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.     

Catabolism of capped (3'-5')- and (2'-5')-adenylates in rat liver nuclei

We describe studies concerning the ability of a nuclear dinucleoside triphosphatase to act as a decapping enzyme in RNA catabolism. The enzymatic release of GMP from the Gp3A moiety was determined in the capped RNA model compounds Gp3A3'pA, Gp3A3'pA-isoprop and Gp3A2'pA in isolated rat liver nuclei; i.e., in the environment in which the dinucleoside triphosphatase operates in vivo. The Gp3A cap moiety is hydrolyzed in (3'-5') linked nucleotides only, whereas an extension of the Gp3A in the 2'-direction prevents the nuclear triphosphatase to operate.     

Adenosine-3':5'-monophosphate phosphodiesterase from Rhizobium

The phosphodiesterase (PDE) activity of adenosine-3':5'-monophosphate was detected in the cells of tubercular bacteria of Rhizobium lupini and Rhizobium japonicum. The specific activity of three Rhizobium forms, e.g. bacteroids from lupine root tubercles, free-nitrogen-fixing culture and vegetative cells grown on a mannitol--yeast agar, were compared. In the bacteroids PDE is represented both by soluble and membrane-bound forms. The optimal enzyme activity is revealed in an alkaline medium, whereas the curve of PDE activity dependence on pH has a broad maximum. PDE is inhibited by methylxanthines, the inhibiting effect being stronger than that of theophylline.     

Activation of 2-5A-dependent RNase by analogs of 2-5A (5'-O-triphosphoryladenylyl(2'----5')adenylyl(2'----5')adenosine ) using 2',5'-tetraadenylate (core)-cellulose

A variety of 2-5A (px(A2'p)nA; x = 2 or 3, n greater than or equal to 2) analogs were assayed for their abilities to activate murine 2-5A-dependent RNase (subsequently "the nuclease") using a recently developed method. This technique consists of immobilizing and partially purifying the nuclease using core-cellulose [A2'p)3A-cellulose) and then monitoring the breakdown of poly(U)-3'-[32P]Cp into acid-soluble fragments. Several 5'-adenosinecapped analogs of 2-5A (containing a tetra-, tri-, or diphosphate) were analyzed, and it was found that reducing the number of phosphoryl groups between the 5' to 5'-diadenosine linkages resulted in a progressive loss of activity. Because A5' pppp(A2'p)3A was a potent activator of the nuclease yet stable during the assay these results suggested that a free 5'-phosphoryl group may not be required for the activation of the nuclease. A number of 8-bromoadenosine-substituted analogs of 2-5A were also studied. Curiously, the brominations decreased the activities of the 5'-di- and triphosphorylated molecules while substantially increasing the activities of the 5'-monophosphorylated species. The results indicated that a tri- or diphosphate moiety on the 5'-end of 2-5A or the presence of ATP is not absolutely required for the nuclease to be active. Furthermore, the ATP analog, beta, gamma-methylene ATP, did not inhibit the activity of the nuclease. Finally, a 3',5'-phosphodiester linkage isomer of 2-5A and a 3'-deoxy (cordycepin) analog of 2-5A were tested, and both were found to be completely without activity.     

A microassay for guanosine 3',5'-monophosphate phosphodiesterase activity

The activity of cyclic GMP phosphodiesterase was determined using a three step procedure. In the first step, cyclic GMP phosphodiesterase catalyzes the conversion of cyclic GMP to 5′-GMP. In the second step, a known amount of ATP and guanylate kinase are incubated with the 5′-GMP formed in the first step. The amount of ATP which remains is inversely related to the amount of 5′-GMP formed. In the third step, the concentration of ATP is measured using the firefly luciferin-luciferase technique. The validity of the assay is confirmed by its ability to show the linearity of the cyclic GMP phosphodiesterase reaction with respect both to time of incubation and concentration of tissue. It is capable of detecting less than 5 pmoles of 5′-GMP in 150 μl, and can be used to measure cyclic GMP phosphodiesterase activity in a supernatant fraction of rat cerebrum which contains less than 25 ng of protein. It has been used to determine the activity and properties of cyclic GMP phosphodiesterase in unpurified supernatant and particulate fractions of several tissues of the rat, as well as in highly purified fractions of rat caudate nucleus.     

