The role of periodic changes in cyclase and protein kinase activity in the mechanism of the proliferative effect of ACTH

A prolonged effect of ACTH on the state of adenylate and guanylate cyclase systems in the adrenal glands of experimental animals was investigated. It was found that in guinea pigs injected with ACTH (4 units daily for 1-50 days) the weight of adrenal glands and the DNA content in these organs increased 2.0-2.5-fold by the end of experiment; the increase in both values was stepwise. The corticosteroid level in the blood varied throughout the experiment: the changes in the DNA content in adrenals and in the corticosteroid content in the blood were oppositely directed. This was accompanied by cyclic changes in the basal and stimulated activities of adenylate and guanylate cyclases and proteinases in the adrenal glands occurring with a periodicity of 6-15 days. The activity peaks for cyclases and protein kinases preceded the rise in the DNA content in the adrenals. A clearcut correlation between the changes in the enzyme activity and the hormone dose was observed. The changes in the basal and stimulated activities of guanylate cyclase seem to be due to the control of cAMP level in the cell (stimulation of cGMP-dependent cAMP phosphodiesterase). Apparently, the periodic changes in the activity of cAMP-dependent protein kinases in the cytoplasmic and nuclear fractions and a relatively high activation of nuclear protein kinases (by 30-60%) in comparison of cytoplasmic ones (8-10%) are related to stimulation of DNA synthesis. It is concluded that the changes in the activity of cyclases and protein kinases play a role in the mechanism of proliferative effect of ACTH.     

Species and organ differences in the activity and regulation of adenylate and guanylate cyclases

The activity of adenylate and guanylate cyclases was determined in adrenal, heart, liver and fat tissues of guinea pigs, mice, rabbits and monkeys. The enzymes activities varied markedly depending both on the species and organs. The highest basal activities of adenylate cyclase was observed in all organs of guinea pigs. It was found that organs with low basal level of adenylate cyclase possess high guanylate cyclase. Species variations of the basal and stimulated adenylate cyclase activity may determine the functional activity of an organ: the higher the adenylate cyclase activity, the more intensive steroidogenesis in adrenals, lipolysis in the fat tissue, muscle contraction and nerve impulse conduction in heart.     

Adenylate cyclase activity and cyclic nucleotide content in human adrenal tumors under Icenko-Cushing syndrome

The adenylate cyclase activity and cyclic nucleotide content in excised human adrenal tumours (Icenko-Cushing syndrome) were determined. The experimental data were compared to those obtained for hyperplastic adrenals. All adrenal tumours under study revealed a decreased cAMP level, an increased cGMP level and a resulting decrease of the cAMP/cGMP ratio. In malignant adrenal tumours the adenylate cyclase activity was sharply increased in comparison with that in hyperplastic adrenals. In the majority of malignant tumours the adenylate cyclase response to ACTH was either altogether absent or sharply decreased. In benign adrenal tumours the basal activity of the enzyme was unchanged and the enzyme response to ACTH was essentially normal. The decrease of adenylate cyclase response to ACTH in malignant tumours is apparently not due to the impaired catalytic activity of the enzyme, since its response to stimulation by sodium fluoride remains unaffected. In some tumours (one malignant and two benign ones) a non-specific stimulation of adenylate cyclase by hormones, which are not natural activators of the enzyme was observed. It was assumed that these changes are due to the damage of hormonal receptors in adrenal tumours.     

Species-specific aggregation factor in sponges. VII. Its effect on cyclic amp and cyclic gmp metabolism in cells of Geodia cydonium.

In dissociated single cells from the sponge Geodia cydonium, DNA synthesis is initiated after incubation with a homologous, soluble aggregation factor. During the DNA-initiation phase the cyclic AMP- and cyclic GMP levels vary drastically; the cyclic AMP content drops from 2.2 pmol/10(6) cells to 0.3 pmol/10(6) cells while the cyclic GMP content increases from 0.6 pmol to 3.7 pmol/10(6) cells. The activity of neither the adenylate cyclase nor of the guanylate cyclase isolated from cells which have been incubated for different periods of time with the aggregation factor, is changed. The soluble as well as the particulate enzyme activities were checked in vitro. The cyclic nucleotide receptors have been isolated from the sponge cells and characterized with respect to their molecular weight, dissociation constant for cyclic AMP or cyclic GMP and intracellular concentration. None of these parameters are altered during aggregation factor-mediated DNA initiation. From these data it is concluded that the regulation of cyclic nucleotide levels is a consequence of a changed activity of nucleotide cyclases or of phosphodiesterases, but this is presumably not caused by a changed rate of synthesis of nucleotide cyclases or of cyclic nucleotide receptors.     

