Marta Mancilla

Properties of two apyrases from Solanum tuberosum

Abstract Two homogeneous isoenzymes of apyrase from Pimpernel and Desiree varieties of Solanum tuberosum were obtained by affinity chromatography on agarose-Cibacron Blue or agarose-ATP-phosphonate columns. Both enzymes split POP bonds of organic and inorganic di- and triphosphates. The ratio of ATPase/ADPase is different for the two apyrases: 10 for Pimpernel and 1 for Desiree. All these activities require bivalent metals. Both isoapyrases have the same MW (49 000) but differ in their p I (8.74 for Pimpernel and 6.69 for Desiree). The optimum pH of hydrolysis of organic di- and triphosphates is 6 (except for Pimpernel ADPase) and 5 for inorganic substrates. Chemical modification of tryptophan, tyrosine, arginine and carboxylic residues decreased all enzymic activities of both enzymes. Protection by substrates and inactivation rates of the individual activities are different for each isoenzyme.

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.

Human placental atp-diphosphohydrolase: Biochemical characterization, regulation and function

1. Kinetic and physico-chemical studies on human placental microsomal fraction confirmed that the ATPase and ADPase activities detected in this fraction correspond to the enzyme ATP-diphosphohydrolase or apyrase (EC 3.6.1.5). These include substrate specificity, and coincident M(r) and pI values of both ATPase-ADPase activities. 2. This enzyme hydrolyses both the free unprotonated and cation-nucleotide complex, the catalytic efficiency for the latter being considerably higher. 3. Microsomal apyrase is insensitive to ouabain and Ap5A. The highly purified enzyme was only inhibited by o-vanadate, DES and slightly by DCCD. 4. Apyrase seems to be a glycoprotein from its interaction with Concanavalin-A. 5. Preliminary studies on the essential amino acid residues suggest the participation of Arg, Lys and His residues, and discard the requirement of -SH, COO-, -OH, and probably also Tyr and Trp. 6. Two kinetic modulatory proteins of apyrase were detected in placental tissue. An activating protein was found in the soluble fraction and an inhibitory protein was loosely bound to the membranes. 7. The proposed in vivo function for apyrase is related to the inhibition of platelet aggregation due to its ADPase activity, which is supported by the direct effect on washed platelets and by its plasma membrane localization.

Identification and subcellular localization of two isoenzymes of apyrase from Solanum tuberosum

Abstract Two forms of ATP-diphosphohydrolase were identified in Solanum tuberosum tuber var. Ultimus. Their hydrolytic activity ratios (ATPase/ADPase) were over 10 for form A and 1 for form B. In the potato tuber homogenate the hydrolytic activity ratio is 3.0, as a result of contributions of the two forms of apyrase. These two apyrases (A and B) were partially separated and the possibility that they are produced as an artifact by partial proteolysis or subunit aggregation was excluded. The subcellular localization of the Ultimus isoapyrases was studied by differential centrifugation. These enzymes are localized in distinct compartments. The high ratio enzyme (A) lies mainly in the soluble fraction, while the low ratio apyrase (B) is principally bound to membranes. The two isoapyrases differ greatly in their kinetic properties and p I , but only slightly in M r . Both enzymes immunocross-react with antiapyrase Desiree, which is important for isoenzyme detection by the immunowestern blot. This is the first example of two isoenzymes of apyrase in the same variety of S. tuberosum .

Purification and characterization of two isoapyrases from Solanum tuberosum var. ultimus

Abstract Two isoenzymes of ATP-diphosphohydrolase (apyrase) were extracted and purified from S. tuberosum var. Ultimus. Their hydrolytic activity ratios (ATPase/ADPase) were 1.0 (apyrase B) and ca 15.0 (apyrase A). They were characterized and compared with apyrases of other varieties of S. tuberosum . Ultimus apyrases, like the other apyrases, did not hydrolyse esteric bonds but only pyrophosphate bonds of organic and inorganic compounds. The optimum pH of all the studied hydrolytic activities of the Ultimus apyrases A and B was 6, except for the ADPase of enzyme A which was 8. Both enzymes require bivalent metal ions for catalytic activity. The activation order for both Ultimus enzymes was: Ca 2+ >Mn 2+ > Mg 2+ >Co 2+ >Zn 2+ . Chemical modification of tryptophan, tyrosine, arginine and carboxylic residues decreased all enzymic activities of both apyrases. The modification of histidine residues reduced the ATPase and ADPase activities of the low ratio apyrase and the ATPase of the high ratio enzyme but did not affect its ADPase activity. Neither of the Ultimus apyrases showed the participation of -SH groups in the active site. The pI values obtained were: 5.45 for apyrase B and 6.56 for apyrase A. The absorption and the fluorescence spectra of the Ultimus isoenzymes were coincident. The amino acid composition of both isoenzymes is very similar, the number of histidines being the most remarkable difference. The amino acid composition of both isoenzymes does not explain the difference of one pH unit in the isoelectric point between the Ultimus enzymes A and B.

