Properties and distribution of Mg2+-HCO−3-ATPase in brush border membranes isolated from rat small intestine

• 1.1. The small intestine was cut into seven segments and properties and distribution of brush border Mg²⁺-HCO⁻₃-ATPase activity in each segment were examined.
• 2.2. The optimal Mg²⁺ concentration was 1.0 mM.
• 3.3. The optimal HCO⁻₃ concentration was 100 mM in the first (duodenal), 50 mM in the 3rd and 40 mM in the 5th segment, respectively.
• 4.4. The optimal pH value was about 9.0.
• 5.5. l-phenylalanine (above 1 mM) and SCN⁻ (above 50 mM) significantly inhibited both Mg²⁺- and Mg²⁺-HCO⁻₃-ATPase activity.
• 6.6. The enzyme activity was found to be highest in the duodenal segment and then gradually decreased in consecutive segments as well as β-glycerophosphatase, Na⁺-K⁺-ATPase and supernatant carbonic anhydrase.
• 7.7. The functional significance of this ATPase and the relationship with carbonic anhydrase was discussed.

     

Ca2+ - and Mg2+-dependent ATPase activities in the deoxycholate-treated rat heart sarcolemma

• 1.1. Isolated rat heart sarcolemma was treated with different concentrations of an ionic detergent, deoxycholate (DOC) and ATP hydrolysis in the presence of Ca²⁺ or Mg²⁺ was determined.
• 2.2. Both Ca²⁺-dependent ATPase and Mg²⁺-dependent ATPase activities were decreased in the DOC-treated membranes; however, the depression of Mg²⁺-dependent ATPase activity was greater than that of Ca²⁺-dependent ATPase.
• 3.3. The differential changes in Ca²⁺-dependent ATPase and Mg²⁺-dependent ATPase activities were apparent when incubations with DOC were carried out for different time intervals and at different temperatures.
• 4.4. In DOC-treated preparations, the K_m value for Ca²⁺-dependent ATPase was decreased whereas that for Mg²⁺-dependent ATPase was increased. The half maximal velocities of the Ca²⁺-dependent ATPase and Mg²⁺-dependent ATPase enzyme reactions in the treated preparations were obtained at a DOC: membrane protein ratio of 3.0 and 0.6, respectively.
• 5.5. In the DOC-treated membranes exhibiting the half maximal velocities of enzyme reactions, the K_i value for Ca²⁺-dependent ATPase was drastically reduced but remained unchanged for Mg²⁺-dependent ATPase.
• 6.6. The DOC treatment was associated with a loss of protein as well as phospholipids and resulted in changes in the ultrastructural integrity of the membrane.
• 7.7. Varying degrees of decreases in the activities of sarcolemmal adenylate cyclase. (Na⁻-K⁺)-ATPase. 5'-nucleotidase and calcium binding were seen upon DOC treatment.
• 8.8. The extent of reduction in Ca²⁺-dependent ATPase and Mg²⁺-dependent ATPase activities were also different when the membrane was treated with a non-ionic detergent, Lubrol PX.
• 9.9. These data suggest that Ca²⁺-dependent ATPase in heart sarcolemma is more resistant than Mg²⁺-dependent ATPase to detergent treatments and further indicate some differences in the properties of these enzymes.

     

The possibility that Ca2+-ATPase from the plasma membrane-rich fraction of bovine parotid gland is ecto-Ca2+-dependent nucleoside triphosphatase

• 1.1. Parotid plasma membrane nonpump low-affinity Ca²⁺-ATPase, which possesses high-affinity (Ca²⁺ + Mg²⁺ )-ATPase activity, was characterized.
• 2.2. Purified Ca²⁺-ATPase hydrolyzed the nucleoside triphosphates, GTP, ITP, CTP, UTP, TTP (67–93% of ATP) and nucleoside diphosphates, ADP. GDP, IDP, CDP, TDP (12–40% of ATP) but not AMP and p-NPP.
• 3.3. The maximum activities of Ca²⁺- and (Ca²⁺ +Mg²⁺ )-ATPases were obtained in the presence of 1 mM and 0.13 μ M Ca²⁺, respectively.
• 4.4. The K_m values for Ca²⁺ in Ca²⁺- and (Ca²⁺+ Mg²⁺ )-ATPases were 0.2 mM and 22 nM. respectively.
• 5.5. The activities of both Ca²⁺- and (Ca²⁺ + Mg²⁺ )-ATPases were found in the right-side-out-vesicles obtained from the plasma membrane-rich fraction.
• 6.6. These features suggest that Ca²⁺-ATPase is an ecto-Ca²⁺-dependent nucleoside triphosphatase.

