Binding of isolectins from red kidney bean (Phaseolus vulgaris) to purified rat brush-border membranes

Ingestion of red kidney bean phytohemagglutinin causes impaired growth and intestinal malabsorption, and facilitates bacterial colonization in the small intestine of weanling rats. We have studied interactions of the highly purified phytohemagglutinin erythroagglutinating (E4) and mitogenic (L4) isolectins with microvillous membrane vesicles prepared from rat small intestines. E4 and L4 were radioiodinated with 125I by the chloramine-T technique. E4 and L4 isolectins both bound to microvillous membrane vesicles. Binding was saturable and reversible. Each mg of membrane protein bound 744 +/- 86 micrograms E4 and 213 +/- 21 micrograms L4. The apparent Ka for E4 and L4 binding was 2.5 x 10(-6) and 13.0 x 10(-6) M-1, respectively. Binding of each 125I-labelled isolectin was abolished by 100-fold excess of unlabelled isolectin. In each case binding also was inhibited by appropriate oligosaccharide inhibitors, indicating that isolectin-microvillous membrane interactions were mediated by carbohydrate recognition. Patterns of saccharide inhibition of isolectin binding were different for E4 and L4. Competitive binding experiments demonstrated mutual noncompetitive inhibition of E4 and L4 binding consistent with steric hindrance. Therefore, E4 and L4 each bound to its own set of receptors. Based on the known saccharide specificities of E4 and L4, these data indicate that there are differences in expression of complex asparagine-linked biantennary and tri- or tetraantennary oligosaccharides at the microvillous surface. The data also provide the possibility that direct interactions of one or more phytohemagglutinin isolectins with intestinal mucosa in vivo may contribute to the antinutritional effects associated with ingestion of crude red kidney beans.     

Seasonal differences in activity of perch (Perca flavescens) gill Na/K ATPase

We measured Na⁺/K⁺ ATPase activity in homogenates of gill tissue prepared from field caught, winter and summer acclimatized yellow perch, Perca flavescens. Water temperatures were 2–4°C in winter and 19–22°C in summer. Na⁺/K⁺ ATPase activity was measured at 8, 17, 25, and 37°C. V_max values for winter fish increased from 0.48±0.07 μmol P mg⁻¹ protein h⁻¹ at 8°C to 7.21±0.79 μmol P mg⁻¹ protein h⁻¹ at 37°C. In summer fish it ranged from 0.46±0.08 (8°C) to 3.86±0.50 (37°C) μmol P mg⁻¹ protein h⁻¹. The K_m for ATP and for Na⁺ at 8°C was ≈1.6 and 10 mM, respectively and did not vary significantly with assay temperature in homogenates from summer fish. The activation energy for Na⁺/K⁺ ATPase from summer fish was 10 309 (μmol P mg⁻¹ h⁻¹) K⁻¹. In winter fish, the K_m for ATP and Na⁺ increased from 0.59±0.08 mM and 9.56±1.18 mM at 8°C to 1.49±0.11 and 17.88±2.64 mM at 17°C. The K_m values for ATP and Na did not vary from 17 to 37°C. A single activation energy could not be calculated for Na/K ATPase from winter fish. The observed differences in enzyme activities and affinities could be due to seasonal changes in membrane lipids, differences in the amount of enzyme, or changes in isozyme expression.     

The response of intact Strongyloides ratti infective (L3) larvae to substrates and inhibitors of respiratory electron transport

Live, intact third-stage larvae (L3s) of Strongyloides ratti in the absence of exogenous substrates consumed oxygen at a rate (E-QO₂) of 181.8 ± 12.4 ng atoms min⁻¹ mg dry weight⁻¹ at 35°C. Respiratory electron transport (RET) Complex I inhibitor rotenone (2 μm) produced 33 ± 6.5% inhibition of the E-QO₂. Unusually the rotenone-induced inhibition was not relieved by 5 μm-succinate. The E-QO₂ of intact L3s was refractory to RET Complex III inhibitor antimycin A at 2 μm; 4 μm-antimycin inhibited ≤ 10% of the E-QO₂. The electron donor couple ascorbate/TMPD augmented the E-QO₂ in the presence of rotenone (2 μm) and antimycin A (4 μm) by 110%. Azide (1 mm) stimulated the antimycin A refractory QO₂ by 36.6 ± 7.2% which was only partially inhibited by 1.0 mm-KCN (IC₅₀ = 0.8 mm). The data suggest the presence of classical (CPW) and alternate (APW) electron transport pathways in S. ratti L3s.     

