The adnosine 3',5'-monophosphate and guanosine 3',5'-monophosphate phosphodiesterases from human lung tissue.

Human lung tissue contains phosphodiesterase enzymes capable of hydrolyzing both adenosine 3′,5′-monophosphate (cyclic AMP) and guanosine 3′,5′-monophosphate (cyclic GMP). The cyclic AMP enzyme exhibits three distinct binding affinities for its substrate (apparent Km = 0.4μM, 3μM, and 40μM) while the cyclic GMP enzyme reveals only two affinities (Km = 5μM and 40μM). The pH optima for the cyclic AMP and cyclic GMP phosphodiesterase are similar (pH 7.6–7.8). Both are inhibited by known inhibitors of phosphodiesterase activity (aminophylline, caffeine, and 3-isobutyl-1-methylxanthine). The divalent cations Mg²⁺ and Mn²⁺ stimulate cyclic AMP phosphodiesterase activity (in the absence of Mg²⁺) while Ca²⁺, Ni²⁺, and Cu²⁺ inhibit the enzyme. Histamine and imidazole slightly stimulate cyclic AMP hydrolytic activity. Thus, human lung tissue does contain multiple forms of both the cyclic AMP and cyclic GMP phosphodiesterase which are influenced by a variety of effectors.     

Comparison of cyclic nucleotide binding to adenosine 3',5'-monophosphate and guanosine 3',5'-monophosphate-dependent protein kinases.

Binding sites for [3H]cAMP on purified regulatory dimers of type II A-kinase (II-R2) are independent as assessed by equilibrium binding (KD = 6 +/- 1.3 nM at pH 7.2, 25 degrees; nH = 1.0) and by the lack of effect of unlabeled cAMP on dissociation rate (kd = 1.0 X 10(-3) sec -1 at pH 7.2, 25 degrees). In contrast, binding sites for [3H]cGMP on purified G-kinase displayed positively cooperative interactions in both equilibrium and dissociation assays with convex upward Scatchard plots, an nH of 1.6 and a dissociation rate (kd = 6.2 X 10(-3) sec-1 at pH 6.8, 0 degree) which was slowed by excess unlabeled cGMP (kd = 1.13 X 10(-3) sec-1 at pH 6.8, degree). Calculated transition state free energies of dissociation revealed that dissociation of nucleotide from G-kinase in the presence of cGMP was restrained by an energy barrier (20.8 kcal.mol-1) similar to that of II-R2 (20.9 kcal.mol-1), whereas dissociation from G-kinase without excess nucleotide occurred more easily (18.9 kcal.mol-1).     

An equilibrium study of the cooperative binding of adenosine cyclic 3',5'-monophosphate and guanosine cyclic 3',5'-monophosphate to the adenosine cyclic 3',5'-monophosphate receptor protein from Escherichia coli

The binding of adenosine cyclic 3',5'-monophosphate (cAMP) and guanosine cyclic 3',5'-monophosphate (cGMP) to the adenosine cyclic 3',5'-monophosphate receptor protein (CRP) from Escherichia coli was investigated by equilibrium dialysis at pH 8.0 and 20 degrees C at different ionic strengths (0.05--0.60 M). Both cAMP and cGMP bind to CRP with a negative cooperativity that is progressively changed to positive as the ionic strength is increased. The binding data were analyzed with an interactive model for two identical sites and site/site interactions with the interaction free energy--RT ln alpha, and the intrinsic binding constant K and cooperativity parameter alpha were computed. Double-label experiments showed that cGMP is strictly competitive with cAMP, and its binding parameters K and alpha are not very different from that for cAMP. Since two binding sites exist for each of the cyclic nucleotides in dimeric CRP and no change in the quaternary structure of the protein is observed on binding the ligands, it is proposed that the cooperativity originates in ligand/ligand interactions. When bound to double-stranded deoxyribonucleic acid (dsDNA), CRP binds cAMP more efficiently, and the cooperativity is positive even in conditions of low ionic strength where it is negative for the free protein. By contrast, cGMP binding properties remained unperturbed in dsDNA-bound CRP. Neither the intrinsic binding constant K nor the cooperativity parameter alpha was found to be very sensitive to changes of pH between 6.0 and 8.0 at 0.2 M ionic strength and 20 degrees C. For these conditions, the intrinsic free energy and entropy of binding of cAMP are delta H degree = -1.7 kcal . mol-1 and delta S degree = 15.6 eu, respectively.     

