Kinetic characteristics of nucleoside mono-, di- and triphosphatase activities of the periplasmic 5'-nucleotidase of Escherichia coli.

Periplasmic 5'-nucleotidase from Escherichia coli, in addition to the monophosphoesterase activity has a diphosphohydrolase activity, acting on nucleoside di- and triphosphates. We proposed that the monophosphoesterase and diphosphohydrolase activities have their own active site. This proposal is based on the different types of bonds being broken. Chemical modification with selective group reagents did not show differences in the essentiality of some residues, like histidyl, carboxyl and arginyl groups, of these two hydrolytic activities. While kinetic approaches employing the competition plot and unidirectional substrate inhibition point to that diphosphohydrolase activity (ATPase-ADPase) do not share the same active site with monophosphoesterase activity. Western blotting developed with polyclonal anti-placental apyrase antibody revealed a single protein in the periplasmic fraction of 66.5 kDa similar to the Mr of the purified enzyme by isoelectrofocusing.

Human placental ecto-enzymes: studies on the plasma membrane anchorage and effect of inhibitors of ATP-metabolizing enzymes.

The human placental microvillar membrane contains several ectoenzymes, including 5'-nucleotidase, alkaline phosphatase and ATP-diphosphohydrolase (ATP-DPH), which might be involved in the extracellular metabolism of nucleotides. The type of anchorage to the plasma membrane of the two first enzymes has been shown to be via a glycosyl-phosphatidylinositol. In the present study, using an enzymatic approach, we show that the ATP-DPH should be attached to the plasma membrane through a different type of anchorage. We were also interested in the search of compounds which could interact differentially with this enzyme to be used as a tool for studying the other two hydrolytic enzymes in the presence of ATP-DPH. Here we report several inhibitors of ecto-ATPases which seem to be a useful tool for studying these three enzymes.

ATP-diphosphophydrolase activity in rat heart tissue.

Extracellular nucleotides interact with specific receptors on the cell surface and are locally metabolized by ecto-nucleotidases. Biochemical characterization of the ATPase and ADPase activities detected in rat heart sarcolemma, under conditions where mitochondrial ATPase and adenylate kinase were blocked, supports our proposal that both activities correspond to a single enzyme, known as ATP-diphosphohydrolase or apyrase. The physiological function of this enzyme could be dephosphorylation of the nucleotides present in the interstitial heart compartment acting together with 5'-nucleotidase. Both hydrolytic activities have similarities in: sarcolemma localization, bivalent metal ion dependence, optimum pH, effect of several amino acid residue modifiers, competitive inhibition of nucleotide analogs, and broad nucleoside di-and triphosphate specificity. The ATPase activity could not be separated from the ADPase either through isoelectrofocusing or electrophoresis under acid conditions.

Comparison of the biochemical properties, regulation and function of ATP-diphosphohydrolase from human placenta and rat kidney.

ATP-diphosphohydrolase (apyrase. EC 3.6.1.5) has both ATPase and ADPase activity that are stimulated by bivalent metals, with Ca2+ being the most effective. The possible physiological function of this enzyme, associated with placental and renal microvilli, is related to the extracellular metabolism of nucleotides. A comparison of the biochemical properties of human placenta and rat kidney apyrase is presented, showing similarities in Mr. bivalent metal stimulation, nucleotide nonspecificity, insensitivity towards specific ATPase inhibitors, and lack of essential sulfhydryl and aliphatic hydroxyl groups. We describe the treatment of membrane preparations from both tissues with different detergents and the isoelectric focusing of the solubilized proteins to partially purify apyrase. An ectoenzyme localization is assigned both in microvillus membranes and in the vasculature on the basis of organ perfusion experiments with nucleotides in the presence of antibodies. Placental and kidney microvillus membranes inhibited ADP-induced platelet aggregation, in agreement with an extracellular role. Initial studies on enzyme regulation suggested the existence of at least two types of modulatory proteins: an activating protein in the cytosol of both tissues, and an inhibitory protein associated with placental microsomes. Possible hormonal regulation was investigated in kidneys using in vivo estradiol treatment, but only slight changes in total apyrase activity were observed.

ATP-diphosphohydrolase activity in rat renal microvillar membranes and vascular tissue.

