Demonstration, in leukemia L-1210 cells, of a phosphodiesterase acting on 3':5'-cyclic CMP but not on 3':5'-cyclic AMP or 3':5'-cyclic GMP.

cCMP-specific phosphodiesterase activity was demonstrated in the 80 to 100% ammonium sulfate fraction obtained from disrupted leukemia L-1210 cells. The activity was linear with time (up to 60 min), was a function of protein concentration, and was markedly stimulated by Mg2+ and by ammonium sulfate. Under identical assay conditions, no significant hydrolysis of cAMP or cGMP was observed, although these cyclic nucleotides served as substrates for phosphodiesterase(s) present in all the fractions obtained by less than 80% ammonium sulfate saturation. This is the first demonstration of a cCMP-specific phosphodiesterase.     

Phototropic sensitivity in Vaucheria geminata regulated by 3',5'-cyclic AMP

Exogenous 3',5'-cyclic AMP in physiological concentrations inhibitsthe phototropic bending of Vaucheria geminata without affectinggrowth. Related substances known to change the intracellularcyclic AMP level also alter phototropic sensitivity. This indicatesthat the steering process (i.e., initial phototropic response)is separate from growth itself. Cyclic AMP extracted from this alga amounted to 0.2–0.3nmole per g fresh weight, or about 3 µM on a cytoplasmvolume basis. This value coincides with expected results fromexperiments with exogenous cyclic AMP. The results suggest thatendogenous cyclic AMP may play an essential role in the gain-controllingprocess of phototropism. ¹ Present address: Institute for Agricultural Research, TohokuUniversity, Sendai 980, Japan. (Received November 26, 1976; )     

Stimulation of virus-induced fusion of Ehrlich ascites tumor cells by 3',5'-cyclic AMP.

HVJ(Sendai virus)-induced fusion of Ehrlich ascites tumor cells was found to be stimulated by treatments which increase the intracellular level of cyclic AMP. This stimulation was optimal at an external concentration of Ca⁺⁺ of about 0.5 mM. During the process of cell fusion, the intracellular concentration of cyclic AMP was increased with a maximum at 2 min after the initiation of the fusion reaction.Evidence is also presented which suggests that the increase of the cyclic nucleotide is a part of control mechanism of HVJ-induced fusion of eukariotic cells. Thus, this cyclic AMP-stimulated process could be one of the step(s) requiring ATP and Ca⁺⁺, both of which are necessary for the overall fusion process of the tumor cells.     

An effect of glucagon on 3', 5'-cyclic AMP phosphodiesterase activity in isolated rat hepatocytes.

Glucagon was found to activate the low Km form of 3′,5′-cyclic AMP phosphodiesterase in intact isolated rat hepatocytes while the high Km phosphodiesterase was unaltered. Activation was concentration dependent and occurred at the same concentration required to observe an increase in 3′,5′-cyclic AMP levels in the cell. The maximal increase in activity occurred within 5 minutes of incubation with glucagon and was sustained for the 35 minutes assayed.     

Role of 3',5'-cyclic AMP in the control of nuclear protein kinase activity

The role of cAMP in the regulation of nuclear protein kinase activity was investigated. Acidic nuclear proteins prepared from rat liver nuclei were separated by phosphocellulose chromatography into four peaks of protein kinase activity and two peaks of cAMP-binding activity. A fraction which bound cAMP also inhibited the most active nuclear protein kinase, K IV, and the inhibition was diminished in the presence of 5 μM cAMP. Further support for the regulation of nuclear kinases by cAMP was obtained using a regulatory subunit prepared from rabbit muscle protein kinase. The muscle regulatory subunit markedly inhibited liver nuclear kinase activities. The addition of cAMP partially restored the activities.     

Activity of tubercidin-, toyocomycin-, and sangivamycin-3',5'-cyclic phosphates and related compounds with some enzymes of adenosine-3',5'-cyclic phosphate metabolism

Rat liver nuclei contain at least two DNA polymerases that can be separated by extracting the nuclei with 5% Brij 58. The loosely-bound activity increases little or not at all after partial hepatectomy and is insensitive to cytosine arabinoside triphosphate (araCTP). The tightly-bound enzyme activity rises along with DNA replication and is inhibited by araCTP.     

Effect of hormones and 3', 5'-cyclic AMP on very low density lipoprotein receptors

Human 125I-labelled VLDL interacts with rat adipocytes in vitro, with properties typical of a ligand-receptor interaction. This VLDL-receptor interaction is modulated by hormones which are known to change cyclic AMP levels. Norepinephrine and isoproterenol, both of which elevate cycle AMP, increase the binding of VLDL to adipocytes. Dibutyryl-cyclic AMP, a derivative of cyclic AMP, also increases the VLDL binding to adipocytes. Insulin reverses the catecholamine-induced increase in VLDL binding. This parallels insulin's effect on the catecholamine-induced changes in cyclic AMP. Direct addition of cyclic AMP itself increases VLDL binding to adipocyte membranes, a system in which no lipolysis of new protein synthesis occurs. Based on the competition between unlabelled VLDL and 125I-labelled VLDL, we conclude that catecholamines act on adipocytes, and cyclic AMP on membrane fractions, by increasing their capacity rather than their affinity to bind VLDL.     

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.     

Identification of 3',5'-cyclic AMP in axenic cultures of Lemna paucicostata by high-performance liquid chromatography.

Nucleotides were purified from the extract of aseptically grown Lemna paucicostata 6746 by low-pressure column chromatography and thin-layer chromatography. In this partially purified sample, a compound has been identified, using a highly sensitive HPLC-fluorometric detection method, with chromatographic properties and substrate specificity for cyclic nucleotide phosphodiesterase similar to authentic 3',5'-cyclic AMP. The level of cAMP has been estimated to be 70-80 pmol/g fresh wt.