A spectrophotometric determination of the binding constants of cyanide by hemin in aqueous ethanol solutions

Binding constants for the coordination of cyanide by monomeric hemin in aqueous ethanol solution were determined in the pH range 8–12 by fitting spectrophotometric data to a binding isotherm using a non-linear least-squares method. Two cyanide ligands are coordinated by Fe(III) without evidence for the intermediate mono-ligated species. The binding constants are strongly pH-dependent because of protonation of the ligand and competition for the metal ion by hydroxide. From the pH dependence of the observed binding constants, a pH-independent value of log K = 17.62±0.03 for the binding of two CN⁻ ligands by monomeric hemin in alkaline solution at 25 °C, μ=0.100 M, is obtained.     

Growth of dinoflagellates, Ceratium furca and Ceratium fusus in Sagami Bay, Japan: The role of temperature, light intensity and photoperiod

Seasonal changes of field populations and growth rates of two dinoflagellates, Ceratium furca and Ceratium fusus, were examined in the temperate coastal water of Sagami Bay, Japan. Weekly field sampling was conducted from August 2002 to August 2003, and laboratory experiments were also carried out to investigate effects of temperature, irradiance and photoperiod on the growth rates of these two Ceratium species. In the field, the abundances of both species increased significantly from April to August 2003, were gradually decreased from November 2002 and were not observed in January 2003. C. fusus was able to increase at lower temperatures in February 2003 compared to C. furca. In the laboratory, the two species did not grow at <10 °C or >32 °C. The highest specific growth rate of C. furca was 0.72 d⁻¹ at 24 °C and 600 μmol m⁻² s⁻¹. Optimum growth rates (>0.4 d⁻¹) of C. furca were observed at temperatures from 18 to 28 °C and at irradiances from 216 to 796 μmol m⁻² s⁻¹. The highest growth rate of C. fusus was 0.56 d⁻¹ at 26 °C and 216 μmol m⁻² s⁻¹. Optimum growth rates of C. fusus were observed at the same irradiance rage of C. furca, whereas optimum temperature range was narrower (26–28 °C). The growth curves of both species indicated saturation of the growth rates when light intensity was above 216 μmol m⁻² s⁻¹, and did not show photoinhibition at irradiances up to 796 μmol m⁻² s⁻¹. The specific growth rates of both Ceratium species were clearly decreased at L:D = 10:14 relative to those at L:D = 14:10 and L:D = 12:12. The present study indicates the two Ceratium species can adapt to a wide range of temperature and irradiance.     

Separation and biological properties of Phaseolus vulgaris isolectins

Two isolectins (L₄E₀-PHA and L₀E₄-PHA) from red kidney beans (Phaseolus vulgaris) were isolated by affinity chromatography on immobilized thyroglobulin and by chromatography on hydroxyapatite. The L₄E₀-PHA siolectin was not retarded on the affinity column and was eluted from the hydroxyapatite column with a 0.1 M Phosphate buffer. This isolectin had no erythroagglutinating activity but had high lymphoagglutinating and lymphocyte stimulating activities. The L₀E₄-PHA isolectin was adsorbed on the affinity column and was eluted from the hydroxyapatite column with a 0.25 M Phosphate buffer. This isolectin has a high erythroagglutinating activity, a very low lymphoagglutinating activity and no lymphocyte stimulating activity. These two isolectins are shown however to be closely related with respect to their oligomeric structure and reactivity towards anti-PHA antibodies. The lack of mitogenic activity of the L₀E₄-PHA isolectin suggests that in the other isolectins, the E monomer is not responsible for their mitogenic activity and that the membrane glycoproteins, which contain the E monomer — specific oligosaccharide, are not involved in the process inducing mitosis.     

