetella, and strain SPB-31 was most closely related to genus Achromobacter. Both strains exhibited high As(III) (15 mM for SPB-24 and 40 mM for SPB-31) and As(V) (>300 mM for both strains) resistance. Both strains oxidized 5 mM As(III) in minimal medium with oxidation rate of 554 and 558 μM h⁻¹ for SPB-24 and SPB-31, respectively. Washed cells of both strains oxidized As(III) over broad pH and temperature range with optimum pH 6 and temperature 42°C for both strains. The As(III) oxidation kinetic by washed cells showed K _m and V _max values of 41.7 μM and 1,166 μM h⁻¹ for SPB-24, 52 μM and 1,186 μM h⁻¹ for SPB-31. In the presence of minimal amount of carbon source, the strains showed high As(III) oxidation rate and high specific arsenite oxidase activity. The ability of strains to resist high concentration of arsenic and oxidize As(III) with highest rates reported so far makes them potential candidates for bioremediation of arsenic-contaminated environment.     

d-Amino acid dehydrogenase from Helicobacter pylori NCTC 11637

Helicobacter pylori is a microaerophilic bacterium, associated with gastric inflammation and peptic ulcers. d-Amino acid dehydrogenase is a flavoenzyme that digests free neutral d-amino acids yielding corresponding 2-oxo acids and hydrogen. We sequenced the H. pylori NCTC 11637 d-amino acid dehydrogenase gene, dadA. The primary structure deduced from the gene showed low similarity with other bacterial d-amino acid dehydrogenases. We purified the enzyme to homogeneity from recombinant Escherichia coli cells by cloning dadA. The recombinant protein, DadA, with 44 kDa molecular mass, possessed FAD as cofactor, and showed the highest activity to d-proline. The enzyme mediated electron transport from d-proline to coenzyme Q₁, thus distinguishing it from d-amino acid oxidase. The apparent K _m and V _max values were 40.2 mM and 25.0 μmol min⁻¹ mg⁻¹, respectively, for dehydrogenation of d-proline, and were 8.2 μM and 12.3 μmol min⁻¹ mg⁻¹, respectively, for reduction of Q₁. The respective pH and temperature optima were 8.0 and 37°C. Enzyme activity was inhibited markedly by benzoate, and moderately by SH reagents. DadA showed more similarity with mammalian d-amino acid oxidase than other bacterial d-amino acid dehydrogenases in some enzymatic characteristics. Electron transport from d-proline to a c-type cytochrome was suggested spectrophotometrically.     

Cleavage of di- and tripeptides by Prevotella ruminicola

The final step in the conversion of protein to amino acids by the common Gram-negative rumen bacterium, Prevotella (formerly Bacteroides) ruminicola , is the cleavage of di- and tripeptides. Dipeptidase and tripeptidase activities were predominantly cytoplasmic, and toluene treatment increased the rate of Ala₂ and Ala₃ hydrolysis by whole cells, suggesting that transport limited the rate of hydrolysis of extracellular di- and tripeptides. The hydrolysis of Ala₂ and Ala₃ by whole cells was not affected by protonophores, ionophores or dicyclohexylcarbodiimide, but Ala₂ hydrolysis by EDTA-treated cells was inhibited by the Ca²⁺/H⁺ ionophore, tetronasin. Ala₃ hydrolysis was not affected by protonophores or ionophores in EDTA-treated cells. The dipeptidase of strain M384 was inhibited > 99% by 1,10-phenanthroline and 39% by EDTA but not other protease inhibitors, consistent with the enzyme being a metalloprotease. Tripeptidase was insensitive to protease inhibitors, except for a 33% inhibition by EDTA. Cleavage of tripeptides occurred at the bond adjacent to the N-terminal amino acid. Distinct di-, tri- and oligopeptidase peaks were obtained by anion-exchange liquid chromatography of disrupted cells. Banding patterns on native PAGE using activity staining also indicated that P. ruminicola M384 had separate single dipeptidase and tripeptidase enzymes which hydrolysed a range of peptides. The dipeptidase of strain M384 was different from other strains of P. ruminicola: strains GA33 and B₁4 had activities which ran at the same R_f; strain GA33 had another band of lower activity; strain 23 had two bands different from those of the other strains. The tripeptidases ran at the same R_f for the different strains. Dipeptidase activity of all strains was inhibited by 1,10-phenanthroline on gels. Gel permeation chromatography indicated that the M_r of the dipeptidases from strains M384 and B₁4 were 115 000 and 114 500 respectively, and 112 500 and 121 500 for the corresponding tripeptidases. Thus the metabolism of small peptides by P. ruminicola involves separate permeases and intracellular peptidases for di- and tripeptides.     

