Characterization of a rat liver cyclic GMP-activated phosphodiesterase by chromatography on hexyl-agarose. Inhibition of phosphodiesterase activity by hexyl-agarose (original) (raw)
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FEBS Letters, 1983
The high affinity (low Km) cyclic GMP phosphodiesterase (PDE) is activated by GTP, while the cyclic AMP PDE is not. GTP and its hydrolysis-resistant analogue, guanylylirnidodiphosphate (GppNHp), display a half-maximal stimulating effect at almost the same concentration (5 x 10e6 M). The GTP stimulating effect is not observed when the socalled cyclic GMP low affinity (high Km) PDE is operative. GTP cooperates with the increase of the substrate concentration on removing the IBMX inhibitory effect. The isolation through a classical chromatographic procedure on a DEAB-cellulose column, of a PDE fraction specific for cyclic GMP, results in the loss of the GTP stimulating effect.
Archives of Biochemistry and Biophysics, 1982
High-affinity cyclic AMP phosphodiesterase purified to homogeneity from dog kidney was studied with respect to its stability, its catalytic and kinetic properties, and its sensitivity to pharmacological agents. The enzyme was shown to rapidly lose activity upon dilution to low protein concentrations in aqueous media, but this activity loss was largely prevented by the presence of bovine serum albumin or ethylene glycol. Similarly, maximum activity required bovine serum albumin to be present during incubation for activity analysis. Enzyme activity required a divalent cation; M%+, Mn2+, and Co2+ each supported activity, but highest activity was obtained with M%+. The temperature optimum ranged from 30 to 45°C and depended on substrate concentration; the E, = 10,600 cal/mol. The pH optimum of the enzyme was broad, with a maximum from pH 8.0 to 9.5. The enzyme exhibits linear Michaelis-Menton kinetics for hydrolysis of cyclic AMP at all substrate concentrations tested and for hydrolysis of cyclic GMP at >20 PM. The K,,, for cyclic AMP hydrolysis was 2 pM, and that for cyclic GMP hydrolysis was 312 PM. The Ki values for the competitive inhibition of hydrolysis of each substrate by the other were similar to their Km values suggesting a single active site. Cyclic AMP hydrolysis was weakly inhibited by cyclic GMP, cyclic IMP, adenine, and adenosine, but was not inhibited by the mono-, di, or trinucleotides of adenosine, guanosine, or inosine. Activity was competitively inhibited with Ki values in the micromolar range by drugs representative of methylxanthines, isoquinolines, pyrazolopyridines, imidazolidinones, triazolopyrimidines, pyridylethylenediamines, phenothiazines, and calcium antagonists. The results are discussed with reference to the similarities and differences between high-and low-affinity phosphodiesterase forms.
Analysis of cyclic nucleotide phosphodiesterase(s) by radioimmunoassay
Archives of Biochemistry and Biophysics, 1982
A high-affinity form of cyclic AMP phosphodiesterase, purified to apparent homogeneity from dog kidney, was labeled with '%I using a solid-state lactoperoxidaseglucose oxidase system and its purity confirmed by acrylamide gel electrophoresis and isoelectric focusing. Sheep anti-cyclic AMP phosphodiesterase immunoglobulin fraction was analyzed for lZI-enzyme binding and covalently bound to agarose A 1.5m for isotopically labeled antigen displacement. Anti-phosphodiesterase antiserum was purified by Sepharose 4B-cAPDE affinity chromatography and used for a radioimmunoassay employing second-antibody precipitation. The specificity of the anti-cyclic AMP phosphodiesterase antibody was established by its use as a covalently bound affinity ligand for cyclic AMP phosphodiesterase purification and analysis of sodium dodecyl sulfate-gel extracts of partially purified and purified dog kidney supernatants. Radioimmunoassay using a monospecific antibody preparation demonstrated the similarity of high-affinity cyclic AMP phosphodiesterase forms of different tissues and species that had been separated by DEAE-cellulose chromatography. Various purified preparations of calmodulin, as well as brain calcineurin, did not cross-react in the high-affinity cyclic AMP phosphodiesterase radioimmunoassay. However, higher molecular weight cyclic GMP/lower affinity cyclic AMP phosphodiesterase enzyme forms, partially purified by anion-exchange chromatography, gel filtration, and Cibacron blue adsorption, were shown to cross-react in the high-affinity CAMP phosphodiesterase radioimmunoassay. These studies suggest immunological similarities between the major forms of this enzyme system and the possibility of higher molecular weight complexes containing both cyclic GMP and cyclic AMP hydrolytic sites.
Structural and functional studies of cyclic GMP phosphodiesterase
Journal of Protein Chemistry, 1989
GTP-binding proteins (e.g., Gs, Gi, Go) play an important part as transmitters in signal transduction chains. These GTP-binding proteins consist of three different subunits (a,/3, Y) and are assumed to exist as heterotrimers in the inactive state. The aim of our experiments was to measure quantitatively dissociation of G-protein subunits after activation with GTP or GTP-analogues using fluorescence energy transfer (FiSrster, 1948). For that purpose, the purified subunits ao and /33 were covalently modified with fluorescein and rhodamine as donor and acceptor, respectively. G0-Protein from bovine brain was purified by the method of Sternweis and Robishaw (1984). Preincubation with [A1F4]-made it possible to separate a-from/33-subunits. The purified a-subunit was then labeled with fluoresceinisothiocyanate while the/33-subunit was labeled with the SH-specific tetramethylrhodaminema-leimid. The molar ratio of dye to protein was 1 : 1 for ao and 3 : 1 for/33. The activity of the subunits was retained following modification, as proven by functional tests (Northup et al., 1982, 1983; Morishita et al., 1988), and was like that measured with unlabeled components. Fluorescence energy-transfer measurements indicated concentration-dependent interaction of labeled subunits. At present, we are trying to label Gs and the /31-adrenergic receptor in order to study the interaction of these components in the course of hormone-stimulated signal transduction in reconstituted liposomes (Feder et al., 1986).
