Cell Cycle-associated Changes of Guanylate Cyclase Activity in Synchronized Tetrahymena: a Possible Involvement of Calmodulin in its Regulation1

Guanylate cyclase activity decreased during the division phase of heat-shock synchronized Tetrahymena pyriformis, strain GL. However, when Ca²⁺ was removed by EGTA to negate the effects of the Ca²⁺-binding protein (calmodulin), which is required for the full activity of guanylate cyclase in this organism, no significant change in the enzymatic activity was observed throughout the cell cycle. On the other hand, the reduced guanylate cyclase activity at division phase was associated with a decreased level of calmodulin content. These results suggest that fluctuations in guanylate cyclase activity during the cell cycle would be dependent on the concentration of calmodulin.     

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.

     

Increase of proton electrochemical potential and ATP synthesis in rat liver mitochondria irradiated in vitro by helium-neon laser

Particulate adenylate cyclase (AC) and guanylate cyclase (GC) activities localized in the ciliary membrane from Paramecium were solubilized by a two-step procedure using the detergents Brij 56 and Lubrol PX. The enzymes remained in the supernatant after a 100 000 × g centrifugation. Upon gel chromatography, AC and GC were almost completely separated proving that each enzyme is a distinct molecular entity. Solubilization of GC was achieved with the calmodulin subunit remaining firmly attached to the catalytic part. Antibodies against calmodulin inhibited the enzyme as did La³⁺ and EGTA. AC activity appeared to be regulated specifically by K⁺, enzyme activity being enhanced up to 100% by 15 mM K⁺. Na⁺ and Li⁺ were inactive.     

The regulatory role of calmodulin-dependent guanylate cyclase in association with hormonal imprinting in Tetrahymena

The primary interaction with insulin accounted for considerable increases in both the calmodulin content and guanylate cyclase activity of Tetrahymena. Both activities were still elevated after 24 h (6-8 generations), but while the calmodulin level showed a decrease, guanylate cyclase activity showed a further significant increase relative to the immediate response. A second treatment with insulin decreased rather than increased both activities, but to dissimilar degrees, in that the calmodulin content returned to the control level, whereas guanylate cyclase activity still increased over the level measured after the first treatment. It appears that insulin imprinting altered the calmodulin-dependent guanylate cyclase regulation in Tetrahymena, and caused a switch-over to an 'energy-saving' system through decelerating the breakdown of cGMP by phosphodiesterase.     

Cyclic nucleotide cyclase variation during development of the insect Ceratitis capitata.

Guanylate and adenylate cyclase activities were estimated in homogenates of the insect Ceratitis capitata at various stages of development. Guanylate cyclase activity was notably higher than adenylate cyclase activity in agreement with both cyclic nucleotide ratio and cyclic nucleotide-dependent protein kinase ratio reported in arthropod tissues. Variations in both enzyme activities during development were coincident in the adult development, while in other biological stages, as the larval development and puparium formation, the most significant changes affected to the activity of guanylate cyclase.     

Separation of soluble adenylate and guanylate cyclases from the mature rat testis

The mature rat testis contains both a soluble guanylate cyclase and a soluble adenylate cyclase. Both these soluble enzymes prefer manganous ion for activity. It is known that guanylate cyclase can, when activated by a variety of agents, catalyze the formation of cyclic AMP. The following experiments were performed to determine whether the testicular soluble adenylate and guanylate cyclase activities were carried on the same molecule. Analysis of supernatants from homogenized rat testis by gel filtration and sucrose density gradient centrifugation showed that the two activities were clearly separable. The molecular weight of guanylate cyclase is 143 000, while that of adenylate cyclase is 58 000.Treatment of the column fractions with 0.1 mM sodium nitroprusside allowed guanylate cyclase activity to be expressed with Mg²⁺ as well as with Mn²⁺. Sodium nitroprusside did not affect the metal ion or substrate specificity of adenylate cyclase.These experiments show that adenylate and guanylate cyclase activities are physically separable.     