Phaseolus vulgaris isolectin binding to human erythrocytes.

The Phaseolus vulgaris isolectins L4,L3E1, L2E2, L1E3, and E4 were isolated by affinity and ion exchange chromatography. Pure isolectins were radiolabeled by the chloramine-T method with Na125IO4 and their binding to human erythrocytes was studied. A normal erythrocyte has approximately 8 times 10(5) receptor sites for each isolectin; however, the association constants (Ka) of binding increased from 1.1 times 10(7) M-1 to 3.8 times 10(8) M-1, with increasing number of E subunits per tetrameric isolectin molecule. Isolectin to erythrocyte binding reached equilibrium rapidly and was reversed by fetuin. All isolectins competed with 125I-E4 for erythrocyte binding sites, with a constant (KI) similar to the Ka calculated for each respective radiolabeled isolectin. When isolectin binding at 0 degrees C, 4 degrees C, or 8 degrees C was compared to that at 25 degrees C, there was no reduction in the number of binding sites per cell, but the Ka of E4 was reduced to 3 times 10(7) M-1. Fixed erythrocytes displayed similar isolectin binding characteristics.     

Arachidonylethanolamide (anandamide) binds with low affinity to dihydropyridine binding sites in brain membranes

The purpose of this study was to explore the hypothesis that the dihydropyridine (DHP) binding site of the L-type calcium channel is a high affinity binding site for the cannabimimetic arachidonylethanolamide (AEA). Binding affinities were determined from competition isotherms using the DHP analog [³H]PN-200. AEA competed for [³H]PN-200 binding with a K₁ of 40 ± 4 μM. Inclusion of phenylmethylsulfonyl fluoride to inhibit an amidohydrolase that converts AEA to arachidonic acid had little effect on the K₁ of AEA (48 ± 6 μM). Arachidonic acid had a slightly higher K₁ (120 ± 11 μM) and other N-acylethanolamides examined (linolenylethanolamide, dihomo-γ-linolenylethanolamide, docosatetraenylethanolamide, and palmitoylethanolamide) had no effect on [³H]PN-200 binding at concentrations as high as 10 μM. Our conclusions are that AEA binds to the DHP binding site with relatively low affinity and its conversion to arachidonic acid is not required for binding.     

Gill (Na+,K+)-ATPase from the blue crab Callinectes danae: modulation of K+-phosphatase activity by potassium and ammonium ions

The kinetic properties of a microsomal gill (Na⁺,K⁺)-ATPase from the blue crab Callinectes danae were analyzed using the substrate p-nitrophenylphosphate. The (Na⁺,K⁺)-ATPase hydrolyzed PNPP obeying cooperative kinetics (n=1.5) at a rate of V=125.4±7.5 U mg⁻¹ with K_0.5=1.2±0.1 mmol l⁻¹; stimulation by potassium (V=121.0±6.1 U mg⁻¹; K_0.5=2.1±0.1 mmol l⁻¹) and magnesium ions (V=125.3±6.3 U mg⁻¹; K_0.5=1.0±0.1 mmol l⁻¹) was cooperative. Ammonium ions also stimulated the enzyme through site–site interactions (n_H=2.7) to a rate of V=126.1±4.8 U mg⁻¹ with K_0.5=13.7±0.5 mmol l⁻¹. However, K⁺-phosphatase activity was not stimulated further by K⁺ plus NH₄⁺ ions. Sodium ions (K_I=36.7±1.7 mmol l⁻¹), ouabain (K_I=830.3±42.5 μmol l⁻¹) and orthovanadate (K_I=34.0±1.4 nmol l⁻¹) completely inhibited K⁺-phosphatase activity. The competitive inhibition by ATP (K_I=57.2±2.6 μmol l⁻¹) of PNPPase activity suggests that both substrates are hydrolyzed at the same site on the enzyme. These data reveal that the K⁺-phosphatase activity corresponds strictly to a (Na⁺,K⁺)-ATPase in C. danae gill tissue. This is the first known kinetic characterization of K⁺-phosphatase activity in the portunid crab C. danae and should provide a useful tool for comparative studies.     

