Biochem. J. (1977) 162,483491 Printed in Great Britain


Adenylate Cyclase Activity in Lymphocyte Subcellular Fractions CHARACTERIZATION OF A NUCLEAR ADENYLATE CYCLASE By H. JAMES WEDNER and CHARLES W. PARKER Department ofMedicine, Washington University School ofMedicine, St. Louis, MO 63110, U.S.A.

(Received 5 July 1976) Nuclei from purified human peripheral lymphocytes were prepared by incubations with Triton X-100 to disrupt the cells, followed by sucrose-density gradient centrifugation. The nuclei were pure as judged by phase-contrast microscopy and had low contents of non-nuclear marker enzymes. In addition, nuclei prepared from lymphocytes surface-labelled with 125I had only 2-7 % of the radioactivity bound to intact lymphocytes. At 3.3mM-Ca2+ and 10M-ATP a fluoride-sensitive adenylate cyclase was demonstrated in nuclei prepared in 0.2% Triton X-100 or 0.33 % Triton X-100. There was linear accumulation of cyclic AMP for 10min in both preparations. The apparent Km for ATP was 90,uM. Adenylate cyclase activity was augmented by 1.0mM-Mn2+ and inhibited at higher concentrations. Ca2+ showed two peaks of stimulation, at 1.0-2.5mM- and above 10mM-Ca2+. Mg2+ was inhibitory at all concentrations. EDTA or EGTA only slightly decreased adenylate cyclase activity, suggesting that another metal ion may be necessary for activity. Adenylate cyclase activity was stimulated by 10mM-isoproterenol and 10,uM-adrenaline in the presence of a phosphodiesterase inhibitor. Phytohaemagglutinin and prostaglandin E1 alone or in combination with isoproterenol had no effect on nuclear adenylate cyclase activity in either nuclei preparation. These results indicate that human lymphocyte nuclei contain one or several adenylate cyclases which differ from adenylate cyclases found in other subcellular fractions of these cells with regard to their bivalentcation requirements and responsiveness to pharmacological agents. Fluoride-sensitive adenylate cyclase activity has been reported in rat ventral prostate nuclei (Liao et al., 1971), and glucagon- and fluoride-sensitive adenylate cyclases have been demonstrated in hepatic nuclei (Soifer & Hechter, 1971). Although rigorous control experiments to exclude the existence of cytoplasmic or plasma-membrane contamination of the nuclear preparations were not presented, these observations are consistent with other evidence indicating that eukaryotic nuclei contain cyclic AMPdependent protein kinases (Langan, 1973; Castagna et al., 1975), raising the possibility that nuclear adenylate cyclases may be involved in the modulation of nuclear function. By using an immunocytotechnical technique for the localization of cyclic AMP (Wedner et al., 1972), we have demonstrated that isoproterenol and adrenaline produce local increases in cyclic AMP in the nucleus of intact human peripheral blood lymphocytes (Wedner et al., 1975a), suggesting that the enzymic machinery for generating cyclic AMP is present in the nuclei of these cells as well. We now report the results of studies of adenylate cyclase in isolated nuclei from these cells. Evidence strongly supporting the existenc Vol. 162

of a highly active adenylate cyclase in human lymphocyte nuclei is presented.

Materials and Methods Materials Reagents and their sources were as follows: Triton X-100, bovine lactoperoxidase, L-adrenaline, and DLnoradrenaline (Sigma Chemical Co., St. Louis, MO, U.S.A.); carrier-free 1251 (Industrial Nuclear Co., St. Louis, MO, U.S.A.); foetal calf serum and Gey's solution (Grand Island Biologicals, Grand Island, NY, U.S.A.); AG1-X2 resin (Bio-Rad, Richmond, CA, U.S.A.); Instagel (Packard, Downers Grove, IL, U.S.A.); L-propranolol (Aldrich, Milwakuee, WI, U.S.A.). Other reagents and their sources are given in the preceding paper (Snider & Parker, 1977). Bivalent cations, propranolol, noradrenaline hydrochloride and theophylline were dissolved directly in 0.25M-sucrose/3.3M-CaC12 (sucrose/Ca2+ medium). Isoproterenol and erythroagglutinating phytohaemagglutinin were dissolved in 0.1M-NaCl. Adrenaline was dissolved at 10mM in 0.1 M-HCI and diluted further in 04 1-NaCl. Prostaglandin Es was


dissolved at 10mg/ml in 95% (v/v) ethanol and diluted to 1 mg/mln 0.1 % Na2C03. Further dilutions were made in 0.1 M-NaCI. NaF, EDTA and EGTA were dissolved in water. Preparation of nuclei