The 2'-5' adenylate (2-5A) system in erythropoiesis

The trimer and tetramer core forms of 2'-5' Adenylate (2-5A) were tested in vitro for their effect on mouse liver CFU-E. Both forms of 2-5A core partially inhibited the CFU-E response to erythropoietin when given as a single dose at time 0 hours. Approximately 25% fewer colonies were seen after 2-days growth following exposure of the CFU-E to 0.1 mM 2-5A core. A 0.01 mM concentration of exogenous 2-5A core was not inhibitory. Endogenous 2-5A synthetase activity was assayed in spleen cell lysates from mice made anemic by several injections of phenylhydrazine. This method of treatment stimulated erythropoiesis, and this was directly correlated with an increase in the rate of enzyme activity measured by lysate conversion of 14C-ATP to 2-5A (our in press data suggests that the same may occur with hypoxic stimulation). This suggested to us that endogenous 2-5A synthetase and its 2-5A, a known inhibitor of DNA synthesis and protein synthesis, may help to regulate some of the late events in erythropoiesis.     

Interaction of 3'-fluoro-3'-deoxyanalogs of a trimer of (2'-5')oligoadenylic acid with (2'-5')A3-antibodies: stereochemical aspect

Mouse antibodies to (2'-5')oligoadenylates were obtained by the immunization of animals with the (2'-5')oligoadenylic acid trimer conjugated with bovine serum albumin through a 2',3'-levulinic acid residue. Using radioimmunoassay, the reactivity of mouse polyclonal antibodies to the (2'-5')oligoadenylic acid trimer was studied for the trimer analogues containing 9-(3-deoxy-3-fluro-beta-D- xylofuranosyl)adenine and 3'-deoxy-3'-fluoro-adenosine in various positions of the chain. It was found that (a) the three-dimensional structure of short oligonucleotides is an important factor in the antibody recognition; (b) antibodies are more sensitive to modifications of the 5'-terminal and central ribose fragments of the (2'-5')oligoadenylic acid trimer; (c) the 3'-hydroxyl group plays a secondary role in the formation of the antigen determinant.     

Adenosine 3'5'-monophosphate phosphodiesterase of buffalo spermatozoa.

The cyclic AMP-phosphodiesterase (EC 3.1.4.17) of buffalo spermatozoa is distributed in the head, mid-piece and tail fractions and has multiple forms, 70% of which is in the bound form. The bound enzyme was not solubilized by Triton X-100, lubrol or hyamine 2389. Kinetic measurements of the soluble enzyme showed two apparent Km values for low and high cAMP concentrations, i.e. 4.5 and 100 micro M with Vmax values of 0.25 and 2.0 nmol cAMP hydrolysed min-1 mg protein-1. The bound enzyme had an apparent Km of 66.6 microM with a Vmax of 0.75 nmol cAMP hydrolysed min-1 mg protein-1. The pH for optimum enzyme activity was 7.5 and Mg2+ was essential for the activity of the soluble and bound enzymes. Methylxanthines, ATP, ADP and ppi inhibited the soluble and bound enzymes, ATP being the most potent inhibitor.     

Induction of (2'-5')An-dependent endoribonuclease activity by interferon in cultured guinea pig macrophages

By a radiobinding assay, an affinity labeling technique, and a column procedure which quantitates mRNA intactness, evidence is presented on the induction of the (2'-5')An-dependent endoribonuclease by interferon in cultured guinea pig macrophages.     

Only one 3'-hydroxyl group of ppp5' A2'p5'A2'p5' A (2-5A) is required for activation of the 2-5A-dependent endonuclease

To investigate the relative importance of each of the ribose 3'-hydroxyl groups of 2-5A (ppp5' A2'p5'A2'-p5' A) in determining binding to and activation of the 2-5A-dependent endonuclease (RNase L), the 3'-hydroxyl functionality of each adenosine moiety of 2-5A trimer triphosphate was sequentially replaced by hydrogen. The analog in which the 5'-terminal adenosine was replaced by 3'-deoxyadenosine (viz. ppp5'(3'dA)-2'p5' A2'p5' A) was bound to RNase L as well as 2-5A itself and was only 3 times less potent than 2-5A as an activator of RNase L. On the other hand, when the second adenosine unit was replaced by 3'-deoxyadenosine (viz. ppp5' A2'p5'(3'dA)2'p5' A), binding to RNase L was decreased by a factor of eight relative to 2-5A trimer and, even more dramatically, there was a 500-1000-fold drop in ability to activate the 2-5A-dependent endonuclease. Finally, when the 3'-hydroxyl substituent was converted to hydrogen in the 2'-terminal residue of 2-5A, a significant increase in both binding and activation ability occurred. We conclude that only the 3'-hydroxyl group of the second (from the terminus) nucleotide residue of 2-5A is needed for effective activation of RNase L.