Immunological distinction between calmodulin-sensitive and calmodulin-insensitive adenylate cyclases

Previous studies using calmodulin-Sepharose affinity chromatography have suggested that bovine brain may contain a mixture of calmodulin-sensitive and -insensitive adenylate cyclase activities (Wescott, K. R., La Porte, D. C., and Storm, D. R. (1979) Proc. Natl. Acad. Sci. U.S.A. 82, 3086-3090). In this study, mice were immunized with a purified preparation of the calmodulin-sensitive adenylate cyclase from bovine brain, and a polyclonal antiserum was obtained which was specific to the calmodulin-sensitive form of the enzyme. The antiserum was not inhibitory and precipitated enzyme activity from a homogeneous preparation of the calmodulin-sensitive adenylate cyclase catalytic subunit. Furthermore, the antiserum did not interact with calmodulin-insensitive adenylate cyclase which was resolved from the calmodulin-sensitive form of the enzyme by calmodulin-Sepharose affinity chromatography. Since the only polypeptide specifically precipitated by the antiserum had an Mr of 135,000, which was identical to the Mr of the catalytic subunit of the enzyme, it is concluded that the antiserum interacted directly and specifically with the catalytic subunit of the calmodulin-sensitive isozyme of adenylate cyclase. Detergent-solubilized membranes from several rat tissues were examined for the presence of calmodulin-sensitive adenylate cyclase using anti-calmodulin-sensitive adenylate cyclase antiserum. Approximately 40-60% of the total adenylate cyclase activity of rat brain and kidney were immunoprecipitated by the antiserum, whereas liver and testes contained no detectable calmodulin-sensitive adenylate cyclase. Approximately 15% of the total adenylate cyclase activity in rat heart and lung was the calmodulin-sensitive form. These data indicate that the calmodulin-sensitive and insensitive adenylate cyclases from bovine brain are immunologically distinct and support the proposal that there may be two or more distinct adenylate cyclase isozymes in brain.     

A novel mechanism for the inhibition of adenylate cyclase via inhibitory GTP-binding proteins. Calmodulin-dependent inhibition of the cyclase catalyst by the beta gamma-subunits of GTP-binding proteins

The adenylate cyclase catalytic protein partially purified from rat brain membranes was activated by the stimulatory GTP-binding protein (Gs), forskolin, and Ca2+-calmodulin. The Ca2+-calmodulin-stimulated activity was markedly, but the Gs- or forskolin-stimulated activity was essentially not, inhibited by low concentrations of the beta gamma-subunits of the inhibitory GTP-binding protein (Gi). The inhibition appeared to be competitive with calmodulin. On the other hand, the association of increasing amounts of beta gamma with the alpha of Gi, which was measured based on the ADP-ribosylation by islet-activating protein, pertussis toxin, was apparently competed by Ca2+-calmodulin. Furthermore, beta gamma bound to calmodulin-Sepharose in the presence of Ca2+, but not in its absence. Thus, the direct interaction of beta gamma with calmodulin is a likely mechanism involved in beta gamma-induced inhibition of the calmodulin-stimulated adenylate cyclase.     

