Mapping adenosine cyclic 3',5'-phosphate binding sites on type I and type II adenosine cyclic 3',5'-phosphate dependent protein kinases using ribose ring and cyclic phosphate ring analogues of adenosine cyclic 3',5'-phosphate

A series of adenosine cyclic 3',5'-phosphate (cAMP) derivatives containing modifications or substitutions in either the 2',3',4', or 5' position or the phosphate were examined for their abilities to activate type I isozymes of cAMP-dependent protein kinase (PK I) from rabbit or porcine skeletal muscle and type II isozymes of cAMP-dependent protein kinase (PK II) from bovine brain and heart. The studies revealed that the activation of both PK I and PK II isozymes requires a 2'-hydroxyl group in the ribo configuration, a 3' oxygen in the ribo configuration, and a charged cyclic phosphate. The two isozymes appeared to differ in those portions of their respective cAMP-binding sites that are adjacent to the 4' position of the ribose ring and the 3' position, 5' position, and phosphate portion of the cyclic phosphate ring.     

Synthesis and conformational properties of O-beta-D-ribofuranosyl-(1'-2')-guanosine and (adenosine)-5'-phosphate

The efficient synthesis of Grp and Arp, minor tRNA components, has been developed.     

Hydrolysis of adenylyl(2'-5')adenosine in chick embryo sera

Similar to adult chick sera in chick embryo sera the enzyme activity hydrolyzing the core of the dimer of A-5A - adenylyl(2' -5')adenosine is present. The enzyme activity possesses the same properties in both sera. Heating of the serum to 60 degrees C for 30 minutes does not affect the enzyme activity. A marked decrease in activity occurs after heating of the serum to above 65 degrees C. Hydrolysis of A2' p5' A in the serum is more efficient under alkaline conditions (pH 9-10) than at physiological pH 7.2-7.4.     

Interaction of "aza" and "deaza" analogs of adenosine cyclic 3', 5'-phosphate with some enzymes of adenosine cyclic 3', 5'-phosphate metabolism: evidence that the lone pair electrons of N-3 are involved in the binding of adenosine cyclic 3', 5'-phosphate to type II adenosine cyclic 3', 5'-phosphate-dependent protein kinase.

Five hetercyclic analogs of adenosine cyclic 3',5'-phosphate (cyclic AMP) were examined for their ability (1) to stimulate type II cyclic AMP-dependent kinases from bovine brain, bovine heart, and rat liver; (2) to serve as substrates for "high Km" (Km for cyclic AMP = 0.13-0.43 mM) cyclic nucleotide phosphodiesterases from bovine heart, rabbit kidney, and rat liver; and (3) to inhibit the hydrolysis of cyclic AMP catalyzed by "low Km" (Km for cAMP = 0.32-1.5 muM) cyclic nucleotide phosphodiesterases from bovine brain, bovine heart, dog heart, rabbit liver, rat brain and rat liver. The analogs all had a purine ring system which had been modified by replacement of a ring carbon with nitrogen or vice versa to yield 2-aza-cAMP (7-amino-4-beta-D-ribofuranosylimidazo [4,5-d] -v-triazine cyclic 3',5'-phosphate); 8-aza-cAMP (7-amino-3-beta-D-ribofuranosyl-v-triazolo-[4,5-d]-pyrimidine cyclic 3',5'-phosphate); 1 deaza-cAMP (7-amino-3-beta-D-ribofuranosylimidazo [4,5-b[pyridine cyclic 3',5'-phosphate); 3-deaza-cAMP (4-amino-1-beta-D-ribofuranosylimidazo[4,5-c]pyridine cyclic 3',5'-phosphate) and 7-deaza-cAMP (7-amino-4-beta-D-ribofuranosylpyrrolo[2,3-d]pyrimidine cyclic 3',5'-phosphate).     

Synthesis of (3'-5'),(2'-5')-linked di- and tri-adenylyl methylphosphonate analogs.

Stereoselectivity was found during the coupling reaction, to form 2',5'- and 3',5'-linked di- and triadenylyl methylphosphonate. The configuration of phosphorus was determined by 1HNMR NOE.     

