Activation of the cyclic nucleotide phosphodiesterase from rat heart cytosol by phospholipase C (original) (raw)
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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.
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...
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.
Characterization of a calcium-calmodulin-stimulated cyclic GMP phosphodiesterase from bovine brain
Biochemistry, 1985
A calmodulin-stimulated form of cyclic nucleotide phosphodiesterase from bovine brain has been extensively purified (1000-fold). Its specific activity is approximately 4 pmol min-' (mg of protein)-' when 1 p M c G M P is used as the substrate. This form of calmodulin-sensitive phosphodiesterase activity differs from those purified previously by showing a very low maximum hydrolytic rate for cAMP vs. cGMP. The purification procedure utilizing ammonium sulfate precipitation, ion-exchange chromatography on DEAE-cellulose, gel filtration on Sephacryl S-300, isoelectric focusing, and affinity chromatography on calmodulin-Sepharose and Cibacron blue-agarose results in a protein with greater than 80% purity with 1% yield. Kinetics of cGMP and cAMP hydrolysis are linear with K , values of 5 and 15 pM, respectively. Addition of calcium and calmodulin reduces the apparent K, for cGMP to 2-3 pM and increases the V,,, by 10-fold. c A M P hydrolysis shows a similar increase in V,,, with an apparent doubling of K,. Both substrates show competitive inhibition with K[s close to their relative K , values. Highly purified preparations of the enzyme contain a major protein band of M , 74 000 that best correlates with enzyme activity. Proteins of M , 59 000 and M , 46 000 contaminate some preparations to varying degrees. An apparent molecular weight of 150000 by gel filtration suggests that the enzyme exists as a dimer of M , 74000 subunits. Phosphorylation of the enzyme preparation by CAMP-dependent protein kinase did not alter the kinetic or calmodulin binding properties of the enzyme. Western immunoblot analysis indicated no cross-reactivity between the bovine brain calmodulin-stimulated cGMP phosphodiesterase and the M , 60 000 high-affinity cAMP phosphodiesterase present in most mammalian tissues. 'These studies were supported by USPHS Grant GM 21361/33538. cGMP phosphodiesterase, 9068-52-4; cGMP, Registry No.
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
Particulate cyclic 3', 5'-nucleotide phosphodiesterase and calmodulin of cardiac muscle
International Journal of Biochemistry, 1984
Cyclic AMP and cyclic GMP phosphodiesterase and calmodulin were measured in purified subcellular fractions of cardiac muscle. 2. Phosphodiesterase activity solubilized by sonication of the nuclear fraction yielded a major 6.6 S form which was calcium-sensitive and cyclic GMP-specific. 3. Phosphodiesterase activity occurring in the nuclear fraction could be further enriched by subfractionation on sucrose density gradients in the presence of MgCl,.
Selective inhibition of cyclic AMP and cyclic GMP phosphodiesterases of cardiac nuclear fraction
Biochemical Pharmacology, 1982
Approximately 60% of the total particulate phosphodiesterase activity occurring in cardiac tissue was associated with the nuclear fraction. Cyclic GMP phosphodiesterase activity of the purified cardiac nuclear fraction was selectively inhibited by trifluoperazine (150 = 19 PM) with negligible inhibition (<15%) of cyclic AMP phosphodiesterase activity. Inhibition of cyclic GMP phosphodiesterase by trifluoperazine was calcium-dependent and suppressed by ethylene glycol bis @-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA). The inhibitory response of both phosphodiesterases to papaverine was similar in the presence of calcium. However, in the presence of EGTA, papaverine inhibition of cyclic GMP but not cyclic AMP phosphodiesterase was reduced significantly. Calmodulin (l-5 &ml) readily reversed the inhibition by 25 fl trifluoperazine of membranous cyclic GMP phosphodiesterase, but had no effect on inhibition by papaverine. With imidazolidinone analogues (Ro 7-2956 and Ro 20-1724), EGTA enhanced the inhibition of cyclic GMP phosphodiesterase without significantly altering the inhibition of cyclic AMP phosphodiesterase. Inhibition of cyclic AMP or cyclic GMP phosphodiesterase activity by l-methyl-3_isobutylxanthine, quinidine, or compound SQ 20,009 was not affected appreciably by calcium or EGTA. The selective inhibitory action of certain pharmacological agents on phosphodiesterases of cardiac nuclear fraction and the modulation of the inhibitory response by calcium suggest an intrinsic and predominant association of calmodulin with cyclic GMP phosphodiesterase activity of these membranes.
Classification of Phosphodiesterases and the Therapeutic Effects of their Inhibitors (Review
—Phosphodiesterase (PDE) is an enzyme that catalyses the hydrolysis of phosphodiester bonds. The enzyme is also takes responsibility for the hydrolysis of cyclic 3',5'adenosine monophosphate (cAMP) and 3',5'cyclic guanosine monophosphate (cGMP). The PDE enzymes in mammals are classified into 11 families, namely PDE1-PDE11. The classification is on the basis of amino acid sequences, substrate specificities, regulatory properties, pharmacological properties, tissue distribution. Various PDE of the same family are related with regards to functionality but differs in their specificities for substrates. Some are hydrolases with selective preferences for cAMP (PDE4, 7 and 8), while the selective preference for some others is for cGMP (PDE5, 6 and 9). Some have the ability to hydrolyse both cAMP and cGMP (PDE1, 2, 3, 10 and 11). cAMP, and cGMP both has important roles in the regulation of inotropic mechanisms in the human myocardium. However, cAMP greatly affects other tissues, and different phosphodiesterase isoenzymes are found in many other tissues. Drugs with inhibitory effects on phosphodiesterase (thus reducing the breakdown of cAMP) have a therapeutic action on the heart, lung, and vasculature as well as on platelet function and inflammatory mechanisms. Inhibitors like these are commonly used as "biochemical tools" to study of role which cyclic nucleotides plays in the cell, but they also may be useful to investigate the structural and functional activities of PDE. As therapeutic agents, they can also be utilized in controlling the pathophysiological changes of responses generated by the cyclic nucleotides in the central nervous system (CNS), cardio-vascular, lung, digestive tract and respectively. PDE enzymes are often targets for inhibition by pharmacological processes due to their unique tissue distribution, structural and functional properties and the inflammatory process. The effect of many of these drugs is evident in more than one isoenzyme, and many tissues possess more than one isoenzyme. As a result, phosphodiesterase inhibitors (PDEI) can have a multiplicity of effects. For example, theophylline has effects on the lung, as well as cardiac and vascular effects; amrinone affects cardiac, vascular and platelet functions. The PDE inhibition, change the intracellular response to extra cellular signals by affecting the processes by the the cyclic nucleotides.