Peripheral lymphocytes from normal human volunteers were prepared by dextran sedimentation and isopycnic centrifugation on a Ficoll/Hypaque gradient as described in the preceding paper (Snider & Parker, 1977). After two washes with 0.15MNaCI/0.01 M-phosphate, pH7.35 (NaCi/phosphate buffer) the lymphocytes were suspended in sucrose/ Ca2+ medium and chiDled to 4°C. All further purification steps were carried out at this temperature. The nuclei were isolated by a modification of the methods of Darzynkiewicz & Jacobson] (1971) and Blobel & Potter (1966). The cells were suspended in sucrose/Ca2+ medium at a density of 106x 106/ml, and an equal volume of sucrose/Ca2+ -medium containing various concentrations of Triton X-400 (usually 0.3 or 0.5%) was added. The suspension was agitated in a VQrtex mixer for 30s and rapidly diluted with 7vol. of sucrose/a2+ medium. Nuclei -and incompletely, disrupted cells were sedimented at lOOgma1. for 4min. The pellets were resuspended in the original volume of sucrose/Ca2+ medium. Then 2vol. of 0.3 or 0.5% Triton X-100 in sucrose/Ca2+ medium were added and the agitation and washing were repeated. After a final wash in sucrose/Ca2+ buffer, the nuclei were resuspended in sucrose/Ca2+ at 10x106-20x106/ml. Then 4ml of the nuclear suspension was underlaid with 2ml of 2.25M-sucrose containing 33mm-Wa2+ in 6ml titrocellulose tubes. The mixture was centrifuged for 3Ominat 10500g.... in a Beckman L5-65B ultracentrifuge. The pellet was resuspended in 30ml of sucrose/Ca2+ medium and washed once with sucrose/Ca2+ medium. The nuclei were counted in a haemocytometer under phase-contrast iluination. The yield of nuclei Was 70.90% of the starting number of purified lymphocytes. Enzyme and protein measurements

Protein,- cytochrome- oxidase, 5'-nucleotidase and NADH oxidoreductase were measured as described in the pecding paper ($nider & Paker 1977). Appropriate controls were caried out,to exclude effects of residual Ca2+ and sucrose in the enzyme assays. Lymphocyte subcellular fractions prepared as described in the preceding paper (Snider &

Parkers, 1977) were used as enzyme standards.

Radioiodination of intact cells As an aid in evaluating the degree of contamination of the nuclear preparations by plasma


membrane, nuclei were prepared from cells that had been enzymically radioiodinated by the technique of Marchalonis et al. (1971) before addition of Triton X-100. Because of possible non-specific lymphocyte damage during iodination and covalent labelling with 1251 inside cells, procedures leading to either light or intermediate degrees of iodination were used. The extent ofiodination was controlled by varying the quantity of 125I and H202 and the incubation time. For light iodination, 40 x 106 lymphocytes in 2mn of NaCI/phosphate buffer were mixed with 80,1 of lactoperoxidase solution (0.3mg/ml in water) and 80p1 of 125J solution (approx. 6 x 108c.p.m./ml) in plastic 'tbes at room temperature. Four 50/d additions of 3 mM-H202 were made at 70s intervals. For heavier iodination a sixfold higher amount of 1251 was used and eight additions of 3mM-H202 were made at 30s intervals. Then 1 min after -the final addition, of H202 the reactions were stopped by the addition of lOml of cold Gey's solution containing 5 % foetal calf serum. All subsequent steps were performed at 4°C. The cells were harvested by centrifugation at lOOOg for 5min. The cell pellet was resuspended in 1 ml of Gey's solution containing 5% (v/v) foetal calf serum and 5ml of cell suspensionwas layered over lOml of 100% foetal calf serum and tubes were centrifuged at lOOOg for lOmin. This procedure was repeated a second time. Finally the cells were washed once in Gey's solution and once in sucrose/Ca2+ medium. A sample was removed for determination of 125I bound to the cells. The nuclei were isolated from the remaining cells as described above and the nuclear preparations were examined for residual 125j. 125I was determined in a Nuclear-Chicago auto-gamma counter. Incubation conditions for nuclei Washed nuclear preparations were suspended in sucrose/Ca2+ medium at a density of 5 x 106 or lOx l00nuclei/ml and maintained at 4°C. Solutions of stimulatory agents, ATP, bivalent cations, chelating agents and buffer control solutions were added to chilled glass tubes (l0mmx 75mm) in afinal volume of 0.2ml or less followed by the addition of 0.5m1l of nuclear suspension (at 40C). This sequence of addition was particularly important with solutions containing EDTA, HTA or Mg2+, which cause nuclear clumping, making it impossible to dispense the nuclei accurately into tubes once mixing has: occurred. The reaction mixtures were incubated at 370C for various periods of time. The reaction was terminated by centrifuging the nuclei at 2200g for 2min, decanting the supernatant and freezing the pellet rapidly in ethanol cooled with solid C02. In selected experiments thd supernatant also was frozen and analysed. All incubations were 1977