Calcium-dependent cyclic nucleotide phosphodiesterase from brain: comparison of adenosine 3',5'-monophosphate and guanosine 3',5'-monophosphate as substrates.

A Ca²⁺-dependent cyclic nucleotide phosphodiesterase has been partially purified from extracts of porcine brain by column chromatography on Sepharose 6 B containing covalently linked protamine residues, ammonium sulfate salt fractionation, and ECTEOLA-cellulose column chromatography. The resultant preparation contained a single form of cyclic nucleotide phosphodiesterase activity by the criteria of isoelectric focusing, gel filtration chromatography on Sephadex G-200, and electrophoretic migration on polyacrylamide gels. When fully activated by the addition of Ca²⁺ and microgram quantities of a purified Ca²⁺-binding protein (CDR), the phosphodiesterase hydrolyzed both adenosine 3′,5′-monophosphate (cyclic AMP) and guanosine 3′,5′-monophosphate (cyclic GMP), with apparent K_m values of 180 and 8 μm, respectively. Approximately 15% of the total enzymic activity was present in the absence of added CDR and Ca²⁺. This activity exhibited apparent K_m values for the two nucleotides identical to those observed for the maximally activated enzyme. Competitive substrate kinetics and heat destabilization studies demonstrated that both cyclic nucleotides were hydrolyzed by the same phosphodiesterase. The purified enzyme was identical to a Ca²⁺-dependent phosphodiesterase present in crude extract by the criteria of gel filtration chromatography, polyacrylamide-gel electrophoresis, and kinetic behavior.Apparent K_m values of the Ca²⁺-dependent phosphodiesterase for cyclic AMP and cyclic GMP were lowered more than 20-fold as CDR quantities in the assay were increased to microgram amounts, whereas the respective maximal velocities remained constant. The apparent K_m for Mg²⁺ was lowered more than 50-fold as CDR was increased to microgram amounts. Half-maximal activation of the phosphodiesterase occurred with lower amounts of CDR as a function of either increasing degrees of substrate saturation or increasing concentrations of Mg²⁺. At low cyclic nucleotide substrate concentrations i.e., 2.5 μm, cyclic GMP was hydrolyzed at a fourfold greater velocity than cyclic AMP. At high substrate concentrations (millimolar range) cyclic AMP was hydrolyzed at a threefold greater rate than cyclic GMP.     

Effect of adenosine 3':5'-monophosphate and guanosine 3':5'-monophosphate on RNA release from isolated nuclei.

The addition of physiological concentrations of either cAMP or cGMP stimulated the release of RNA from isolated prelabeled rat liver nuclei to a fortified cytosol in a cell-free system. The released RNA was shown to be primarily mRNA by its binding to oligo(dT)-cellulose and its sedimentation profile. Treatment of rats with cAMP or cGMP 30 min prior to the preparation of cyclic nucleotides on the cell-free system. Cyclic nucleotides stimulation of RNA release occurred in systems prepared from resting rat liver, Novikoff hepatoma, and Morris hepatoma 5123D, but not the 18-h regenerating liver. The response of the cell-free system to added cyclic nucleotides reflected the in vivo concentration of these substances in the tissues from which the system was prepared. Those with high in vivo levels were not stimulated while those with lower levels did respond to added cyclic nucleotides. Neither cAMP nor cGMP had an appreciable effect on rRNA release.     

Self-phosphorylation of cyclic guanosine 3':5'-monophosphate-dependent protein kinase from bovine lung. Effect of cyclic adenosine 3':5'-monophosphate, cyclic guanosine 3':5'-monophosphate and histone.