Domoic acid inhibits adenylate cyclase activity in rat brain membranes

Adenylate cyclase activity measured by the formation of cyclic AMP in rat brain membranes was inhibited by a shellfish toxin, domoic acid (DOM). The inhibition of enzyme was dependent on DOM concentration, but about 50% of enzyme activity was resistant to DOM-induced inhibition. Rat brain supernatant resulting from 105,000×g centrifugation for 60 min, stimulated adenylate cyclase activity in membranes. Domoic acid abolished the supernatant-stimulated adenylate cyclase activity. The brain supernatant contains factors which modulate adenylate cyclase activity in membranes. The stimulatory factors include calcium, calmodulin, and GTP. In view of these findings, we examined the role of calcium and calmodulin in DOM-induced inhibition of adenylate cyclase in brain membranes. Calcium stimulated adenylate cyclase activity in membranes, and further addition of calmodulin potentiated calcium-stimulated enzyme activity in a concentration dependent manner. Calmodulin also stimulated adenylate cyclase activity, but further addition of calcium did not potentiate calmodulin-stimulated enzyme activity. These results show that the rat brain membranes contain endogenous calcium and calmodulin which stimulate adenylate cyclase activity. However, calmodulin appears to be present in membranes in sub-optimal concentration for adenylate cyclase activation, whereas calcium is present at saturating concentration. Adenylate cyclase activity diminished as DOM concentration was increased, reaching a nadir at about 1 mM. Addition of calcium restored DOM-inhibited adenylate cyclase activity to the control level. Similarly, EGTA also inhibited adenylate cyclase activity in brain membranes in a concentration dependent manner, and addition of calcium restored EGTA-inhibited enzyme activity to above control level. The fact that EGTA is a specific chelator of calcium, and that DOM mimicked adenylate cyclase inhibition by EGTA, indicate that calcium mediates DOM-induced inhibition of adenylate cyclase activity in brain membranes. While DOM completely abolished the supernatant-, and Gpp (NH)p-stimulated adenylate cyclase activity, it partly blocked calmodulin-, and forskolin-stimulated adenylate cyclase activity in brain membranes. These results indicate that DOM may interact with guanine nucleotide-binding (G) protein and/or the catalytic subunit of adenylate cyclase to produce inhibition of enzyme in rat brain membranes.     

Activation and stabilization of the catalytic unit of adenylate cyclase.

The requirements for stability and activity of the catalytic unit (C) of adenylate cyclase were investigated. After solubilization of bovine brain membranes in the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]propane-1-sulphonate (Chaps), the catalytic unit was separated from the stimulatory guanine-nucleotide-binding protein (Gs) by gel filtration on Ultrogel AcA-34. The partially purified C unit was rapidly inactivated at 30 degrees C; 0.25 mM-ATP stabilized activity. Although C-unit activity was dependent on Mg2+ or Mn2+, stabilization by ATP did not require bivalent cations. Activity of the Ultrogel-AcA-34-purified C unit was increased by Ca2+ plus calmodulin and by phosphatidylcholine plus lysophosphatidylcholine; activity in the presence of both activators was significantly greater than with each alone. Calmodulin plus Ca2+ and phospholipids also stabilized C unit. The column-purified C unit was activated by forskolin; the effect of forskolin was additive to those of calmodulin plus Ca2+ and phospholipids. p[NH]ppG-stimulated adenylate cyclase activity was reconstituted by mixing samples from the gel-filtration column containing Gs with C unit. Activation by Ca2+ plus calmodulin and Gs plus p[NH]ppG was additive; Ca2+ plus calmodulin did not alter the concentration of p[NH]ppG required for half-maximal activation. Results were similar with forskolin and Gs plus p[NH]ppG; the presence of one activator did not alter the effect of the other. These studies define conditions for separation of C unit and Gs from brain adenylate cyclase and demonstrate that ATP (in the absence of bivalent cations), phospholipids, calmodulin plus Ca2+, and forskolin all interact with C unit in a manner that is independent of functional Gs.     

Immunoprecipitation of adenylate cyclase with an antibody to a carboxyl-terminal peptide from Gs alpha

An antibody (RM) raised against the carboxyl-terminal decapeptide of the alpha subunit of the stimulatory guanine nucleotide regulatory protein (Gs alpha) has been used to study the interaction of Gs alpha with bovine brain adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1]. RM antibody immunoprecipitated about 60% of the solubilized adenylate cyclase preactivated with either GTP-gamma-S or AlF4-. In contrast, RM antibody immunoprecipitated about 5% of the adenylate cyclase not preactivated (control) and 15% of the adenylate cyclase pretreated with forskolin. Adenylate cyclase solubilized from control membranes or GTP-gamma-S preactivated membranes was partially purified by using forskolin-agarose affinity chromatography. The amount of Gs alpha protein in the partially purified preparations was determined by immunoblotting with RM antibody. There was 3-fold more Gs alpha detected in partially purified adenylate cyclase from preactivated membranes than in the partially purified adenylate cyclase from control membranes. Partially purified adenylate cyclase from preactivated membranes was immunoprecipitated with RM antibody and the amount of adenylate cyclase activity immunoprecipitated (65% of total) corresponded to the amount of Gs alpha protein immunoprecipitated. Only 15% of the partially purified adenylate cyclase from control membranes was immunoprecipitated. The presence of other G proteins in the partially purified preparations of adenylate cyclase was investigated by using specific antisera that detect Go alpha, Gi alpha, and G beta. The G beta protein was the only subunit detected in the partially purified preparations of adenylate cyclase and the amount of G beta was about the same in adenylate cyclase from preactivated membranes and from control membranes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Adenylate cyclase from bovine brain cortex: purification and characterization of the catalytic unit.