Ecto-nucleotidases may have a role in the regulation of purinoceptor-mediated responses. ATP-diphosphohydrolase or apyrase has been described as an ecto-nucleotidase, which is characterized by a low specificity for its substrates and bivalent cations. The aim of this work was to demonstrate the presence of apyrase as an ecto-enzyme in the rat kidney. ATPase-ADPase activities of the renal microvillar membrane preparation, which correspond to "right side out' membranes, were characterized. The detection of ATP-diphosphohydrolase in the renal vasculature was done through perfusion of isolated rat kidney. ATPase-ADPase activities of the microvillar membrane preparation and apyrase share similar kinetic properties. These include: low substrate and bivalent metal specificities and insensitivity towards inhibitors like: oligomycin, ouabain, verapamil, levamisole and Ap5A. The M(r) or native ATPase and ADPase activities was determined by the 60Co irradiation-inactivation technique being around 65 kDa for both hydrolytic activities. Immunowestern blot analysis also supports the presence of apyrase in microvilli. Perfusion of isolated rat kidney with ATP and ADP, in the presence or absence of different inhibitors or apyrase antibodies indicated the existence of this enzyme in the vascular endothelium. The identification of ATP-diphosphohydrolase as an ecto-enzyme both in microvilli and vasculature support the proposal that the enzyme may have an important role in the extracellular metabolism of nucleotides.

Comparison of the biochemical properties, regulation and function of ATP-diphosphohydrolase from human placenta and rat kidney

ATP-diphosphohydrolase (apyrase, EC 3.6.1.5) has both ATPase and ADPase activity that are stimulated by bivalent metais, with Ca2+ being the most effective. The possible physiological function of this enzyme, associated with placental and renal microvilli, is related to the extracellular metabolism of nucleotides. A comparison of the biochemical properties of human placenta and rat kidney apyrase is presented, showing similaiities in Mr, bivalent metal stimulation, nucleotide nonspecificity, insensitivity towards specifjc ATPase inhibitors, and lack of essential sulfhydryl and aliphatic hydroxyl groups. We describe the treatment of membrane preparations from both tissues with different detergents and the isoelectric focusing of the solubilized proteins to partially purify apyrase. An ectoenzyme localization is assigned both in microvillus membranes and in the vasculature on the basis of organ perfusion experiments with nucleotides in the presence of antibodies. Placental and kidney microvillus membranes inhibited ADP-induced platelet aggregation, in agreement with an extracellular role. Initial studies on enzyme regulation suggested the existence of at least two types of modulatory proteins: an activating protein in the cytosol of both tissues, and an inhibitory protein associated with placental microsomes. Possible hormonal regulation was investigated in kidneys using in vivo estradiol treatment, but only slight changes in total apyrase activity were observed.

Characterization of ATP-diphosphohydrolase from rat mammary gland.

ATP-diphosphohydrolase (or apyrase) hydrolyses nucleoside di- and triphosphates in the presence of millimolar concentration of divalent cations. It is insensitive towards sulfhydryl and aliphatic hydroxyl-selective reagents and to specific inhibitors of ATPases. We present further evidence that ATPase and ADPase activities present in rat mammary gland correspond to apyrase. Two kinetic approaches have been employed, competition plot and chemical modification with group-selective reagents. The M(r) of these activities was determined by 60Co radiation-inactivation. The kinetic approaches employed, competition plot (which discriminate whether competitive reactions occur at the same site) and chemical modification, point to the presence of a single protein which hydrolyses ATP and ADP. The similar M(r) values of ATPase and ADPase activities also support this proposal. ATPase and ADPase activities of mammary gland show a similar sensitivity or insensitivity towards several chemical modifiers. These results suggest that this enzyme is ATP-diphosphohydrolase, also known as apyrase. The results obtained are compared with the ones obtained by us and other authors with the enzyme isolated from other sources.

Changes in apyrase activity in uterus and mammary gland during the lactogenic cycle.

1. The purpose of this present research was to explore the possible roles of ATP-diphosphohydrolase (apyrase) in two tissues with high energetic demands during cell proliferation and differentiation. 2. Changes in apyrase activities during the pregnancy lactation cycle were examined in the rat uterus and mammary gland. 3. A significant decrease in apyrase activity (ATPase-ADPase) was observed in the pregnant uterus; this observation correlates with a minor inhibitory effect on platelet aggregation. 4. In mammary gland, the enzyme activity increases during lactation in parallel with an increase in blood supply, synthesis of glycoproteins and cell proliferation. 5. Apyrase activity did not change during the estrous cycle. Estradiol administration to rats slightly increased (20%) both ATPase-ADPase activities. 6. The probable function of apyrase is finally discussed, based on its substrate specificity and subcellular localization.

