Extraction, purification, identification and metabolism of 3'',5''-cyclic UMP, 3'',5''-cyclic IMP and 3'',5''-cyclic dTMP from rat tissues.

The large-scale extraction and partial purification of endogenous 3',5'-cyclic UMP, 3',5'-cyclic IMP and 3',5'-cyclic dTMP are described. Rat liver, kidney, heart, spleen and lung tissues were subjected to a sequential purification procedure involving freeze-clamping, perchlorate extraction, alumina and Sephadex ion-exchange chromatography and preparative electrophoresis. The samples thus obtained co-chromatographed with authentic cyclic UMP, cyclic IMP and cyclic dTMP on t.l.c. and h.p.l.c. and the u.v. spectra of the extracted samples were identical with those of the standards. Fast atom bombardment of the three cyclic nucleotide standards yielded mass spectra containing a molecular protonated ion in each case; mass-analysed ion kinetic-energy spectrometry ('m.i.k.e.s') of these ions produced a spectrum unique to the parent cyclic nucleotide. The extracted putative cyclic UMP, cyclic IMP and cyclic dTMP each produced a m.i.k.e.s. identical with that obtained with the corresponding cyclic nucleotide standard. Rat liver, heart, kidney, brain, intestine, spleen, testis and lung protein preparations were each found capable of the synthesis of cyclic UMP, cyclic IMP and cyclic dTMP from the corresponding nucleoside triphosphate, of the hydrolysis of these cyclic nucleotides and of their binding, with the exception that cyclic dTMP was not synthesized by the kidney preparation.     

Absolute configuration of Rp-uridine 3'',5''-cyclic phosphorothioate.

Triethylammonium uridine-3',5'-cyclic phosphorothioate crystallizes in space group P2(1)2(1)2(1), a = 7.177(1), b = 13.155(6), c = 21.114(7) A, C15H26N3O7PS, MW 423.4, Z = 4, dx = 1.41g/cm3. The crystal structure was solved by direct methods on the basis of 1493 counter X-ray diffraction data (CuK alpha) and refined to R = 5.1%. The configuration of the thiophosphate group is Rp; conformational parameters are: glycosyl torsion angle anti, -151.9(5) degrees, sugar pucker C(3')-endo with P = 27.3 degrees, vmax = 45.5 degrees, six-membered cycle in chair form. The bond distances in the non-esterified P-S and P-O suggest that the negative charge is distributed between the groups. As illustrated in this and other studies, P-O has a much higher affinity for hydrogen bonds than P-S, indicated here by interactions with triethyl-ammonium N-H and O(2')-H as donors. One additional hydrogen bond N(3)-H---0(4) ties the bases which form a ribbon-like structure. 0(2) and S are not engaged in hydrogen bonds. The triethylammonium ion is two-fold disordered.     

Polyamines and cellular adenosine 3'' :5''-cyclic monophosphate.

The effect of polyamines on the cellular concentrations of cyclic AMP was studied. It was shown that 1 microM-spermine caused a decrease in cyclic AMP in chick-embryo heart cells, chick-embryo fibroblasts, neuroblastoma, glioma and neuroblastoma-glioma hybrid cells, grown in culture. A similar decrease was observed when polyamines were added to cells in the presence of a phosphodiesterase inhibitor or after stimulating the cells with various hormones. Noradrenaline was used in cultures of heart cells, prostaglandin E1 and adenosine for neuroblastoma and neuroblastoma-glioma hybrids, whereas isoproterenol was used for the stimulation of glioma cells. Polyamines at higher concentrations were either without effect or caused a slight increase in cyclic AMP. Spermidine (10 microM) also caused a decrease in cellular cyclic AMP, as did 0.1 microM-putrescine. It is suggested that the effect of polyamines on cellular cyclic AMP may be explained by the effect of these polycations on the activity of cellular phosphodiesterase.     

Acetylation and cleavage of purine nucleosides. Synthesis of 6-azauridine, 5-fluorouridine, and 5-methyluridine.

Inosine (I) when acetylated with acetic anhydride in the presence of acetyl chloride in acetic acid solution (the so called "acid acetylation"), affords an acetylated nucleoside III (75%) along with cleavage products of the nucleoside (hypoxanthine, 19%). The reaction of I with acetyl chloride (7 days) results in the formation of hypoxanthine (95%) and triacetylribofuranosyl chloride (IV) isolated in the form of tetraacetylribofuranose (47%). The acetylated purine nucleoside affords a similar result by reaction with acetyl chloride or acetyl bromide. 2'-Deoxyuridine gives a diacetyl derivative (80%) by reaction with acetyl bromide. On treatment with acetyl bromide, the nucleoside bond of purine nucleosides is quantitatively cleavaged (4 h, 20 degrees C) with the formation of tri-O-acetyl-D-ribofuranosyl bromide (X). The halogenose X affords pure beta-anomers, namely, 1,2,3,5-tetra-O-acetyl-beta-D-ribofuranose (75%), the triacetyl derivatives of 5-methyluridine (XVIIa; 75%, referred to guanosine), 6-azauridine (XVIII; 71%), and 5-fluorouridine (XIXa; 75%).     