The non-stimulated (basal) adenylate cyclase from bovine brain cortical membranes was purified 10 000-fold to apparent homogeneity by Lubrol PX extraction and two cycles of affinity chromatography on forskolin-agarose. The final product appears as one major band (mol. wt. 115 000) on SDS-polyacrylamide gels. Further identification was achieved by affinity cross-linking using Gs (stimulatory GTP-binding protein) that was [32P]ADP-ribosylated by cholera-toxin/[32P]NAD: cross-linking with disuccinimidyl suberate gave products with mol. wts. of 160 000, approximately 270 000 and higher. The distribution of these products was dependent on the concentration of cross-linker, suggesting aggregation of two or more adenylate cyclase complexes. In contrast, photo-affinity cross-linking with 4-azidobenzoyl-[32P]Gs yielded a single product with a mol. wt. of 160 000. Purified adenylate cyclase was completely unresponsive towards stimulators (GTP-analogs, NaF) acting via Gs suggesting that this component was removed during purification. On the other hand, stimulation by forskolin and by added activated Gs was preserved but to a smaller degree as compared with the crude enzyme. In contrast, the stimulation of Ca2+/calmodulin was only marginal. Purified adenylate cyclase reversibly bound to wheat germ agglutinin-Sepharose. This suggests that bovine brain adenylate cyclase is a glycoprotein.     

Identification of calmodulin-sensitive and calmodulin-insensitive adenylate cyclase in rat kidney

Adenylate cyclase from rat kidney membranes solubilized with Lubrol-PX, was resolved into calmodulin-insensitive and calmodulin-sensitive forms using DEAE-Sephacel and calmodulin-Sepharose affinity chromatography. The major fraction, 90% of the activity recovered, did not bind to the calmodulin-Sepharose in the presence of Ca2+, and was insensitive to activation by calmodulin. The calmodulin-sensitive enzyme, approximately 10% of the recovered activity, bound to the affinity column and was eluted with buffer containing 2 mM EGTA. In the presence of free Ca2+, calmodulin increased the specific activity of the calmodulin-sensitive adenylate cyclase from 15.2 to 60.4 pmol/mg protein-1 min-1. Maximum stimulation occurred at 0.035-0.076 mM Ca2+. The apparent Ka for calmodulin was 8 nM. The calmodulin-mediated increase in activity was inhibited by trifluoperazine, but not by its analog trifluoperazine-5-oxide. In contrast, trifluoperazine did not inhibit the calmodulin-insensitive activity. The GTP analog, guanyl-5'-yl imidodiphosphate, did not activate either fraction. Furthermore, activation by calmodulin did not require the presence of a guanyl nucleotide. The present finding of a calmodulin-sensitive form of adenylate cyclase in kidney raises the possibility that a calmodulin-mediated mechanism is involved in the formation of cAMP in this organ.     

Direct interaction between the catalytic subunit of the calmodulin-sensitive adenylate cyclase from bovine brain with 125I-labeled wheat germ agglutinin and 125I-labeled calmodulin

A calmodulin-sensitive adenylate cyclase has been purified to apparent homogeneity from bovine cerebral cortex using calmodulin-Sepharose followed by forskolin-Sepharose and wheat germ agglutinin-Sepharose. The final product appeared as one major polypeptide of approximately 135,000 daltons on sodium dodecyl sulfate-polyacrylamide gels. This polypeptide was a major component of the protein purified through calmodulin-Sepharose. The catalytic subunit was stimulated 3-4-fold by calmodulin (CaM) with a turnover number greater than 1000 min-1 and was directly inhibited by adenosine. The catalytic subunit of the enzyme interacted directly with 125I-CaM on a sodium dodecyl sulfate-polyacrylamide gel overlay system, and this interaction was Ca2+ concentration dependent. In addition, the catalytic subunit was shown to directly bind 125I-labeled wheat germ agglutinin using a sodium dodecyl sulfate-polyacrylamide gel overlay technique, and N-acetylglucosamine inhibited binding of the lectin to the catalytic subunit. Calmodulin did not inhibit binding of wheat germ agglutinin to the catalytic subunit, and the binding of calmodulin was unaffected by wheat germ agglutinin. These data illustrate that the catalytic subunit of the calmodulin-sensitive adenylate cyclase is a glycoprotein which interacts directly with calmodulin and that adenosine can inhibit the enzyme without intervening receptors or G coupling proteins. It is concluded that the catalytic subunit of adenylate cyclase is a transmembrane protein with a domain accessible from the outer surface of the cell.     