ATPase-ADPase activities of rat placental tissue.

1. Calcium-stimulated ATPase-ADPase activities were studied in a microsomal fraction of rat placental tissue. 2. The kinetic characteristics correspond to those of ATP-diphosphohydrolase, also known as apyrase (E.C. 3.6.1.5). 3. These characteristics include the lack of specificity towards nucleoside di- and triphosphates, activation by Ca2+, Mg2+ or Mn2+, insensitivity to specific inhibitors of some ATPase and absence of an effect of sulphydryl reagents. 4. Chemical modification of tyrosine, tryptophan, arginine and carboxylic residues decreases both ATPase and ADPase activities. 5. The substrate analogue, 5'-(beta, gamma-methylene)triphosphate, protected both enzyme activities against all the modifying amino acid reagents tested. 6. Placental fractions (homogenate and microsomes) inhibit ADP-dependent platelet aggregation. 7. The solubilized microsomal enzyme has a molecular mass of 67 kDa by size-exclusion chromatography; the pI is 9.36. 8. A differential effect is observed on the activation produced by Concanavalin A on microsomal and solubilized fractions when treated in the presence and absence of alpha-methylmannoside.

Comparative subcellular distribution of apyrase from animal and plant sources. Characterization of microsomal apyrase.

1. Apyrase (ATP: diphosphohydrolase) has been found in the microsomal fraction of rat salivary gland, mammary gland and uterus. 2. This enzyme, already described in plant tissue, is mainly present as a soluble polypeptide in tubers of Solanum tuberosum. 3. A fraction of this enzyme is associated with the microsomal fraction with a higher specific activity than the soluble one, for either ATP or ADP as substrate. 4. Apyrase bound to microsomes from rat and potato tissues was characterized in its substrate specificity and effect of inhibitors. 5. The Km values for ATP and ADP, optimum pH and metal ion requirement were determined. 6. A characteristic common to the microsomal and soluble apyrases is the stimulatory effect of a potato activator protein of soluble plant apyrase. 7. The microsomal-bound apyrase from rat and potato tissues were solubilized and subjected to size-exclusion chromatography. 8. The mammary gland and salivary gland apyrases eluted as molecular aggregates, in contrast to the uterus and potato enzyme.

Hydrolysis of synthetic pyrophosphoric esters by an isoenzyme of apyrase from Solanum tuberosum.

A highly purified isoenzyme of apyrase obtained from potatoes (Solanum tuberosum var. Pimpernel) exhibits a low specificity for the organic moiety of synthetic pyro- and triphosphates. Methyl di- and tri-phosphates were hydrolysed at higher rates than ADP and ATP, but their Km values were also higher. Steric hindrance at the carbon atom linked to the pyrophosphate chain decreases both binding and maximum rate, whereas length or polarity of the organic chain do not have systematic effects. t-Butyl diphosphate, inorganic pyrophosphate, adenosine 5'-[alpha,beta-methylene]triphosphate and adenosine 5'-[beta,gamma-methylene]triphosphate are competitive inhibitors of the hydrolysis of ATP and ADP.

Effects of protein-modifying reagents on an isoenzyme of potato apyrase.

Treatment of an isoenzyme of potato apyrase of high adenosine triphosphatase/adenosine diphosphatase (ATPase/ADPase) ratio with iodine, N-acetylimidazole or tetranitromethane inactivates the ATPase activity of this enzyme faster than its ADPase activity. There was protection by substrates with the two last-named substances. This and the appearance of nitrotyrosine suggests the participation of tyrosyl residues in both enzymic activities of potato apyrase. The participation of thiol groups is excluded by the insensitivity of apyrase to p-chloromercuribenzoate. Also, 2-hydroxy-5-nitrobenzyl bromide or carboxymethylation produce the same rate of inactivation of ATPase and ADPase activities. Substrates protect both activities from inactivation. Hydrogen peroxide and photo-oxidation inactivate ATPase activity faster than ADPase activity. There is no protection by substrates. Analysis of pH effects on V(max.) and K(m) suggest different pK values for the amino acid residues at the ATP and ADP sites.