2,4-Dichlorophenoxyacetic Acid (2,4-D) Utilization by Delftia acidovorans MC1 at Alkaline pH and in the Presence of Dichlorprop is Improved by Introduction of the tfdK Gene

Growth of Delftia acidovorans MC1 on 2,4-dichlorophenoxyacetic acid (2,4-D) and on racemic 2-(2,4-dichlorophenoxy)propanoic acid ((RS)-2,4-DP) was studied in the perspective of an extension of the strain’s degradation capacity at alkaline pH. At pH 6.8 the strain grew on 2,4-D at a maximum rate (μ_max) of 0.158 h⁻¹. The half-maximum rate-associated substrate concentration (K_s) was 45 μM. At pH 8.5 μ_max was only 0.05 h⁻¹ and the substrate affinity was mucher lower than at pH 6.8. The initial attack of 2,4-D was not the limiting step at pH 8.5 as was seen from high dioxygenase activity in cells grown at this pH. High stationary 2,4-D concentrations and the fact that μ_max with dichlorprop was around 0.2 h⁻¹ at both pHs rather pointed at limited 2,4-D uptake at pH 8.5. Introduction of tfdK from D. acidovorans P4a by conjugation, coding for a 2,4-D-specific transporter resulted in improved growth on 2,4-D at pH 8.5 with μ_max of 0.147 h⁻¹ and K_s of 267 μM. Experiments with labeled substrates showed significantly enhanced 2,4-D uptake by the transconjugant TK62. This is taken as an indication of expression of the tfdK gene and proper function of the transporter. The uncoupler carbonylcyanide m-chlorophenylhydrazone (CCCP) reduced the influx of 2,4-D. At a concentration of 195 μM 2,4-D, the effect amounted to 90% and 50%, respectively, with TK62 and MC1. Cloning of tfdK also improved the utilization of 2,4-D in the presence of (RS)−2,4-DP. Simultaneous and almost complete degradation of both compounds occurred in TK62 up to D = 0.23 h⁻¹ at pH 6.8 and up to D = 0.2 h⁻¹ at pH 8.5. In contrast, MC1 left 2,4-D largely unutilized even at low dilution rates when growing on herbicide mixtures at pH 8.5.     

The bacteriophages of ruminal prevotellas

Rumen bacteriophage-lyzed bacterial strains of the genusPrevotella were isolated and preliminarily characterized. The strain TC1-1 the speciesP. bryantii was the only prevotella strain successfully infected with filter sterilized rumen fluid from a black-and-white Holstein cow. Two types of plaques were observed, both rather small and turbid. Preliminary electron microscopy observation showed that several morphologically different bacteriophages were present in these plaques. The plaque eluates were further used for the infection of other prevotella strains. The plaques produced by the bacteriophages were observed with two strains,i.e. P. bryantii B₁4 andP. brevis GA33. The bacteriophages from both strains were examined by transmission electron microscopy and several morphologically different bacteriophages were observed, among others also a large virion with an icosahedral head with the diameter of approximately 120 nm. The bacteriophage was identified in plaques of bacterial cells of the strain GA33 and has an approximately 800 nm long helical tail, which places it among the largest ruminal bacteriophages described to date. Other bacteriophages from the same indicator strain as well as fromP. bryantii B₁4 strain were smaller and tail structures were not observed in all of them.     

