Mn2+-dependent ADP-ribose/CDP-alcohol pyrophosphatase: a novel metallophosphoesterase family preferentially expressed in rodent immune cells

ADPRibase-Mn (Mn2+-dependent ADP-ribose/CDP-alcohol pyrophosphatase) was earlier isolated from rat liver supernatants after separation from ADPRibase-I and ADPRibase-II (Mg2+-activated ADP-ribose pyrophosphatases devoid of CDP-alcohol pyrophosphatase activity). The last mentioned are putative Nudix hydrolases, whereas the molecular identity of ADPRibase-Mn is unknown. MALDI (matrix-assisted laser-desorption ionization) MS data from rat ADPRibase-Mn pointed to a hypothetical protein that was cloned and expressed and showed the expected specificity. It is encoded by the RGD1309906 rat gene, which so far has been annotated simply as 'hydrolase'. ADPRibase-Mn is not a Nudix hydrolase, but it shows the sequence and structural features typical of the metallophosphoesterase superfamily. It may constitute a protein family of its own, the members of which appear to be specific to vertebrates, plants and algae. ADP-ribose was successfully docked to a model of rat ADPRibase-Mn, revealing its putative active centre. Microarray data from the GEO (Gene Expression Omnibus) database indicated that the mouse gene 2310004I24Rik, an orthologue of RGD1309906, is preferentially expressed in immune cells. This was confirmed by Northern-blot and activity assay of ADPRibase-Mn in rat tissues. A possible role of ADPRibase-Mn in immune cell signalling is suggested by the second-messenger role of ADP-ribose, which activates TRPM2 (transient receptor potential melastatin channel-2) ion channels as a mediator of oxidative/nitrosative stress, and by the signalling function assigned to many of the microarray profile neighbours of 2310004I24Rik. Furthermore, the influence of ADPRibase-Mn on the CDP-choline or CDP-ethanolamine pathways of phospholipid biosynthesis cannot be discounted.     

Rat liver glucocorticoid receptor isolated by affinity chromatography is not a Mg2+- or Ca2+-dependent protein kinase

The glucocorticoid hormone receptor (92 kDa), purified 9000-fold from rat liver cytosol by steroid affinity chromatography and DEAE-Sephacel chromatography, was assayed for the presence of protein kinase activity by incubations with [gamma-32P]ATP and the photoaffinity label 8-azido-[gamma-32P]ATP. Control preparations isolated by affinity chromatography in the presence of excess steroid to prevent the receptor from binding to the affinity matrix were assayed for kinase activity in parallel. The receptor was not labeled by the photoaffinity label under photoactivation conditions in the presence of Ca2+ or Mg2+. A Mg2+-dependent protein kinase (48 kDa) that could be photoaffinity labeled with 8-azido-ATP copurified with the receptor. This kinase was also present in control preparations. The kinase could phosphorylate several minor contaminants present in the receptor preparation, including a protein (or proteins) of similar molecular weight to the receptor. The phosphorylation of 90-92-kDa proteins was independent of the state of transformation or steroid-binding activity of the receptor. These experiments provide direct evidence that neither the glucocorticoid receptor nor the 90-92-kDa non-steroid-binding protein associated with the molybdate-stabilized glucocorticoid receptor possesses intrinsic Ca2+- or Mg2+-dependent protein kinase activity.     

Mg2+- or Mn2+-dependent p-nitrophenylphosphatase activity is present in Ehrlich ascites tumor cells

The presence of a soluble, Mg2+- or Mn2+-dependent p-nitrophenylphosphatase activity in Ehrlich ascites tumor cell homogenates is reported. The crude homogenate was fractionated over Sephadex G-150 gel-filtration and DEAE-Sephacel anion-exchange columns, and two p-nitrophenylphosphatase activities were resolved. The most active fraction, Peak I, was characterized and found to be similar to phosphotyrosyl-protein phosphatases characterized elsewhere in that it has optimal activity at neutral pH; it is inhibited by phosphate, Zn2+, and vanadate; and it is not inhibited by levamisole. However, Peak I differs from phosphotyrosyl-protein phosphatases in that Mg2+ or Mn2+ is required for activity, fluoride is an inhibitor, and pyrophosphate is not inhibitory. Inhibition by the phosphorylated compounds phosphotyrosine, phosphoserine, phosphothreonine, ATP, CTP, GTP, ITP, NADP, fructose 6-phosphate, glucose 1-phosphate, galactose 1-phosphate, 2-phosphogluconic acid, and 6-phosphogluconic acid was also observed. Ehrlich ascites tumor cell p-nitrophenylphosphatase is shown to be sensitive to inactivation by trypsin, N-ethylmaleimide, or heat treatments.     

