Ontogenetic changes in levels of phosphodiesterase for adenosine 3':5'-monophosphate and glucosine 3':5'-monophosphate in the lung, brain and heart from guinea pigs.

Changes in tissue levels of the low Km phosphodiesterase for adenosine 3':5'-monophosphate (cyclic AMP) and guanosine 3':5'-monophosphate (cyclc GMP) in the lung, liver, heart and brain from developing guinea pigs were studied. It was found that the contents of the soluble (cytosol) phosphodiesterase for both cyclic AMP and cyclic GMP were higher in the lung from the fetus than from the neonate and adult. The ontogenetic changes seen in the liver were qualitatively similar to thos in the lung with respect to cyclic GMP hydrolysis, while a reversed pattern of change was noted in the brain. The level of cyclic AMP phosphodiesterase was highest in the fetal heart. Throughout the fetal stage, the levels of the enzyme for cyclic GMP hydrolysis were higher than those for cyclic AMP in the lung. At or around birth, a reversal in the relative levels of the two enzymes took place; two days after birth, the level of the enzyme for cyclic AMP was 2-3times higher than thos for cyclic GMP. Kinetic analysis showed that phohphodiesterases from extracts of the lung from all developmental stages of guinea pigs had the same Km (2.6 muM) for cyclic AMP and the same Km (6.6 muM) for cyclic GMP. The relative values of V, based on assays using the same amount of enzyme protein, in decreasing order, were fetus greater than neonate greater than adult. The present findings suggest that metabolism of the two cyclic nucleotides may be closely related to developmental processes of the tissues. Moreover, the actions involving cyclic GMP may be more predominent in the fetal lung and adult brain.     

Accumulation of adenosine 3',5'-monophosphate in rat brain slices: effects of prostaglandins.

Prostaglandin E₁ and E₂ and 15(S)-15-methyl PGE₂ methyl ester stimulate the accumulation of radioactive cyclic AMP in brain slices from Sprague-Dawley rats, labelled during a prior incubation with [¹⁴C] adenine. Prostaglandins A₁ and B₁ have marginal effects and prostaglandin F_1α has no effect. Relatively high concentrations of about 80 μM PGE₁, PGE₂ and 15(S)-15-methyl PGE₂ are required to elicit a maximal 2–5 fold increase in accumulation of cyclic AMP in slices from cerebrum, but significant increases are elicited by 3.5 μM prostaglandin. Similar increases are elicited in slices from neocortex, striatum or midbrain-thalamus-hypothalamus, while lesser increases pertain in slices from cerebellum, medulla-pons or hippocampus. The accumulation of cyclic AMP elicited by PGE₁ in slices from cerebrum was not blocked by naloxone, propranololphentolamine, tetracaine, theophylline, or by nearly equimolar concentrations of either of two prostaglandin antagonists, 7-oxa-13-prostynoic acid and the dibenzoxazepine hydrazide, SC 19220. Morphine potentiated the effects of PGE₁. The combination of 85 μM PGE₁ with either isoproterenol, norepinephrine, adenosine or veratridin did not increase the accumulation of cycli AMP significantly above those elicited by the isoproterenol, norepinephrine, adenosine or veratridine alone. The combined effect of PGE₁ and norepinephrine in the presence of a β-adrenergic antagonist, sotalol, was, however, additive. The results indicate that PGE₁ stimulates cyclic AMP formation in rat brain slices, but that it either has antagonist activity with respect to accumulations of cyclic AMP-elicited by other agents or has no detectable agonist activity when cyclases are maximally stimulated by other agents.     

Effect of glucose on adenosine 3', 5'-monophosphate levels in rat pancreatic islets.

Time course of the changes in insulin release and cyclic AMP levels in isolated rat islets incubated in media containing 5 or 16.7 mM of glucose were followed. The higher glucose concentration caused a slight but significant increase of cyclic AMP levels after 10 min incubation, but not 5 min incubation, whereas the stimulation of insulin release by 16.7 mM of glucose was apparent in both incubation times. Theophylline increased cyclic AMP levels markedly but did not stimulate insulin release when the glucose concentration was 5 mM. A slight augmentation by theophylline of insulin release was observed in the incubation medium containing 16.7 mM glucose. All these findings suggest that the elevation of cyclic AMP in islets may not play a role for the initiation of the insulin release induced by glucose, though it may act to modulate the glucose effect.     