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.     

Characterization of rat testicular guanylate cyclase during development

The biochemical characteristics of rat testicular guanylate cyclase were investigated and the activity and subcellular distribution of the enzyme was determined during testicular development. Examination of the effects of metal ions, nucleotides, detergents and other in vitro activators on the activity of guanylate cyclase revealed that the testicular enzyme is similar in most respects to guanylate cyclase isolated from other mammalian tissues. Changes in the total activity of guanylate cyclase during testicular development paralleled changes in the tissue concentration of cyclic GMP; i.e. guanylate cyclase activity and tissue cyclic GMP were highest during the early stages of development. Subcellular fractionation revealed that the activity of the soluble form of guanylate cyclase was best correlated with tissue cyclic GMP. Bichemical analysis of the soluble enzyme prepared from testes of neonatal and adult rats did not reveal any significant differences in the characteristics of the enzyme during ontogeny with the exception of a 2.5 fold increase in V noted in the neonatal testis. The results of this study are consistent with a molecular mechanism that allows independent regulation of the different forms of guanylate cyclase.     

The activity of guanylate cyclase and cyclic GMP phosphodiesterase during synchronous growth of the acellular slime mould Physarum polycephalum.

Guanylate cyclase (EC 4.6.1.2.) and cyclic GMP phosphodiesterase (EC 3.1.4.-.) activity were measured in three subcellular fractions of Physarum polycephalum macroplasmodia isolated at intervals during synchronous growth. In a particulate fraction prepared by high-speed centrifugation guanylate cyclase activity was twice to ten times that of other fractions and highest in mid S and late G2. Two-thirds of the cyclic GMP phosphodiesterase activity was in a soluble fraction but there was no significant change in enzyme activity or distribution during the mitotic cycle.     

Enzymes of cyclic nucleotide metabolism in invertebrate and vertebrate sperm.

Sperm from several invertebrates contained guanylate cyclase activity several-hundred-fold greater than that in the most active mammalian tissues; the enzyme was totally particulate. Activity in the presence of Mn²⁺ was up to several hundred-fold greater than with Mg²⁺ and was increased 3–10-fold by Triton X-100. Sperm from several vertebrates did not contain detectable guanylate cyclase. Sperm of both invertebrates and vertebrates contained roughly equal amounts of Mn²⁺-dependent adenylate cyclase activity; in invertebrate sperm, this enzyme was generally several hundred-fold less active than guanylate cyclase. Adenylate cyclase was particulate, was unaffected by fluoride, and was generally greater than 10-fold more active with Mn²⁺ than with Mg²⁺. Invertebrate sperm contained phosphodiesterase activities against 1.0 μm cyclic GMP or cyclic AMP in amounts greater than mammalian tissues. Fish sperm, which did not contain guanylate cyclase, had high phosphodiesterase activity with cyclic AMP as substrate but hydrolyzed cyclic GMP at a barely detectable rate. In sea urchin sperm, phosphodiesterase activity against cyclic GMP was largely particulate and was strongly inhibited by 1.0% Triton X-100. In contrast, activity against cyclic AMP was largely soluble and was weakly inhibited by Triton. The cyclic GMP and cyclic AMP contents of sea urchin sperm were in the range of 0.1–1 nmol/g. Sea urchin sperm homogenates possessed protein kinase activity when histone was used as substrate; activities were more sensitive to stimulation by cyclic AMP than by cyclic GMP.⁵     

Cell cycle-associated changes of guanylate cyclase activity in synchronized Tetrahymena: a possible involvement of calmodulin in its regulation

Guanylate cyclase activity decreased during the division phase of heat-shock synchronized Tetrahymena pyriformis, strain GL. However, when Ca2+ was removed by EGTA to negate the effects of the Ca2+-binding protein (calmodulin), which is required for the full activity of guanylate cyclase in this organism, no significant change in the enzymatic activity was observed throughout the cell cycle. On the other hand, the reduced guanylate cyclase activity at division phase was associated with a decreased level of calmodulin content. These results suggest that fluctuations in guanylate cyclase activity during the cell cycle would be dependent on the concentration of calmodulin.     

