A case of serendipity*

An account is given of how a sensitive bioassay system for measurement of the neurotransmitter acetylcholine serendipitously led to the identification of adenosine triphosphate (ATP) released in vitro from active skeletal muscle. Subsequent application of the identification procedures to exercising human muscle in vivo, cardiac muscle cells in vitro, and human erythrocytes exposed to hypoxia gave rise to the general concept of ATP as a molecule that could influence cell function from the extracellular direction. Mechanisms of ATP release from cells in terms of “trigger” events such as mechanical distortion of the membrane, depolarization of the membrane, and exposure to hypoxia are discussed. Potential therapeutic uses of extracellular ATP in cancer therapy, radiation therapy, and a possible influence upon aging are discussed. Possible roles (distant and local) of extracellular ATP released from muscle during whole body exercise are discussed

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

Síntesis de análogos de ATP unidos a Sepharosa en la purificación de una pirofosfohidrolasa.

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ATP fosfonato y azul de cibacron unidos a poliextrano para purificar pirofosfohidrolasas

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

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 pI, but only slightly in Mr. Both enzymes immunocross-react with antiapyrase Desirée, which is important for isoenzyme detection by the immunowestern blot. This is t

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

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

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

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 Mr 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. © 1987

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

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 Desirée. 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 i