Separation of the Mg-ATPases from the Ca-Phosphatase Activity of Microsomal Membranes Prepared from Barley Roots

Two methods for preparing membrane fractions from barley (Hordeum vulgare cv California Mariout 72) roots were compared in order to resolve reported differences between the characteristics of the plasma membrane ATPase of barley and that of other species. When microsomal membranes were prepared by a published procedure and applied to a continuous sucrose gradient, the membranes sedimented as a single broad band with a peak density of 1.16 grams per cubic centimeter (g/cm³). Activities of NADH cytochrome (Cyt) c reductase, Ca²⁺-ATPase, and Mg²⁺-ATPase were coincident and there was little ATP-dependent proton transport anywhere on the gradient. When the homogenization procedure was modified by increasing the pH of the buffer and the ratio of buffer to roots, the microsomal membranes separated as several components on a continuous sucrose gradient. A Ca²⁺-phosphatase was at the top of the gradient, NADH Cyt c reductase at 1.08 g/cm³, a peak of ATP-dependent proton transport at 1.09 to 1.12 g/cm³, a peak of nitrate-inhibited ATPase at 1.09 to 1.12 g/cm³, and of vanadate-inhibited ATPase at 1.16 g/cm³. The Ca²⁺-phosphatase had no preference for ATP over other nucleoside di- and tri-phosphates and was separated from the vanadate-inhibited ATPase on a sucrose gradient; approximately 70% of the Ca²⁺-phosphatase was removed from the microsomes by washing with 150 millimolar KCl. The vanadate-sensitive ATPase required Mg²⁺, was highly specific for ATP, and was not affected by the KCl wash. These results show that barley roots have a plasma membrane ATPase similar to that of other plant species.     

Localization of a Mg2+- or Ca2+-activated ("basic") ATPase in skeletal muscle.

A chicken pectoralis muscle membrane fraction enriched in a Mg²⁺- or Ca²⁺-activated (‘basic’) ATPase was obtained by sucrose gradient centrifugation. Enzymatic properties of the ‘basic’ ATPase were determined and used to localize its enzymatic activity in situ by ultrastructural cytochemistry. The enzyme was activated by Mg²⁺ or Ca²⁺ but not by Sr²⁺, Ba²⁺, Co²⁺, Ni²⁺ or Pb²⁺. It was present in a membranous fraction with a buoyant density of 1.10-1.12 (24–27.5% ($\text{w}\text{w}$) sucrose). ‘Basic’ ATPase activity had a sedimentation pattern similar to the putative plasma membrane enzymes, 5′-nucleotidase and leucyl β-naphthylamidase, but different from that of sarcoplasmic reticulum Ca²⁺ ATPase. Also unlike sarcoplasmic reticulum Ca²⁺ ATPase, ‘basic’ ATPase was resistant to N-ethylmaleimide and aldehyde fixatives, was active in a medium containing a high Ca²⁺ concentration (3 mM), and was lost when exposed to Triton X-100 or deoxycholate. In cytochemical studies, a low Pb²⁺ concentration was used to capture the enzymatically released phosphate ions. Under conditions which eliminated interfering (Na⁺ + K⁺) ATPase and sarcoplasmic reticulum Ca²⁺ ATPase activities, electron-dense lead precipitates were present at the plasmalemma and T-system membranes. These studies suggest that ‘basic’ ATPase activity is associated with plasmalemma and T-system membranes of skeletal muscle.     

Novel Microbial Inhibitors of Angiotensin-converting Enzyme,Aspergillomarasmines A and B

Brush border membrane vesicles (BBMV) from the midgut epithelial cells of silkworm larvae were prepared. ATP hydrolyzing activity (ATPase activity) was associated with the BBMV. ATPase activity without Mg^{2 +} was not observed at pH 7 but substantial ATP hydrolyzing activity was observed at pH 7 with Mg^{2 +}. The enzyme required Mn^{2 +}, Mg^{2 +}, or Ca²⁺ ions. The enzyme also hydrolyzed ITP and GTP but not p-NPP, ADP, or AMP. KNO₃ and NEM strongly inhibited the ATPase activity. Behaviours of the ATPase against inhibitors suggested that it resembled vacuolar type ATPase.     