Biochemical pharmacology of total retro-inverso analogues of bradykinin and angiotensin II: Molecular recognition by G-protein-coupled receptors and angiotensin converting enzyme

Summary Total retro-inverso (TRI) analogues of bradykinin (BK), the B_2a -selective kinin antagonistd-Arg⁰[Hyp³,d-Phe⁷,Leu⁸]BK, angiotensin II (AT II) and the AT II antagonist Saralasin ([Sar¹, Val⁵, Ala⁸]AT II) were prepared by conventional solid-phase synthesis. Molecular recognition of TRI peptidomimetics by G-protein-coupled receptors was studied by competitive radioligand displacement experiments. TRI analogues ofd-Arg⁰[Hyp³,d-Phe⁷,Leu⁸]BK specifically bound to the kidney medulla B_2a bradykinin receptor with affinities (K _d ) ranging from 64 μM to 4 μM. Conversely, TRI analogues of BK, AT II and Saralasin did not bind to either the B_2a bradykinin receptor or the rat AT_1a AT II receptor, respectively. These studies indicate that the TRI strategy is more compatible with the synthesis of antagonists than ‘agonists’. Three TRI peptidomimetics ofd-Arg⁰[Hyp³,d-Phe⁷,Leu⁸]BK were weak inhibitors of angiotensin converting enzyme. All other TRI peptidomimetics had no effect upon ACE activity. These data endorse the utility of the TRI strategy for the synthesis of protease-resistant antagonists of peptide hormones and neuropeptides.     

The Ca2+-extruding ATPase of the human platelet creates and responds to cytoplasmic pH changes,consistent with a 2 Ca2+/nH+ exchange mechanism

The Ca²⁺-extruding ATPase pump of the human platelet was studiedin situ by measuring Ca²⁺ extrusion from quin2-overloaded platelets (Johansson, J.S., Haynes, D.H. 1988.J. Membrane Biol. 104:147–163). Cytoplasmic pH (pH_cyt) was measured by BCECF fluorescence in parallel experiments. The pump was studied by raising the cytoplasmic free Ca²⁺ to 2.5 μM and monitoring active Ca²⁺ extrusion into a Ca²⁺-free medium. The pump was shown to perturb pH_cyt, to not respond to changes in membrane potential and to respond to imposed changes in pH_cyt in a manner consistent with the Ca²⁺ pump acting as a 2 Ca²⁺/nH⁺ exchanger. (i) Raising the external pH (pH_ext) from 7.40 to 7.60 lowers the V_max of the pump in basal condition (V_max,1) from 110±18 to 73±12 μM/min (=μmol/liter cell volume/min). (ii) Lowering pH_ext to 7.13 raised V_max,1 to 150±15 μM/min. (iii) In an N-methyl-d-glucamine (NMDG⁺) medium, the pump operation against high [Ca²⁺]_cyt acidifies the cytoplasm by −0.36±0.10 pH units, and the pump becomes self-inhibited. (iv) Use of nigericin to drive pH_cyt down to 6.23 reduces the V_max,1 to 18±11 μM/min. (v) Alkalinization of the cytoplasm by monensin in the presence of Na⁺ raises the V_max,1 (basal state withK _m,1=80 nM) to 136±24 μM/min, but also activates the pump fourfold (V_max,2=280±28 μM/min;K _m,2=502±36 nM). (vi) Transient elevation of pH_cyt by NH₄Cl at high [Ca²⁺]_cyt activates the pump eightfold (V_max,2≥671±350 μM/min). The large activation by alkaline pH_cyt at high [Ca²⁺]_cyt can be explained by Ca²⁺-calmodulin activation of the pump (Valant, P.A., Adjei, P.N., Haynes, D.H. 1992.J. Membrane Biol. 130:63–82) and by increased Ca²⁺ affinity of calmodulin at high pH.     