     

Evidence that both Ca2+-ATPase and (Ca2+ + Mg2+)-ATPase activities in the plasma membrane-rich fraction from bovine parotid gland reside on the same enzyme molecule

• 1.1. Evidence was obtained that activities of both low-affinity Ca²⁺-ATPase and high-affinity (Ca²⁺ + Mg²⁺)-ATPase in the plasma membrane-rich fraction from bovine parotid gland reside on the same enzyme.
• 2.2. Two solubilized ATPases were purified by four steps of HPLC; and both activities eluted at the same fractions from each column, and the specific activity ratio of the two enzymes at each step was constant.
• 3.3. By non-denaturing PAGE, the final preparation gave a single band for both protein staining and activity staining for the two ATPases; and the Ca²⁺-ATPase activity comigrated with that of (Ca²⁺ + Mg²⁺)-ATPase.
• 4.4. In SDS-PAGE, each activity staining for the ATPases also gave a single band, and both activities comigrated.
• 5.5. These findings suggest that Ca²⁺-ATPase and (Ca²⁺ + Mg²⁺)-ATPase are a single enzyme.

     

Erythrocyte membrane anion-sensitive Mg2+-ATPase—Identity with monovalent cation sensitive Ca2+-Mg2+-ATPase

• 1.1. Activation of Mg²⁺-ATPase of rabbit and guinea-pig erythrocyte membrane by bicarbonate or chloride could be completely abolished by ethylene-glycol-bis-(β-aminoethylether)-N,N'-tetraacetic acid. The anion stimulation was actually an activation of contaminating Ca²⁺ -stimulated Mg²⁺-ATPase by monovalent cations associated with the anions.
• 2.2. Guinea-pig red cell Ca²⁺-Mg²⁺-ATPase could be activated by both sodium and potassium while the rabbit enzyme was sensitive only to sodium. The concentrations of monovalent cations for half-maximal stimulation of Ca²⁺-Mg²⁺-ATPase are: k_na+ = 40.8 mM, k_k+ = 12.2 mM (guinea-pig); K_Na+ = 13.3mM (rabbit).
• 3.3. Potassium enhanced activation of rabbit erythrocyte membrane Ca²⁺-Mg²⁺-ATPase by red cell Ca²⁺-Mg²⁺-ATPase activator protein. With the guinea pig enzyme, neither sodium nor potassium enhanced activator stimulation of Ca²⁺-Mg²⁺-ATPase.
• 4.4. Ca²⁺-Mg²⁺-ATPase of aged rabbit erythrocyte membrane responded to sodium but not to activator protein.
• 5.5. Triton X-100 solubilized rabbit erythrocyte membrane Ca²⁺-Mg²⁺-ATPase has an apparent molecular weight of 371,000. It did not respond to the activator.
• 6.6. One major and three minor proteins, visualized by SDS-polyacrylamide gel electrophoresis, were extracted from rabbit erythrocyte membrane by 50 μM chlorpromazine.

     

High-affinity Ca2+-Mg2+-ATPase in kidney of euryhaline Gillichthys mirabilis: kinetics,subcellular distribution and effects of salinity

• 1.1. Two components of Ca²⁺-Mg²⁺-ATPase are observed in kidneys of G. mirabilis. The high-affinity component has a K_0.5Ca of 0.23μM; the low-affinity activity K_0.5Ca is 90–110μM. The high-affinity activity requires Mg²⁺, displays Michaelis-Menten kinetics, has peak activity at 1.2 μM Ca²⁺, and is insensitive to ouabain and Na⁺ azide.
• 2.2. In subcellular fractions, the high-affinity component segregates with Na⁺-K⁺-ATPase and is localized predominantly in BLM. The low-affinity component is broadly distributed among membranous organelles, including brush border, and may be equivalent to alkaline phosphatase.
• 3.3. Specific activity of the high-affinity Ca²⁺-Mg²⁺-ATPase is modestly increased following adaptation of fish to FW, but total renal high-affinity activity is greatest in the hypertrophied kidneys of FW-adapted fish and is least in kidneys of fish adapted to 200% SW.
• 4.4. High-affinity Ca²⁺-Mg²⁺-ATPase may be associated with active Ca²⁺ transport or with regulation of intracellular Ca²⁺ concentration of tubular cells.

