Ouabain sensitive Na+/K+-transporting ATPase from the brain of Poekilocerus bufonius

• 1.1. The properties of Na⁺/K⁺-transporting ATPase in microsomal fractions from the nervous tissue of the grasshopper, Poekilocerus bufonius were investigated.
• 2.2. Two components of ATPase activity are present.
• 3.3. Inclusion of 1 mM ouabain in the incubation media reduced the activity of total and Na⁺/K⁺-ATPase by 57 and 79%, respectively.
• 4.4. The maximum velocity (V_max) was decreased by the addition of 1 mM ouabain, whereas the apparent K_m value was not affected indicating a non-competitive type of inhibition.
• 5.5. The calculated value of the pI₅₀ was 6.4 (I₅₀ = 3.98 × 10⁻⁷M) for ouabain inhibition of the enzyme showing great sensitivity to the cardiac glycoside ouabain.
• 6.6. The present results show that the physicochemical properties of Na⁺/K⁺-transporting ATPase from the brain of P. bufonius are essentially the same as for the enzyme prepared from the excretory system of the insect which has been previously investigated.
• 7.7. Dissimilarities were also observed between these tissues in the way that the enzyme from the brain was sensitive to ouabain inhibition with a non-competitive type rather than a ouabain-resistance and a competitive type of inhibition for the enzyme from the excretory system.
• 8.8. These dissimilarities are probably due to different isoenzyme patterns available in the same insect.

     

Purification and some properties of an atypical alkaline p-nitrophenylphosphate phosphatase activity from Halobacterium halobium

• 1.1. An alkaline p-nitrophenylphosphate phosphatase has been purified 440-fold from extracts of Hatobacterium halobium.
• 2.2. The enzyme has an apparent molecular weight of 24,000.
• 3.3. A K_m value for p-nitrophenylphosphate of 1.12mM has been found under optimal conditions.
• 4.4. The enzyme is selectively activated and stabilized by Mn²⁺.
• 5.5. It requires high salt concentrations for stability and maximum activity.
• 6.6. It displays an unusual restricted substrate specificity of 25 phosphate esters tested, only phosphotyrosine and casein were hydrolysed besides p-nitrophenylphosphate.

     

The effect of mercury and aluminum on sodium-potassium-Mg2+ dependent-adenosine triphosphatase activity of Electrophorus electricus (L.) electrocyte

• 1.1. Na⁺,K⁺-ATPase, which mediates the active transport of Na⁺ and K⁺ across the plasma membrane, is found in equivalent amounts in both plasma membranes of the electrocyte, the anterior, non-innervated (fraction P₂) and the posterior, innervated (fraction P₃) obtained by differential centrifu gation of Electrophorus electricus (L.) electric organ.
• 2.2. The kinetic effects of Hg²⁺ and A1³⁺, described as neurotoxic metals, on the Na⁺,K⁺-ATPase activity of the two membrane fractions (P₂ and P₃) were analysed with respect to Na⁺ and K⁺ ions, after the I₅₀ estimation of each metal.
• 3.3. Mercury is a potent Na⁺,K⁺-ATPase inhibitor in the nanomolar range. In all cases, it behaved as a mixed partial hyperbolic inhibitor.
• 4.4. Aluminum was shown to be a poor enzyme inhibitor. Changing the K⁺ concentration, it behaved as a mixed linear inhibitor (P₂ fraction) and as a non-essential mixed activator (fraction P₃). Aluminum behaved as a partial hyperbolic inhibitor for both P₂ and P₃ fractions with respect to Na⁺ concentration.
• 5.5. The observation of the variable kinetic behaviour of P₂ and P₃ led us to attribute these differences to the Na⁺,K⁺-ATPase electrocyte isoenzymes which occur in different proportions in these fractions (Gomes-Quintana et al., 1992 Comp. biochem. Physiol.103B/3 623–628).