The effect of bivalent metal ions on ATPase-ADPase activities of apyrase from Solanum tuberosum

Abstract The purpose of this study was the elucidation of the possible importance of bivalent metal ions in controlling the activity of apyrase (ATP: diphosphohydrolase EC 3.6.1.5) purified from tubers of Solanum tuberosum cv Desiree. Similarities between the Km and Vmm values for ADP and Ca2+ suggest that the true substrate of this enzyme is the metal ion-nueleotide complex. The association constant of the Ca-ADP complex was measured under the same conditions of pH and ionic strength as in the enzymatic assay system in order to calculate the true concentration of this complex. In contrast, [Mn(H2O)6]2+ spin resonance spectroscopy (ESR) showed that apyrase binds this paramagnetic metal ion in the absence of ATP or ADP. The spectrum of [Mn(H2O)6]2+ showed a transition at low field after the addition of apyrase. This result indicates that the binding of the enzyme produces a distortion in the electronic symmetry of [Mn(H2O)6]2+. Apyrase binds other bivalent cations because hysteretic behaviour is observed when the enzyme is preincubated with bivalent metal ions in the absence of nucleotide; this hysteretic behaviour can be interpreted as a displacement of a ligand strongly bound to the enzyme (M2+) by the substrate (Ca-ATP or Ca-ADP). The contents in vivo of Ca, Mg, Mn, Co, Zn, ATP and ADP were determined.

Structural studies of two apyrases from Solanum tuberosum

Abstract The proportion of acid and basic amino acid residues obtained for two homogeneous isoenzymes of apyrase isolated from different clonal varieties of Solanum tuberosum (Pimpernel and Desiree) was essentially the same. This does not agree with the difference in pI values observed. Treatment with asparaginase and glutaminase caused partial inactivation of both enzyme activities in both isoenzymes, and pI values were changed, but not equalized. The differences in pI values of the native isoenzymes may still be attributed to different proportions of glutamine and asparagine in the primary structure. Leucine is the amino-terminal residue in both isoenzymes. Both have two disulphide bridges and one buried sulphydryl group which is not essential for enzyme activity. Differences in pI values should thus be attributed to factors other than amino acid composition.

Apyrase activity and changes in metabolites during germination and tuberization of Solanum tuberosum

Abstract Levels of adenine nucleotides, inorganic phosphate, starch and apyrase were measured during the growth of Solanum tuberosum tuber. The data obtained from potatoes sown in summer were compared with the data already reported for tubers sown in winter. Two major differences were found between the sowings. The first leads us to conclude that tubers from the summer sowing mature 21 days before the winter one, because starch and inorganic phosphate reach constant values earlier. The second difference was found in apyrase activity, which in tubers from the summer sowing reached a maximum after 106 days, while in winter tubers its specific activity and amount increased continously until maturity. The same types of metabolites and apyrase activity were investigated in shoot and mother tuber during germination of potatoes. In the mother tuber the amounts of metabolites and enzyme activity remain almost constant except for starch which showed a tendency to decrease at the end of the period analysed. Apyrase activity in the shoot increased in the first stages studied, decreasing later to levels lower than those in the mother tuber. Apyrase was partially purified from potato shoots and some of its kinetic and physicochemical properties compared with those of the same enzyme isolated from tubers. Potato calluses were grown and, despite their low protein content, apyrase of high specific activity could be detected.

Identification of an apyrase activating protein and of calmodulin in Solanum tuberosum

Abstract Three types of effector proteins have been isolated from a partially purified protein preparation of potato tuber. One of the proteins is a typical calmodulin which has no effect on apyrase. The two other proteins modulate ATPase and ADPase activities; one of them with an activating and the other with an inhibitory effect on apyrase. Calmodulin from potato tuber purified to homogeneity had a M r of 17 500 and an isoelectric point of 4.4. Although the apyrase activating protein is not a pure fraction it differs from calmodulin because unlike this protein it is independent of calcium and does not activate cyclic nucleotide phosphodiesterase from bovine heart. Treatment of the activating protein with tetranitromethane reduces its effect on apyrase, while no change was detected upon treatment with bisdithionitrobenzoic acid.

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.