Site-specific mutagenesis of the alpha-helices of calmodulin. Effects of altering a charge cluster in the helix that links the two halves of calmodulin

Alteration of residues 82-84 in the alpha-helix that links the two halves of calmodulin results in a differential effect on activator activity. Previous studies (Lukas, T. J., Burgess, W. H., Prendergast, F. G., Lau, W., and Watterson, D. M. (1986) Biochemistry 25, 1458-1464) indicated the importance of positive charge clusters in the calmodulin-binding protein, myosin light chain kinase. This suggested the possible importance of complementary negative charge clusters in calmodulin. By using an efficient cassette mutagenesis approach and a synthetic calmodulin gene (Roberts, D. M., Crea, R., Malecha, M., Alvarado-Urbina, G., Chiarello, R. H., and Watterson, D. M. (1985) Biochemistry 24, 5090-5098), this possibility was directly addressed by engineering a new calmodulin, VU-8 calmodulin, in which the glutamate cluster at residues 82-84 in the synthetic gene product (VU-1 calmodulin) was replaced by three lysines. VU-8 calmodulin activated phosphodiesterase to the same maximal extent as VU-1 calmodulin, although there was an alteration in the concentration of calmodulin required for half-maximal stimulation. In contrast, myosin light chain kinase was activated to only 30% of maximal activity and NAD kinase was not activated. These results provide insight into the functional role of the unusual central helix structure found in the calmodulin family of proteins and indicate that different, although possibly overlapping, chemical complementarities are employed in the interaction between calmodulin and its various physiological targets.     

Characterization of ATP and calmodulin-binding properties of a truncated form of Bacillus anthracis adenylate cyclase

The Bacillus anthracis cya gene encodes a calmodulin-dependent adenylate cyclase. A deletion cya gene product obtained by removing 261 codons at the 5' end was expressed in a protease-deficient lon- E. coli strain and purified to homogeneity. This truncated enzyme (CYA 62) exhibits catalytic and calmodulin-binding properties similar to the properties of wild-type adenylate cyclase from B. anthracis culture supernatants, i.e., a kcat of 1100 s-1 at 30 degrees C and pH 8, an apparent Km for ATP of 0.25 mM, and a Kd for bovine brain calmodulin of 23 nM. The calmodulin-binding domain of the CYA 62 truncated enzyme was labeled with a cleavable radioactive photoaffinity cross-linker coupled to calmodulin. The labeled CYA 62 protein was then cleaved with cyanogen bromide and N-chlorosuccinimide. We show that the calmodulin-binding domain of B. anthracis adenylate cyclase is located within the last 150 amino acid residues of the protein. A further deletion at the 3' end of the CYA 62 coding sequence yielded an adenylate cyclase species (CYA 57) lacking 127 C-terminal amino residues. CYA 57, still sensitive to activation by high concentrations of calmodulin, exhibits less than 0.1% of the specific activity of CYA 62. Binding of 3'dATP (a competitive inhibitor) to CYA 62 was determined by equilibrium dialysis. In the absence of calmodulin, binding of the ATP analogue to this truncated protein was severely impaired, which explains, at least in part, the absolute requirement for calmodulin for the catalytic activity of B. anthracis 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.     

Insertional mutagenesis of Bordetella pertussis adenylate cyclase.

We developed an improved method of linker insertion mutagenesis for introducing 2 or 16 codons into the Bordetella pertussis cyaA gene which encodes a calmodulin-dependent adenylate cyclase. A recombinant kanamycin resistance cassette, containing oligonucleotide linkers, was cloned in plasmids which carried a truncated cyaA gene, fused at its 3' end to the 5' end of the Escherichia coli lacZ gene, specifying the alpha-peptide. This construction permitted a double selection for in-frame insertions by using screening for kanamycin resistance and for lactose-positive phenotype, resulting from alpha-complementation. We showed that most of the two-amino acid insertions within the N-terminal moiety of the catalytic domain of adenylate cyclase abolished enzymatic activity and/or altered the stability of the protein. All two-amino acid insertions within the C-terminal part of adenylate cyclase resulted in fully stable and active enzymes. These results confirm the modular structure of the catalytic domain of adenylate cyclase, previously proposed on the basis of proteolytic studies. Two-amino acid insertions between residues 247-248 and 335-336 were shown to affect the calmodulin responsiveness of adenylate cyclase, suggesting that the corresponding region in the enzyme is involved in the binding of calmodulin or in the process of calmodulin activation. In addition, we have identified within the primary structure of adenylate cyclase several permissive sites which tolerate 16-amino acid insertions without interfering with the catalytic activity or calmodulin binding. By inserting foreign antigenic determinants into these permissive sites the resulting recombinant adenylate cyclase toxin could be used to deliver specific epitopes into antigen-presenting cells.     