Entamoeba histolytica: localization and characterization of ca2+-dependent nucleotidases

An activity of Ca²⁺-dependent nucleotidase was detected in axenically-cultivated trophozoites of Entamoeba histolytica. The enzyme was concentrated by differential and sucrose density gradient centrifugation and catalyzed hydrolysis of nucleoside tri- and diphosphates and also thiamine pyrophosphate. Hydrolysis of nucleoside mono-phosphates was not affected by Ca²⁺. Among substrates tested, ATP was most active. Addition of Zn²⁺ or heat treatment almost abolished the enzyme activity. The enzyme exhibited almost the identical activity at acid and neutral pH. Among 6 bands isolated by polyacrylamide gel electrophoresis, 4 were stained with ATP, UTP, CTP and ADP, whereas the other 2 were stained only with ATP, UTP and CTP. The concentrated enzyme preparation, primarily composed of membrane fragments, also had activities of acid phosphatase, acid inorganic pyrophosphatase, 5'-nucleotidase and Mg²⁺-dependent ATPase. These observations suggest that E. histolytica has 2 Ca²⁺-dependent nucleotidases, i.e. one Ca²⁺-dependent ATPase and the other Ca²⁺-dependent nucleoside diphosphatase or an apyrase-like enzyme, and that these nucleotidases are at least partially associated with the plasma membrane or an organelle of lysosomal nature in this parasite.     

Isolation and properties of ion-stimulated ATPase activity associated with cauliflower plasma membranes

The association of K⁺-stimulated, Mg²⁺-dependent ATPase activity with plasma membranes from higher plants has been used as a marker for the isolation and purification of a plasma membrane-enriched fraction from cauliflower (Brassica oleraceae L.) buds. Plasma membranes were isolated by differential centrifugation followed by density gradient centrifugation of the microsomal fraction. The degree of purity of plasma membranes was determined by increased sensitivity of Mg²⁺-ATPase activity to stimulation by K⁺ and by assay of approximate marker enzymes. In the purified plasma membrane fraction, Mg²⁺-ATPase activity was stimulated up to 700% by addition of K⁺. Other monovalent cations also markedly stimulated the enzyme, but only in the presence of the divalent cation Mg²⁺. Ca²⁺ was inhibitory to enzyme activity. ATPase was the preferred substrate for hydrolysis, there being little hydrolysis in the presence of ADP, GTP, or p-nitrophenylphosphate. Monovalent cation-stimulated activity was optimum at alkaline pH. Enzyme activity was inhibited nearly 100% by AgNO₃ and about 40% by diethylstilbestrol.     

Anaerobic transport of amino acids coupled to the glycerol-3-phosphate-fumarate oxidoreductase system in a cytochrome-deficient mutant of Escherichia coli

The uptake of proline and glutamine by cytochrome-deficient cells of Escherichia coli SASX76 grown aerobically on glucose or anaerobically on pyruvate was stimulated by these two substrates. Pyruvate could not stimulate transport in the glucose-grown cells. Uptake of these amino acids energized by glucose was inhibited by inhibitors of the Ca²⁺, Mg²⁺-stimulated ATPase such as DCCD, pyrophosphate, and azide, and by the uncouplers CCCP and 2,4-dinitrophenol. Glycerol (or glycerol 3-phosphate) in the presence of fumarate stimulated the transport of proline and glutamine under anaerobic conditions in cytochrome-deficient cells but not in membrane vesicles prepared from these cells although glycerol 3-phosphate-fumarate oxidoreductase activity could be demonstrated in the vesicle preparation. In contrast, in vesicles prepared from cytochrome-containing cells of E. coli SASX76 amino acid transport was energized under anaerobic conditions by this system. Inhibitors of the Ca²⁺, Mg²⁺-activated ATPase and uncoupling agents inhibited the uptake of proline and glutamine in cytochrome-deficient cells dependent on the glycerol-fumarate oxidoreductase system. Ferricyanide could replace fumarate as an electron acceptor to permit transport of phenylalanine in cytochrome-deficient or cytochrome- containing cells under anaerobic conditions. It is concluded that in cytochrome-deficient cells using glucose, pyruvate, or glycerol in the presence of fumarate, transport of both proline and glutamine under anaerobic conditions is energized by ATP through the Ca²⁺, Mg²⁺-activated ATPase. In cytochrome-containing cells under anaerobic conditions electron transfer between glycerol and fumarate can also drive transport of these amino acids.     

