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)

Analyses were done on a human type-IV cyclic AMP (cAMP) phosphodiesterase (hPDE-IVA-h6.1) expressed in an engineered strain of Saccharomyces cerevisiae. This strain (YMS6) expressed soluble PDE activity, together with an insoluble activity which was not released by re-homogenization, treatment with high-ionic-strength solutions or with the detergent Triton X-100. Pellet and soluble PDE activities were typical of type-IV PDE. They were cAMP-specific, insensitive to the addition of either cGMP (1 microM) or Ca2+/calmodulin, and inhibited by rolipram. Thermostability studies showed both activities to decay as single exponentials, indicating the presence of homogeneous PDE protein species in each fraction. Pellet PDE activity was more thermostable than the soluble enzyme. Mg2+ and Mn2+ dose-dependently increased PDE activity and reversed the inactivating effect of EDTA.h6.1 was engineered to express a C-terminal five-histidine motif (h6.1his5). This allowed purification of the PDE to apparent homogeneity in a simple two-step process involving a rolipram affinity column and a Ni2(+)-chelate column. A single monomeric protein of subunit molecular mass approximately 73 kDa and native molecular mass approximately 74 kDa resulted after a approximately 53000-fold purification. This exhibited a Km for cAMP of 8 microM, a true Vmax. of 0.8 mumol of cAMP hydrolysed/min per mg of PDE protein, a kcat. of 3702 s-1, and a value of the specificity constant kcat/Km of 4.6 x 10(8) M-1.s-1, the last implying a diffusion controlled reaction. Rolipram (Ki 0.4 soluble; 0.7 microM pellet) and 3-isobutyl-1-methylxanthine (Ki 15 soluble; 19 microM pellet) served as simple competitive inhibitors for both soluble and pellet forms of h6.1, respectively.
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PMID:Purification, characterization and analysis of rolipram inhibition of a human type-IVA cyclic AMP-specific phosphodiesterase expressed in yeast. 752 9

1. We have investigated the inhibitory potency of RP 73401, a novel, highly selective and potent inhibitor of cyclic AMP-specific phosphodiesterase (PDE IV), against partially-purified PDE isoenzymes from smooth muscle and the particulate PDE IV from guinea-pig eosinophils. The inhibitory effects of RP 73401 on the generation of superoxide (.O2-), major basic protein (MBP) and eosinophil cationic protein (ECP) from guinea-pig eosinophils have also been studied. 2. RP 73401 potently inhibited partially-purified cyclic AMP-specific phosphodiesterase (PDE IV) from pig aortic smooth muscle (IC50 = 1.2 nM); it was similarly potent against the particulate PDE IV from guinea-pig peritoneal eosinophils (IC50 = 0.7 nM). It displayed at least a 19000 fold selectivity for PDE IV compared to its potencies against other PDE isoenzymes. Rolipram was approximately 2600 fold less potent than RP 73401 against pig aortic smooth muscle PDE IV (IC50 = 3162 nM) and about 250 times less potent against eosinophil PDE IV (IC50 = 186 nM). 3. Solubilization of the eosinophil particulate PDE IV increased the potency of rolipram 10 fold but did not markedly affect the potency of RP 73401. A similar (10 fold) increase in the PDE IV inhibitory potency of rolipram, but not RP 73401, was observed when eosinophil membranes were exposed to vanadate/glutathione complex (V/GSH). 4. Reverse transcription polymerase chain reaction (RT-PCR), using primer pairs designed against specific sequences in four distinct rat PDE IV subtype cDNA clones (PDE IVA-D), showed only mRNA for PDE IVD in guinea-pig eosinophils. PDE IVD was also the predominant subtype expressed in pig aortic smooth muscle cells. 5. RP 73401 (Kiapp = 0.4 nM) was 4 fold more potent than (+/-)-rolipram (Kiapp = 1.7 nM) in displacing[3H]-(+/-)-rolipram from guinea-pig brain membranes.6. In intact eosinophils, RP 73401 potentiated isoprenaline-induced cyclic AMP accumulation(EC50 = 79 nM). RP 73401 also inhibited leukotriene B4-induced generation of *02- (IC50 = 25 nM), and the release of major basic protein (ICo = 115 nM) and eosinophil cationic protein (IC50 = 7 nM). Rolipram was 3-14 times less potent than RP 73401.7. Thus RP 73401 is a very potent and selective PDE IV inhibitor which suppresses eosinophil function suggesting that it may be a useful agent for the treatment of inflammatory diseases such as asthma. The greatly different inhibitory potencies of rolipram against PDE IV from smooth muscle and eosinophils(in contrast to the invariable effects of RP 73401) are unlikely to be attributable to diverse PDE IV subtypes but suggest distinct interactions of the two inhibitors with the enzyme.
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PMID:Suppression of eosinophil function by RP 73401, a potent and selective inhibitor of cyclic AMP-specific phosphodiesterase: comparison with rolipram. 764 82