Guanylate cyclase in the accessory gland of the cricket, Acheta domesticus

Guanylate cyclase (E.C. 4.6.1.2.) was investigated in the accessory reproductive gland of the male house cricket, Acheta domesticus, which is known to accumulate exceptionally high levels of guanosine 3′,5′-cyclic monophosphate (cyclic GMP). Accessory gland guanylate cyclase activity was linear with time for at least one hour, and with enzyme concentration to about 5 mg soluble protein per ml. Activity was dependent on Mn²⁺ and was maximal at pH 7.3 to 8.0. Sodium fluoride had no effect on activity, but sodium azide was slightly stimulatory. About 80% of the activity was sedimentable at 16,000 g, and both soluble and particulate activities were increased slightly in the presence of Triton X-100. Kinetic analysis indicated half-maximal velocity at 85 μM GTP in the presence of excess Mn²⁺, and reciprocal plots were concave upward. Changes in activity during maturation of the gland were small, and did not provide evidence for a regulatory role of guanylate cyclase in the accumulation of accessory gland cyclic GMP. The regulation and rôle of cyclic GMP in the accessory gland are discussed.     

Metabolic and cyclic nucleotide enzyme activities in muscle and nonmuscle cells of rat heart during perinatal development

Enzyme activities related to aerobic metabolism and cyclic nucleotides were evaluated in muscle and nonmuscle cells of rat heart. The perinatal period from weaning to adult was studied. Malate dehydrogenase, citrate synthase, and 3-hydroxyacyl-CoA dehydrogenase activities of nonmuscle cells equal or exceed muscle cell activities in the weanling heart. Aerobic enzymes remain unchanged in nonmuscle cells during growth; however, muscle cell activities are enhanced. Adenylate cyclase and guanylate cyclase activities are higher in heart homogenates of weanling than adult rats. Despite elevated adenylate cyclase activity, cyclic AMP levels are identical in weanling and adult rats. Cyclic GMP levels are twofold higher in weanling than in adult rats. Muscle cell metabolism and cyclic nucleotide levels are associated with growth-related changes in heart function and cellularity, respectively.     

Subcellular localizations of guanylate cyclase and 3',5'-cyclic nucleotide phosphodiesterase in sea urchin sperm.

The subcellular localizations of guanylate cyclase and 3',5'-cyclic nucleotide phosphodiesterase in sea urchin sperm were examined. Both the specific and total activities of these two enzymes were much higher in sperm flagella (tails) than in the heads. In addition to the observation that guanylate cyclase in the flagella was particulate-bound and solubilized by Triton X-100, more than 80% of the cyclase activity in the flagella was found in the plasma membrane fraction, whereas the activity of cyclic nucleotide phosphodiesterase was observed in both the axonemal and plasma membrane fractions. The observations indicated that the cyclase in the flagella appeared to be associated with the plasma membrane. Cyclic nucleotide phosphodiesterase in the plasma membrane fraction as well as the axonemal fraction hydrolyzed both cyclic GMP and cyclic AMP; however, the rates of hydrolysis for cyclic GMP were obviously higher than those for cyclic AMP. The enzymic properties of guanylate cyclase and cyclic nucleotide phosphodiesterase in sperm flagella were also briefly described.     

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.     