Membrane-bound ATPase in chloroplasts of Euglena gracilis

Membrane-bound ATPase activities in chloroplasts of Euglena were examined. Ca²⁺- and Mg²⁺-dependent activities were relatively high in membrane preparations and could not be further activated by a number of procedures. The enzyme was found to be highly specific for purine nucleotides and was inhibited by the usual inhibitors of photophosphorylation. K_m values of Ca²⁺ and Mg²⁺ ATPase for ATP were 2.5 and 2.1 mM, respectively. Both activities were competitively inhibited by ADP and inorganic phosphate. A relationship was found between Ca²⁺- or Mg²⁺-dependent ATPase activities and chloroplast completeness. The possibilities that these activities result from one enzyme depending on Ca²⁺ or Mg²⁺ or from two different enzymes are discussed.     

Characterisation of a Ca2+-dependent ATP hydrolysis associated with the vacuolar membrane of wheat roots

Microsomal fractions from wheat tissues exhibit a higher level of ATP hydrolytic activity in the presence of Ca²⁺ than Mg²⁺. Here we characterise the Ca²⁺-dependent activity from roots of Triticum aestivum lev. Troy) and investigate its possible function. Ca²⁺-dependent ATP hydrolysis in the microsomal fraction occurs over a wide pH range with two slight optima at pH 5.5 and 7.5. At these pHs the activity co-migrates with the major peak of nitrate-inhibited Mg²⁺. Cl-ATPase on continuous sucrose gradients indicating that it is associated with the vacuolar membrane. Ca²⁺-dependent ATP hydrolysis can be distinguished from an inhibitory effect of Ca²⁺ on the plasma membrane K⁺, Mg²⁺-ATPase following microsomal membrane separation using aqueous polymer two phase partitioning. The Ca²⁺-dependent activity is stimulated by free Ca²⁺ with a K_m of 8.1 μM in the absence of Mg²⁺ ([CaATP] = 0.8 mM). Vacuoiar membrane vacuolar preparations contain a higher Ca²⁺-dependent than Mg²⁺-dependent ATP hydrolysis, although the two activities are not directly additive. The nucleotide specificity of the divalent ion-dependent activities in vacuolar membrane-enriched fractions was low. hydrolysis of CTP and UTP being greater than ATP hydrolysis with both Ca²⁺ and Mg²⁺ The Ca²⁺-dependent activity did discriminate against dinucleotides, and mononucleotides. and failed to hydrolyse phosphatase substrates. Despite low nucleotide specificity the Mg²⁺-dependent activity functioned as a bafilomycin sensitive H⁺-pump in vacuolar membrane vesicles. Ca²⁺-dependent ATP hydrolysis was not inhibited by the V-, P-, or F-type ATPase inhibitors bafilomycin. vanadate and azide, respectively. nor by the phosphatase inhibitor molybdate, but was inhibited 20% at pH 7.5 by K⁺. Possible functions of Ca²⁺-dependent hydrolysis as a H⁺-pump or a Ca²⁺-pump was investigated using vacuolar membrane vesicles. No H⁺ or Ca²⁺ translocating activity was observed under conditions when the Ca²⁺-dependent ATP hydrolysis was active.     

Characterization of Plasma Membrane-associated Adenosine Triphosphase Activity of Oat Roots

ATPase activity of plasma membranes isolated from oat (Avena sativa L. cv. Goodfield) roots was activated by divalent cations (Mg²⁺ = Mn²⁺ > Zn²⁺ > Fe²⁺ > Ca²⁺) and further stimulated by KCl and a variety of monovalent salts, both inorganic and organic. The enzyme exhibited greater specificity for cations than anions. The presence of Mg²⁺ was necessary for KCl stimulation. Ca²⁺ was ineffective in replacing Mg²⁺ for activation of plasma membrane ATPase, but it did activate other membrane-bound ATPases. The pH optima for Mg²⁺ activation and KCl stimulation of the plasma membrane ATPase were 7.5 and 6.5, respectively.     