Relationship Among Calmodulin-, Forskolin-, and Guanine Nucleotide-Dependent Adenylate Cyclase Activities in Cerebellar Membranes: Studies by Limited Proteolysis

Adenylate cyclase activity in bovine cerebellar membranes is regulated by calmodulin, forskolin, and both stimulatory (Ns) and inhibitory (Ni) guanine nucleotide-binding components. The susceptibility of the enzyme to chymotrypsin proteolysis was used as a probe of structure-function relationships for these different regulatory pathways. Pretreatment of membranes with low concentrations of chymotrypsin (1-2 micrograms/ml) caused a three- to fourfold increase in basal adenylate cyclase activity and abolished the Ca2+-dependent activation of the enzyme by calmodulin. In contrast, the stimulation of the enzyme by GTP plus isoproterenol was strongly potentiated after protease treatment, an effect that mimics the synergistic activation of adenylate cyclase by Ns and calmodulin in unproteolyzed membranes. Limited proteolysis revealed low- and high-affinity components in the activation of adenylate cyclase by forskolin. The low-affinity component was readily lost on proteolysis, together with calmodulin stimulation of the enzyme. The activation via the high-affinity component was resistant to proteolysis and nonadditive with the Ns-mediated activation of the enzyme, suggesting that both effectors utilize a common pathway. The inhibitory effect of low concentrations (10(-7) M) of guanyl-5'-yl imidodiphosphate [Gpp(NH)p] on forskolin-activated adenylate cyclase was retained after limited proteolysis of the membranes, indicating that the proteolytic activation does not result from an impairment of the Ni subunit. Moreover, in the rat cerebellum, proteolysis as well as calmodulin was found to enhance strongly the inhibitory effect of Gpp(NH)p on basal adenylate cyclase activity. Our results suggest that calmodulin and Ns/Ni interact with two structurally distinct but allosterically linked domains of the enzyme. Both domains appear to be involved in the mode of action of forskolin.     

Stimulation of rat platelet adenylate cyclase by an endogenous calcium-dependent protease-like activity

The stimulation of adenylate cyclase by various exogenous proteases has been described in several tissues. In this study, we describe a 2 to 7-fold increase of adenylate cyclase activity in a particulate preparation from rat platelets following prior exposure of the homogenate to calcium. Calmodulin alone was unable to increase the adenylate cyclase activity and trifluoperazine only partially inhibited the calcium-dependent activation. On the other hand, calcium had a slight stimulatory effect on the particulate preparation but this activation was greatly enhanced by the addition of supernatant. Only the combined addition of calcium, supernatant and calmodulin to washed particulate preparations reconstituted the activation seen in homogenates. The activation was significantly inhibited by leupeptin and thiol reagents. It is concluded that platelets contain a calcium-dependent protease-like activity that is able to increase adenylate cyclase activity in membrane fractions. This phenomenon may be involved in the regulation of adenylate cyclase activity in platelets.     

Functional consequences of single amino acid substitutions in calmodulin-activated adenylate cyclase of Bordetella pertussis.

Calmodulin-activated adenylate cyclase of Bordetella pertussis and Bacillus anthracis are two cognate bacterial toxins. Three short regions of 13-24 amino acid residues in these proteins exhibit between 66 and 80% identity. Site-directed mutagenesis of four residues in B. pertussis adenylate cyclase situated in the second (Asp188, Asp190) and third (His298, Glu301) segments of identity were accompanied by important decrease, or total loss, of enzyme activity. The calmodulin-binding properties of mutated proteins showed no important differences when compared to the wild-type enzyme. Apart from the loss of enzymatic activity, the most important change accompanying replacement of Asp188 by other amino acids was a dramatic decrease in binding of 3'-anthraniloyl-2'-deoxyadenosine 5'-triphosphate, a fluorescent analogue of ATP. From these results we concluded that the two neighbouring aspartic acid residues in B. pertussis adenylate cyclase, conserved in many other ATP-utilizing enzymes, are essential for binding the Mg(2+)-nucleotide complex, and for subsequent catalysis. Replacement of His298 and Glu301 by other amino acid residues affected the nucleotide-binding properties of adenylate cyclase to a lesser degree suggesting that they might be important in the mechanism of enzyme activation by calmodulin, rather than being involved directly in catalysis.     