Dinuclear complexes of transition metals containing carbonate ligands. VIII. Kinetics and mechanism of carbon dioxide uptake by the tri-μ-hydroxo-bis (1,5-diamino-3-aza-pentane)cobalt(III) ion in weakly basic aqueous carbonate solution

The kinetics of formation of the complex ion, μ-carbonato-di-μ-hydroxo-bis((1,5-diamino-3-aza-pentane) cobalt(III), from the tri-μ-hydroxo-bis((1,5-diamino-3-aza-pentane(III)cobalt(III)) ion in aqueous buffered carbonate solution have been studied spectrophotometrically at 295 nm over the ranges 20.0θ°C34.8, 8.03pH9.44, 5 mM [CO₃²⁻35 mM and at an ionic strength of 0.1 M (LiClO₄). On the basis of the kinetic results a mechanism, involving rapid cleavage of an hydroxo bridge followed by carbon dioxide uptake with subsequent bridge formation, has been proposed. At 25 °C, the rate of the carbon dioxide uptake is 0.58 M⁻¹ s⁻¹ with ΔH≠ = (13.2±0.7) kcal mol⁻¹ and ΔS≠ = (−15.1 ± 0.7) cal deg⁻¹ mol⁻¹. The results are composed with those obtained for several mononuclear cobalt(III) and one dinuclear cobalt(III) complexes.     

Visualization of lactotransferrin brush-border receptors by ligand-blotting

The uptake of iron (III) mediated by lactotransferrin to human biopsies from upper intestine has suggested the presence of specific receptors for human lactotransferrin at the brush border (Cox, T., Mazurier, J., Spik, G., Montreuil, J. and Peters, T.J. (1979) Biochim. Biophys. Acta 588, 120–128). In the present data, using ¹²⁵I-radiolabeled transferrins, we have demonstrated that a preparation of microvillous membrane vesicles, from rabbit jejunal brush-border specifically binds human lactotransferrin. This binding is specific, saturable and calcium dependent. Scatchard plots analysis of lactotransferrin binding indicates 1.5 · 10¹³ sites per mg of membrane proteins with an equilibrium constant of 1.2 · 10⁶ M⁻¹. Sodium dodecyl sulfate solubilization of the brush-border proteins allows the lactotransferrin receptor to retain its binding activity. Moreover, the ligand blotting of the detergent solubilized membrane proteins on nitrocellulose sheet and after incubation with ¹²⁵I-labeled lactotransferrin, has shown that the receptor is a protein of about 100 kDa. In the same experimental conditions, the rabbit microvillous membrane vesicles do not specifically bind rabbit serotransferrin indicating the absence of serotransferrin receptors at the brush border.     

Properties and purification of the catalytic subunit of cyclic AMP-dependent protein kinase of adipose tissue

The catalytic subunit of cAMP-dependent protein kinase from rat adipose tissue was purified to apparent homogeneity by making use of the differential binding of the holoenzyme and the free catalytic subunit to CM-Sephadex and by gel chromatography. Stability and yield was improved by inclusion of nonionic detergent in all steps after dissociation of the holoenzyme. Isoelectric focusing separated enzyme species with pI values of 7.8 and 8.6–8.8. The amino acid composition was similar to the enzyme purified from other tissues. Enzyme activity was markedly unstable in dilute solutions (<5 μg/ml). Additions of nonionic detergent, glycerol, bovine serum albumin and, especially, histones stabilized the enzyme. With protamine, the catalytic subunit had an apparent K_m of 60 μM and V_max of 20 μmol·min⁻¹·mg⁻¹, corresponding values with mixed histones were 12 μM and 1.2 μmol·min⁻¹·mg⁻¹. With both protein substrates the apparent K_m for ATP was 11 μM. Concentrations of Mg²⁺ above 10 mM were inhibitory. Histone phosphorylation was inhibited by NaCl (50% at 0.5 M NaCl) while protamine phosphorylation was stimulated (4-fold at 1 M NaCl). Inorganic phosphate inhibited both substrates (histones: 50% at 0.3 M, and protamine: 50% at 0.5 M). pH optimum was around pH 9 with both substrates. The catalytic subunit contained 2.0 (range of three determinations, 1.7–2.3) mol phosphate/mol protein. It was autophosphorylated and incorporated ³²P_i from [γ-³²P]ATP in a time-dependent process, reaching saturation when approx. 0.1 mol phosphate/mol catalytic subunit was incorporated.     