     

Taurine regulation of Ca2+ uptake and (Ca2+ + Mg2+)-ATPase in developing chick B-cells

• 1.1. The objective of the present study was to determine the effect of age and taurine on chick B cell calcium uptake and membrane (Ca²⁺ + Mg²⁺)-ATPase activity in 1–4-week-old chicks.
• 2.2. The calcium uptake rate decreased with age (P < 0.05) and was further decreased by taurine (P < 0.05).
• 3.3. (Ca²⁺ + Mg²⁺)-ATPase activity increased with age (P < 0.05) and was stimulated by taurine (P < 0.05).
• 4.4. The data demonstrate that the flux of calcium across the B-cell membrane changes during early post-hatch development, and that taurine regulates both the influx and efflux of calcium in chick B-cells.

     

Mg2+-dependent (Na+ + K+)- and Na+-ATPases in the kidneys of the gilthead bream (Sparus auratus L.)

• 1.1. The (Na⁺ + K⁺)- and Na⁺-ATPases, both present in kidney microsomes of Sparus auratus L., have different activities and optimal assay conditions as, in the first of the two stocks of fish used (A), the spec. act. of the former is 51.7 μmol P_i mg prot⁻¹ hr⁻¹ at pH 7.5, 100 mM Na⁺, 10 mM K⁺, 17.5 mM Mg²⁺, 7.5 mM ATP and that of the latter is 6.5 μmol P_i mg prot⁻¹ hr⁻¹ at pH 6.5, 40 mM Na⁺, 4.0 mM Mg²⁺, 2.5 mM ATP.
• 2.2. Ouabain and vanadate specifically inhibit the (Na⁺ + K⁺)-ATPase but not the Na⁺-ATPase that is preferentially inhibited by ethacrynic acid.
• 3.3. While the (Na⁺ + K⁺)-ATPase is strictly specific for ATP and Na⁺, Na⁺-ATPase can be activated by various monovalent cations and, apart from ATP, hydrolyses CTP, though less efficiently.
• 4.4. The second stock B, subjected to higher salinity than A, shows an acidic shifted Na⁺-ATPase optimal pH, opposed to the stability of that of the (Na⁺ + K⁺)-ATPase, a decreased (Na⁺ + K⁺)-ATPase and a strikingly depressed Na⁺-ATPase.
• 5.5. The results are compared with literature data and discussed on the basis of the presumptive different roles as well as functional prevalence in various salinities of the two ATPases.

     

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.

     

Myofibril and sarcoplasmic reticulum changes during muscle development: Activity vs inactivity

• 1.1. The purpose of this study was to determine whether biochemical changes of skeletal muscle that occur as a result of exercise in young rats persist into adulthood.
• 2.2. Littermates (10 days old) were assigned to a 3, 6 and 12 week control or training group. In addition, a rest-exercise group (R-E) and exercise-rest (E-R) group were included.
• 3.3. The rest-exercise and exercise-rest rats were maintained for the 12 weeks with the first 6 weeks being either rest or exercise and the condition reversed during the last 6 weeks of the experiment.
• 4.4. Myofibril ATPase activity of rat plantaris increased from the 10d to 12 week animals (P < 0.05). As anticipated, training resulted in a lowered activity at 6 and 12 weeks compared to controls.
• 5.5. The Ca²⁺ uptake and Ca²⁺-ATPase activity of the sarcoplasmic reticulum followed a similar pattern.
• 6.6. With regard to the exercise-rest rats, the myofibril and SR ATPase activities at 12 weeks were comparable to the 12 weeks control rats.
• 7.7. The rest-exercise group approximated the 12 week training group with regard to myofibril and SR ATPase activities (P > 0.05).
• 8.8. The results suggest that the training adaptations that occur during development of skeletal muscle return to normal, when training ceases in the adult rat.
• 9.9. Furthermore, animals that started to train prior to puberty do not have a greater capacity to adapt than animals which initiated training during adulthood.

     

Biochemical characterization of the plasma membrane Ca2+ pumping ATPase activity present in the gill cells of Mytilus galloprovincialis LAM