Characterization of an ATPase Associated with the Inner Envelope Membrane of Amyloplasts from Suspension-Cultured Cells of Sycamore (Acer pseudoplatanus L.)

Amyloplast envelope membranes isolated from cultured, white-wild cells of sycamore (Acer pseudoplatanus L.) have been found to contain a Mg²⁺-ATPase, ranging in specific activity from 5 to 30 nanomoles per minute per milligram protein. This ATPase hydrolyzes a broad range of nucleoside triphosphates, whereas it hydrolyzes nucleoside mono- and diphosphates poorly, if at all. The ATPase activity was stimulated by several divalent cations, including Mg²⁺, Mn²⁺ and Ca²⁺, whereas it was not affected by Sr²⁺, K⁺, or Na⁺. The K_m for total ATP was 0.6 millimolar, and the activity showed a broad pH optimum between 7.5 and 8.0. The ATPase was insensitive to N,N′-dicyclohexylcarbodiimide and oligomycin, but it was inhibited by vanadate. All these characteristics are basically similar to those reported previously for the Mg²⁺-ATPase of the chloroplast inner-envelope membrane. Likewise, the amyloplast envelope enzyme was shown to be located specifically on the inner envelope membrane. The amyloplast envelope membranes were chemically modified with a series of unique affinity labeling reagents, the adenosine polyphosphopyridoxals (M Tagaya, T Fukui 1986 Biochemistry 25: 2958-2964). About 90% of the ATPase activity was lost when the envelope membranes were preincubated with 0.1 millimolar adenosine triphosphopyridoxal. Notably, the enzyme was protected completely from inactivation in the presence of its substrate, ATP. In contrast, both adenosine diphosphopyridoxal and pyridoxal phosphate caused much less of an inhibitory effect. This greater relative reactivity of the triphosphopyridoxal analog is similar to that reported previously with Escherichia coli F₁ ATPase (T Noumi et al. 1987 J Biol Chem 262: 7686-7692).     

Membrane ATPase of Bacillus subtilis. I. Purification and properties

The membrane ATPase (EC 3.6.1.3) of Bacillus subtilis can be solubilized by a shock-wash process. Two procedures for purifying the solubilized enzyme are reported. A protease inhibitor, phenylmethane sulfonylfluoride, was introduced in the solubilization and purification step.The resultant ATPase purified by density gradient centrifugation has a molecular weight of 315 000, an s_20,w of 13,4 and an ámino acid composition very similar to bacterial ATPases already studied.After exposure to polyacrylamide gel electrophoresis in presence of sodium dodecyl sulphate (SDS), or 8 M urea or SDS-urea, the purified ATPase can be dissociated in two non-identical subunits of molecular weights 59 000 (α) and 57 000 (β) with different charges.Kinetic studies showed that Ca²⁺ or Zn²⁺ are required for ATPase activity, although Mg²⁺ was uneffective. At optimal Ca²⁺ concentration, the Mg²⁺ has an inhibitory effect. The K_m for ATP is 1.3 mM. Inhibitors of the oxydative phosphorylation, of the mitochondrial ATPase and of the (Na⁺ + K⁺)-ATPase are studied.     