An antiserum was generated against a dodecapeptide whose sequence is found at the C-terminus of a cyclic AMP (cAMP)-specific, type-IVA phosphodiesterase encoded by the rat 'dunc-like' cyclic AMP phosphodiesterase (RD1) cDNA. This antiserum identified a single approximately 73 kDa protein species upon immunoblotting of cerebellum homogenates. This species co-migrated upon SDS/PAGE with a single immunoreactive species observed in COS cells transfected with the cDNA for RD1. Native RD1 in cerebellum was found to be predominantly (approximately 93%) membrane-associated and could be found in isolated synaptosome populations, in particular those enriched in post-synaptic densities. Fractionation of lysed synaptosomes on sucrose density gradients identified RD1 as co-migrating with the plasma membrane marker 5'-nucleotidase. Laser scanning confocal and digital deconvolution immunofluorescence studies done on intact COS cells transfected with RD1 cDNA showed RD1 to be predominantly localized to plasma membranes but also associated with the Golgi apparatus and intracellular vesicles. RD1-specific antisera immunoprecipitated phosphodiesterase activity from solubilized cerebellum membranes. This activity had the characteristics expected of the type-IV cAMP phosphodiesterase RD1 in that it was cAMP specific, exhibited a low Km cAMP of 2.3 microM, high sensitivity to inhibition by 4-[3-(cyclopentoxyl)-4-methoxyphenyl]-2-pyrrolidone (rolipram) (Ki approximately 0.7 microM) and was unaffected by Ca2+/calmodulin and low concentrations of cyclic GMP. The phosphodiesterase activities of RD1 solubilized from both cerebellum and transfected COS cell membranes showed identical first-order thermal denaturation kinetics at 50 degrees C. Native RD1 from cerebellum was shown to be an integral protein in that it was solubilized using the non-ionic detergent Triton X-100 but not by either re-homogenization or high NaCl concentrations. The observation that hydroxylamine was unable to cause the release of RD1 from either cerebellum or COS membranes and that [3H]palmitate was not incorporated into the RD1 protein immunoprecipitated from COS cells transfected with RD1 cDNA, indicated that RD1 was not anchored by N-terminal acylation. The engineered deletion of the 25 residues forming the unique N-terminal domain of RD1 caused both a profound increase in its activity (approximately 2-fold increase in Vmax) and a profound change in intracellular distribution. Thus, immunofluorescence studies identified the N-terminal truncated species as occurring exclusively ion the cytosol of transfected COS cells. The cDNA for RD1 thus appears to encode a native full-length type-IVA phosphodiesterase that is expressed in cerebellum.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Identification and characterization of the type-IVA cyclic AMP-specific phosphodiesterase RD1 as a membrane-bound protein expressed in cerebellum. 770 77