• 1.1. In the plasma membrane of mussel gill cells an ouabain insensitive, Ca²⁺-activated ATPase activity is present. The ATPase has high Ca²⁺ affinity (K_ma = 0.3 μM).
• 2.2. The optimum assay conditions to evaluate the enzymatic activity of the Ca²⁺-stimulated ATPase at 19°C are: 120–300 mM KCl ionic strength, pH 7.0 and 2 mM ATP. As for mammalian enzymes, the Ca²⁺ ATPase activity is stimulated by DTT (0.5–1 mM) and it is inhibited by low concentrations of vanadate (10–50 μM) and -SH inhibitors such as PCMB and PCMBS (10 μM); the enzyme appears to be calmodulin insensitive.
• 3.3. Electrophoretic analyses of plasma membrane proteins demonstrate that: (a) Ca²⁺ at n-μM concentrations is necessary to activate ATP hydrolysis with consequent formation of the enzyme-phosphate complex; (b) the steady state concentration of the phosphorylated intermediate is increased in the presence of La³⁺; (c) the mol. wt of Ca²⁺ ATPase is about 140 kDa.
• 4.4. Low Ca²⁺ concentrations (n-μM) are sufficient to stimulate the ATP-dependent Ca²⁺ uptake by plasma membrane inside-out vesicles.
• 5.5. The results indicate that the Ca²⁺ pump present in the gill plasma membranes could be responsible for Ca²⁺ extrusion and therefore involved in maintaining the cytosolic Ca²⁺ concentration within physiological levels.

     

Osmolality and electrolyte concentration of hemolymph and the problem of ion and volume regulation of cells in higher insects

• 1.1. The study was carried out on 22 species of insects from 5 orders. The osmolality of their hemolymph varied from 319 to 421 mOsm/kg H₂O, concentration of Na⁺ 4.6 to 118 mM/l, K⁺ 6.3 to 73mM/l, Ca²⁺ 3.6 to 12.9 mM/l, Mg²⁺ 2.3 to 76 mM/l. The most abundant cation in the hemolymph of insects from higher orders is either K⁺ or Mg²⁺.
• 2.2. In the muscles of lower and higher insects K⁺ is usually within 80–120 mM/kg wet wt.
• 3.3. Most Ca²⁺ and Mg²⁺ in hemolymph is bound with protein and low molecular anions, concentration of free Ca²⁺ is 0.9-2.1mM/l Mg²⁺ 3.7–8.0 mM/l.
• 4.4. It is concluded that, in insects, potassium hemolymph, cell volume regulation and accumulation of ions in the cell, are ensured by an increased osmolality of hemolymph due to a high percentage contribution of low molecular organic substances which are retained in the hemolymph due to the absence of filtration apparatus in the Malpighian tubules.

     

Thermostability and activation by divalent cations of the membrane-bound inorganic pyrophosphatase of Rhodospirillum rubrum

• 1.1. The activation energy of the membrane bound H⁺-pyrophosphatase is 44.9 k J·mol⁻¹, for the detergent solubilized enzyme is 55.9 kJ·mol⁻¹.
• 2.2. The Arrhenius plots obtained for pyrophosphatases of Rhodospirillum rubrum show no breaks.
• 3.3. At 70°C, the membrane-bound pyrophosphatase is more stable in the presence of either Mg²⁺ or Zn²⁺ than in their absence.
• 4.4. At 65°C, an activator effect of Mg²⁺ or Zn²⁺ was observed. Nevertheless, at 70°C no activation was obtained.
• 5.5. The activator effects of Mg²⁺ or Zn²⁺ were depended of their concentration.

     

Evidence for the presence of ion pumps in the photophores of two mesopelagic fishes: Argyropelecus and maurolicus

• 1.l. Adenosine triphosphatase (ATPase) activity has been measured on homogenates of photophores from the two mesopelagic fishes Argyropelecus and Maurolicus. This activity is equivalent for both fishes when reported to the protein content as is their O₂ consumption.
• 2.2. The activity is optimal at pH 6.8–7.5. It is not specific for ATP since GTP, ITP, UTP and CTP are also hydrolyzed to a significant extent. It is also not specific for Mg²⁺, the activity being equivalent (Argyropelecus) or higher (Maurolicus) with Ca²⁺ and high also with Co²⁺ and Mn²⁺
• 3.3. Twenty to 30 per cent of the activity measured at pH 7.4 is probably due to the mitochondrial ATPase as it is shown by oligomycin and venturicidin inhibition.
• 4.4. Activities of both fishes photophores are partly inhibited by N-N'-dicyclohexylcarbodiimide (DCCD), azide, LaCl₃, vanadate, diethylstilbestrol (DES) and N-ethylmaleimide (NEM) which are all inhibitors of ionic pumps.
• 5.5. Argyropelecus activity is sensitive to ouabaïn.
• 6.6. Our results show the presence of ionic pumps in Argyropelecus and Maurolicus photophores. If there is evidence for the absence or very low activity of a H⁺ pump, it is sure that Argyropelecus at least possess a Na⁺K⁺-ATPase.
• 7.7. The significance of a high protein content in Maurolicus photophores and of a large inorganic phosphate concentration in Argyropelecus is discussed.