Purification and composition of Ca2+/Mg2+ ATPase from rat heart plasma membrane

The Ca²⁺/Mg²⁺ ATPase, which is activated by millimolar concentrations of Ca²⁺ or Mg²⁺, was solubilized from rat heart plasma membrane by employing lysophosphatidylcholine, CHAPS, Nal, EDTA and Tris-HCI at pH 7.4. The enzyme was purified by sucrose density gradient, Affi-Gel Blue column and Sepharose 6B column chromatography. The purified enzyme was seen as a single peptide band in the sodium dodecyl sulfate polyacrylamide gel electrophoresis with a molecular weight of about 90,000. The apparent molecular weight of the holoenzyme as determined under non-dissociating conditions by gel filtration on Sepharose 6B column was about 180,000 indicating two subunits. The enzyme was insensitive to ouabain, verapamil, vanadate, oligomycin, N,N-dicyclohexylcarbodiimide and NaN₃, but was markedly inhibited by 20 µM gramacidin S and 50 µM trifluoperazine. Analysis of the purified Ca²⁺/Mg²⁺ ATPase revealed the presence of 17 amino acids where leucine, glutamic acid and aspartic acid were the major components and histidine, cysteine and methionine were the minor components. The purified enzyme was associated with 19.7 µmol phospholipid/mg protein which was 60 times higher than the phospholipid content in plasma membrane. The cholesterol content in the purified enzyme preparation was 0.75 µmol/mg protein and this represented an 8-fold enrichment over plasma membrane. The glycoprotein nature of the enzyme was evident from the positive periodic acid-Schiff staining of the purified Cau2+/Mg^ATPase in the sodium dodecyl sulfate polyacrylamide gel. The polysaccharide content of the enzyme was enriched 8-fold over plasma membrane; neurominidase treatment decreased the polysaccharide content. Concanavalin A prevented the ATP-dependent inactivation of the purified Ca²⁺/Mg²⁺ ATPase and was found to bind to the purified enzyme with a K_D of 576 nM and Bmax of 4.52 nmol/mg protein. The results indicate that Ca²⁺/Mg²⁺ ATPase is a glycoprotein and contains a large amount of lipids.     

Ca2+-dependent ATPase associated with plasma membrane from a calcareous alga, Serraticardia maxima (Corallinaceae, Rhodophyta)

The plasma membrane was isolated from a calcareous red alga, Serraticardia maxima (Yendo) Silva (Corallinaceae), by aqueous two-phase partitioning. Its purity was examined with marker enzymes, Mg²⁺-dependent ATPase, inosine diphosphatase, cytochrome c oxidase and NADH-cytochrome c reductase, as well as the sensitivity of Mg²⁺-dependent ATPase to vanadate, azide and nitrate. The results showed that the isolated plasma membrane was purified enough to study its functions. Electron microscopic observations on thin tissue sections revealed that most vesicles of the isolated plasma membrane were stained by the plasma membrane specific stain, phosphotungstic acid-chromic acid. Mg²⁺- or Ca²⁺-dependent ATPases were associated with the plasma membrane. Ca²⁺-dependent ATPase was activated at physiological cytoplasmic concentrations of Ca²⁺ (0.1–10 μmol/L). However, calmodulin (0.5 μmol/L) did not affect its activity. The pH optimum was 8.0, in contrast to 7.0 for Mg²⁺-dependent ATPase. The isolated plasma membrane vesicles were mostly right side-out. To test for H⁺-translocation, right side-out vesicles were inverted; 27% of vesicles were inside-out after treatment with Triton X-100. The inside-out plasma membrane vesicles showed reduction of quinacrine fluorescence in the presence of 1 mmol/L ATP and 100 μmol/L Ca²⁺. The reduced fluorescence was recovered with the addition of 10 mmol/L NH₄Cl, or 5 μmol/L nigericin plus 50 mmol/L KCl. UTP and CTP substituted for ATP, but ADP did not. Ca²⁺-dependent ATPase might pump H⁺ out in the physiological state. The acidification by this pump might be coupled with alkalinization at the calcifying sites, which induces calcification.     