A novel plasmid was generated which allowed the expression of the cytosolic bacterial enzyme chloramphenicol acetyl transferase (CAT) in COS-7 cells. Upon transfection, the majority of the novel CAT activity was found in the cytosol fraction of COS cells. Chimeric molecules were made between N-terminal portions of the type IVA cyclic AMP-specific rat 'dunce-like' phosphodiesterase (RD1) (RNPDE4A1A; rPDE-IVA1) fused to CAT at its N-terminus. Expression in COS-7 cells of chimeras formed from 1-100RD1-CAT and 1-25RD1-CAT now showed CAT activity associated with the membrane fraction. In contrast, a chimera formed from 26-100RD1-CAT showed an identical expression pattern to native CAT, with the major fraction of CAT activity occurring in the cytosol fraction. Membrane-bound CAT activity provided by 1-100RD1-CAT and 1-25RD1-CAT was not released by either high-salt or washing treatments but was solubilized in a dose-dependent fashion by the non-ionic detergent Triton X-100. Subcellular fractionation of COS-7 cells showed that, as with RD1, the membrane-bound activity of the RD1-CAT chimera followed that of the plasma membrane marker 5'-nucleotidase. Plasmids containing chimeric cDNAs were exposed to a coupled transcription-translation system that, in addition to the full-length chimeras, was found to generate a range of N-terminal truncated species due to initiation at different methionine residues. Incubation of the mature protein products formed in this system with a COS cell membrane fraction showed that only those chimeric CAT constructs containing the first 25 amino acids of RD1 became membrane-associated. The unique 25 amino acid N-terminal domain of RD1 contains structural information that can confer membrane association upon an essentially soluble protein.
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PMID:Chimeric constructs show that the unique N-terminal domain of the cyclic AMP phosphodiesterase RD1 (RNPDE4A1A; rPDE-IVA1) can confer membrane association upon the normally cytosolic protein chloramphenicol acetyltransferase. 777 57

Screening a human T lymphocyte cDNA library with a phosphodiesterase (PDE) specific probe resulted in the isolation of two overlapping cDNA clones, h2.2 and h6.1, that encode a type IV, rolipram inhibited cAMP-specific PDE. Clones h2.2 and h6.1 were 1015 bp and 2288 bp in length, respectively, and overlapped for 984 bp with only one nucleotide difference. The h6.1 cDNA was extended at the 5'-end by 1304 bp, with respect to h2.2, and encoded an incomplete ORF (lacking an initiation codon) of 668 amino acids. The merged nucleotide sequence of h6.1/h2.2 exhibited 99.5% homology in the ORF (ten nucleotide changes resulting in six amino acid changes), and 95% homology in the 3'-untranslated region, with the previously reported human PDE-IVA cDNA [Livi G. P., Kmetz P., Mchale M. M., Cieslinski L. B., Sathe G. M., Taylor D. P., Davis R. L., Torphy T. J. and Balcarek J. M. (1990) Mol. Cell Biol. 10, 2678-2686]. The sequence reported for h6.1/h2.2 matched that found for IVA clones isolated from three other human cDNA libraries, a human genomic cosmid clone and pcr amplified products of the exon covering these differences in two individuals. The h6.1 cDNA was engineered to generate a complete ORF by building in the 56 bp, including the initiation codon, present in hPDE-IVA-Livi and missing from the 5'-end of h6.1, producing a cognate ORF encoding a protein of 687 amino acids but differing in five amino acids which lay in or adjacent to the putative catalytic domain. The complete h6.1 ORF was engineered for expression in both Saccharomyces cerevisiae and in COS-1 cells. Integration of a single copy of the engineered ORF of h6.1, under the transcriptional control of a constitutive yeast promoter, at the pep4 locus of a S. cerevisiae strain lacking both yeast PDE genes resulted in functional complementation of the yeast pde-phenotype. Yeast strains with functional PDE were a light creamy white colour, while strains devoid of PDE activity were a dull brown colour. Expression of h6.1 in COS-1 cells led to the production of a typical type IV PDE activity in that cAMP, but not cGMP, served as substrate and its activity was insensitive to either Ca2+/CaM or cGMP but was inhibited by low concentrations of rolipram.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Molecular cloning and expression, in both COS-1 cells and S. cerevisiae, of a human cytosolic type-IVA, cyclic AMP specific phosphodiesterase (hPDE-IVA-h6.1). 788 6