     

Ca2+-binding and protein-protein interaction domains of the sea urchin extraembryonic coat protein hyalin

• 1.1. As reported previously (Hopper and Robinson, 1990; Int. J. Biochem. 22, 1165–1170) the sea urchin extraembryonic coat protein hyalin undergoes a Ca²⁺-induced self-association into an insoluble gel (gelation) in the presence of Mg²⁺ and/or NaCl.
• 2.2. A 275 kDa peptide fragment, generated by limited tryptic digestion of hyalin, binds Ca²⁺+ but does not undergo gelation in the presence of Ca²⁺, Mg²⁺ and NaCl.
• 3.3. Comparisons between the capacities of hyalin and the 275 kDa peptide fragment to bind Ca²⁺ indicate that the latter binds 88% less Ca²⁺ than hyalin.
• 4.4. However, the presence of Ca²⁺ alone, at a concentration of 5 mM, protects the 275 kDa peptide fragment from further digestion by trypsin mimicking the effect of this cation in protecting hyalin.
• 5.5. Gel exclusion Chromatographie analyses of the 275 kDa peptide fragment, both in the presence and absence of 5 mM Ca²⁺, indicate that this cation does induce self-association of the fragment.
• 6.6. These results provide information on the organization of the functional domains on hyalin which are required for gel formation.

     

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.

     

The sea urchin extraembryonic hyaline layer: Ionic parameters controlling the hyalin gelation reaction

• 1.1. As reported previously (Robinson, 1988) the Ca²⁺-induced self-association reaction of the protein hyalin, purified from the sea urchin extraembryonic hyaline layer, was modulated by both Mg²⁺ and NaCl.
• 2.2. In the presence of 400 mM NaCl the apparent dissociation constant (Ca²⁺) decreased five-fold from 4.8 ± 1.1 mM in the absence to 0.9 ± 0.5 mM in the presence of 20 mM Mg²⁺.
• 3.3. The potentiating effect of Mg²⁺ occurred with an apparent dissociation constant (Mg²⁺) of 4.6 ± 0.5mM.
• 4.4. In the absence of Ca²⁺ or NaCl hyalin dissociated from isolated hyaline layers indicating that the behavior of hyalin within the layer is predictable from results obtained with the purified protein.

     

Bioelectrical potentials across the mantle epithelium of Achatina fulica

• 1.1. The shell side of the mantle of Achatina fulica is several millivolts positive to the blood side in vitro.
• 2.2. The electrical potential does not depend on Na⁺, Ca²⁺, Mg²⁺, K⁺ or HCO₃⁻ but requires the presence of chloride on the shell side.
• 3.3. The potential difference and short-circuit current ranged from 3.0 to 30.0 mV and 15.0 to 75 μA/cm² with averages at 10m V and 50 μA/cm² respectively.
• 4.4. The electrical gradient is reduced by 2,4-dinitrophenol, thiocyanate and furosemide but not by ouabain, CO₂ or acetozolamide.
• 5.5. It is suggested that the nature and mechanism of electrogenesis in Achatina parallels that of the Helix mantle.

     

Some properties of ATPase activity in the intact cells of yeast Saccharomycopsis fibuligera

• 1.1. The properties of ATPase activity were studied with the cells at the early stationary phase of Saccharomycopsis fibuligera.
• 2.2. Optimal pH for the activity was approximately 7.
• 3.3. The activity was stimulated by Mg²⁺.
• 4.4. The activity was inhibited by NaF, DCCD, oligomycin, NaN₃, NaVO₃, or PCMB but not inhibited by ouabain.

     

Characterization of NaK ATPase from the gills of the freshwater prawn Macrobrachium rosenbergii (de Man)

• 1.1. The specific activity of Na-K ATPase was determined from the microsomal preparation of gills dissected from adult Macrobrachium rosenbergii.
• 2.2. Maximal ATPase activity was achieved at a substrate concentration of 0.5 mM ATP.
• 3.3. Optimal enzyme activity was obtained at pH of 7.5.
• 4.4. The Arrhenius plot of Na-K ATPase activity revealed a marked discontinuity at 30°C. “Mg” ATPase activity did not exhibit a marked discontinuity.
• 5.5. The E_a for Na-K ATPase and “Mg” ATPase was 14.6 kCal/mole and 9.31 kCal/mole respectively. Q₁₀ values for Na-K ATPase was 2.34 and for “Mg” ATPase 1.65.
• 6.6. ATPase activity and gill homogenate protein concentration exhibited a linear relationship up to 130 μg protein/ml.
• 7.7. Na-K ATPase activity was inhibited by 10⁻³ M ouabain. It was equally inhibited by the removal of K⁺ from the reaction medium.