The Ca-Transport ATPase of Plant Plasma Membrane Catalyzes a nH/Ca Exchange

Microsomal vesicles from 24-hour-old radish (Raphanus sativus L.) seedlings accumulate Ca²⁺ upon addition of MgATP. MgATP-dependent Ca²⁺ uptake co-migrates with the plasma membrane H⁺-ATPase on a sucrose gradient. Ca²⁺ uptake is insensitive to oligomycin, inhibited by vanadate (IC₅₀ 40 micromolar) and erythrosin B (IC₅₀ 0.2 micromolar) and displays a pH optimum between pH 6.6 and 6.9. MgATP-dependent Ca²⁺ uptake is insensitive to protonophores. These results indicate that Ca²⁺ transport in these microsomal vesicles is catalyzed by a Mg²⁺-dependent ATPase localized on the plasma membrane. Ca²⁺ strongly reduces ΔpH generation by the plasma membrane H⁺-ATPase and increases MgATP-dependent membrane potential difference (Δψ) generation. These effects of Ca²⁺ on ΔpH and Δψ generation are drastically reduced by micromolar erythrosin B, indicating that they are primarily a consequence of Ca²⁺ uptake into plasma membrane vesicles. The Ca²⁺-induced increase of Δψ is collapsed by permeant anions, which do not affect Ca²⁺-induced decrease of ΔpH generation by the plasma membrane H⁺-ATPase. The rate of decay of MgATP-dependent ΔpH, upon inhibition of the plasma membrane H⁺-ATPase, is accelerated by MgATP-dependent Ca²⁺ uptake, indicating that the decrease of ΔpH generation induced by Ca²⁺ reflects the efflux of H⁺ coupled to Ca²⁺ uptake into plasma membrane vesicles. It is therefore proposed that Ca²⁺ transport at the plasma membrane is mediated by a Mg²⁺-dependent ATPase which catalyzes a nH⁺/Ca²⁺ exchange.     

Immunolocalization of the sarcolemmal Ca2+/Mg2+ ecto-ATPase (myoglein) in rat myocardium

Cardiac plasma membrane Ca²⁺/Mg²⁺ ecto-ATPase (myoglein) requires millimolar concentrations of either Ca²⁺ or Mg²⁺ for maximal activity. In this paper, we report its localization by employing an antiserum raised against the purified rat cardiac Ca²⁺/Mg²⁺ ATPase. As assessed by Western blot analysis, the antiserum and the purified immunoglobulin were specific for Ca²⁺/Mg²⁺ ecto-ATPase; no cross reaction was observed towards other membrane bound enzymes such as cardiac sarcoplasmic reticulum Ca²⁺-pump ATPase or sarcolemmal Ca²⁺-pump ATPase. On the other hand, the cardiac Ca²⁺/Mg²⁺ ecto-ATPase was not recognized by antibodies specific for either cardiac sarcoplasmic reticulum Ca²⁺-pump ATPase or plasma membrane Ca²⁺-pump ATPase. Furthermore, the immune serum inhibited the Ca²⁺/Mg²⁺ ecto-ATPase activity of the purified enzyme preparation. Immunofluorescence of cardiac tissue sections and neonatal cultured cardiomyocytes with the Ca²⁺/Mg²⁺ ecto-ATPase antibodies indicated the localization of Ca²⁺/Mg²⁺ ecto-ATPase in association with the plasma membrane of myocytes, in areas of cell-matrix or cell-cell contact. Staining for the Ca²⁺/Mg²⁺ ecto-ATPase was not cardiac specific since the antibodies detected the presence of membrane proteins in sections from skeletal muscle, brain, liver and kidney. The results indicate that Ca²⁺/Mg²⁺ ecto-ATPase is localized to the plasma membranes of cardiomyocytes as well as other tissues such as brain, liver, kidney and skeletal muscle.     

Characterization of a partially purified adenosine triphosphatase from a corn root plasma membrane fraction