Adenosine 3'':5''-cyclic monophosphate in higher plants: Isolation and characterization of adenosine 3'':5''-cyclic monophosphate from Kalanchoe and Agave.

The activity of the putative ketogenic beta-oxoacyl-CoA thiolase from mitochondria of rat liver increases with starvation, during neonatal life, and after the injection of glucagon. These changes are associated with alteration in ketonaemia. The changes in activities of this species of thiolase are not associated with significant alterations in the apparent affinity (Km) for the ketogenic substrate, acetyl-CoA. These results support a role for thiolase in the regulation of ketogenesis.     

Turnover of adenosine 3'':5''-cyclic monophosphate in chicken erythrocytes.

Turnover of cyclic AMP was studied in intact chicken erythrocytes. Production of cyclic AMP was stimulated by adrenaline and then blocked by propranolol. The decline in the cyclic AMP concentration under these conditions is solely due to its intracellular degradation, whereas efflux of the nucleotide, although existing in these cells, does not contribute significantly to the change in its concentration. Intracellular degradation of cyclic AMP follows a first-order kinetics with a half-life of about 6 min. Similar half-lives were obtained at widely different adrenaline concentrations or when the ration of propranolol to adrenaline was varied by 25-fold. Theoretical equations were applied to calculate the rates of cyclic AMP synthesis and degradation in the intact cells under different experimental conditions. Maximal adrenaline concentrations raise the rate of cyclic AMP synthesis and its steady-state concentration by about 10-fold. The addition of caffeine causes a further 33% increase in intracellular concentration of the nucleotide, which is in good agreement with the theoretical increase computed from its slowed-down degradation.     

Adenosine 3'',5''-cyclic monophosphate and morphology in Neurospora crassa: drug-induced alterations.

Grown in liquid culture in the presence of a variety of structurally unrelated drugs, mycelia of wild-type Neurospora assume a colonial or semicolonial growth habit similar to that of known morphological mutants. Drugs that produce these morphological changes include atropine, theophylline, histamine, and several of the quinoline-containing antimalarials. Each of these drugs decrease the endogenous adenosine 3',5'-cyclic monophosphate (cAMP) concentration of mycelia as a result of their effect on the activity of adenyl cyclase, the cAMP-dependent phosphodiesterase, or both. The evidence indicates a relationship between the degree of morphological abnormality, the degree to which intracellular cAMP is reduced, and the action of the drugs on the adenyl cyclase and phosphodiesterase.     

Adenosine 3'':5''-cyclic monophosphate-binding proteins in bovine and rat tissues.

1. At least two classes of high-affinity cyclic AMP-binding proteins have been identified: those derived from cyclic AMP-dependent protein kinases (regulatory subunits) and those that bind a wide range of adenine analogues (adenine analogue-binding proteins). 2. In fresh-tissue extracts, regulatory subunits could be further subdivided into 'type I or 'type II' depending on whether they were derived from 'type I' or 'type II' protein kinase [see Corbin et al. (1975) J. Biol. Chem. 250, 218-225]. 3. The adenine analogue-binding protein was detected in crude tissue supernatant fractions of bovine and rat liver. It differed from the regulatory subunit of cyclic AMP-dependent protein kinase in many of its properties. Under the conditions of assay used, the protein accounted for about 45% of the binding of cyclic AMP to bovine liver supernatants. 4. The adenine analogue-binding protein from bovine liver was partially purified by DEAE-cellulose and Sepharose 6B chromatography. It had mol.wt. 185000 and was trypsin-sensitive. As shown by competition and direct binding experiments, it bound adenosine and AMP in addition to cyclic AMP. At intracellular concentrations of adenine nucleotides, binding of cyclic AMP was essentially completely inhibited in vitro. Adenosine binding was inhibited by only 30% under similar conditions. 5. Rat tissues were examined for the presence of the adenine analogue-binding protein, and, of those examined (adipose tissue, heart, brain, testis, kidney and liver), significant amounts were only found in the liver. The possible physiological role of the adenine analogue-binding protein is discussed. 6. Because the adenine analogue-binding protein or other cyclic AMP-binding proteins in tissues may be products of partial proteolysis of the regulatory subunit of cyclic AMP-dependent protein kinase, the effects of trypsin and aging on partially purified protein kinase and its regulatory subunit from bovine liver were investigated. In all studies, the effects of trypsin and aging were similar. 7. In fresh preparations, the cyclic AMP-dependent protein kinase had mol.wt. 150000. Trypsin treatment converted it into a form of mol.wt 79500. 8. The regulatory subunit of the protein kinase had mol.wt. 87000. It would reassociate with and inhibit the catalytic subunit of the enzyme. Trypsin treatment of the regulatory subunit produced a species of mol.wt. 35500 which bound cyclic AMP but did not reassociate with the catalytic subunit. Trypsin treatment of the protein kinase and dissociation of the product by cyclic AMP produced a regulatory subunit of mol.wt. 46500 which reassociated with the catalytic subunit. 9. These results may be explained by at least two trypsin-sensitive sites on the regulatory subunit. A model for the effects of trypsin is described.     