     

Modulation of active potassium transport by aldosterone

• 1.1. The role of aldosterone on active potassium transport across lizard colon under voltage-clamped conditions has been investigated.
• 2.2. Control colons exhibited no net potassium flux (J^k_net) despite of the existence of active opposite unidi ectional fluxes.
• 3.3. An important net secretory potassium flux was found in short-circuited aldosterone-stimulated colons.
• 4.4. Mucosal amiloride did not change (J^k_net) either in control or aldosterone-stimulated colons.
• 5.5. Luminal barium alters K ⁺ transport in a manner consistent with the presence of barium-sensitive conductances at the apical membrane of both control and aldosterone-treated colons.
• 6.6. The effects of ouabain and barium on control and aldosterone-induced potassium flows were consistent with a model involving basolateral uptake by an Na ⁺-K ⁺-ATPase and conductive exit across the apical membrane.
• 7.7. The stimulatory effect of aldosterone on potassium secretion is associated with parallel increases of both basolateral K ⁺ entry and the apical conductive pathway.

     

Oxidation of 6-hydroxydopamine catalyzed by tyrosinase

• 1.1. The oxidation of 3,4-dihydroxyphenylethylamine (dopamine) by O₂ catalyzed by tyrosinase yields 4-(2-aminoethyl)-1,2-benzoquinone, with its amino group protonated (o-dopaminequinone-H⁺, which evolves non-enzymatically through two branches or sequences of reactions, whose respective operations are determined by the pH of the medium.
• 2.2. The cyclization branch of o-dopaminequinone-H⁺ takes place in the entire range of pH and is the only significant branch at pH ⩾ 6.
• 3.3. The hydroxylation branch of o-dopaminequinone-H⁺ only operates significantly at pH < 6, and involves the accumulation of 2,4,5-trihydroxyphenylethylamine (6-hydroxydopamine), identified by high performance liquid chromatography (HPLC).
• 4.4. 6-hydroxydopamine is also a substrate of tyrosinase. The identification and evolution of the oxidation products of 6-hydroxydopamine has been carried out by spectrophotometry and HPLC assays.

     

Effect of Li+ and Ba2+ on the electrocyte membrane-bound (Na+ + K+)-ATPase

• 1.1. Membrane-bound (Na⁺ + K⁺)-ATPase activity from the non-innervated and innervated faces of Electrophorus electricus (L.) electric organ, obtained by differential centrifugation, was measured using AChE as an enzyme marker for membranes derived from the post-synaptic area (fraction P₃) of the electrolyte.
• 2.2. The effect of Li⁺ and Ba²⁺ on (Na⁺ + K⁺)-ATPase activity of the two membrane fractions (P₂ and P₃) was analysed with respect to K⁺ and Mg²⁺ ions, after the I₅₀ estimation.
• 3.3. The kinetics of the reactions with these cations were investigated showing that Li⁺ inhibits P₂ uncompetitively and for P₃ presented a mixed type inhibition.
• 4.4. Ba²⁺ behaved as an hyperbolic mixed type inhibitor for P₂ and a linear mixed type inhibitor for P₃ fraction.

     

Cationic and anionic channels of apical receptive membrane in a taste cell contribute to generation of salt-induced receptor potential

• 1.1. After ionic composition of superficial fluid (ISF) and interstitial fluid (ISF) of the frog Rana catesbeiana) tongue had mostly been changed with a low Na⁺ saline solution, the relations between membrane potentials and receptor potentials in a frog taste cell evoked by various concentrations of NaCl and various types of salts were analyzed to examine permeability of the taste receptive membrane to cations and anions.
• 2.2. The mean reversal potentials for depolarizing potentials of a taste cell in response to 0.05 M, 0.2 M and 0.5 M Nad were -40.0, 6.4 and 28.8 mV, respectively.
• 3.3. When adding an anion channel blocker, SITS, to a NaCl solution the reversal potential for receptor potential with NaCl plus SITS became about twice as large than with NaCl alone.
• 4.4. Reversal potentials for 0.2 M NaCl, LiCl, KCl and NaSCN were 6.4, 25.4, −1.0 and −7.8 mV, respectively, indicating that permeability of the apical taste receptive membrane to cations of Cl⁻ salts is arranged in the order of Li⁺ > Na⁺ > K⁺ and that the permeability to anions of Na⁺ salts is arranged as SCN⁻ > Cl⁻
• 5.5. It is concluded that in the case of NaCl stimulation, Na⁺ and Cl⁻ of NaCl stimulus permeate NaCl-gated cationic and anionic channels at the apical taste receptive membrane in generating receptor potentials.