Purification and characterization of c-Ki-ras p21 from bovine brain crude membranes

In the present studies, we attempted to purify the native molecular forms of the c-ras proteins (c-ras p21s) from bovine brain crude membranes and separated at least three GTP-binding proteins (G proteins) cross-reactive with the antibody recognizing all of Ha-, Ki-, and N-ras p21s. Among them, one G protein with a Mr of about 21,000 was highly purified and characterized. The Mr 21,000 G protein bound maximally about 0.6 mol of [35S]guanosine 5'-(3-O-thio)triphosphate (GTP gamma S)/mol of protein with a Kd value of about 30 nM. [35S]GTP gamma S-binding to Mr 21,000 G protein was inhibited by GTP and GDP, but not by other nucleotides such as ATP, UTP, and CTP. [35S]GTP gamma S-binding to Mr 21,000 G protein was inhibited by pretreatment with N-ethylmaleimide. Mr 21,000 G protein hydrolyzed GTP to liberate Pi with a turnover number of about 0.01 min-1. Mr 21,000 G protein was not copurified with the beta gamma subunits of the G proteins regulatory for adenylate cyclase. Mr 21,000 G protein was not recognized by the antibody against the ADP-ribosylation factor for Gs. The peptide map of Mr 21,000 G protein was different from those of the G proteins with Mr values of 25,000 and 20,000, designated as smg p25A and rho p20, respectively, which we have recently purified from bovine brain crude membranes. The partial amino acid sequence of Mr 21,000 G protein was identical with that of human c-Ki-ras 2B p21. These results indicate that Mr 21,000 G protein is bovine brain c-Ki-ras 2B p21 and that c-Ki-ras 2B p21 is present in bovine brain membranes.     

Purification and characterization of a calmodulin-sensitive adenylate cyclase from Bordetella pertussis

Bordetella pertussis, the bacterium responsible for whooping cough, releases a soluble, calmodulin-sensitive adenylate cyclase into its culture medium. B. pertussis mutants deficient in this enzyme are avirulent, indicating that the adenylate cyclase contributes to the pathogenesis of the disease. It has been proposed that B. pertussis adenylate cyclase may enter animal cells and increase intracellular adenosine cyclic 3',5'-phosphate (cAMP) levels. We have purified the enzyme extensively from culture medium using anion-exchange chromatography in the presence and absence of calmodulin and gel filtration chromatography. The enzyme was purified 1600-fold to a specific activity of 608 mumol of cAMP min-1 mg-1 and was free of islet activating protein. The molecular weight of the enzyme was 43 400 in the absence of calmodulin and 54 200 in the presence of calmodulin. The Km of the bacterial enzyme for adenosine 5'-triphosphate was 2.0 mM, whereas the Km of the calmodulin-sensitive adenylate cyclase from bovine brain was 0.07 mM. Although the enzyme was not purified to homogeneity, its turnover number of 27 000 min-1 is the highest documented for any adenylate cyclase preparation.     

Calmodulin-stimulated cyclic nucleotide phosphodiesterases in plasma membranes of bovine epididymal spermatozoa

Cyclic nucleotide phosphodiesterase in the plasma membranes of bovine epididymal spermatozoa was stimulated by added Ca2+ and calmodulin. The rate of hydrolysis and responsiveness toward calmodulin was greater for cAMP than for cGMP. The kinetic analysis of the activity revealed two forms of phosphodiesterase with apparent Km values of 7.5 and 95 microM for cAMP. Calmodulin stimulated both of the activities by increasing the Vmax without affecting the Km's. The activity response with respect to Ca2+ concentration appears to be biphasic in both the absence and presence of added calmodulin. Trifluoperazine inhibited the Ca2+- and calmodulin-sensitive enzyme activity in a dose-dependent manner. The calmodulin-stimulated phosphodiesterase activity in the sperm plasma membranes can be solubilized and absorbed to a Calmodulin-Sepharose affinity column in the presence of Ca2+.     