Interaction of anthelmintic benzimidazoles and benzimidazole derivatives with bovine brain tubulin

The binding and inhibitory properties of 11 benzimidazoles for bovine brain tubulin were investigated. The effects of the benzimidazoles on the initial rates of microtubule polymerization were determined by a turbidimetric assay. The median inhibitory concentrations (I₅₀) for nocodazole, oxibendazole, parbendazole, mebendazole and fenbendazole ranged from 1.97 · 10⁻⁶ to 6.32 · 10⁻⁶ M. Benomyl, cambendazole and carbendazim had I₅₀ values from 5.83 · 10⁻⁵ to 9.01 · 10⁻⁵ M. Thiabendazole had an I₅₀ value of 5.49 · 10⁻⁴ M. Inhibitor constants (K_i) were determined by the colchicine binding assay. Oxibendazole, fenbendazole, and cambendazole had K_i values of 3.20 · 10⁻⁵, 1.73 · 10⁻⁵ and 1.10 · 10⁻⁴ M, respectively. Oxibendazole and fenbendazole were competitive inhibitors of colchicine. In contrast, cambendazole was a noncompetitive inhibitor of colchicine. The ability of these benzimidazoles to inhibit microtubule polymerization and the mode of action for the anthelmintic benzimidazoles is discussed.     

Effects of a synthetic prostaglandin analogue, cloprostenol, on the corpus luteum of the guinea pig

The effects of a synthetic prostaglandin analogue, cloprostenol, on luteal function in a guinea pig were studied. At a dose of 250μg, cloprostenol administered I-P on day 9 of the oestrous cycle caused a reduction in the length of the oestrous cycle from 17.4±s.d. 0.9 to 14.5±1.1 days (p<0.01). Lower doses were ineffective, and post-treatment cycles were not different in length from pre-treatment cycles. Cloprostenol also caused a dose-dependent reduction in luteal weight, which fell from 3.52±0.82 to 1.82±0.4mg (<0.01) 48 h after administation of a 250μg dose on day 9. Plasma progesterone, measured by radioimmunoassay, was reduced from 4.67±0.59 to 2.69±0.66 ng ml⁻¹(p<0.01) 48 h after administration of 250μg cloprostenol on day 9. 250μg cloprostenol also reduced blood flow per corpus luteum, measured by ⁸⁵Sr-labelled 15μm microspheres, both at 3 h (20.20±10.36 to 9.40±4.2μ1 min⁻¹; p0.05) and at 48 h 18.47±8.27 to 5.23±1.90μl min⁻¹; p<0.01) after administration on day 9. No adverse side-effects were observed at any dose level of cloprostenol used. It was concluded that cloprostenol is a useful experimental luteolysin in the guinea pig.     

Opioid peptides are substrates for the bifunctional enzyme LTA4 hydrolase/aminopeptidase

We determined if any naturally occurring peptides could act as substrates or inhibitors of the bifunctional, Zn²⁺ metalloenzyme LTA₄ hydrolase/aminopeptidase (E.C. 3.3.2.6). Several opioid peptides including met⁵-enkephalin, leu⁵-enkephalin, dynorphin_1–6, dynorphin_1–7, and dynorphin_1–8 competitively inhibited the hydrolysis of L-proline-p-nitroanilide by leukotriene A₄ hydrolase/ aminopeptidase, consistent with an interaction at its active site. The enzyme catalyzed the N-terminal hydrolysis of tyrosine from met⁵-enkephalin with K_m =450 ± 58 μM and V_max =4.9 ± 0.6 nmol-hr⁻¹-ug⁻¹ and from leu⁵-enkephalin with K_m =387 ± 90 μM and V_max =6.2 ± 2.5 nmol-hr⁻¹-ug⁻¹. Bestatin, captopril and carnosine inhibited the hydrolysis of the enkephalins. It is noteworthy that the bifunctional catalytic traits of this enzyme include generation of an hyperalgesic substance, LTB₄, and inactivation of analgesic opioid peptides.     