Extraction, purification and identification of cytidine 3'',5''-cyclic monophosphate from rat tissues.

The large-scale extraction and purification to homogeneity of cyclic CMP and its unequivocal identification are described. Rat liver, kidney, heart, spleen and lung tissues were subjected to a sequential purification procedure involving freeze-clamping, perchlorate extraction, alumina and boronate column chromatography, polyacrylamide-gel column electrophoresis and high-voltage paper electrophoresis. The purified sample co-chromatographed with authentic cyclic CMP on t.l.c. and high-pressure liquid chromatography and was positive in a cyclic CMP radio-immunoassay. The u.v., i.r. and p.m.r. spectra were each essentially identical with those of authentic cyclic CMP. Fast-atom bombardment of authentic cyclic CMP yielded a mass spectrum containing a molecular protonated ion: mass-ion-kinetic-energy scanning of this ion produced a spectrum unique to 3',5'-cyclic CMP. The extracted nucleotide produced an identical mass-ion-kinetic-energy spectrum.     

The modified nucleosides of tRNAs. II. Synthesis of 2''-O-methylcytidylyl (3''-5'') cytidine.

The synthesis of 2'-O-methylcytidylyl (3'-5')cytidine by the triester method using as protecting groups, 2,2,2-trichloroethyl for phosphate hydroxyl group, p-chlorophenyoxyacetyl for 5-hydroxyl group, methoxymethylidene for 2',3'-cis-diol system, and benzoyl for the exo-amino group of cytidine is presented. The obtained product was characterised by UV, electrophoresis, chromatography and an enzymatic digestion.     

Guanosine 3'',5''-cyclic monophosphate and the in vitro physiology of frog photoreceptor membranes

Frog rod outer segments freshly detached from dark-adapted retinas contain approximately 1-2 molecules of guanosine 3',5'-cyclic monophosphate (cyclic GMP) for every 100 molecules of visual pigment present. This cyclic GMP decays to 5'-GMP, and the conversion is accelerated upon illumination of the outer segments. Bleaching one rhodopsin molecule can lead to the hydrolysis of 1,000-2,000 molecules of cyclic GMP within 100-300 ms. The decline in cyclic GMP concentration becomes larger as illumination increases, and varies with the logarithm of light intensity at levels which bleach between 5 X 10(2) and 5 X 10(5) rhodopsin molecules per outer segment-second. Light suppression of plasma membrane permeability, assayed in vitro as light suppression of outer segment swelling in a modified Ringer's solution, occurs over this same range of light intensity. The correlation between cyclic GMP and permeability or swelling is maintained in the presence of two pharmacological perturbations: papaverine, a phosphodiesterase inhibitor, increases both cyclic GMP levels and the dark permeability of the plasma membrane; and beta,gamma-methylene ATP increases the effectiveness of light in suppressing both permeability and cyclic GMP levels.     

2'',3''=Carbonates in the synthesis of uridine 5''-deoxy and 2'',5''-dideoxy derivatives.

Detritylation of 2',3'-O-carbonyl-5'-O-trityluridine (Ia) with ethereal hydrogen chloride affords 2',3'-O-carbonyluridine (Ib; 83%) which is converted by mesylation to the 5'-mesylcarbonate Ic (75%). Reaction of compound, Ic with tetrabutylammonium bromide in DMF affords the 5'-bromo carbonate Id (77%) which is reduced with tributyltin hydride to the 5'-deoxyuridine 2',3'-cyclic carbonate Ie (70%). When heated with imidazole, compound Ie affords the 2,2'-anhydro derivative IIa (76%) which is converted to the 2'-chloro derivative IIIa (88%) on heating with HC1/DMF. The tributyltin hydride reduction of compound IIIa gives 2',5'-dideoxyuridine (IIIb; 68%). When heated with NaHCO3 in DMF, the 5'-bromo carbonate Id affords the anhydro bromo derivative IIb (50%) which is converted to the 2',5'-dichloro derivative IIIc (86%) on heating with HC1/DMF. The tributyltin hydride reduction of compound IIIc affords the 2',5'-dideoxy derivative IIIb (59%). Alkaline hydrolysis of the 2,2'-anhydro derivative IIa affords the arabinosyl derivative IVa which is converted to the diacetyl derivative IVb (34%) by acetylation. When refluxed in water, the 2',3'-cyclic carbonates Ib, Id, and Ie are hydrolysed to the parent nucleosides, namely, uridine (Va; 81%), 5'-bromo-5'-deoxyuridine (Vb; 78%), and 5'-deoxyuridine (Vc; 83%). Hydrolysis of carbonates Ib and Ie is accompanied by the formation of the 2,2'-anhydro derivatives IIc (10%) and IIa (5%) as by-products.     