Purification of the catalyst of adenylate cyclase

The catalytic moiety of hormone-sensitive adenylate cyclase has been purified from bovine brain. It is isolated largely without its guanine nucleotide-binding regulatory protein, Gs, by affinity chromatography on 7-O-hemisuccinyldeacetylforskolin-agarose. It appears to be a single polypeptide which migrates on sodium dodecyl sulfate-polyacrylamide gels with an apparent Mr of approximately 120,000. When subjected to electrophoresis on gradient (5-10%) sodium dodecyl sulfate-polyacrylamide gels, it displays a larger apparent Mr of 150,000. The adenylate cyclase activity of the preparation can be stimulated by the addition of Gs, forskolin, or calcium-calmodulin. The preparation has been reconstituted with purified beta-adrenergic receptors and Gs to form a hormone-stimulated adenylate cyclase system (May, D., Ross, E.M., Gilman, A.G., and Smigel, M.D. (1985) J. Biol. Chem. 260, 15829-15833). In contrast to its stimulation by Gs, inhibition by the alpha subunits of Gi and Go, G proteins known to be coupled to inhibitory receptors (Sternweis, P., and Florio, V. (1985) J. Biol. Chem. 260, 3477-3483), is not seen. Preparations of adenylate cyclase show varying degrees of inhibition by added G protein beta . gamma subunit. This inhibition can be explained as reflecting a variable, small (under 5%) contamination of the preparation by Gs alpha which would be deactivated by complexing with the added beta . gamma subunit.     

Characterization of a Ca2+-calmodulin-stimulated cyclic GMP phosphodiesterase from bovine brain

A calmodulin-stimulated form of cyclic nucleotide phosphodiesterase from bovine brain has been extensively purified (1000-fold). Its specific activity is approximately 4 mumol min-1 (mg of protein)-1 when 1 microM cGMP is used as the substrate. This form of calmodulin-sensitive phosphodiesterase activity differs from those purified previously by showing a very low maximum hydrolytic rate for cAMP vs. cGMP. The purification procedure utilizing ammonium sulfate precipitation, ion-exchange chromatography on DEAE-cellulose, gel filtration on Sephacryl S-300, isoelectric focusing, and affinity chromatography on calmodulin-Sepharose and Cibacron blue-agarose results in a protein with greater than 80% purity with 1% yield. Kinetics of cGMP and cAMP hydrolysis are linear with Km values of 5 and 15 microM, respectively. Addition of calcium and calmodulin reduces the apparent Km for cGMP to 2-3 microM and increases the Vmax by 10-fold. cAMP hydrolysis shows a similar increase in Vmax with an apparent doubling of Km. Both substrates show competitive inhibition with Ki's close to their relative Km values. Highly purified preparations of the enzyme contain a major protein band of Mr 74 000 that best correlates with enzyme activity. Proteins of Mr 59 000 and Mr 46 000 contaminate some preparations to varying degrees. An apparent molecular weight of 150 000 by gel filtration suggests that the enzyme exists as a dimer of Mr 74 000 subunits. Phosphorylation of the enzyme preparation by cAMP-dependent protein kinase did not alter the kinetic or calmodulin binding properties of the enzyme. Western immunoblot analysis indicated no cross-reactivity between the bovine brain calmodulin-stimulated gGMP phosphodiesterase and the Mr 60 000 high-affinity cAMP phosphodiesterase present in most mammalian tissues.     