Peptidoleukotriene binding in guinea pig uterine membrane preparations

Peptidoleukotrienes are known to be potent smooth muscle contractile agents in many tissues, including guinea pig uterus. In order to characterize the receptors at which the leukotrienes interact, guinea pig uteri were homogenized in 50nM Tris-HCl, pH 7.4 at 40°C and centrifuged at 1000xg fpr 10 min. The supernatant was centrifuged at 40,000 xg and the washed pellet was used to measure the binding of ³H-LTC₄ and ³H-LTD₄. Specific binding of ³H-LTD₄ was not detected, but specific, saturable binding of ³H-LTC₄ was measured at 40°C, was complete in 10 min. and was rapidly reversible on addition of unlabeled LTC₄. Binding was linear with protein concentration and stimulated by CaCl₂ and L-serine borate. Scatchard and kinetic analysis of binding in the presence of calcium suggested a K_d of 10–12 nM. LTC₄ was a more potent competitor of binding than LTD₄ (IC₅₀ − 40nM and 30 μM, respectively). FPL 55712 inhibited binding from 10–100 μM but stimulated binding at lower concentrations. Thus, the guinea pig uterus has specific receptors for LTC₄, but not LTD₄, that can be demonstrated by radioligand binding.     

High-performance liquid chromatographic analysis of AQ4N, an alkylaminoanthraquinone N-oxide

A simple, highly selective and reproducible reversed-phase high-performance liquid chromatography method has been developed for the analysis of the new anti-cancer pro-drug AQ4N. The sample pre-treatment involves a simple protein precipitation protocol, using methanol. Chromatographic separations were performed using a HiChrom HIRPB (25 cm×4.6 mm I.D.) column, with mobile phase of acetonitrile–ammonium formate buffer (0.05 M) (22:78, v/v), with final pH adjusted to 3.6 with formic acid. The flow-rate was maintained at 1.2 ml min⁻¹. Detection was via photodiode array performed in the UV range at 242 nm and, since the compounds are an intense blue colour, in the visible range at 612 nm. The structurally related compound mitoxantrone was used as internal standard. The validated quantification range of the method was 0.05–10.0 μg ml⁻¹ in mouse plasma. The inter-day relative standard deviations (RSDs) (n=5) ranged from 18.4% and 12.1% at 0.05 μg ml⁻¹ to 2.9% and 3.3% at 10.0 μg ml⁻¹ for AQ4N and AQ4, respectively. The intra-day RSDs for supplemented mouse plasma (n=6) ranged from 8.2% and 14.2% at 0.05 μg ml⁻¹ to 7.6% and 11.5% at 10.0 μg ml⁻¹ for AQ4N and AQ4, respectively. The overall recovery of the procedure for AQ4N was 89.4±1.77% and 76.1±7.26% for AQ4. The limit of detection was 50 ng ml⁻¹ with a 100 μl sample volume. The method described provides a suitable technique for the future analysis of low levels of AQ4N and AQ4 in clinical samples.     

Effect of renoguanylin on hydrogen/bicarbonate ion transport in rat renal tubules