Subcellualr localizations of guanylate cyclase and 3′,5′-cyclic nucleotide phophodiesterase in sea urchin sperm

The subcellular localizations of guanylate cyclase and 3′,5′-cyclic nucleotide phophodiesterase in sea urchin sperm were examined. Both the specific and total activities of these two enzymes were much higher in sperm flagella (tails) than in the heads. In addition to the observation that guanylate cyclase in the flagella was particulate-bound and solubilized by Triton X-100, more than 980% of the cyclase activity in the flagella was found in the plasma membrane fraction, whereas the activity of cyclic nucleotide phosphodiesterase was observed in both the axonemal and plasma membrane fractions. The observations indicated that the cyclase in the flagella appeared to be associated with the plasma membrane. Cyclic nucleotide phosphodiesterase in the plasma membrane fraction as well as the axonemal fraction hydrolyzed both cyclic GMP and cyclic AMP; however, the rates of hydrolysis for cyclic GMP were obviously higher than those for cyclic AMP. The enzymic properties of guanylate cyclase and cyclic nucelotide phosphodiesterase in sperm flagella were also briefly described.     

Adenylate cyclase in cilia from Paramecium: Localization and partial characterization

A particulate adenylate cyclase was identified in the excitable ciliary membrane from Paramecium tetraurelia. MnATP was preferentially used as substrate, the K_m was 67 μM, V_max was 1 nmol cAMP.min^?1.mg^?1, a marked temperature optimum of 37°C was observed. Adenylate cyclase was not inhibited by 100 μM EGTA or 100 μM La³⁺, whereas under these conditions guanylate cyclase activity was abolished. Fractionation of ciliary membrane vesicles by a Percoll density gradient yielded two vesicle populations with adenylate cyclase activity. In contrast, calmodulin/Ca-dependent guanylate cyclase was associated with vesicles of high buoyant density only.     

Studies On Cyclic Nucleotide Metabolism In Tetrahymena Pyriformis: Partial Characterization of Cyclic Amp- and Cyclic Gmp-Dependent Phosphodiesterases*

SYNOPSIS. Cyclic nucleotide phosphodiesterase [EC 3.1.4.17] was examined in Tetrahymena pyriformis strain NT-1. Enzymic activity was associated with the soluble and the particulate fractions, whereas most of the cyclic GMP phosphodiesterase activity was localized in the soluble fraction: the activities were optimal at pH 8.0–9.0. Although very low activities were detected in the absence of divalent cations, they were significantly increased by the addition of either Mg²⁺ or Mn^2-. A kinetic analysis of the properties of the enzymes yielded 2 apparent K_III values ranging in concentration from 0.5 to 50 μM and from 0.1 to 62 μ M for cyclic AMP and GMP. respectively. A Ca²⁺-dependent activating factor for cyclic nucleotide phosphodiesterase was extracted from Tetrahymena cells, but this factor did not stimulate guanylate cyclase [EC 4.6.1.2] activity in this organism. On the other hand, Tetrahymena also contained a protein activator which stimulated guanylate cyclase in the presence of Ca²⁺, although this activator did not stimulate the phosphodiesterase. the results suggested that Tetrahymena might contain 2 types of Ca²⁺-dependent activators, one specific for phosphodiesterase and the other for guanylate cyclase.     

Activation of the renal cortical and hepatic guanylate cyclase-guanosine 3′,5′-monophosphate systems by nitrosoureas divalent cation requirements and relationship to thiol reactivity