Characterisation of Ca2+ or Mg(2+)-dependent nucleoside triphosphatase from rat mesenteric small arteries.

When isolated rat mesenteric small arteries were submitted to 2 s of sonication, a nucleoside triphosphatase activity was released to the medium, mainly from the plasma membrane of the vascular smooth muscle cells. The activity was kinetically characterized: It hydrolysed ATP, UTP and GTP with the same substrate affinity and the same specific activity. CaATP, as well as MgATP were substrates for the enzyme with an apparent Km in the micromolar range. ATPase inhibitors: ouabain, vanadate, AlF4-, oligomycin and N-ethylmaleimide were without effect on the hydrolytic activity. Among other modifiers tested only N,N'-dicyclohexylcarbodiimide caused significant (greater than 30%) inhibition. In the presence of micromolecular concentrations of Ca2+ and Mg2+, small (less than 20 mM) concentrations of Na+, K+, Rb+, Cs+ and choline+, irrespective of the nature of the anion, activated the hydrolysis with an equilibrium ordered pattern, but concentrations of monovalent cation salts above 20 mM decreased the hydrolysis rate. No activation by monovalent cation salts was seen at millimolar concentrations of divalent cations and substrate. On the basis of the results a standard mixture is proposed, which allows a sensitive assay of the specific enzyme activity.     

Ca2+- or Mg2+-dependent ATPase in plasma membrane of cultured endothelial cells from bovine carotid artery

ATPase was found in plasma membrane of cultured endothelial cells from bovine carotid artery. The activity of the enzyme solubilized by octaethyleneglycol mono-n-dodecyl ether was enhanced by the addition of Ca2+ or Mg2+ and was not affected by F-actin and ouabain. Vmax was 2.8 and 10.0 mumol Pi/mg protein per h for Ca2+- and Mg2+-dependent activity, respectively, and the corresponding Km was 4.8 X 10(-4) M and 3.2 X 10(-4) M. Molecular weight of the protein was estimated to be approx. 250 000, as determined by activity-staining electrophoresis with polyacrylamide gels.     

Rat liver plasma membrane Ca2+- or Mg2+-activated ATPase. Evidence for proton movement in reconstituted vesicles

The Ca2+- or Mg2+-activated ATPase from rat liver plasma membrane was partly purified by treatments with sodium cholate and lysophosphatidylcholine, and by isopycnic centrifugation on sucrose gradients. The ATPase activity had high sensitivity to detergents, poor nucleotide specificity and broad tolerance for divalent cations. It was insensitive to mitochondrial ATPase inhibitors such as oligomycin and to transport ATPase inhibitors such as vanadate and ouabain. Using the cholate dialysis procedure, the partly purified enzyme was incorporated into asolectin vesicles. Upon addition of Mg2+-ATP, fluorescence quenching of 9-amino-6-chloro-2-methoxyacridine (ACMA) was observed. The quenching was abolished by a protonophore, carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP). Asolectin vesicles or purified ATPase alone failed to promote quenching. These data suggest that the Ca2+- or Mg2+-activated ATPase from rat liver plasma membrane is able of H+-translocation coupled to ATP hydrolysis.     

Mn2+/Mg2+-dependent pyruvate kinase from a d-lactic acid-producing bacterium Sporolactobacillus inulinus: characterization of a novel Mn2+-mediated allosterically regulated enzyme

Sporolactobacillus inulinus has attracted scientific and commercial interest due to its high efficiency in d-lactic acid production. Pyruvate kinase (PYK) is one of the key regulatory points in glycolysis, and well-activated PYK can improve d-lactic acid production. A novel Mn²⁺/Mg²⁺-dependent PYK from S. inulinus was expressed in Escherichia coli and purified to homogeneity. Kinetic characterization demonstrated that the S. inulinus PYK had drastically higher activity and affinity toward substrates in the presence of Mn²⁺ compared to those of the common PYK cofactor Mg²⁺, and the circular dichroism spectra of the S. inulinus PYK suggested a Mn²⁺-mediated allosteric activation. The S. inulinus PYK was also allosterically regulated by ribose-5-phosphate or AMP activation and inorganic phosphate or ATP inhibition. The inhibition could be marked reduced or fully eliminated in the presence of activators. The result of fermentations by S. inulinus Y2-8 showed that the extracellular-added MnSO₄ and KH₂PO₄ significantly affected glycolysis flux and d-lactic acid production, which is consistent with the allosteric regulation of Mn²⁺ and inorganic phosphate on PYK. The sophisticated regulatory role of PYK would establish the foundation of substantial disturbance or restructuring of cellular metabolism for improving the S. inulinus d-lactic acid production.     

