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Heterogeneity of cyclic nucleotide phosphodiesterases in liver endoplasmic reticulum.

ABSTRACT: A microsomal fraction from rat liver was subfractionated into three rough endoplasmic reticulum fractions RIII, RII and RI, together with a smooth endoplasmic reticulum plus Golgi fraction. Cyclic nucleotide phosphodiesterase activity was found in all fractions. Subsequently it was shown that Golgi fractions were essentially devoid of cyclic AMP phosphodiesterase activity and the activity resided in the smooth endoplasmic reticulum fraction. The activity of the endoplasmic reticulum constituted some 20% of the homogenate activity, with the major fraction of this being associated with the RII fraction and the least with the RI fraction. With the exception of the activity of the RI fraction, which was a peripheral enzyme, all of the other enzyme activities were integral, requiring detergent or repeated freeze-thawing to effect solubilization. All of the activities appeared to be exposed at the external surface of the endoplasmic reticulum, as they were inactivated by trypsin under conditions where glucose 6-phosphatase was not. All of these activities displayed distinct sensitivities to both thermal and trypsin inactivation, yielding activity decays consistent with a single enzyme species being present in each case. The freeze-thaw-solubilized enzymes yielded single symmetrical peaks on sucrose-density-gradient centrifugation and polyacrylamide-gel electrophoresis. The sedimentation coefficients for the enzymes in the smooth-endoplasmic-reticulum-plus-Golgi, RIII, RII and RI fractions were 3.2S, 4.2S, 4.5S and 4.5S respectively. Whereas the activity in the smooth-endoplasmic-reticulum-plus-Golgi fraction exhibited normal Michaelis kinetics, those in the other fractions yielded kinetics indicative of apparent negative co-operativity. All of the enzymes exhibited low Km values towards cyclic AMP. The enzymes did not appear to be regulated by Ca2+ or calmodulin. ZnCl2 was found to be a potent non-competitive inhibitor of the enzyme in all fractions. NaF was a weak non-competitive inhibitor. The bilayer fluidizing agent benzyl alcohol exerted dissimilar effects on the enzyme activities. It is concluded that the endoplasmic reticulum displays lateral heterogeneity, with single, rather distinct, cyclic AMP phosphodiesterases being found in the different fractions.

SUBMITTER: Cercek B

PROVIDER: S-EPMC1152094 | biostudies-other | 1983 Jul

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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Heterogeneity of cyclic nucleotide phosphodiesterases in liver endoplasmic reticulum.

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