Streptozotocin, 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) and N-methyl nitrosourea, compounds with both oncogenic and cytotoxic properties, increased guanylate cyclase activity in the 100 000 × g soluble fractions of rat renal cortex and liver 35- to 65-fold over basal values. Particulate enzyme activities of these tissues were increased 2- to 4-fold by a maximally effective concentration of the nitrosoureas. In the presence of the cyclic nucleotide phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine, maximally effective concentrations of these nitrosoureas increased cyclic GMP accumulation of hepatic and renal cortical slices to peak levels 7- to 10-fold over control in 30 min. By contrast, with the structurally related carcinogen N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) peak increases occurred in 5–10 min and were 40- to 70-fold over control levels in renal cortex and liver, respectively. Unlike the Ca²⁺-dependent actions of cholinergic stimuli on cyclic GMP, the nitrosoureas and MNNG increased cyclic GMP in either the presence or absence of extracellular Ca²⁺. Moreover, while basal soluble guanylate cyclase of renal cortex was highly Mn²⁺-dependent and decreased 85% when either Mg²⁺ or Ca²⁺ was employed as sole divalent cation in reaction mixtures, the actions of nitrosoureas on enzyme activity were well expressed with either Mn²⁺ or Mg²⁺, but not with Ca²⁺, as sole divalent cation. Improved utilization of Mg²⁺ by guanylate cyclase in the presence of nitrosoureas would favor enhanced enzyme activity under cellular conditions where Mg²⁺ is abundant. In the presence of maximally stimulatory concentrations of streptozotocin or BCNU, high concentrations of Mg²⁺ or Mn²⁺ further increased soluble guanylate cyclase, suggesting important differences in metal and nitrosourea stimulation of enzyme activity.Preincubation of supernatant fractions with nitrosoureas plus dithiothreitol inhibited the action of the N-nitroso compounds to increase renal cortical guanylate cyclase. Glutathione and cysteine were also inhibitory, but less effective than dithiothreitol. Initial incubation of nitrosoureas with dithiothreitol in buffer alone similarly suppressed the subsequent action of the N-nitroso compounds on guanylate cyclase, and implicated direct chemical interactions. Prior incubation of renal cortical supernatant fractions with the SH blockers N-ethylmaleimide or maleimide significantly suppressed guanylate cyclase activation mediated by streptozotocin or BCNU. Direct drug interactions seemed unlikely, since effects of the inhibitors were optimally expressed by initial exposure of the supernatant fraction of tissue to the SH blockers and were not potentiated by a 30 min preincubation of the SH blockers and nitrosoureas in buffer alone.Thus, nitrosoureas activate and alter the metal requirements of soluble guanylate cyclase and increase cellular cyclic GMP in the presence or absence of extracellular Ca²⁺. Activation of soluble guanylate cyclase by nitrosoureas may involve an interaction of these agents with tissue SH groups, and possibly SH to SS transformation. Stimulation of the guanylate cyclase system by nitrosoureas could be related to the oncogenic actions of these agents.     

Blockade by N-methylhydroxylamine or activation of guanylate cyclase and elevations of guanosine 3′,5′-monophosphate levels in nervous tissues

Hydroxylamine and N-methylhydroxylamine prevented the activation of soluble guanylate cyclase by the endogenous activator as well as by nitroso compounds such as N-methyl-N′-nitro-N-nitroguanidine or nitroprusside, while the other derivaties of hydroxylamine were ineffective. Hydroxylamine and N-methylhydroxylamine did not alter the basal guanylate cyclase activity of purified enzyme preparations. Kinetics analysis indicated that N-methylhydroxylamine competes with N-methyl-N′nitro-N-nitrosuguanidine for guanylate cyclase. The activation of guanylate cyclase by N-methyl-N′-nitro-N-nitrosoguanidine and its inhibition by N-methylhydroxylamine were reversible reactions. These efects of N-methyl-N′-nitro-N-nitrosoguanine and N-methylhydroxylamine were observed with guanylate cyclase from other tissues.N-Methylhydroxylamine preveneed the increase of guanosine 3′,5′-monophosphate (cyclic GMP) levels in cerebellar slices of guinea pig by N-methyl-N′-nitro-N-nitroguanidine, veratridine and adenosine, while the elevalations of adenosine 3′,5′-monophosphate by these agents were not affected. N-Methylhyroxylamine also blocked the increased of cyclic GMP levels by carbachol, prostaglandin E₁ and N-methyl-N′-nitro-N-nitrosoguanidine in neuroblastoma N1E 115 cells. Thus N-methylhydroxylamine prevents the activation of guanylate cyclase and the increased synthesis of cyclic GMP in responses to transmitters without blocking the synthesis of cyclic GMP via basal enzyme activity.