Incubation of purified cyclic guanosine 3':5'-monophospate-dependent protein kinase with [gamma-32P]ATP and Mg2+ led to formation of one 32P-labeled protein, Mr = 75,000, which corresponded to the single protein band detected after polyacrylamide gel electrophoresis in sodium dodecyl sulfate. When electrophoresis was performed without detergent, the labeled protein coincided with the position of cGMP-dependent protein kinase activity. Phosphorylation was enhanced severalfold by either histone or cAMP and was inhibited by the addition of cGMP. Low concentrations of cGMP blocked the stimulatory effects of cAMP or histone (or both). Since neither cAMP-dependent protein kinase nor cGMP-dependent phosphoprotein phosphatase activities were detected in the purified enzyme, we concluded that the cGMP-dependent protein kinase is a substrate for its own phosphotransferase activity and that other protein substrates (histone) and cyclic nucleotides modulate the process of self-phosphorylation.     

Bidirectional regulation of lysosomal enzyme secretion and phagocytosis in human neutrophils by guanosine 3',5'-monophosphate and adenosine 3',5'-monophosphate.

The biologic roles of guanosine 3',5'-monophosphate (cyclic GMP) and adenosine 3',5'-monophosphate (cyclic AMP) in the secretion of lysosomal enzymes from, and in phagocytosis by, human neurtrophils were studied. Contact between neurophils and particulate immunologic reactants results in both phagocytosis of the particles and secretion of lysosomal enzymes. These cellular events are accompanied by the accumulation of cyclic GMP and require the presence of extracellular caclium. Acetylcholine, pilocarpine, and cyclic GMP enhance, whereas epinephrine, cyclic AMP, and/or dibutyryl cyclic AMP inhibit, both phagocytosis and lysosomal enzyme secretion. The stimulatory action of cholinergic agents and the inhibitory action of epinephrine are accompanied by the accumulation of cyclic GMP and cyclic AMP, respectively, in human neutrophils. The data suggest that cyclic GMP mediates whereas cyclic AMP inhibits the major functions of human neutrophils. Moreover, by virtue of their effects of cyclic nucleotide accumulation, autonomic neurohormones are capable of modulating human neutrophil function.     

Compartmentalization of adenosine 3':5'-monophosphate and adenosine 3':5'-monophosphate-dependent protein kinase in heart tissue.

In rabbit heart homogenates about 50% of the cAMP-dependent protein kinase activity was associated with the low speed particulate fraction. In homogenates of rat or beef heart this fraction represented approximately 30% of the activity. The percentage of the enzyme in the particulate fraction was not appreciably affected either by preparing more dilute homogenates or by aging homogenates for up to 2 h before centrifugation. The particulate enzyme was not solubilized at physiological ionic strength or by the presence of exogenous proteins during homogenization. However, the holoenzyme or regulatory subunit could be solubilized either by Triton X-100, high pH, or trypsin treatment. In hearts of all species studied, the particulate-bound protein kinase was mainly or entirely the type II isozyme, suggesting isozyme compartmentalization. In rabbit hearts perfused in the absence of hormones and homogenized in the presence of 0.25 M NaCl, at least 50% of the cAMP in homogenates was associated with the particulate fraction. Omitting NaCl reduced the amount of particulate-bound cAMP. Most of the particulate-bound cAMP was probably associated with the regulatory subunit in this fraction since approximately 70% of the bound nucleotide was solubilized by addition of homogeneous catalytic subunit to the particulate fraction. The amount of cAMP in the particulate fraction (0.16 nmol/g of tissue) was approximately one-half the amount of the regulatory subunit monomer (0.31 nmol/g of tissue) in this fraction. The calculated amount of catalytic subunit in the particulate fraction was 0.18 nmol/g of tissue. Either epinephrine alone or epinephrine plus 1-methyl-3-isobutylxanthine increased the cAMP content of the particulate and supernatant fractions. The cAMP level was increased more in the supernatant fraction, possibly because the cAMP level became saturating for the regulatory subunit in the particulate fraction. The increase in cAMP was associated with translocation of a large percentage of the catalytic subunit activity from the particulate to the supernatant fraction. The distribution of the regulatory subunit of the enzyme was not significantly affected by this treatment. The catalytic subunit translocation could be mimicked by addition of cAMP to homogenates before centrifugation. The data suggest that the regulatory subunit of the protein kinase, at least that of isozyme II, is bound to particulate material, and theactive catalytic subunit is released by formation of the regulatory subunit-cAMP complex when the tissue cAMP concentration is elevated. A model for compartmentalized hormonal control is presented.