Protamine kinase independent of adenosine 3',5'-monophosphate from rat brain cytosol

A protein kinase was obtained from rat brain cytosol which phosphorylated preferentially protamine and to some extent histone. This enzyme was independent of adenosine 3′,5′-monophosphate (cyclic AMP) and was not identical with the catalytic unit of cyclic AMP-dependent protein kinase. The enzyme and cyclic AMP-dependent protein kinase from this tissue were distinguishable from each other in their kinetic and catalytic properties, and phosphorylated different seryl and threonyl residues of protamine and histone.     

Cyclic adenosine 3':5'-monophosphate and the induction of deoxyribonucleic acid synthesis in liver.

A mixture containing glucagon and thyroid hormone was previously devised that enhances markedly nuclear DNA replication and mitosis in the parenchymal liver cells of the unoperated rat. It is now shown that the glucagon of the stimulatory solution can be completely replaced by a mixture of a butyryl derivative of cyclic adenosine 3':5'-monophosphate and theophylline. Cyclic guanosine 3':5'-monophosphate and its butyryl derivatives and insulin and high levels of glucose are inactive. The inactivity of N2-monobutyryl cyclic guanosine 3':5'-monophosphate cannot be ascribed to rapid breakdown in the animal or to the impenetrability of the liver cell since the coumpound elevates the rate of hepatic amino acid transport and the activity of ornithine decarboxylase. The observation of others (MacManus, J.P., Franks, D.J., Youdale, T. & Braceland, B.M. (1972) Biochem. Biophys. Res. Commun. 49, 1201-1207) that the level of cylcic adenosine 3':5'-monophosphate is raised during most of the prereplicative period after 70% hepatectomy is confirmed. The evidence supports a positive role for adenosine 3':5-monophosphate in regulating DNA synthesis in the liver.     

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.     

Guanosine 3',5'-monophosphate receptor protein: separation from adenosine 3',5'-monophosphate receptor protein.

A specific cGMP receptor protein has been identified and separated from the cAMP receptor protein by chromatography on 8-(6-aminohexyl)-amino-cAMP-Sepharose. Scatchard analysis of cGMP binding indicates a single affinity class of receptor sites with KD = 1.4 × 10^?8 M. The specificity of the cGMP receptor site has been defined by using a number of nucleotides as competitors for cGMP binding. The cGMP receptor protein sediments at 7S in glycerol density gradients.     

Conversion of guanosine 3', 5'-monophosphate to adenosine 3', 5'-monophosphate in frog myocardial tissue.

One of the transformation products of tritiated cyclic GMP in frog heart tissue homogenate was identified with cyclic AMP. The purified product met all criteria tested for: solubility in ZnCO₃, behavior on various ion-exchangers and sensitivity to cyclic nucleotide phosphodiesterase. Our findings may provide insight into the understanding of heart pacemaker activity.     

Separation of pigeon liver apo- and holo- fatty acid synthetases by affinity chromatography.

Apo- and holo-fatty acid synthetases of pigeon liver were separated by affinity gel chromatography under conditions similar to, but not identical to, those used in separating subunits I and II of [¹⁴C]pantetheine-labeled fatty acid synthetase complex [Lornitzo $\text{et al.}$, J. Biol. Chem. $\text{249}$, 1654 (1974)]. When [¹⁴C]pantetheine-labeled fatty acid synthetases were separated, the enzymatically active holo form contained all of the [¹⁴C] label. Incubation of the apo-pigeon liver fatty acid synthetase complex with CoA, ATP and a partially purified pigeon liver soluble enzyme system, from which fatty acid synthetase had been removed, resulted in the formation of holo-enzyme. Activation of apo-fatty acid synthetase could also be achieved by replacing the apo-(4′-phosphopantetheine-less) acyl carrier protein with holo-acyl carrier protein. It is evident, therefore, that the inactive apo-fatty acid synthetase lacks a 4′-phosphopantetheine group.