Characterization of Ca2+- or Mg2+-ATPase of the excitable ciliary membrane from Paramecium tetraurelia: comparison with a soluble Ca2+-dependent ATPase

We have characterized divalent-cation-stimulated nucleoside triphosphate hydrolase activity of the excitable ciliary membrane and compared it with a soluble Ca2+-ATPase released upon deciliation of Paramecium. The membrane-bound activity is strongly dependent on a divalent cation; calcium stimulates the basal activity of this enzyme at least 10-fold; magnesium and manganese stimulate less well, and strontium and barium, although less effective, also give measurable stimulation. This membrane-bound activity prefers ATP and GTP as substrates but also hydrolyzes UTP and CTP at measurable rates. The maximum velocity at saturating ATP concentrations and optimal calcium concentrations is 0.3 mumol/min per mg. The pH optimum for the membrane-bound activity is broad and centers around pH 7. From the temperature dependence of ATP hydrolysis, we calculate activation energies of 14 and 11 kcal/mol for the Ca2+- and Mg2+-stimulated activities, respectively. The Arrhenius plot is linear over the temperature range of 4 to 25 degrees C. The membrane ATPase is relatively insensitive to ouabain, oligomycin, N,N'-dicyclohexylcarbodiimide, vanadate, Ruthenium red and two calmodulin antagonists. Polyclonal antisera raised against the purified soluble ATPase from the deciliation supernatant show low reactivity with the membrane-bound ATPase. We conclude from the comparison of properties of the two activities that the ciliary membrane-bound ATPase is distinct from the soluble ATPase released by deciliation.     

Purification and characteristics of Ca2+,Mg2+- and Ca2+,Mn2+-dependent and acid DNases from spermatozoa of the sea urchin Strongylocentrotus intermedius

Ca²⁺,Mg²⁺- and Ca²⁺,Mn²⁺-dependent and acid DNases were isolated from spermatozoa of the sea urchin Strongylocentrotus intermedius. The enzymes have been purified by successive chromatography on DEAE-cellulose, phenyl-Sepharose, Source 15Q, and by gel filtration, and the principal physicochemical and enzymatic properties of the purified enzymes were determined. Ca²⁺,Mg²⁺-dependent DNase (Ca,Mg-DNase) is a nuclear protein with molecular mass of 63 kD as the native form and its activity optimum is at pH 7.5. The enzyme activity in the presence of bivalent metal ions decreases in the series (Ca²⁺ + Mg²⁺) > Mn²⁺ = (Ca²⁺ + Mn²⁺) > (Mg²⁺ + EGTA) > Ca²⁺. Ca,Mg-DNase retains its maximal activity in sea water and is not inhibited by G-actin and N-ethylmaleimide, whereas Zn²⁺ inhibits the enzyme. The endogenous Ca,Mg-DNase is responsible for the internucleosomal cleavage of chromosomal DNA of spermatozoa. Ca²⁺,Mn²⁺-dependent DNase (Ca,Mn-DNase) has molecular mass of 25 kD as the native form and the activity optimum at pH 8.5. The enzyme activity in the presence of bivalent metal ions decreases in the series (Ca²⁺ + Mn²⁺) > (Ca²⁺ + Mg²⁺) > Mn²⁺ > (Mg²⁺ + EGTA). In seawater the enzyme is inactive. Zinc ions inhibit Ca,Mn-DNase. Acid DNase of spermatozoa (A-DNase) is not a nuclear protein, it has molecular mass of 37 kD as a native form and the activity optimum at pH 5.5, it is not activated by bivalent metal ions, and it is inhibited by N-ethylmaleimide and iodoacetic acid. Mechanisms of the endonuclease cleavage of double-stranded DNA have been established for the three enzymes. The possible involvement of DNases from sea urchin spermatozoa in programmed cell death is discussed.     

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.

     

Effects of substrate structure on the kinetics of circle opening reactions of the self-splicing intervening sequence from Tetrahymena thermophila: evidence for substrate and Mg2+ binding interactions.

The self-splicing intervening sequence from the precursor rRNA of Tetrahymena thermophila cyclizes to form a covalently closed circle. This circle can be reopened by reaction with oligonucleotides or water. The kinetics of circle opening as a function of substrate and Mg2+ concentrations have been measured for dCrU, rCdU, dCdT, and H2O addition. Comparisons with previous results for rCrU suggest: (1) the 2' OH of the 5' sugar of a dinucleoside phosphate is involved in substrate binding, and (2) the 2' OH of the 3' sugar of a dimer substrate is involved in Mg2+ binding. Evidently, the binding site for a required Mg2+ ion is dependent on both the ribozyme and the dimer substrate. The apparent activation energy and entropy for circle opening by hydrolysis are 31 kcal/mol and 50 eu, respectively. The large, positive activation entropy suggests a partial unfolding of the ribozyme is required for reaction.