     

Ionic dependence and vanadate sensitivity of (Na+, K+)-ATPase isolated from branchial sac of ascidians

• 1.1. Ion dependence and vanadium-induced inhibition on branchial sac ATPase in five species of ascidian Phlebobranchiata (vanadium-accumulating) and Stolidobranchiata (iron-accumulating) were studied.
• 2.2. The ATPase was obtained from the microsomal fraction, which was prepared from each ascidian branchial sac.
• 3.3. The ATPase was dependent on Mg²⁺ and activated by exogenous Na⁺ + K⁺.
• 4.4. Ouabain inhibited the ATPase activity in vitro, 10 μM to 100 μM vanadate, in vitro, suppressed the (Na⁺, K⁺)-ATPase.

     

Effects of Na+ and/or K+ on the Mg2+-dependent ATPase activities in shrimp (Macrobrachium amazonicum) gill homogenates

• 1.1. Homogenates of gills from the freshwater shrimp M. amazonicum exhibit the following ATPase activities: (i) a basal, Mg²⁺-dependent ATPase; (ii) an ouabain-sensitive, Na⁺ + K⁺-stimulated ATPase; (iii) an ouabain-insensitive, Na⁺-stimulated ATPase; and (iv) an ouabain-insensitive, K⁺-stimulated ATPase.
• 2.2. K⁺ suppresses the Na⁺-stimulated ATPase activity in a mixed-type kind of inhibition, whereas Na⁺ does not exert any noticeable effect on the K⁺-stimulated ATPase activity.
• 3.3. The Na⁺- and the K⁺-stimulated ATPase activities are totally inhibited by 5 mM ethacrynic acid in the incubation medium.
• 4.4. The Na⁺- and the K⁺-stimulated ATPase activities are not expressions of the activation of a Ca-ATPase.
• 5.5. The possible localization and roles of the described ATPases within the gill epithelium are briefly discussed and evaluated.

     

Sodium and potassium balance of captive meadow voles (Microtus pennsylvanicus) fed laboratory chow and vegetation diets

• 1.1. Mineral balance was studied in meadow voles (Microtus pennsylvanicus) maintained in the laboratory.
• 2.2. Urine and fecal Na⁺ contents of voles on low-Na⁺ diets were comparable to those reported for other herbivore species, but urine and fecal K levels were higher.
• 3.3. Voles approached Na⁺ balance (input = output) on diets with Na⁺ content as low as 56 ppm.
• 4.4. There was not a clearcut hypertrophy of the adrenal-gland zona glomerulosa in voles maintained on low-Na⁺ diets.
• 5.5. Plasma K content and bone water content were higher in voles maintained on high-Na ⁺ vegetation diets, suggesting expansion of extracellular fluid volume.

     

Na+/H+ exchange in the kidneys of eels (Anguilla anguilla) adapted to sea water or to freshwater environments: Studies with brush border membrane vesicles

• 1.1. In brush border membrane vesicles isolated from eel kidneys, adapted either to sea water or freshwater environments, a Na⁺/H⁺ antiporter is present.
• 2.2. Using a calibration plot it is possible to evaluate the amount of protons that this antiporter can accumulate inside the vesicular space.
• 3.3. The activity of the antiporter seems to be affected by the salinity of the water; it is higher in animals adapted to seawater.
• 4.4. This adaptation seems to occur by a J_max regulation of the antiporter.

     

Behaviour and haemolymphatic ionic composition of the intertidal crab Chasmagnathus granulata dana, 1851 (Crustacea: Decapoda) during emersion

• 1.1. Behavioural observations and haemolymphatic measurements of Na⁺ K⁺ and Ca⁺ were performed in Chasmagnalhus granulata during emersion.
• 2.2. Activity levels were found to be higher during voluntary emersion periods than when the animals were submerged. A lt₅₀ of 39.45 hr was observed when no access to water was allowed.
• 3.3. The Na⁺ and K⁺ and Ca⁺ levels increased during aerial exposure. The Na⁺ and K⁺ levels were restored prior the end of the experimental period. Mechanisms for such regulation are therefore discussed. The Ca²⁺ levels, remaining high during emersion, are probably a result of acid-base balance adjustments.