Biochemical Pharmacology, 1987
Extraction of frozen canine cardiac muscle rendered soluble over 90% of the cyclic AMP phosphodiesterase activity. The residual activity was membrane-bound. Ion exchange chromatography of the soluble activity on DE-52 allowed for the resolution of three distinct cyclic AMP phosphodiesterase fractions termed PDE-I. PDE-II and PDE-III in order of elution from the column bv a linear NaCl gradient. The relative ratio of cyclic AMP phosphodiesterase activity exhibited by thkse three peaks was 1: 0.65 : 0.82 and of cvclic GMP ohosohodiesterase activitv was 1: 0.52: 0.05 for PDE-I. PDE-II and PDE-III respectively. Pi)E-II and PDi-III were further p&ified by re-chromatograph; on DE-52. Fractions PDE-II and PDE-III were thermolabile at 50", decaying as single exponentials with half lives of 180 set and 77 set respectively. All three species exhibited non-linear Lineweaver-Burke plots for the hvdrolysis of cyclic AMP, exhibiting both high and low affinity components. Hydrolysis of cyclic GMP-by all three-components obeyed normal-kinetics, yielding-linea; plots. PdE-I &as a da2*/ calmodulin-activated species which exhibited a low Km for both cyclic AMP and cyclic GMP but hydrolysed cyclic GMP with a higher V
European journal of biochemistry / FEBS, 1989
A cyclic AMP phosphodiesterase form of rat brain cytosol was purified by means of affinity chromatography on an immobilized analog of the specific inhibitor rolipram, followed by an exclusion chromatography step. The resulting preparation presented two protein bands in polyacrylamide gel electrophoresis, both with phosphodiesterase activity. Kinetics of cyclic AMP hydrolysis by the purified enzyme proved of the Michaelis type, with a Km of 3 microM, while hydrolysis of cyclic GMP displayed anomalous negatively cooperative kinetics. At micromolar concentrations, this enzyme from hydrolyzed highly specifically cyclic AMP (50-fold faster than cyclic GMP). Cyclic GMP proved a poor competitor of cyclic AMP hydrolysis (Ki 1.04 mM). The neurotropic compound, rolipram, strongly inhibited the enzyme, in a competitive manner (Ki 0.9 microM). This enzyme displayed a molecular mass of around 44 kDa as determined by exclusion chromatography, but two molecular masses of 42 kDa and 89 kDa were obs...
Journal of Biological Chemistry, 2005
Cyclic diguanylic acid (c-di-GMP) is a global second messenger controlling motility and adhesion in bacterial cells. Synthesis and degradation of c-di-GMP is catalyzed by diguanylate cyclases (DGC) and c-di-GMP-specific phosphodiesterases (PDE), respectively. Whereas the DGC activity has recently been assigned to the widespread GGDEF domain, the enzymatic activity responsible for c-di-GMP cleavage has been associated with proteins containing an EAL domain. Here we show biochemically that CC3396, a GGDEF-EAL composite protein from Caulobacter crescentus is a soluble PDE. The PDE activity, which rapidly converts c-di-GMP into the linear dinucleotide pGpG, is confined to the C-terminal EAL domain of CC3396, depends on the presence of Mg2+ ions, and is strongly inhibited by Ca2+ ions. Remarkably, the associated GGDEF domain, which contains an altered active site motif (GEDEF), lacks detectable DGC activity. Instead, this domain is able to bind GTP and in response activates the PDE activity in the neighboring EAL domain. PDE activation is specific for GTP (K(D) 4 microM) and operates by lowering the K(m) for c-di-GMP of the EAL domain to a physiologically significant level (420 nM). Mutational analysis suggested that the substrate-binding site (A-site) of the GGDEF domain is involved in the GTP-dependent regulatory function, arguing that a catalytically inactive GGDEF domain has retained the ability to bind GTP and in response can activate the neighboring EAL domain. Based on this we propose that the c-di-GMP-specific PDE activity is confined to the EAL domain, that GGDEF domains can either catalyze the formation of c-di-GMP or can serve as regulatory domains, and that c-di-GMP-specific phosphodiesterase activity is coupled to the cellular GTP level in bacteria.
Journal of Neurochemistry, 1981
The enzyme 2':3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) was isolated from bovine brain white matter by a rapid (72 h) procedure. The minimum molecular weight (MW) of the enzyme was approximately 52,500 as estimated by sucrose density gradient analysis. When this isolated enzyme was stimulated with bovine serum albumin (BSA), the peak of activity was shifted to approximately 90,000 MW. Prior treatment by trypsin blocked the expression of the higher MW form of CNPase, but not the BSA activation of the enzyme. If the trypsin digestion was allowed to progress, the MW was gradually lowered to a broad peak sedimenting between 20,000 and 50,000 MW. An apparently soluble form of CNPase found in serum is described. Kinetic and MW comparisons between the serum soluble enzyme and CNPase isolated from bovine brain, as well as an analysis of substrate specificity, were made and it was concluded that the two enzymes were identical.