Changes in the system of cyclic nucleotides in irradiated body tissues

It has been shown that the content of cAMP in the rat thymus, spleen, and liver 1 and 3 days after gamma-radiation with 7.5 Gy decreases, and that of cGMP increases. Analogous dynamics has been revealed when studying adenylate cyclase and guanylate cyclase activities. The activity of cAMP and cGMP phosphodiesterases increased during the first period of study but subsequently it showed no distinction from the initial data level. The revealed postradiation changes in the content of cyclic nucleotides seem to be basically caused by the cyclases activity alterations.     

Rhizobium meliloti adenylate cyclase is related to eucaryotic adenylate and guanylate cyclases.

A gene from Rhizobium meliloti coding for an adenylate cyclase was sequenced, and the deduced protein sequence was compared with those of other known adenylate cyclases. No similarity could be detected with the procaryotic counterparts. However, striking similarity was found with the catalytic region of Saccharomyces cerevisiae adenylate cyclase, the cytoplasmic domains of bovine adenylate cyclase, and two mammalian guanylate cyclases. The gene was fused to the enteric beta-galactosidase, and the chimeric protein was purified by affinity chromatography. This fusion protein was found to direct the synthesis of cyclic AMP in vitro. This activity was strongly inhibited by the presence of GTP, but no cyclic GMP synthesis could be detected in conditions permitting cyclic AMP synthesis.     

Role of Silicon in Diatom Metabolism : Cyclic Nucleotide Levels, Nucleotide Cyclase, and Phosphodiesterase Activities during Synchronized Growth of Cylindrotheca fusiformis

Adenylate cyclase, guanylate cyclase, and the cyclic nucleotide phosphodiesterases of Cylindrotheca fusiformis were characterized in crude and partially purified preparations. Both cyclases were membrane-bound and required Mn²⁺ for activity, though Mg²⁺ gave 50% activity with adenylate cyclase. Properties of adenylate cyclase were similar to those of higher eukaryotic cyclases in some respects, and in other respects were like lower eukaryotic cyclases. Guanylate cyclase was typical of other lower eukaryotic enzymes.

Two phosphodiesterase activities were found, one selective for cyclic AMP, the other for cyclic GMP. The 5′-nucleoside monophosphate was the major product of both activities and each of the enzymes had distinctive divalent cation requirements, pH optima, and kinetic parameters. Both phosphodiesterases were similar to those of other lower eukaryotes with one notable difference: the cyclic AMP enzyme was inhibited by calcium.

Changes in the cyclic nucleotide levels were quantitated in light-dark and silicon-starvation synchronized cultures using a more sensitive radioimmunoassay than used in a previously published study (Borowitzka and Volcani 1977 Arch Microbiol 112: 147-152). Contrary to the previous report, the cyclic GMP level did not change significantly in either synchrony. The cyclic AMP level increased dramatically very early in the period of DNA replication with the peak cyclic AMP accumulation substantially preceding that of DNA synthesis in both synchronies. There was no significant change in the activity of either cyclase or either phosphodiesterase during either synchrony. Thus, the mechanism for the rise in cAMP level remains unclear.

     

Species-Specific Aggregation Factor In Sponges

In dissociated single cells from the sponge Geodia cydonium, DNA synthesis is initiated after incubation with a homologous, soluble aggregation factor. During the DNA -initiation phase the cyclic AMP - and cyclic GMP levels vary drastically; the cyclic AMP content drops from 2.2 pmol/10⁶ cells to 0.3 pmol/10⁶ cells while the cyclic GMP content increases from 0.6 pmol to 3.7 pmol/10⁶ cells. the activity of neither the adenylate cyclase nor of the guanylate cyclase isolated from cells which have been incubated for different periods of time with the aggregation factor, is changed. the soluble as well as the particulate enzyme activities were checked in vitro. the cyclic nucleotide receptors have been isolated from the sponge cells and characterized with respect to their molecular weight, dissociation constant for cyclic AMP or cyclic GMP and intracellular concentration. None of these parameters are altered during aggregation factor-mediated DNA initiation. From these data it is concluded that the regulation of cyclic nucleotide levels is a consequence of a changed activity of nucleotide cyclases or of phosphodiesterases, but this is presumably not caused by a changed rate of synthesis of nucleotide cyclases or of cyclic nucleotide receptors.     

From adenylate cyclase to guanylate cyclase. Mutational analysis of a change in substrate specificity.

