Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.1.4.1 (phosphodiesterase)
 18,767 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The subcellular distribution of PI-PLC beta 1, gamma 1, and delta 1 has been investigated in rat liver by western blot and immunohistochemical analysis with a panel of isoform-specific antibodies. The data obtained in situ on cryo-sectioned tissue indicate that PI-PLC beta 1 is predominantly nuclear, while gamma 1 is largely cytoplasmic and delta 1 is sharply restricted to the cytoplasm. In fractionation experiments, the Western blot analysis indicated that the recovery of the nuclear isoforms beta 1 and gamma 1 was not affected by the removal of the nuclear membrane, and that the two enzymes persisted in nuclear matrix and lamina, obtained after nuclease digestion and extraction with high salt and detergent. The assay of the phosphodiesterase activity in different cell fractions correlates with the observed relative abundance of the enzymes, and specific inhibition with neutralizing anti-beta 1 and -gamma 1 isoforms confirms that these are the enzymes active at the nuclear level. These results demonstrate that in rat liver cells, as in other cell types, different members of the PI-PLC family show a discrete intracellular distribution, and suggest that PI-PLC beta 1 and gamma 1 play a central role in modulating the nuclear phosphoinositide cycle.
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PMID:Identification of PI-PLC beta 1, gamma 1, and delta 1 in rat liver: subcellular distribution and relationship to inositol lipid nuclear signalling. 851 96

A Zn(2+)-glycerophosphocholine cholinephosphodiesterase was purified with a specific activity of 4.6 mumole/min.mg protein from bovine brain membranes by procedures involving PI-PLC solubilization, concanavalin A affinity chromatography, CM-sephadex chromatography and Sephadex G-150 chromatography. Based on molecular weight determination gel chromatography and SDS polyacrylamide gel electrophoresis, the phosphodiesterase activity appears to be a dimeric protein (110 kDa) composed of two subunits with a molecular weight of approximately 54 kDa. The K(m) value for p-nitrophenylphosphocholine and the optimum pH were found to be 16 microM and pH 10.5, respectively. The phosphodiesterase was inhibited by Cu2+, but not the other divalent metal ions. The activity of the apoenzyme was remarkably activated by Co2+ or Zn2+, but not Mn2+ or Mg2+. In addition, the inactivation of the enzyme in glycine buffer was prevented by Mn2+ or Zn2+, but not Co2+ or Mg2. In a separate experiment, comparing properties of the purified and membrane-bound phosphodiesterases, the forms of two enzymes were quite similar except in stability. Both enzymes were more stable at pH 7.4 than pH 5 or 10. However, the membrane-bound enzyme was more stable than the soluble enzyme at all three pHs. These data suggest that the activity of the phosphodiesterase may be stabilized in-vivo.
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PMID:Properties of a Zn(2+)-glycerophosphocholine cholinephosphodiesterase from bovine brain membranes. 892 80

Large enhancements (maximum of 82-fold in terms of enzyme efficiency, Vmax/Km) of bacterial PI-PLC cyclic phosphodiesterase activity were observed in the presence of organic solvents miscible in water (dimethyl sulfoxide, dimethylformamide, and 2-propanol). In general, organic solvents lowered the Km for myo-inositol 1,2-cyclic phosphate (cIP) and increased Vmax substantially. This kinetic effect was similar to that obtained with phosphatidylcholine micelles and bilayers in an aqueous assay system for cyclic inositol phosphate hydrolysis [Zhou, C., et al. (1997) Biochemistry 36, 347-355]. Solvent properties were examined to determine which ones correlated with the activation of PI-PLC toward cIP in each solvent. Activation correlated best with the solvent polarity as measured by ET(30); no significant correlation was observed with solution surface tension, the bulk dielectric constant (epsilon), 1/epsilon (a measure of the strength of charge interactions), or the Hildebrand solubility parameter. The sigmoidal curve of the enzyme activity versus solvent polarity was consistent with the solvent promoting a transition in the enzyme from a low-activity to a high-activity form. Possible candidates for this change, including enzyme dimerization, helix B/loop stabilization, and dehydration of the active site, are discussed.
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PMID:Phosphatidylinositol-specific phospholipase C cyclic phosphodiesterase activity depends on solvent polarity. 921 96

The kinetics of full-length and PH domain truncated cloned PI-PLC delta1 from rat toward soluble substrates [inositol 1, 2-(cyclic)-phosphate (cIP) and glycerophosphoinositol phosphates (GPIPx)] as well as PI in detergent micelles provide the following insights into the mechanism of this enzyme. (i) That cIP is a substrate for the enzyme implies a two-step mechanism for PI hydrolysis [intramolecular phosphotransferase reaction to form cIP followed by cyclic phosphodiesterase activity to form inositol-1-phosphate (I-1-P)]. The dependence of enzyme activity on cIP is sigmoidal, suggesting a transition between less active and more active forms of the enzyme that is affected by substrate. (ii) Interfaces increase the kcat for cIP (but do not affect the cooperativity), and this allosteric activation requires an intact PH domain. (iii) Phosphorylation of the soluble inositol phosphodiesters GPI, GPIP, and GPIP2 enhances PI-PLC delta1 activity by dramatically increasing kcat and decreasing Km. For these phosphodiesters, the substrate saturation curve is no longer sigmoidal but hyperbolic, indicating the phosphorylated substrate can shift the enzyme to the activated form. (iv) Given the kinetic parameters for cIP hydrolysis and the constant ratio of cIP/I-1-P generated during PI hydrolysis, the cIP produced in situ is either released (and not readily rebound since its concentration is well below Km) or attacked by a water molecule for the generation of the acyclic product.
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PMID:Phosphoinositide-specific phospholipase C delta1 activity toward micellar substrates, inositol 1,2-cyclic phosphate, and other water-soluble substrates: a sequential mechanism and allosteric activation. 928 65