     

Purification and some properties of NAD+-dependent glutamate dehydrogenase from Halobacterium halobium

• 1.1. A NAD⁺-dependent glutamate dehydrogenase (EC 1.4.1.2.) was purified 126-fold from Halobacterium halobium.
• 2.2. Activity and stability of the enzyme were affected by salt concentration. Maximum activity of the NADH-dependent reductive amination of 2-oxoglutarate occurs at 3.2 M NaCl and 0.8 M KCl, and the NAD⁺-dependent oxidative deamination of l-glutamate occurs at 0.9 M NaCl and 0.4 M KCl. The maximum activity is higher with Na⁺ than with K⁺ in the amination reaction while the reverse is true in the deamination reaction.
• 3.3. The apparent K_m values of the various substrates and coenzymes under optimal conditions were: 2-oxoglutarate, 20.2 mM; ammonium, 0.45 M; NADH, 0.07 mM; l-glutamate, 4.0 mM; NAD⁺, 0.30 mM.
• 4.4. No effect of ADP or GTP on the enzyme activity was found. The purified enzyme was activated by some l-amino acids.

     

Pigment migration in fish erythrophores is controlled by alpha2-adrenoceptors

• 1.1. The aggregation of erythrosomes within erythrophores of the squirrel fish (Myripristis occidentalis; belonging to the family Holocentridae) was, on pharmacological grounds, shown to be mediated by alpha₂-adrenoceptors.
• 2.2. The erythrophores were shown to be controlled by adrenergic nerves activating the alpha₂-adrenoceptors.
• 3.3. The erythrophores themselves were found to possess a K⁺-sensitive mechanism of aggregation.
• 4.4. Some similarities and differences of the alpha₂-adrenoceptor-mediated chromatosome aggregation in melanophores and erythrophores are also discussed.

     

l-lysinamidase from Cryptococcus laurenti 112. Purification and properties

• 1.1. The purified enzyme hydrolyzes the linear l-lysinamide and the cycle amide of l-lysine—l-α-amino-ϵ-caprolactam.
• 2.2. The apparent relative molecular mass is 180,000. The enzyme consists of four subunits and the molecular mass of a single subunit was found to be 47,000.
• 3.3. The coefficient of molecular sedimentation equals 8.3 S, the isoelectric point was determined to be pH 4.3
• 4.4. The enzyme is not a glycoprotein. p-Mercuribenzoate binds 10 SH-groups of the native enzyme molecule and 20 SH-groups in the presence of 0.7% SDS.
• 5.5. pH- optimum for the hydrolysis of l-lysine amides was observed to be 7.5–7.7. The enzyme is strictly dependent on Mn²⁺ and Mg²⁺.
• 6.6. The kinetic parameters for the hydrolysis of l-lysinamide where K_m = 3.8 mM and k_cat = 3000 sec⁻¹ For the hydrolysis of cyclic L-lysinamide K_m = 4.8 mM and k_cat = 2600 sec⁻.

     

Effects of long- and medium-chain triglycerides on amino acid uptake in rat intestinal brush border membrane vesicles

• 1.1. Uptake of l-leucine, l-phenylalanine, l-proline and l-lysine into brush border membrane vesicles from rats fed either a medium-chain triglyceride (MCT) or a long-chain triglyceride (LCT) diet was studied under conditions of the presence or absence of a Na⁺ gradient.
• 2.2. From the results of initial rate, Na⁺-dependent transport in LCT feeding were lower than in feeding MCT. The Na⁺-independent transport did not vary in either group except for l-lysine uptake.
• 3.3. For l-leucine, l-phenylalanine and l-proline in Na⁺ dependence, kinetic analysis revealed 4–6-fold smaller V_max values in LCT group than in MCT group. l-Lysine in Na⁺-independent transport was 10-fold lower in LCT group than in MCT group. The K_m values were not affected by feeding the LCT or MCT diet.
• 4.4. It is clear that amino acid transport is regulated by different types of dietary fat. We consider that the alteration of transport activity is attributable to the changes in number of membrane-bound transport carriers but not to their affinity.