Renoguanylin (REN) is a recently described member of the guanylin family, which was first isolated from eels and is expressed in intestinal and specially kidney tissues. In the present work we evaluate the effects of REN on the mechanisms of hydrogen transport in rat renal tubules by the stationary microperfusion method. We evaluated the effect of 1 μM and 10 μM of renoguanylin (REN) on the reabsorption of bicarbonate in proximal and distal segments and found that there was a significant reduction in bicarbonate reabsorption. In proximal segments, REN promoted a significant effect at both 1 and 10 μM concentrations. Comparing control and REN concentration of 1 μM, JHCO₃⁻, nmol cm^− 2 s^− 1 − 1,76 ± 0,11_control × 1,29 ± 0,08_{REN 10 μM}; P < 0.05, was obtained. In distal segments the effect of both concentrations of REN was also effective, being significant e.g. at a concentration of 1 μM (JHCO₃⁻, nmol cm^− 2 s⁻ 1 − 0.80 ± 0.07_control × 0.60 ± 0.06_{REN 1 μM}; P < 0.05), although at a lower level than in the proximal tubule. Our results suggest that the action of REN on hydrogen transport involves the inhibition of Na⁺/H⁺exchanger and H⁺-ATPase in the luminal membrane of the perfused tubules by a PKG dependent pathway.     

Dopamine-stimulated cyclic AMP and binding of [H]dopamine in acini from dog pancreas

Dispersed acini from dog pancreas were used to examine the ability of dopamine to increase cyclic AMP cellular content and the binding of [³H]dopamine. Cyclic AMP accumulation caused by dopamine was detected at 1·10⁻⁸ M and was half-maximal at 7.9±3.4·10⁻⁷M. The increase at 1·10⁻⁵ M, (7.5-fold) was equal to the half-maximal increase caused by secretin at 1·10⁻⁹ M. Haloperidol, a dopaminergic receptor antagonist inhibited cyclic AMP accumulation caused by dopamine. The IC₅₀ value for haloperidol, calculated from the inhibition of cyclic AMP increase caused by 1·10⁻⁵ M dopamine was 2.3±0.9·10⁻⁶M. Haloperidol did not alter basal or secretin-stimulated cyclic AMP content. [³H]Dopamine binding was studied on the same batch of cells as cyclic AMP accumulation. At 37°C, it was rapid, reversible, saturable and stereospecific. The K_d value for high affinity binding sites was 0.43±0.1·10⁻⁷M and 4.7±1.6·10⁻⁷M for low affinity binding sites. The concentration of drugs necessary to inhibit specific binding of dopamine by 50% was 1.2±0.4·10^/t-7M noradrenaline, 2·10^/t-7 M epinine, 4.1±1.8·10^/t-6M fluphenazine, 8.0±1.6·10^/t-6M haloperidol, 4.2±1.2·10⁻⁶Mcis-flupenthixol, 2.7±0.4·10⁻⁵Mtrans-flupenthixol, >1·10⁻⁵M apomorphine, sulpiride, naloxone and isoproterenol.     

Ammonium excretion by a mutant of the nitrogen-fixing cyanobacterium Anabaena siamensis

Anabaena siamensis isolated from rice fields in Thailand is a fast growing cyanobacterium with a high nitrogen-fixing activity. Mutant strains resistant to the l-glutamate analogue, l-methionine sulfoximine (MSX) were isolated by ethyl methanesulfonate mutagenesis. A stable mutant named A. siamensis SS1, which released ammonium to the medium, was studied further. In batch cultures the rate of ammonium production peaked at the early log phase and gradually decreased until the 4th day of growth when the cultures reached a density of 90 μg chl ml⁻¹. To obtain constant release of ammonium by SS1, continuous culture experiments were performed at a cell density of 5 μg chl ml⁻¹ and the following results were obtained: (1) growth rate as the parent (μ:0·123 h⁻¹) in the presence and absence of 500 μm MSX; (2) 48% GS transferase activity when compared with the parent; (3) ammonium excretion at a rate of 8 μmol (mg chl)⁻¹ h⁻¹ as measured up to 20 generations (120 h); (4) depressed nitrogenase activity; and (5) 30% higher nitrogenase activity than that of the parent. SS1 immobilized in alginate beads (5 μg chl ml⁻¹) exhibited values of glutamine synthetase and nitrogenase activity similar to those of free cells. However, ammonium excretion at the rate of 11·61 μmol (mg chl)⁻¹ h⁻¹ was obtained only up to 20 h after loading in bioreactors, due to the fast growth of SS1 as also occurred in batch cultures.