Synthesis and biological activity of 5'-capped derivatives of 5'-triphosphoadenylyl(2'----5')adenylyl(2'----5')adenosine

The oligonucleotides A5'pp5'A2'p5'A2'p5'A and A5'ppp5'A2'p5'A2'p5'A were prepared by reaction of AMP or ADP, respectively, with the 5'-(phosphoimidazolidate) of A2'p5'A2'p5'A. A5'pppp5'A2'(p5'A)n (n = 1-3) were synthesized by reaction of p5'A2'(p5'A)n (n = 1-3) with adenosine 5'-trimetaphosphate. All structures were confirmed by enzyme digestion and 1H and 31P nuclear magnetic resonance (NMR). The products A5'pppp5'A2'p5'A and A5'pppp5'A2'p5'A2'p5'A were found to be identical with two of the products of the 2-5A synthetase catalyzed reaction of Ap4A with ATP, thus confirming the structural assignments made by earlier investigators. In extracts of mouse L cells programmed with encephalomyocarditis virus RNA, A5'pppp5'A2'p5'A2'p5'A2'p5'A and A5'pppp5'A2'p5'A2'p5'A were equipotent with 2-5A itself as inhibitors of translation. The oligomers A5'ppp5'A2'p5'A2'p5'A and A2'pppp5'A2'p5'A were about 100 times less active than 2-5A, and A5'pp5'A2'p5'A2'p5'A was without translational inhibitory activity. When affinity for the 2-5A-dependent endonuclease was determined (by displacement of 2-5A[32P]pCp from endonuclease), all of the analogues, as well as 2-5A itself, had similar affinities for the endonuclease except for A5'pppp5'A2'p5'A, which was bound approximately 100 times less effectively. Under conditions of the radiobinding assay, A5'pppp5'A2'p5'A2'p5'A was degraded (t1/2 = 2 h) to ATP, ADP, AMP, ppp5'A2'p5'A2'p5'A, and p5'A2'p5'A2'p5'A.(ABSTRACT TRUNCATED AT 250 WORDS)     

Prostaglandin-E2 and cyclic adenosine 3'-5' monophosphate levels in the hypertrophied rat heart.

To assess whether prostaglandin-E2 (PGE2) and cyclic adenosine 3'-5'-monophosphate (cAMP) are involved in the cardiac response to chronic pressure overload, we measured by specific radioimmunoassay method the cardiac tissue and plasma concentrations of PGE2 and cAMP in an animal model of left ventricular hypertrophy. The cardiac hypertrophy was accompanied by a significant increase in PGE2 content, and a significant decrease in cAMP content, in the heart. In addition, we found elevated PGE2 and cAMP levels in arterial plasma samples from the rats with hypertrophied hearts compared to normal rats. These findings suggest a link between cardiac and vascular PGE2 and cAMP generation and the hemodynamic stresses of advanced cardiac overload.     

Synthesis and use of 3'-(azidoiodosalicyl) derivatives of 2', 5'-dideoxyadenosine as photoaffinity ligands for adenylyl cyclase

3'-[(4-Azidosalicyl)glycyl]-2',5'-dideoxyadenosine (1), 3'- [(4-azidosalicyl)-gamma-aminobutyryl]-2',5'-dideoxyadenosine (2), and the (125)I-labeled mono- and diiodinated analogs of 1 were synthesized and tested as photoaffinity probes for adenylyl cyclases. Kinetics for inhibition of purified type I enzyme by 1 was noncompetitive with respect to Mn(*)5'-ATP in the absence of light, implying a P-site mechanism of inhibition. In a UV-dependent manner both 1 and 2 and the iodinated derivative of 1 irreversibly inactivated membrane-bound and purified forms of recombinant type I bovine adenylyl cyclase expressed in ovarian cells of either the fall armyworm (Sf9) or Trichoplasia ni (High Five). Irreversible inactivation was independent of 5'-ATP and was prevented by 2', 5'-dideoxyadenosine. Adenylyl cyclase, whether purified from bovine brain or in membranes from High Five cells expressing type I enzyme, when subjected to UV irradiation in the presence of (125)I-labeled 1 resulted in radioactive incorporation into protein migrating at approximately 116 kDa. The cross-linking of 1 and its iodinated derivative with adenylyl cyclase suggests potential for such compounds to be useful in structural studies of adenylyl cyclases or of other proteins for which adenine nucleosides are substrates or allosteric regulators.     