Adenylate and guanylate cyclases, having different but related substrates, are a paradigm for the study of substrate discrimination. A prokaryotic adenylate cyclase gene, phylogenetically related to eukaryotic counterparts, was screened for mutants remodelling the enzyme's specificity. In a first step, a mutant was selected displaying a significant level of guanylate cyclase activity. This was due to a point mutation destroying most of the adenylate cyclase activity. A second selection step restored most of the original activity. This resulted from an additional mutation in the same region, thus permitting the first identification of a functional domain in adenylate and guanylate cyclases.     

Cyclic nucleotides and platelet aggregation. Effect of aggregating agents on the activity of cyclic nucleotide-metabolizing enzymes.

The activities of adenylate and guanylate cyclase and cyclic nucleotide 3':5'-phosphodiesterase were determined during the aggregation of human blood platelets with thrombin, ADP, arachidonic acid and epinephrine. The activity of guanylate cyclase is altered to a much larger degree than adenylate cyclase, while cyclic nucleotide phosphodiesterease activity remains unchanged. During the early phases of thrombin-and ADP-induced platelet aggregation a marked activation of the guanylate cyclase occurs whereas aggregation induced by arachidonic acid or epinephrine results in a rapid diminution of this activity. In all four cases, the adenylate cyclase activity is only slightly decreased when examined under identical conditions. Platelet aggregation induced by a wide variety of aggregating agents including collagen and platelet isoantibodies results in the "release" of only small amounts (1-3%) of guanylate cyclase and cyclic nucleotide phosphodiesterase and no adenylate cyclase. The guanylate cyclase and cyclic nucleotide phosphodiesterase activities are associated almost entirely with the soluble cytoplasmic fraction of the platelet, while the adenylate cyclase if found exclusively in a membrane bound form. ADP and epinephrine moderately inhibit guanylate and adenylate cyclase in subcellular preparations, while arachidonic and other unsaturated fatty acids moderately stimulate (2-4-fold) the former. It is concluded that (1) the activity of platelet guanylate cyclase during aggregation depends on the nature and mode of action of the inducing agent, (2) the activity of the membrnae adenylate cyclase during aggregation is independent of the aggregating agent and is associated with a reduction of activity and (3) cyclic nucleotide phosphodiesterase remains unchanged during the process of platelet aggregation and release. Furthermore, these observations suggest a role for unsaturated fatty acids in the control of intracellular cyclic GMP levels.     

Growth-dependent changes of guanylate and adenylate cyclase activities in cilia and cell bodies of Tetrahymena pyriformis

The calmodulin-dependent guanylate cyclase of Tetrahymena pyriformis was shown previously to be localized in surface membranes (ciliary and pellicular membranes) (Kudo, S, Nakazawa, K, Nagao, S & Nozawa, Y, Japan j exp med 52 (1952) 193) [21], whereas in a recent report Schultz et al, (Schultz, J E, Schonefeld, U & Klumpp, S, Eur j biochem 137 (1983) 89) [12] demonstrated the localization of this enzyme in ciliary membrane, arguing against its presence in pellicular membrane. To examine the discrepancy, the activities of guanylate and adenylate cyclases were examined in cilia and cell bodies of Tetrahymena pyriformis during transition from early log to stationary growth phase. The guanylate cyclase activity in the cell bodies increased significantly with growth of age, while in cilia the activity was rather consistent. In contrast, adenylate cyclase did not show any growth-dependent activity changes in both cilia and cell bodies. The increase of guanylate cyclase activity was not related to the increase of its activator calmodulin, because the change in enzyme activity could not be negated by addition of a saturating amount of calmodulin. These results suggest that the content of guanylate cyclase itself would be increased in the cell bodies during growth.     

Identification of bacterial guanylate cyclases

The ability of bacteria to use cGMP as a second messenger has been controversial for decades. Recently, nucleotide cyclases from Rhodospirillum centenum, GcyA, and Xanthomonas campestris, GuaX, have been shown to possess guanylate cyclase activities. Enzymatic activities of these guanylate cyclases measured in vitro were low, which makes interpretation of the assays ambiguous. Protein sequence analysis at present is insufficient to distinguish between bacterial adenylate and guanylate cyclases, both of which belong to nucleotide cyclases of type III. We developed a simple method for discriminating between guanylate and adenylate cyclase activities in a physiologically relevant bacterial system. The method relies on the use of a mutant cAMP receptor protein, CRP_G, constructed here. While wild‐type CRP is activated exclusively by cAMP, CRP_G can be activated by either cAMP or cGMP. Using CRP‐ and CRP_G‐dependent lacZ expression in two E. coli strains, we verified that R. centenum GcyA and X. campestris GuaX have primarily guanylate cyclase activities. Among two other bacterial nucleotide cyclases tested, one, GuaA from Azospillrillum sp. B510, proved to have guanylate cyclase activity, while the other one, Bradyrhizobium japonicum CyaA, turned out to function as an adenylate cyclase. The results obtained with this reporter system were in excellent agreement with direct measurements of cyclic nucleotides secreted by E. coli expressing nucleotide cyclase genes. The simple genetic screen developed here is expected to facilitate identification of bacterial guanylate cyclases and engineering of guanylate cyclases with desired properties. Proteins 2015; 83:799–804. © 2015 Wiley Periodicals, Inc.     