The (K⁺,Mg²⁺)-ATPase was partially purified from a plasma membrane fraction from corn roots (WF9 × Mol7) and stored in liquid N₂ without loss of activity. Specific activity was increased 4-fold over that of the plasma membrane fraction. ATPase activity resembled that of the plasma membrane fraction with certain alterations in cation sensitivity. The enzyme required a divalent cation for activity (Co²⁺ > Mg²⁺ > Mn²⁺ > Zn²⁺ > Ca²⁺) when assayed at 3 millimolar ATP and 3 millimolar divalent cation at pH 6.3. When assayed in the presence of 3 millimolar Mg²⁺, the enzyme was further activated by monovalent cations (K⁺, NH₄⁺, Rb⁺ Na⁺, Cs⁺, Li⁺). The pH optima were 6.5 and 6.3 in the absence and presence of 50 millimolar KCl, respectively. The enzyme showed simple Michaelis-Menten kinetics for the substrate ATP-Mg, with a K_m of 1.3 millimolar in the absence and 0.7 millimolar in the presence of 50 millimolar KCl. Stimulation by K⁺ approached simple Michaelis-Menten kinetics, with a K_m of approximately 4 millimolar KCl. ATPase activity was inhibited by sodium orthovanadate. Half-maximal inhibition was at 150 and 35 micromolar in the absence and presence of 50 millimolar KCl. The enzyme required the substrate ATP. The rate of hydrolysis of other substrates, except UDP, IDP, and GDP, was less than 20% of ATP hydrolysis. Nucleoside diphosphatase activity was less than 30% of ATPase activity, was not inhibited by vanadate, was not stimulated by K⁺, and preferred Mn²⁺ to Mg²⁺. The results demonstrate that the (K⁺,Mg²⁺)-ATPase can be clearly distinguished from nonspecific phosphohydrolase and nucleoside diphosphatase activities of plasma membrane fractions prepared from corn roots.     

Adenosine triphosphatase activity in the neural lobe of the bovine pituitary gland

1. Homogenates of neural lobes of bovine pituitary glands were fractionated by differential and density-gradient ultracentrifugation and the distribution of adenosine triphosphatase (ATPase) activity was studied. It was shown that all the activity was membrane-bound. 2. On the basis of ionic requirements the ATPase activity was grouped into three categories: (a) Mg²⁺-dependent, (b) Ca²⁺-dependent and (c) Mg²⁺+Na⁺+K⁺-dependent (ouabain-sensitive) ATPases. The activity in the absence of bivalent cations was negligible. The ratio between the activities of the three ATPases varied between the different subcellular fractions. 3. Preincubation of the subcellular fractions with deoxycholate increased the activity of the Mg²⁺+Na⁺+K⁺-dependent enzyme, whereas the Mg²⁺- and Ca²⁺-activated ATPases were either unaffected or slightly inhibited. Triton X-100 solubilized the Mg²⁺- and Ca²⁺-ATPases; however, the activity of the Mg²⁺+Na⁺+K⁺-ATPase was abolished by the concentration of Triton X-100 used. 4. All the subfractions displayed unspecific nucleotide triphosphatase activity towards GTP, ITP and UTP. These substrates inhibited the hydrolysis of ATP by all three ATPases. ADP also inhibited the ATPases. 5. Polyacrylamide-gel electrophoresis of extracts containing the Mg²⁺- and Ca²⁺-dependent ATPase activity solubilized by Triton X-100 revealed the presence of two enzymes; one activated by either Mg²⁺ or Ca²⁺ and the other activated only by Ca²⁺. 6. In sucrose density gradients the distribution of vasopressin was different from that of all three types of ATPases. It is therefore suggested that the neurosecretory granules do not possess ATPase activity.     

Purification and characterization of a Ca2+/Mg2+ ecto-ATPase from rat heart sarcolemma

The Ca²⁺/Mg²⁺ ATPase of the rat heart sarcolemmal particles was solublized with Triton X-100 after treating the membranes with trypsin and purified by high speed centrifugation, ammonium sulfate fractionation, hydrophobic chromatography and gel filtration. The purified enzyme was seen as a single protein band in nondenaturing polyacrylamide gel electrophoresis and its molecular weight by gel filtration was found to be about 240000. The enzyme utilized Ca-ATP or Mg-ATP as a substrate with high affinity sites (Km = 0.12 – 0.16 mM) and low affinity sites (Km = 1 mM). The enzyme also utilized CTP, GTP, ITP, UTP and ADP as substrates but at a lower rate in comparison to ATP. The enzyme was activated by Ca²⁺ (Ka = 0.4 mM) and Mg²⁺ (Ka = 0.2 mM) as well as by other cations in the order Ca^2– > Mg²⁺ > Mn²⁺ > Sr²⁺ > Ba²⁺ > Ni²⁺ > Cu²⁺. The ATPase activity in the presence of Ca²⁺ was markedly inhibited by Mg²⁺, Mn²⁺, Ni²⁺ and Cu²⁺ whereas the monovalent cations such as Na⁺ and K⁺ were without effect. The enzyme did not exhibit Ca²⁺ stimulated Mg²⁺ dependent ATPase activity and was insensitive to calmodulin, ouabain, verapamil, D-600, oligomycin, azide and vanadate. Optimum pH for Ca²⁺ or Mg²⁺ ATPase activity was 8.5 – 9.0. In view of the possible ectoenzyme nature of the ATPase, its role in adenine nucleotide and Ca²⁺ metabolism in the myocardium is discussed.     