Reconstitution of types I and II adenosine cyclic 3',5'-phosphate dependent protein kinase

Fluorescence intensity and anisotropy measurements using the fluorescent adenosine cyclic 3',5'-phosphate (cAMP) analogue 1,N6-ethenoadenosine cyclic 3',5'-phosphate (epsilon-cAMP) are sensitive to the dissociation of epsilon-cAMP which occurs when either the type I or the type II regulatory subunit (RI or RII) of cAMP-dependent protein kinase associates with the catalytic subunit. Studies using epsilon-cAMP show that MgATP has opposite effects on the reconstitution of both types of protein kinase: MgATP strongly stabilizes the type I holoenzyme while it slightly destabilizes the type II holoenzyme. The synthetic substrate Kemptide has a small inhibitory effect on the reconstitution of both holoenzymes when tested at 10 microM concentration. The protein kinase inhibitor has a larger effect which is especially pronounced in the reassociation of the type I enzyme. The diminished relative ability of the type I regulatory subunit to compete with the protein kinase inhibitor suggests that the combined effects of the two opposing equilibria (epsilon-cAMP and catalytic subunit binding) are different for the two types of regulatory subunits. Displacement experiments show that cAMP and epsilon-cAMP bind about equally well to the type I subunit. Slow conformational changes accompanying the binding of epsilon-cAMP by both regulatory subunits are greatly accelerated with the holoenzymes, suggesting that dissociation of the holoenzymes occurs via ternary complexes. The time courses of epsilon-cAMP binding also show the heterogeneity of binding characteristics of RII. The 37 000-dalton fragment of type II subunit retains the epsilon-cAMP binding properties of the native subunit. However, only a fraction of the fragment preparation (approximately 32% estimated from sedimentation measurements) binds the catalytic subunit well, suggesting heterogeneity of cleavage.     

Mediation of macrophage collagenase production by 3'-5' cyclic adenosine monophosphate

The production of collagenase by lipopolysaccharide-(LPS) activated guinea pig macrophages is mediated by prostaglandins (PG) of the E series. After stimulation of guinea pig macrophages with LPS, extracellular PGE levels and cellular cAMP levels are elevated. Indomethacin inhibits not only PG synthesis, but also cAMP and collagenase production in LPS-stimulated macrophage cultures. In these indomethacin-inhibited cultures containing LPS, dibutyryl (dB) cAMP, or cholera toxin can restore macrophage collagenase production but not PG synthesis. Moreover, dBcAMP and cholera toxin enhance collagenase production in LPS-activated cultures. Initial activation of the macrophages by an agent such as LPS is a prerequisite for synthesis of collagenase, since in the absence of LPS, dBcAMP or cholera toxin alone are ineffective stimuli. These findings clearly demonstrate a role for PG-induced elevations of cAMP in the production of collagenase by LPS-activated macrophages.     

In vivo activation of cyclic adenosine 3':5'-phosphate-dependent protein kinase in Chinese hamster ovary cells treated with N-6, O-2'-diburyl cyclic adenosine 3':5'-phosphate.

Treatment of Chinese hamster ovary cells with dibutyryl cyclic AMP, which results in a net increase of the intracellular cyclic AMP level, converts the epithelial-like cells to a fibroblast-like shape. Protein kinase activity in cells treated with 1 mM dibutyryl cyclic AMP show a 3-fold increase in V_max but no appreciable changes in the apparent K_m for ATP. When cells are treated with dibutyryl cyclic AMP, there is a time-dependent conversion of cyclic AMP-stimulable protein kinase to cyclic AMP-independent catalytic subunits, as demonstrated by Sephadex G-100 gel filtration. These experiments demonstrate the activation of the cyclic AMP-dependent protein kinase $\text{in vivo}$. This activation may lead to phosphorylation of certain cellular constituent(s) and thus may be involved in the observed morphological transformation.     