The interaction of Ca2+ with the calmodulin-sensitive adenylate cyclase from Bordetella pertussis

Bordetella pertussis, the etiologic agent of whooping cough, produces a calmodulin-sensitive adenylate cyclase which elevates intracellular cAMP in a variety of eucaryotic cells. Exogenous calmodulin added to the partially purified adenylate cyclase has been shown to inhibit invasion of animal cells by this enzyme (Shattuck, R. L., and Storm, D. R. (1985) Biochemistry 24, 6323-6328). In this study, several properties of the calmodulin-sensitive adenylate cyclase are shown to be influenced by Ca2+ in the absence of calmodulin. The presence or absence of Ca2+ during QAE-Sephadex ion exchange chromatography produced two distinct chromatographic patterns of adenylate cyclase activity. Two different forms of the enzyme (Pk1 and Pk2EGTA) were isolated by this procedure. Pk1 adenylate cyclase readily elevated intracellular cAMP levels in mouse neuroblastoma cells (N1E-115) while Pk2EGTA adenylate cyclase had no effect on cAMP levels in these cells. Gel exclusion chromatography of Pk1 adenylate cyclase gave apparent Stokes radii (RS) of 43.5 A (+/- 1.3) in the presence of 2 mM CaCl2 and 33.8 A (+/- 0.94) in the presence of 2 mM EGTA [( ethylenebis (oxyethylenenitrilo)]tetraacetic acid). These Stokes radii are consistent with molecular weights of 104,000 (+/- 6,400) and 61,000 (+/- 3,600), respectively. Pk2EGTA adenylate cyclase had an apparent RS of 33.0 (+/- 1.2) (Mr = 60,600 (+/- 2,800] in the presence of Ca2+ or excess EGTA. At 60 degrees C, Pk1 adenylate cyclase exhibited a Ca2+-dependent heat stability with a half-life for loss of enzyme activity of 10.3 min in 5 mM CaCl2 and a half-life of 2.8 min in the presence of 0.1 microM CaCl2. The stability of Pk2EGTA adenylate cyclase was not affected by changes in free Ca2+. The adenylate cyclase preparations described above were submitted to sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, and enzyme activity was recovered from gel slices by extraction with detergent containing buffers. The catalytic subunit isolated from SDS-polyacrylamide gels was activated 7-fold in the presence of Ca2+ with maximum activity observed at 1 microM free Ca2+. With both preparations, the apparent molecular weight of the catalytic subunit on SDS gels was 51,000 in the presence of 2 mM CaCl2 and 45,000 in the presence of 2 mM EGTA. The catalytic subunit of the enzyme was purified to apparent homogeneity by preparative SDS-polyacrylamide gel electrophoresis and resubmitted to SDS gel electrophoresis in the presence or absence of free Ca2+. The purified catalytic subunit also exhibited a Ca2+-dependent shift in its mobility on SDS gels.(ABSTRACT TRUNCATED AT 400 WORDS)     

Regulation of signal transfer from beta 1-adrenoceptor to adenylate cyclase by beta gamma subunits in a reconstituted system

The beta gamma subunits of guanine nucleotide binding proteins from bovine brain and bovine rod outer segments have different structural and immunochemical properties. In spite of these structural differences, beta gamma subunits from these sources have been found to be fully interchangeable in terms of their interaction with alpha subunits of pertussis-toxin-sensitive G proteins. In contrast, however, there are striking differences between these beta gamma subunits with regard to their ability to deactivate fluoride-stimulated Gs. These profound differences were also observed when the interaction of the purified components of the adenylate cyclase system was studied after reconstitution into phospholipid vesicles. Addition of beta gamma purified from bovine brain to vesicles containing beta-receptor and Gs results in a biphasic effect on receptor-stimulated GTPase activity, whereas addition of transducin beta gamma was virtually without any effect. Likewise, beta gamma from bovine brain, but not transducin beta gamma, affected adenylate cyclase activity of a reconstituted system consisting of three purified components (R, Gs, C). Thus, the alpha subunit of Gs, but not the alpha subunits of pertussis-toxin-sensitive G proteins discriminate between structurally different beta gamma subunits.     