     

Changes in size,hydration and low molecular weight osmotic effectors during meiotic maturation of Fundulus oocytes in vivo

• 1.1. Gains in the water content of the Fundulus heteroclitus follicle account for over 90% of the total wet weight increase, and consequentially most of the size increase, associated with meiotic maturation.
• 2.2. Increases in intracellular Na⁺ and K⁺ actually exceed the accompanying increases in oocyte water, resulting in net gains in the concentration of these solutes. Changes in oocyte osmolality during maturation are mostly closely paralleled by variations in the concentration of K⁺.
• 3.3. Concentrations of various free amino acids, including taurine, remain constant or decline during maturation.
• 4.4. Taken together, these results suggest that an influx of K⁺, followed by osmotically-obliged water, is a primary cause of water uptake during oocyte maturation in F. heteroclitus.

     

Biomines-stimulated phosphorylation and (Na+, K+-ATPase in the gills of the chinese crab,Eriocheir sinensis

• 1.1. Subcellular distribution of (NA⁺, K⁺-ATPase and ouabain-insensitive ATPase (Mg²⁺-ATPase) are compared in branchial tissues of the euryhaline crab, Eriocheir sinensis, acclimated to fresh water.
• 2.2. Both the anterior and posterior gills contain cAMP-dependent protein kinase and endogenous protein substrate for phosphorylation.
• 3.3. Phosphorylation occurs in both “particulate” and “soluble” subcellular fractions but its stimulation by cAMP is restricted to the “soluble” fraction.
• 4.4. serotonin (5-HT) and dopamine receptors are present only in the “light particulate” fraction isolated from the posterior gills.

• 1.(a) Serotonin and dopamine have no effect on the phosphorylation observed in a subcellular fraction alone.
• 2.(b) Activation of the phosphorylation by serotonin and dopamine is found when the soluble fraction (source of cAMP-dependent protein kinase) is added to the fraction P₃ from the posterior gills.
• 3.(c) No activation occurs with the fractions P₃ as well as P₁ or P₂ (not shown) from anterior gills of fresh water crab.
• 4.(d) Cyproheptadine, a serotonin receptor antagonist, inhibits the 5-HT dependent increase in phosphorylation.
• 5.(e) The dopamine receptor antagonist, chlorpromazine, inhibits dopamine-stimulated phosphorylation.
• 6.5. Ouabain mimics the effect of cyproheptadine on the serotonin-stimulated phosphorylation found in the posterior gills.

     

Intestinal l-methionine transport in the cultured gilthead bream (Sparus aurata)

• 1.1. The influx and transepithelial movements of l-methionine and its effects on the electrophysiology and Na-Cl-transport in upper and lower intestine of the cultured fish, Spanis aurata, were measured.
• 2.2. The K_m and V_max of l-methionine influx into the tissues were higher in lower intestine than in upper intestine. A prominent diffusion-like transport component was also measured in both segments during influx experiments.
• 3.3. Net transepithelial fluxes of l-methionine (1 mM) were observed in both upper and lower intestine, this transport being Na⁺-dependent.
• 4.4. The two intestinal segments exhibited an electrical potential difference (PD) and a short circuit current (I_sc) serosa negative or near zero. Tissue conductance (G_t) was higher in posterior than in lower intestine.
• 5.5. Addition of l-methionine to the mucosal side of lower or upper intestine did not induce changes in PD in either part.
• 6.6. Isotopic fluxes of Cl⁻ or Na⁺ measurements under short circuit conditions showed that there were no net Cl⁻ or Na⁺ transport in either part.
• 7.7. l-Methionine additions to the mucosa did not induce changes in unidirectional fluxes of Cl⁻ or Na⁺ or in the (I_sc) in either the anterior or posterior intestine.

     

The regulation of glucose 6-phosphate dehydrogenase from Dicentrarchus labrax (bass) liver

• 1.1. Kinetic constant values of the reaction catalyzed by bass liver glucose 6-phosphate dehydrogenase show to be modified between 10 and 40°C.
• 2.2. The Arrhenius plot between 10 and 50°C shows two slopes with different activation energies.
• 3.3. These results suggest a regulation of this enzyme by environmental temperature.
• 4.4. Kinetics of ATP inhibition were examined between pH 6.2 and 7.8: patterns and K_i values obtained are affected by the pH variation.
• 5.5. NADH is an effective inhibitor of bass glucose 6-phosphate dehydrogenase but this enzyme does not show NAD-linked activity.
• 6.6. Kinetics of pyridoxal 5′-phosphate inhibition have indicated the presence of a lysine in the catalytic site for NADP⁺.