The effects of magnesium and calcium ions on phosphatase activities at alkaline pH in the molar region of newborn mice

Summary The hydrolysis of ATP, AMP and glycerophosphate (GP) at alkaline pH in mineralizing bone and teeth of young mice has been studied histochemically. The substrates were visibly hydrolyzed to the same degree in osteoblasts, cells of stratum intermedium, odontoblasts and subodontoblasts at Ca²⁺ concentrations ranging from 10 mM to 600 mM. In the ameloblasts, however, only ATP was hydrolyzed. The ATPase activities gradually decreased at increasing Mg²⁺/Ca²⁺ ratios. The AMPase and GPase activities, on the other hand, were visibly unaffected. Marked cellular staining, including the nuclei was seen with AMP and GP as substrates when only Mg²⁺ ions were added. No ATPase activity at all could be recorded in media containing Mg²⁺ but no Ca²⁺ ions. The different phosphatase activities in cells involved in hard tissue formation were identically affected by preincubations with solutions containing various concentrations of Ca²⁺ or Mg²⁺ ions. The ATPase activity in striated muscle fibres and blood vessel walls, however, was affected differently by the same procedure.The results indicate that the phosphatase activities recorded in osteoblasts, cells of stratum intermedium, odontoblasts and subodontoblasts at alkaline pH belong to one single enzyme. The results also imply that CaATP is the preferred substrate in the enzymatic hydrolysis of ATP in hard-tissue-forming cells.     

Highly purified sarcoplasmic reticulum vesicles are devoid of Ca2+-independent (‘basal’) ATPase activity

On solubilization with Triton X-100 of sarcoplasmic reticulum vesicles isolated by differential centrifugation, the Ca²⁺-ATPase is selectively extracted while approximately half of the initial Mg²⁺-, or ‘basal’, ATPase remains in the Triton X-100 insoluble residue. The insoluble fraction, which does not contain the 100 000 dalton polypeptide of the Ca²⁺-ATPase, contains high levels of cytochrome c oxidase. Furthermore, its Mg²⁺-ATPase activity is inhibited by specific inhibitors of mitochondrial ATPase, indicating that the ‘basal’ ATPase separated from the Ca²⁺-ATPase by detergent extraction originates from mitochondrial contaminants.To minimize mitochondrial contamination, sarcoplasmic reticulum vesicles were fractionated by sedimentation in discontinuous sucrose density gradients into four fractions: heavy, intermediate and light, comprising among them 90–95% of the initial sarcoplasmic reticulum protein, and a very light fraction, which contains high levels of Mg²⁺-ATPase. Only the heavy, intermediate and light fractions originate from sarcoplasmic reticulum; the very light fraction is of surface membrane origin. Each fraction of sarcoplasmic reticulum origin was incubated with calcium phosphate in the presence of ATP and the loaded fractions were separated from the unloaded fractions by sedimentation in discontinuous sucrose density gradients. It was found that vesicles from the intermediate fraction had, after loading, minimal amounts of mitochondrial and surface membrane contamination, and displayed little or no Ca²⁺-independent basal ATPase activity. This shows conclusively that the basal ATPase is not an intrinsic enzymatic activity of the sarcoplasmic reticulum membrane, but probably originates from variable amounts of mitochondrial and surface membrane contamination in sarcoplasmic reticulum preparations isolated by conventional procedures.