Interaction of 2',3'-di-O-nitro-5'-(N-ethylcarboxamido)adenosine with adenosine receptors in intestinal smooth muscle

The adenosine derivative, 2'3'-di-O-nitro-(5'-N-ethylcarboxamido)adenosine (DINECA), caused relaxation in several isolated smooth muscle preparations including guinea pig taenia caeci, beef coronary arteries, and rabbit small intestine. In rabbit small intestine the response profile of DINECA action differed from that of established adenosine receptor agonists and, in contrast with the latter, its relaxant effect was only partially reversed by the antagonist 8-p-sulfophenyltheophylline. Concentration-response curves to 5'-(N-ethylcarboxamido)adenosine (NECA), but not those to DINECA, were significantly shifted to the right by 100 microM of 8-sulfophenyltheophylline. Tissues exposed previously to DINECA became refractory to adenosine, an effect not observed with tissues exposed to NECA, suggesting that DINECA became bound to adenosine receptors. Adenylate cyclase from neuroblastoma cells, containing Ra-type adenosine receptors, was stimulated by 2-chloroadenosine and NECA but not by DINECA. The results suggest that most of the smooth muscle relaxant actions of DINECA are not due to interaction with adenosine receptors but are probably due to its function as a nitrate. However, DINECA appears to interact with adenosine receptors, causing long lasting inhibition of adenosine action in rabbit intestine. Such actions may contribute to the overall response to DINECA application in vivo, although lowering of blood pressure due to the high reactivity of the vasculature to nitrates may be the initial and major effect.     

A bioluminescence method for determining adenosine 3'-phosphate 5'-phosphate (PAP) and adenosine 3'-phosphate 5'-sulfatophosphate (PAPS) in biological materials.

A rapid, sensitive, bioluminescence technique for detecting PAPS (adenosine 3′-phosphate 5′-sulfatophosphate) in biological materials is described. PAPS is first hydrolysed in 0.2 n HCl to PAP (adenosine 3′-phosphate 5′-phosphate) and is then assayed by the luciferin-luciferase system of the sea pansy, Renilla reniformis, which is specific for PAP. This bioluminescence system produces light at a rate that is proportional to the amount of PAP present. Light emission is measured in a liquid scintillation spectrometer with the two photomultipliers out of coincidence.Very low amounts of PAPS (10–100 pmoles) have been determined in extracts of yeast and various plant tissues by this method. The production of PAPS in extracts of young wheat leaves is enhanced by including either 5′-AMP or 3′-AMP in the reaction mixture. It is possible that these nucleotides protect PAPS from enzymes that degrade this compound, e.g., a nucleotidase.     

Synthesis of RNA containing O-beta-D-ribofuranosyl-(1'-2')-adenosine-5'-phosphate and 1-methyladenosine, minor components of tRNA

tRNA is best known for its function as amino acid carrier in the translation process, using the anticodon loop in the recognition process with mRNA. However, the impact of tRNA on cell function is much wider, and mutations in tRNA can lead to a broad range of diseases. Although the cloverleaf structure of tRNA is well-known based on X-ray-diffraction studies, little is known about the dynamics of this fold, the way structural dynamics of tRNA is influenced by the modified nucleotides present in tRNA, and their influence on the recognition of tRNA by synthetases, ribosomes, and other biomolecules. One of the reasons for this is the lack of good synthetic methods to incorporate modified nucleotides in tRNA so that larger amounts become available for NMR studies. Except of 2'-O-methylated nucleosides, only one other sugar-modified nucleoside is present in tRNA, i.e., 2'-O-beta-D-ribofuranosyl nucleosides. The T loop of tRNA often contains charged modified nucleosides, of which 1-methyladenosine and phosphorylated disaccharide nucleosides are striking examples. A protecting-group strategy was developed to introduce 1-methyladenosine and 5'-O-phosphorylated 2'-O-(beta-D-ribofuranosyl)-beta-D-ribofuranosyladenine in the same RNA fragment. The phosphorylation of the disaccharide nucleoside was performed after the assembly of the RNA on solid support. The modified RNA was characterized by mass-spectrometry analysis from the RNase T1 digestion fragments. The successful synthesis of this T loop of the tRNA of Schizosaccharomyces pombe initiator tRNA(Met) will be followed by its structural analysis by NMR and by studies on the influence of these modified nucleotides on dynamic interactions within the complete tRNA.