Spectrophotometric assay, solubilization and purification of brain 2'':3''-cyclic nucleotide 3''-phosphodiesterase.

1. A spectrophotometric assay of 2':3'-cyclic nucleotide 3'-phosphodiesterase (EC 3.1.4.37) based on the use of an acid-base indicator and a buffer having identical pKa values is described. The assay is simple and rapid; it was particularly convenient for monitoring the enzyme activity at various stages of purification. 2. Several proteinases were examined for their ability to solubilize 2':3'-cyclic nucleotide 3'-phosphodiesterase from delipidated brain white matter. Trypsin (EC 3.4.21.4) and elastase (EC 3.4.21.11) appeared to be more effective than the other proteinases examined. Trypsin, however, caused inactivation; elastase was therefore chosen to solubilize 2':3'-cyclic nucleotide 3'-phosphodiesterase. When a partially purified preparation of 2':3'-cyclic nucleotide 3'-phosphodiesterase was treated with elastase, 2':3'-cyclic nucleotide 3'-phosphodiesterase was solubilized nearly quantitatively. Elastatinal, a specific inhibitor of elastase, specifically inhibited the solubilization with elastase. 3. 2':3'-cyclic nucleotide 3'-phosphodiesterase was purified from bovine brain white matter by: (i) delipidation; (ii) solubilization with hexadecyltrimethylammonium bromide; (iii) gel chromatography on Sepharose; (iv) ethanol precipitation and resolubilization by digestion with elastase; (v) chromatography on DEAE-Sephadex; (vi) affinity chromatography on 8-(6-aminohexyl)amino-2'-AMP-Sepharose. 4. The purified enzyme migrated as a single protein band on polyacrylamide-gel electrophoresis at pH 4.3 and on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis; the estimated mol.wt. in the latter electrophoresis was 27000-31000. Gel filtration of the purified enzyme through Sephadex G-150 indicated a mol.wt. of 31000. Therefore the purified enzyme is a monomer protein with a mol.wt. of approx. 30000.     

Isolation, characterization and distribution of adenosine 3'':5''-cyclic monophosphate from Pinus radiata.

Cyclic AMP was extracted in 0.1 M-HCl from tissues of Pinus radiata and purified by gel filtration on Sephadex G-10, and chromatography on Dowex AG1 (X2) and polyethyleneimine-cellulose in two separate solvent systems. Presumptive cyclic AMP from 10kg batches of pine needles was characterized by countercurrent distribution in the presence of cyclic [8-3H]AMP. Statistical analysis of the curves for radioactivity and mass (determined by the Gilman competitive-binding assay) showed that the fit of the curves was highly significant for seven degrees of freedom. The distribution of cyclic AMP within P. radiata and various other plant tissues was determined by the Gilman procedure. The results suggest that there is no relationship between variations in cyclic AMP concentrations and the known function of the tissue in which it was measured.     

Regulation of synthesis of guanosine 3'':5''-cyclic monophosphate in neuroblastoma cells.

The increase in intracellular cyclic GMP concentrations in response to muscarinic-receptor activation in N1E-115 neuroblastoma cells is dependent on extracellular Ca2+ ion. The calcium ionophore A23187 can also evoke an increase in cyclic GMP in the presence of Ca2+ ion. Most (about 85%) of the guanylate cyclase activity of broken-cell preparations is found in the soluble fraction. The soluble enzyme can utilize MnGTP (Km = 55 micrometer), MgGTP (Km = 310 micrometer) and CaGTP (Km greater than 500 micrometer) as substrates. Free GTP is a strong competitive inhibitor (Ki approximately 20 micrometer). The enzyme possesses an allosteric binding site for free metal ions (Ca2+, Mg2+ and Mn2+). The membrane-bound guanylate cyclase is qualitatively similar to the soluble form, but has lower affinity for the metal-GTP substrates. Entry of Ca2+ into cells may increase cyclic GMP concentration by activating guanylate cyclase through an indirect mechanism.