NUCLEAR ADENYLATE CYCLASE done in duplicate or triplicate unless otherwise noted. The results presented are the means of three or more experiments. Intra-experimental variability was usually less than 10% and the standard deviation for three experiments was 15 % or less. Assay of cyclic AMP

Frozen nuclear pellets or supematants were stored at-90°C until assay. Samples were dilutedwith 50mM-sodium acetate buffer, pH6.10, boiled, sonicated (lOs at 35% of output, Bronson Biosonik III, Bronwill Scientific, Rochester, NY, U.S.A.), and evaluated by radioimmunoassay as described in the preceding paper (Snider & Parker, 1977). In experiments in which EDTA or EGTA was used, the acetate buffer contained 5mM-CaCh. In the absence of excess of Ca2+, these two agents inhibited the binding of cyclic AMP and iodinated cyclic AMP marker to the anti-(cyclic AMP) antibody. This could be completely reversed by the addition of Ca2+ to the assay mixture. None of the other agents interfered with the radioimmunoassay of cyclic AMP. The immunoassay was validated by adding known amounts of cyclic AMP to nuclear pellets or extracts, by incubation of samples with cyclic nucleotide phosphodiesterase and column chromatography in the presence of tracer amounts of cyclic [3H]AMP (Wedner et al., 1975b).

Effect of Triton X-100 concentrations Essentially pure nuclear preparations with adenylate cyclase activity were obtained by successive treatments with-0.15 and 0.2% Triton X-100 (0.2% Tx nuclei) or with 0.25 and 0.33% Triton X-100 (0.33% Tx nuclei) followed by sucrose-densitygradient centrifugation. These two preparations had comparable resting cyclic AMP concentrations (6-12pmol/107 nuclei), but their responses to fluoride and catecholamines-differed markedly. The 0.33 % Tx nuclei had a threefold greater response to fluoride but a considerably decreased (or absent) response to catecholamines. For this reason most of the subsequent experiments with fluoride were


carried out with 0.33% Tx nuclei, whereas with catecholamines 0.2% Tx nuclei were used. Nuclei obtained at final Triton X-100 concentrations above 0.33 % had greatly decreased or absent adenylate cyclase activity. The effect of Triton X-100 on responsiveness to fluoride and catecholamine in lymphocyte nuclei is similar to that observed with detergents in other tissues, where the typical result is an increase in fluoride responsiveness and a decrease in hormonal responsiveness (Bimbaumer et al., 1971). Nuclear purity

After purification as described above, no 5'nucleotidase, NADH oxidoreductase or cytochrome c oxidase activity was found in sonicated or intact purified nuclei (enzyme activities of less than 1 % of those present in whole lymphocyte sonicates when expressed per cell or per nucleus). However, it was possible that marker-enzyme inactivation might have occurred during the exposure of the lymphocytes to detergent, or because of failure to remove residual Triton X-100, giving a falsely low estimate of nuclear contamination. This proved not to be the case. Plasma-membrane and microsomal-rich fractions were obtained by homogenization and densitygradient centrifugation (method B) as described in the preceding paper (Snider & Parker, 1977). When fractions enriched in plasma membranes were combined and exposed to 0.33% Trition X-100 in sucrose/Ca2+ medium the 5'-nucleotidase activity increased from 180nmol/min per mg to 310nmol/min per mg in membranes with 0.33 % Triton X-100 in sucrose/Ca2+ medium. When fractions PF-2 and PF-3 (enriched in microsomal particles) were combined and studied in the same way for changes in NADH oxidoreductase activity, no effect of the detergent was observed. As another control for plasma-membrane contamination, cells were radioiodinated by the lactoperoxidase technique before disruption and nuclear purification. Since this procedure selectively iodinates the cell surface, any radioactivity associated with isolated nuclei would presumably be of plasmamembrane origin. The results of two experiments are shown in Table 1. When cells are heavily iodinated

Table 1. Iodinatlonofhuman lymphocytes andresidualiodinepresent on washed lymphocyte nucki Purified human peripheral lymphocytes were iodinated by the technique of Marchalonis et al. (1971) as described in the text. Nuclei were then isolated from the iodinated lymphocytes using 0.2% Triton X-100. Expt. 1, intermediate iodination, 1.84% residual iodine counts; Expt. 2, light iodination, 7.14% residual iodine counts.