The crystal structure of the catalytic domain of a eukaryotic guanylate cyclase

Background

Soluble guanylate cyclases generate cyclic GMP when bound to nitric oxide, thereby linking nitric oxide levels to the control of processes such as vascular homeostasis and neurotransmission. The guanylate cyclase catalytic module, for which no structure has been determined at present, is a class III nucleotide cyclase domain that is also found in mammalian membrane-bound guanylate and adenylate cyclases.

Results

We have determined the crystal structure of the catalytic domain of a soluble guanylate cyclase from the green algae Chlamydomonas reinhardtii at 2.55 Å resolution, and show that it is a dimeric molecule.

Conclusion

Comparison of the structure of the guanylate cyclase domain with the known structures of adenylate cyclases confirms the close similarity in architecture between these two enzymes, as expected from their sequence similarity. The comparison also suggests that the crystallized guanylate cyclase is in an inactive conformation, and the structure provides indications as to how activation might occur. We demonstrate that the two active sites in the dimer exhibit positive cooperativity, with a Hill coefficient of ~1.5. Positive cooperativity has also been observed in the homodimeric mammalian membrane-bound guanylate cyclases. The structure described here provides a reliable model for functional analysis of mammalian guanylate cyclases, which are closely related in sequence.     

Selective sensitivity of synaptosomal cyclases to glutamic acid

Electron cytochemical studies have been made of the effect of various concentrations of the glutamic acid on localisation of adenylate and guanylate cyclases in synaptosomes from the brain cortex of rats. It was found that the glutamic acid (10(-3) M) stimulates the activity of intrasynaptosomal adenylate cyclase, but does not affect postsynaptic pool of the enzyme. The effect of glutamate on guanylate cyclase results in the increase of the frequency of the reaction both in synaptosomal and postsynaptic membranes. It is suggested that in the conduction of glutamate signal, guanylate cyclase--cGMP, but not adenylate cyclase--cAMP, system may be involved, although activation of intrasynaptosomal adenylate cyclase indicates its participation in presynaptic processes.     

Disorders in the system of cyclic nucleotides in atherosclerosis: cyclic AMP and cyclic GMP content and activity of related enzymes in human aorta

Cyclic AMP and cyclic GMP content and activities of cyclic nucleotide metabolic enzymes were determined in intima and media of atherosclerotic and unaffected human aorta obtained shortly after death due to myocardial infarction. Cyclic AMP content in fatty streaks and atherosclerotic plaques was lower by three- and five-fold, respectively, as compared with uninvolved intima. Cyclic GMP level in atherosclerotic lesions was estimated to be three-fold higher than in grossly normal area. Basal activity of adenylate cyclase in fatty streaks and plaques was two- to six-fold lower than in unaffected intima. Besides, the ability of adenylate cyclase to be stimulated by the stable analogue of prostacyclin, carbacyclin, was suppressed in plaques. Guanylate cyclase activity in fatty streaks was 1.5- to three-fold higher than in normal tissue. The thiol-reducing agent, dithiothreitol, decreased the enzyme activity to normal level, suggesting the oxidative nature of guanylate cyclase activation in the lesion zone. There were no significant changes in cyclic AMP phosphodiestease activity in the regions of the atherosclerotic lesion. Cyclic GMP phosphodiesterase activity in atherosclerotic plaques was two-fold lower than in the intima of unaffected areas. We did not find differences in the content of cyclic nucleotides or related enzyme activities in the media of uninvolved areas of human aorta nor in the media underlying atherosclerotic lesions. Our findings suggest that development of human atherosclerotic lesions is accompanied by dramatic changes in the cyclic nucleotide metabolism featuring gradual hormonal receptor uncoupling from adenylate cyclase, activation of guanylate cyclase in fatty streaks and inhibition of cyclic GMP phosphodiesterase in plaques.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulation of cyclic nucleotides in retinal photoreceptors

Summary The distribution of cyclases in retinal photoreceptors of dark- and light-adapted brook trout was studied by means of a cytochemical method (lead precipitation). It confirms earlier reports that retinal photoreceptors contain high levels of cyclic nucleotides, and that cAMP predominates in cones and cGMP in rods. There is an apparent difference in the level of the cyclases with the adaptive states. In addition, the catalytic unit of cyclase is interlamellar in cones. In rods, adenylate cyclase is intradiscal, while the location of guanylate cyclase varies with the adaptive state. The variation of cyclase with adaptation indicates that this enzyme has a role in the process of visual transduction.     