Ca2+-dependent regulation of calmodulin binding and adenylate cyclase activation in bovine cerebellar membranes

The binding parameters of 125I-labeled calmodulin to bovine cerebellar membranes have been determined and correlated with the activation of adenylate cyclase by calmodulin. In the presence of saturating levels of free Ca2+ calmodulin binds to a finite number of specific membrane sites with a dissociation constant (Kd) of 1.2 nM. Furthermore, Scatchard analysis reveals a second population of binding sites with a 100-fold lower affinity for calmodulin. The Ca2+-dependence of calmodulin binding and of adenylate cyclase activation varies with the amount of calmodulin present, as can be inferred from the model of sequential equilibrium reactions which describes the activation of calmodulin-dependent enzymes. On the basis of this model, a quantitative analysis of the effect of free Ca2+ and of free calmodulin concentration on both binding and activation of adenylate cyclase was carried out. This analysis shows that both processes take place only when calmodulin is complexed with at least three Ca2+ atoms. The concentration of the active calmodulin X Ca2+ species required for half-maximal activation of adenylate cyclase is very similar to the Kd of the high affinity binding sites on brain membranes. A Hill coefficient of approx. 1 was found for both processes indicating an absence of cooperativity. Phenothiazines and thioxanthenes antipsychotic agents inhibit calmodulin binding to membranes and calmodulin-dependent activation of adenylate cyclase with a similar order of potency. These results suggest that the Ca2+-dependent binding of calmodulin to specific high affinity sites on brain membranes regulates the activation of adenylate cyclase by calmodulin.     

The stimulatory guanine-nucleotide regulatory unit of adenylate cyclase from bovine cerebral cortex. ADP-ribosylation and purification.

Hormonal stimulation of adenylate cyclase from bovine cerebral cortex is mediated by a guanine-nucleotide regulatory protein (Gs). This protein contains at least three polypeptides: a guanine nucleotide-binding alpha s component and a beta X gamma component, which modulates the function of alpha s. The alpha s component from many tissues can be ADP-ribosylated with cholera toxin, but has been unusually difficult to modify in brain. We have improved incorporation of ADP-ribose by including isonicotinic acid hydrazide to inhibit the potent NAD glycohydrolase activity of brain. ADP-ribosylation is further improved by addition of detergent to render the substrates accessible and 20 mM-EDTA to chelate metal ions. Although Mg2+ is absolutely required for activation of adenylate cyclase by the GTP analogue guanosine 5'-[beta gamma-imido]triphosphate (p[NH]ppG), it is not obligatory for p[NH]ppG-stimulated ADP-ribosylation by cholera toxin. Under these conditions, the ADP-ribosylation of brain membranes is not enhanced by a cytosolic protein. We find that there are two major sizes of brain alpha s, which we have named 'alpha sL', with an apparent Mr of 42,000-45,000, and 'alpha sH' with an apparent Mr of 46,000-51,000 depending on the gel-electrophoretic system used. The alpha sL and alpha sH components can incorporate different amounts of ADP-ribose depending on the reaction conditions, so that one or the other may appear to predominate. Thus we show that incomplete ADP-ribosylation by cholera toxin is not a good indication of the relative amounts of alpha s units. Functionally, however, both forms of alpha s appear to be similar. Both forms associate with the catalytic unit of adenylate cyclase, but neither of them does so preferentially. There is an excess of each of them over the amount associated with catalytic unit. We have now substantially purified Gs from brain by a modification of the method of Sternweis et al. [(1981) J. Biol. Chem. 256, 11517-11526] as well as by a new, simplified, procedure. On SDS/polyacrylamide-gel electrophoresis, the purified brain Gs contains both the 45 and 51 kDa alpha s polypeptides revealed by ADP-ribosylation and a beta X gamma component. Activation of purified alpha s by guanine nucleotides or fluoride can be reversed by addition of purified beta X gamma component. The activated form of purified brain Gs has an Mr of 49,000 as determined by hydrodynamic measurements, which is consistent with the idea that the active form of brain Gs is the dissociated one.     