Cation-dependent nutrient transport in shrimp digestive tract

Purified epithelial brush border membrane vesicles (BBMV) were produced from the hepatopancreas of the Atlantic White shrimp, Litopeneaus setiferus, using standard methods originally developed for mammalian tissues and previously applied to other crustacean and echinoderm epithelia. These vesicles were used to study the cation dependency of sugar and amino acid transport across luminal membranes of hepatopancreatic epithelial cells. ³H-d-glucose uptake by BBMV against transient sugar concentration gradients occurred when either transmembrane sodium or potassium gradients were the only driving forces for sugar accumulation, suggesting the presence of a possible coupled transport system capable of using either cation. ³H-l-histidine transport was only stimulated by a transmembrane potassium gradient, while ³H-l-leucine uptake was enhanced by either a sodium or potassium gradient. These responses suggest the possible presence of a potassium-dependent transporter that accommodates either amino acid and a sodium-dependent system restricted only to l-leucine. Uptake of ³H-l-leucine was significantly stimulated (P < 0.05) by several metallic cations (e.g., Zn²⁺, Cu²⁺, Mn²⁺, Cd²⁺, or Co²⁺) at external pH values of 7.0 or 5.0 (internal pH 7.0), suggesting a potential synergistic role of the cations in the transmembrane transfer of amino acids. ³H-l-histidine influxes (15 suptakes) were hyperbolic functions of external [zinc] or [manganese], following Michaelis–Menten kinetics. The apparent affinity constant (e.g., K _m) for manganese was an order of magnitude smaller (K _m = 0.22 μM Mn) than that for zinc (K _m = 1.80 μM Zn), while no significant difference (P > 0.05) occurred between their maximal transport velocities (e.g., J _max). These results suggest that a number of cation-dependent nutrient transport systems occur on the shrimp brush border membrane and aid in the absorption of these important dietary elements.     

Day–night specific binding of 2-[125I]Iodomelatonin and melatonin content in gill, small intestine and kidney of three fish species

Some of melatonin’s (Mel) well-established physiological effects are mediated via high-affinity cell-membrane receptors belonging to the superfamily of G-protein-coupled receptors. Specific binding of ligand 2-[¹²⁵I]iodomelatonin, using membrane preparations from osmoregulatory tissues of flounder, rainbow trout and sea bream, together with Mel concentrations in the tissues and plasma were studied. The kidney, gill and small intestine samples were collected during the day and at night. The dissociation constants (K _d) and maximal binding densities (B _max) were calculated for each tissue at 11:00 and 23:00 h. The binding sites with K _d values in the tissues in the picomolar range indicated the high affinity. K _d and B _max values were tissue- and species-dependent. The GTP analogue [Guanosine 5′-O-(3-thiotriphosphate)] treatment significantly reduced the B _max value, indicating that the 2-[¹²⁵I]iodomelatonin-binding sites are probably coupled to a G-protein. No daily variations in K _d and B _max values were observed. These are the first studies of the presence of 2-[¹²⁵I]iodomelatonin-binding sites in the small intestine, kidney tubule and gill of fish. The data strongly suggest new potential targets for Mel action and the influence of Mel on water/ion balance in fish. The intestine seems to be a site of Mel synthesis and/or an active accumulation of the hormone.     

Pharmacological properties of the potassium-activated inward current in pyramidal hippocampal neurons

Elevation of the external potassium concentration induced a two-phase inward current in freshly isolated pyramidal hippocampal neurons. This current was voltage-dependent and demonstrated strong inward rectification. The current consisted of a leakage current and a time-dependent current (τ=40–50 msec at 21°C); the latter was designated asI _ΔK. As was shown earlier, K⁺ is a major charge carrier in the development of slow potassium-activated current. The pharmacological properties ofI _ΔK were studied using a patch-clamp technique.I _ΔK was completely blocked by external 10 mM TEA or 5 mM Ba²⁺ (IC₅₀=480±90mM) and exhibited low sensitivity to extracellular Cs⁺ (2 mM). This current was not affected by 1 mM 4-aminopyridine and was insensitive to a muscarinic agonist, carbachol (50 μM), and to 1 mM extracellular Cd²⁺. Elevation of external Ca²⁺ from 2.5 mM to 10 mM did not changeI _ΔK. Our data indicate that the pharmacological properties ofI _ΔK differ from those of other voltage-gated potassium currents, but more specific blockers must be used to make this evidence conclusive.