Expt. 1 Radioactivity added (c.p.m./107 cells) 29.5 x 106 Radioactivity bound to washed cells (c.p.m./107 cells) 3.8 x 105 Radioactivity bound to washed 0.2% Tx nuclei (c.p.m./107 nuclei) 7x103

Vol. 162

Expt. 2 5.Ox 106 2.1 x 104 1.5 x 103


486 Table 2. Cyclic AMP accumulation in washed lymphocyte nuclei from iodinated human lymphocytes Nuclei from Expt. 2, Table 1, containing 7.14% residual 1251 counts, were incubated with 10mMNaCl, lOnM-isoproterenol or 1 M-isoproterenol as described and cyclic AMP accumulation was determined as described in the text. Cyclic AMP (pmol/107 nuclei) 13.7 lOmM-NaCI (control) 104.0 lOmM-Isoproterenol 52.0 1 mM-Isoproterenol

140 r0

120 1 100 t-


60 [ -4t

Table 3. Effect of fluoride and ATP on cyclic AMP response in human lymphocyte nuclei The indicated agents or lOmM-NaCl, (control) were incubated with 5.0x 106 0.2% Tx nuclei for 15min. Cyclic AMP accumulation was measured as described in-the text. Results are means±S.E.M. for three experiments. Cyclic AMP Stimulating agent (pmol/107 nuclei) 9.6+ 2.0 Control 13.1+ 2.3 Fluoride (10mM) 23.0+ 5.0 ATP (1 mM) 115.5+23.1 Fluoride (lOmM)+ATP (1 mM)

(Expt. 1) only 2% of the radioactivity bound to intact lymphocytes was found in the final washed nuclear preparation. When the cells were lightly iodinated 7 % of the radioactivity was associatetwith the nuclei. The nuclei from this experiment -were stimulated with lOmm-isoproterenol in the absence of ATP (see below). The intranuclear concentration of cyclic AMP rose from 13.7 to 104pmol/107 nuclei (Table 2), which is equivalent to the degree of stimulation of cyclic AMP accumulation by isoproterenol in the same number of intact cells (Parker & Smith, 1973). fluoride stimulation In nuclei prepared in medium with final concentrations of either 0.2 or 0.33% Triton X-100. and incubated with fluoride and ATP, there. was a timerelated increase in intranuclear cyclic AMP. In the absence of ATP no increase in cyclic AMP occurred (Fig. 1 and Table 3). The response to fluoride was linear with time for at least lOmin (15min with 0.2% Tx nuclei). Intranuclear cyclic AMP increased to a lesser extent in nuclei not incubated at 37°C presumably owing to cyclic AMP formation during the 2min centrifugation step at room temperature. Almost all of the generated cyclic AMP remained associated with the nuclei, as indicated by a virtual absence of cyclic AMP from the medium. The


40 20 -B-~~~~~~~-

_---_-_-_---_7 -_L


0 0









Time (min) Fig. 1. Tinm-course for cyclic AMP accumulation in .fluoride-stimulated nuclei Incubations were carried out with 5 x 106 0.2% Tx nuclei in the presence of lOnM-NaCl (control, 0), lOmm-NaF (U) or lOnM-NaF+1mM-ATP (o). Reactions were terminated as described in the text.