Oscillating levels of adenylate and guanylate cyclase activities in rat embryo fibroblasts stimulated to divide.

Basal activities of membrane-bound adenylate and guanylate cyclase were determined in confluent rat embryo cells stimulated to proliferate by either the renewal of serum-supplemented growth medium or the addition of a mitogen, the 12-0-tetradecanoyl-phorbol-13-acetate (TPA). A transient increase in guanylate cyclase activity was observed within minutes following either treatment while adenylate cyclase activity either abruptly declined in serum-stimulated cells or remained unaffected in TPA-treated cells. In response to both mitogenic treatment, adenylate and guanylate cyclase activities varied reciprocally throughout the pre-replicative phase up to DNA synthesis. The lower levels of guanylate over adenylate activity ratio occurred prior to the onset of the replicative phase whereas the higher levels were coincident with DNA synthesis. A similar pattern of oscillating levels of sodium-fluoride-stimulated adenylate and lubrol-treated guanylate cyclase activities was observed.     

Effect of pituitary hormones on phosphodiesterase and adenyl cyclase activity of brain tissue in vitro

Effects of seiwhale somatotropin (STH), its biologically active fragment 77--107, porcine corticotropin (ACTH) and seiwhale prolactin on phosphodiesterase and adenylate cyclase activity of glial cells and synaptosomes isolated from the rat brain cortex were investigated. As compared with control, ACTH increased phosphodiesterase activity of glial cells by 392%, of synaptosomes by 123%, while STH by 49 and 77%, respectively, somatotropin fragment by 455 and 74%, and prolactin by 30 and 37%, respectively. Adenylate cyclase activity was significantly changed only by ACTH and only in synaptosomes (a 50% decrease). STH, its fragment and prolactin virtually failed to alter adenylate cyclase activity. The data obtained indicate that some of pituitary hormones, primarily ACTH and STH, may play the role of neuromodulators in some brain structures by decreasing the cyclic AMP level, by activating phosphodiesterase (STH and ACTH) and inhibiting adenylate cyclase (ACTH in synaptosomes).     

Characterization and subcellular localization of nucleotide cyclases in calf thymus lymphocytes

The role of cyclic nucleotides in the regulation of lymphocyte growth and differentiation remains controversial, as an adequate characterization of the key enzymes, adenylate cyclase and guanylate cyclase, in the plasma membrane of lymphocytes is still lacking. In this study, calf thymus lymphocytes were disrupted by nitrogen cavitation and various cellular fractions were isolated by differential centrifugation and subsequent sucrose density ultracentrifugation. As revealed by the chemical composition and the activities of some marker enzymes, the plasma membrane fraction proved to be highly purified. Nucleotide cyclases were present in the plasma membranes in high specific activities, basal activities of adenylate cyclase being 13.7 pmol/mg protein per min and 34.0 pmol/mg protein per min for the guanylate cyclase, respectively. Adenylate cyclase could be stimulated by various effectors added directly to the enzyme assay, including NaF, GTP, 5'-guanylyl imidodiphosphate, Mn2+ and molybdate. Addition of beta-adrenergic agonists only showed small stimulating effects on the enzyme activity in isolated plasma membranes. Basal activity of adenylate cyclase as well as activities stimulated by NaF or 5'-guanylyl imidodiphosphate exhibited regular Michaelis-Menten kinetics. Activation by both agents only marginally affected the Km values, but largely increased Vmax. The activity of the plasma membrane-bound guanylate cyclase was about 10-fold enhanced by the nonionic detergent Triton X-100 and high concentrations of lysophosphatidylcholine, but was slightly decreased upon addition of the alpha-cholinergic agonist carbachol. Basal guanylate cyclase indicated to be an allosteric enzyme, as analyzed by the Hill equation with an apparent Hill coefficient close to 2. In contrast, Triton X-100 solubilized enzyme showed regular substrate kinetics with increasing Vmax but unaffected Km values. Thus the lymphocyte plasma membrane contains both adenylate cyclase and guanylate cyclase at high specific activities, with properties characteristic for hormonally stimulated enzymes.