We have previously demonstrated that rat liver nuclei contain PI-PLC beta1 and gamma1 in the inner nuclear matrix and lamina associated with specific phosphodiesterase activity (Bertagnolo et al., 1995, Cell Signall. 7, 669-678). Since compensatory hepatic growth is an informative and well characterized model for natural cell proliferation, the presence of specific PI-PLC isoforms and their activity as well as PIP2 recovery were studied at various regenerating times, ranging from 3 to 22 h after partial hepatectomy. Three PI-PLC isoforms (beta1, gamma1, delta1) were examined in control and regenerating liver cells by using specific antibodies. By means of in situ immunocytochemistry and confocal microscopy, PI-PLC beta1 was found mainly in the nucleoplasm and this pattern was not modified after hepatectomy. On the contrary, the nuclear gamma1 isoform showed a marked decrease at 3 and 16 h after hepatectomy, but a clear increase at 22 h covering with bright intensity the whole nucleus. The PI-PLC delta1 isoform, which is exclusively cytoplasmic, was not altered during rat liver regeneration. By western blotting analysis on whole cell homogenates, none of the PI-PLC isozymes under study showed proliferation-linked modification. However, analyses of isolated nuclei identified changes in the nucleus associated PI-PLC gamma1 that paralleled the in situ observation whereas the beta1 isoform was unmodified at all the times examined. Nuclear phosphodiesterase activity on PIP2 was lower at 3 and 16 h, in comparison with sham operated rats, increased at 6 h and reached the highest value after 22 h. Consistently, the recovery of PIP2, obtained in conditions that optimise PIP-kinase activity, showed a marked decrease at 3 h and an increase up to 16 h of liver regeneration, followed by a further decrease at 22 h. These data are consistent with a close relationship between cell proliferation and the nuclear inositide cycle, depending, in rat liver, predominantly on the modulation of the gamma1 isoform of PI-PLC.
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PMID:Changes of nuclear PI-PLC gamma1 during rat liver regeneration. 937 14

Here we investigated whether cADPR and NAADP are synthesized in mitochondria. We found that ADPR-cyclase activity is present in mitochondria. In addition, we describe for the first time synthesis of NAADP in this intracellular organelle. ADPR-cyclase activities (V(MAX)) and NAADP synthesis in mitochondria were about 4-fold lower than that in plasma membranes. Otherwise, ADPR-cyclases in mitochondria and in plasma membranes have similar catalytic properties in terms of apparent K(m) for the substrate NGD and K(i) values for inhibition by dithiotreitol, beta-NAD, and nicotinamide. ADPR-cyclase in plasma membranes and to a lesser degree mitochondrial enzyme, was inhibited by Zn(2+) and Cu(2+); ADPR-cyclase from mitochondria was more stable upon thermal inactivation. CD38 antigen, determined by Western blot, was well-expressed in plasma membranes but was far less so (17-fold less) in mitochondria. The major difference between ADPR-cyclase activity in mitochondria and plasma membranes is that mitochondrial cyclase activity was increased by incubation with nonionic detergents. Conversely, the incubation with phosphatidylinositol-specific phosphodiesterase C (PI-PLC) released ADPR-cyclase activity from plasma membranes, but not from mitochondria. We conclude that ADPR-cyclase in mitochondria and in plasma membranes are both multifunctional enzymes with similar catalytic properties; however, the two ADPR-cyclases differ in the mode of anchoring to the membrane: by glycosylphosphoinositol anchor in plasma membranes and by hydrophobic interactions in mitochondria. In addition, synthesis of NAADP can also be found in intracellular organelles via mitochondria. We propose that independent mitochondrial cADPR and NAADP systems may have an intracrine signaling function that is not dependent on direct input by extracellular hormonal stimuli, but rather responds to changes of intermediary cellular metabolism.
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PMID:Synthesis of NAADP and cADPR in mitochondria. 1054 20

The interactions of PI-PLC with nonsubstrate zwitterionic [phosphatidylcholine (PC)] and anionic [phosphatidylmethanol (PMe), phosphatidylserine, phosphatidylglycerol, and phosphatidic acid] interfaces that affect the catalytic activity of PI-PLC have been examined. PI-PLC binding is strongly coupled to vesicle curvature and is tighter at acidic pH for all of the phospholipids examined. PI-PLC binds to small unilamellar vesicles (SUVs) of anionic lipids with much higher affinity (K(d) is 0.01-0.07 microM for a site consisting of n = 100 +/- 25 lipids when analyzed with a Langmuir adsorption isotherm) than to zwitterionic PC SUVs (K(d) is 5-20 microM and n = 8 +/- 3). The binding to PC surfaces is dominated by hydrophobic interactions, while binding to anionic surfaces is dominated by electrostatic interactions. The contributions of specific cationic side chains and hydrophobic groups at the rim of the alpha beta-barrel to zwitterionic and anionic vesicle binding have been assessed with mutagenesis. The results are used to explain how PC activates the enzyme for both phosphotransferase and cyclic phosphodiesterase activities.
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PMID:Investigating the interfacial binding of bacterial phosphatidylinositol-specific phospholipase C. 1289 24