0.33% Tx nuclei had a much higher cyclic AMP accumulation in response to fluoride than the 0.2% Tx nuclei [276.6 (S.E.M. ± 33.6) and 115.5 (s.E.M.+ 23.1)pmol/15min per 107 nuclei respectively]. The kinetics of cyclic AMP, accumulation were studied in 0.33 % Tx nuclei by using an incubation time of only 3min to assure linearity of the reaction. In the standard sucrosefCa2+medium the apparent Km for ATP was 90juM. An identical K. value was noted in nuclei incubated with 2mM-EDTA and 10mM-Ca2+ (see below). For this reason subsequent experiments were done at ATP concentrations of 100pM or greater. The importance of bivalent cations in adenylate cyclase activity has been demonstrated in a variety of cellular systems. In most of these Mg2+ seems to be necessary for optimum activity, but stimulatory effects of Mn2+ and Ca2+ have also been reported. The Ca2+-containing solution used in the isolation of nuclei was studied initially because it provided the most homogeneous preparation of nuclei and could be used without nuclear clumping. When bivalent cations were added to nuclei suspended in sucrose/Ca2+ medium (Fig. 2), Mn2+ produced the most marked increase in fluoride responsiveness, with maximum stimulation at l mM-Mn2+ concentrations. Higher concentrations of Mn2+ (above 1977



360 280

320 F



__ 280 A

z 200 F ,^ 240 F

9 160 F




120 F-

S 160 F

80 F




120 80 F


40h 0





Bivalent cation (mM) 2. Fig. Effect of bivalent-cation concentration on cyclic AMP accumulation in fluoride-stimulated nucki Incubations were performed with 2.5 x 106 0.33%Y Tx nuclei in the presence of lOmM-NaF, lOO1M-ATP and various concentrations of Ca2+ (O), Mn2+ (0) and Mg2+ (o). The incubations were for 10min at 37°C and the reactions were terminated and cyclic AMP assayed as described in the text. Results are the means of three experiments.

2.5mM) were inhibitory. When Ca2+ was increased above the 3.3 mm-Ca2+ concentrations already present in sucrose/Ca2+ medium, a more complicated pattern emerged. Two peaks of stimulation were observed at total Ca2+ concentrations of 5mM and above 12mM. Mg2+ was inhibitory at all concentrations tested, with maximum inhibition at concentrations in the 1-10mM range. The effects of Mg2+ were not due to changes in ionic strength, since equivalent quantities of NaCl had no effect on adenylate cyclase activity (not shown). Similar results were seen in the presence of EDTA (Fig. 3). EDTA decreased the adenylate cyclase activity (compare Figs. 2 and 3), but the shape of the curves with added Mn2+, Ca2+ and Mg2+ were similar. In this case Mn2+ was clearly the best stimulator at low concentrations. EGTA also caused an overall decrease in adenylate cyclase activity without altering the shape of the bivalentcation curves. The biphasic response seen with Ca2+ and the rise in activity seen with Mn2+ might logically be attriVol. 162







Bivalent cation (mM) Fig. 3. Effect ofEDTA and bivalent cations on cyclic AMP accumulation in fluoride-stimulated nucki Incubations were performed with 2.5x 106 0.33%Y Tx nuclei in the presence of 2mM-EDTA, lOmM-NaF, lOO1uM-ATP and various concentrations of Ca2+ (e), Mn2+ (0) or Mg2+ (0). The incubations were for 1Omin at 37°C, and the reactions were terminated and cyclic AMP was measured as described in the text. Results shown are the means of three experiments.

buted to two separate enzymes with different bivalent-cation requirements. However, the fluoride dose-response curve in sucrose/Ca2+ medium was the same in 2.0mM-EDTA or 2.0mM-EDTA/5mMMg2+ (Fig. 4). Similarly studies of ATP-concentration-dependence of the fluoride response at 3.3 and 1OmM-Ca2+ revealed a difference in the Vmax. value but not in the Km value for stimulation by ATP. Thus, by the above two criteria, no conclusive evidence for two separate enzymes was obtained.

Catecholamine stimulation With 10mm-isoproterenol, there was a marked and consistent increase in cyclic AMP over a lOmin timeperiod, with essentially no change in the control (Table 3). The increase in cyclic AMP was linear with time for approx. 10min, after which the curve paralleled the slow rise in cyclic AMP seen in control nuclei. In contrast with the response to fluoride, the stimulation of cyclic AMP accumulation by isoproterenol did not require exogenous ATP. The addition of ATP had no effect whatsoever on the magnitude or duration of the response, even when the ATP was added after 15mMi, the time at which specific isoproterenol stimulation seems to stop.












Adenylate cyclase activity in lymphocyte subcellular fractions. Characterization of a nuclear adenylate cyclase.

Biochem. J. (1977) 162,483491 Printed in Great Britain 483 Adenylate Cyclase Activity in Lymphocyte Subcellular Fractions CHARACTERIZATION OF A NUCL...
1MB Sizes 0 Downloads 0 Views