Purification of the calmodulin-sensitive adenylate cyclase from bovine cerebral cortex

A calmodulin-sensitive adenylate cyclase was purified 3000-fold from bovine cerebral cortex using DEAE-Sephacel, calmodulin-Sepharose, and two heptanediamine-Sepharose column steps. The purified enzyme activity was stimulated by calmodulin, forskolin, 5'-guanylyl imidodiphosphate, and NaF. The molecular weight of the protein component was estimated as 328 000 with a smaller form of Mr 153 000 obtained in the presence of Mn2+. The most highly purified preparations contained major polypeptides of 150 000, 47 000, and 35 000 daltons on sodium dodecyl sulfate (SDS) gels. Photoaffinity labeling of the preparation with azido[125I]iodocalmodulin gave one product of 170 000 daltons on SDS gels. It is proposed that the catalytic subunit of the calmodulin-sensitive enzyme is 150 000 +/- 10 000 daltons and that the enzyme exists as a complex of one catalytic subunit and the stimulatory guanyl nucleotide regulatory complex. These data are consistent with the previous report that the catalytic subunit of this enzyme has a molecular weight of 150 000 +/- 10 000 [Andreasen, T.J., Heideman, W., Rosenberg, G.B., & Storm, D.R. (1983) Biochemistry 22,2757].     

Distinct interactions between Ca2+/calmodulin and neurotransmitter stimulation of adenylate cyclase in striatum and hippocampus

1. Ca2+ and cAMP both act as intracellular second messengers of receptor activation. In neuronal tissue, Ca2+ acting via calmodulin can elevate cAMP levels. This regulation by Ca2+ provides a means whereby the elevation of intracellular [Ca2+] might modulate cAMP generation. 2. In the present studies, the impact of the Ca2+/calmodulin regulation on receptor-mediated stimulation of activity is compared in striatum and hippocampus--regions of differing sensitivity to Ca2+/camodulin. Ca2+/calmodulin stimulated striatal and hippocampal adenylate cyclase activity by 1.4- and 2.7-fold respectively, while dopamine and vasoactive intestinal peptide (VIP) stimulated the enzyme activity of these respective regions by 1.3- and 2-fold. 3. In the presence of Ca2+/calmodulin, the dopamine dose-response curve in the striatum was shifted upward, without alteration of the slope of the curve or of the maximal stimulation of activity elicited by dopamine. In the hippocampus, the ability of VIP to stimulate adenylate cyclase activity was reduced by the presence of calmodulin. 4. The dose dependence of these actions of calmodulin was examined. In the striatum, the stimulation of adenylate cyclase activity by 0.1 to 0.3 microM calmodulin obscured dopamine stimulation, while 1 to 10 microM was additive with the dopamine stimulation. In the hippocampus, all concentrations of calmodulin (0.1 to 10 microM) reduced VIP-mediated stimulation of enzyme activity. 5. These data suggest that the ratio of calmodulin-sensitive to calmodulin-insensitive adenylate cyclase activity varies in different rat brain regions and that, in those regions in which this ratio is low (e.g., rat striatum and most peripheral systems), calmodulin- and receptor-mediated activation of adenylate cyclase activity will be additive, while in those systems in which this ratio is high (e.g., most of the central nervous system), calmodulin will reduce receptor-mediated stimulation of enzyme activity.     

Calmodulin plays a dominant role in determining neurotransmitter regulation of neuronal adenylate cyclase

Ca2+, through the mediation of calmodulin, stimulates the activity of brain adenylate cyclase. The growing awareness that fluctuating Ca2+ concentrations play a major role in intracellular signalling prompted the present study, which aimed to investigate the implications for neurotransmitter (receptor) regulation of enzymatic activity of this calmodulin regulation. The role of Ca2+/calmodulin in regulating neurotransmitter-mediated inhibition and stimulation was assessed in a number of rat brain areas. Ca2+/calmodulin stimulated adenylate cyclase activity in EGTA-washed plasma preparations from each region studied--from 1.3-fold (in striatum) to 3.4-fold (in cerebral cortex). The fold-stimulation produced by Ca2+/calmodulin was decreased in the presence of GTP, forskolin, or Mn2+. In EGTA-washed membranes, receptor-mediated inhibition of adenylate cyclase was strictly dependent upon Ca2+/calmodulin stimulation in all regions, except striatum. A requirement for Mg2+ in combination with Ca2+/calmodulin to observe neurotransmitter-mediated inhibition was also observed. In contrast, receptor-mediated stimulation of activity was much greater in the absence of Ca2+/calmodulin. The findings demonstrate that ambient Ca2+ concentrations, in concert with endogenous calmodulin, may play a central role in dictating whether inhibition or stimulation of adenylate cyclase by neurotransmitters may proceed.