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Higher-plant cyclic nucleotide phosphodiesterases. Resolution, partial purification and properties of three phosphodiesterases from potato tuber.

ABSTRACT: 1. Three phosphodiesterases that are capable of hydrolysing 3':5'-cyclic nucleotides were purified from potato tubers. 2. The phosphodiesterases were fractionated by (NH4)2SO4 precipitation and CM-cellulose chromatography. The phosphodiesterases were resolved from each other and further purified by gel filtration in high- and low-ionic-strength conditions. 3. All three enzymes lacked significant nucleotidase activity. 4. Enzymes I and II had mol. wts. 240,000 and 80,000 respectively, determined by gel filtration, whereas enzyme III showed anomalous behaviour on gel filtration, behaving as a high- or low-molecular-weight protein in high- or low-ionic-strength buffers respectively. 5. All enzymes hydrolysed 2':3'-cyclic nucleotides as well as 3':5'-cyclic nucleotides. The enzymes also had nucleotide pyrophosphatase activity, hydrolysing NAD+ and UDP-glucose to various extents. Enzymes I and II hydrolyse cyclic nucleotides at a greater rate than NAD+, whereas enzyme III hydrolyses NAD+ at a much greater rate than cyclic nucleotides. All three enzymes hydrolysed the artificial substrate bis-(p-nitro-phenyl) phosphate. 6. The enzymes do not require the addition of bivalent cations for activity. 7. Both enzymes I and II have optimum activity at pH6 with 3':5'-cyclic AMP and bis-(p-nitrophenyl) phosphate as substrates. The products of 3':5'-cyclic AMP hydrolysis were 3'-AMP and 5'-AMP, the ratio of the two products being different for each enzyme and varying with pH. 8. Theophylline inhibits enzymes I and II slightly, but other methyl xanthines have little effect. Enzymes I and II were competitively inhibited by many nucleotides containing phosphomonoester and phosphodiester bonds, as well as by Pi. 9. The possible significance of these phosphodiesterases in cyclic nucleotide metabolism in higher plants is discussed.

SUBMITTER: Ashton AR

PROVIDER: S-EPMC1165626 | biostudies-other | 1975 Aug

REPOSITORIES: biostudies-other

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Project description:BACKGROUND: Leishmania represent a complex of important human pathogens that belong to the systematic order of the kinetoplastida. They are transmitted between their human and mammalian hosts by different bloodsucking sandfly vectors. In their hosts, the Leishmania undergo several differentiation steps, and their coordination and optimization crucially depend on numerous interactions between the parasites and the physiological environment presented by the fly and human hosts. Little is still known about the signalling networks involved in these functions. In an attempt to better understand the role of cyclic nucleotide signalling in Leishmania differentiation and host-parasite interaction, we here present an initial study on the cyclic nucleotide-specific phosphodiesterases of Leishmania major. RESULTS: This paper presents the identification of three class I cyclic-nucleotide-specific phosphodiesterases (PDEs) from L. major, PDEs whose catalytic domains exhibit considerable sequence conservation with, among other, all eleven human PDE families. In contrast to other protozoa such as Dictyostelium, or fungi such as Saccharomyces cerevisiae, Candida ssp or Neurospora, no genes for class II PDEs were found in the Leishmania genomes. LmjPDEA contains a class I catalytic domain at the C-terminus of the polypeptide, with no other discernible functional domains elsewhere. LmjPDEB1 and LmjPDEB2 are coded for by closely related, tandemly linked genes on chromosome 15. Both PDEs contain two GAF domains in their N-terminal region, and their almost identical catalytic domains are located at the C-terminus of the polypeptide. LmjPDEA, LmjPDEB1 and LmjPDEB2 were further characterized by functional complementation in a PDE-deficient S. cerevisiae strain. All three enzymes conferred complementation, demonstrating that all three can hydrolyze cAMP. Recombinant LmjPDEB1 and LmjPDEB2 were shown to be cAMP-specific, with Km values in the low micromolar range. Several PDE inhibitors were found to be active against these PDEs in vitro, and to inhibit cell proliferation. CONCLUSION: The genome of L. major contains only PDE genes that are predicted to code for class I PDEs, and none for class II PDEs. This is more similar to what is found in higher eukaryotes than it is to the situation in Dictyostelium or the fungi that concomitantly express class I and class II PDEs. Functional complementation demonstrated that LmjPDEA, LmjPDEB1 and LmjPDEB2 are capable of hydrolyzing cAMP. In vitro studies with recombinant LmjPDEB1 and LmjPDEB2 confirmed this, and they demonstrated that both are completely cAMP-specific. Both enzymes are inhibited by several commercially available PDE inhibitors. The observation that these inhibitors also interfere with cell growth in culture indicates that inhibition of the PDEs is fatal for the cell, suggesting an important role of cAMP signalling for the maintenance of cellular integrity and proliferation.

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Higher-plant cyclic nucleotide phosphodiesterases. Resolution, partial purification and properties of three phosphodiesterases from potato tuber.

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