Differential Regulation by Calmodulin of Basal, GTP-, and Dopamine-Stimulated Adenylate Cyclase Activities in Bovine Striatum

The concentration requirements of calmodulin in altering basal, GTP-, and dopamine-stimulated adenylate cyclase activities in an EGTA-washed particulate fraction from bovine striatum were examined. In the bovine striatal particulate fraction, calmodulin activated basal adenylate cyclase activity 3.5-fold, with an EC50 of 110 nM. Calmodulin also potentiated the activation of adenylate cyclase by GTP by decreasing the EC50 for GTP from 303 +/- 56 nM to 60 +/- 10 nM. Calmodulin did not alter the maximal response to GTP. The EC50 for calmodulin in potentiating the GTP response was only 11 nM as compared to 110 nM for activation of basal activity. Similarly, calmodulin increased the maximal stimulation of adenylate cyclase by dopamine by 50-60%. The EC50 for calmodulin in eliciting this response was 35 nM. These data demonstrate that calmodulin can both activate basal adenylate cyclase and potentiate adenylate cyclase activities that involve the activating GTP-binding protein, Ns. Mechanisms that involve potentiation of Ns-mediated effects are much more sensitive to calmodulin than is the activation of basal adenylate cyclase activity. Potentiation of GTP-stimulated adenylate cyclase activity by calmodulin was apparent at 3 and 5 mM MgCl2, but not at 1 or 10 mM MgCl2. These data further support a role for calmodulin in hormonal signalling and suggest that calmodulin can regulate cyclic AMP formation by more than one mechanism.     

Calmodulin activation of rat lung adenylate cyclase is independent of the cytoplasmic factors modulating the enzyme.

Adenylate cyclase activity in the rat lung membranes washed with 150 microM-EGTA was stimulated by calmodulin in the presence of 100 microM-Ca2+. The calmodulin activation of the enzyme was concentration-dependent; however, at high concentrations the activation was diminished. Activation of adenylate cyclase by calmodulin was immediate, reversible and due to an increase in the Vmax. without apparent effect on the affinity of the enzyme for ATP. The rat lung supernatant produced additive activation of the adenylate cyclase that was already maximally stimulated by calmodulin, indicating that either calmodulin and cytoplasmic factors act at different sites on adenylate cyclase or different adenylate cyclases may be involved. The data further support our previous conclusion that calmodulin is not involved in the activation of adenylate cyclase by cytoplasmic factors in rat lungs.     

Involvement of calmodulin in the regulation of adenylate cyclase activity in guinea-pig enterocytes

The involvement of calmodulin as an activator of adenylate cyclase activity was examined in isolated guinea-pig enterocytes and in a membrane preparation. In enterocytes, which responded to prostaglandin E1, vasoactive intestinal peptide and cholera toxin with a significant increase in the rate of cAMP formation trifluoperazine, a calmodulin antagonist, completely inhibited cAMP formation. In a membrane preparation adenylate cyclase activity was stimulated 10-20-fold by the GTP analog, guanosine 5'-[beta-imido]5'-triphosphate (Gpp[NH]p). Prostaglandin E1 and vasoactive intestinal peptide enhanced cAMP formation in this system by 2-3- and 1.2-1.6-fold. respectively. Addition of 200 nM calmodulin to membranes, in which endogenous calmodulin was decreased from 1.4 microgram/mg protein to 0.5 microgram/mg protein by washing with buffer containing EGTA and EDTA, resulted in a 3-4-fold increase of adenylate cyclase activity. The absolute increment in adenylate cyclase activity caused by calmodulin (10-15 pmol cAMP/min per mg protein) was approximately the same in the absence or presence of Gpp[NH]p. The apparent Ka for Gpp[NH]p (6 . 10-7 M) was not significantly changed by the addition of calmodulin. Although endogenous calcium (approx. 10 microM) in the enzyme assay was adequate to affect stimulation by calmodulin, a maximal effect was observed at a calcium concentration of 100 microM. These findings indicate that a calmodulin-sensitive form of adenylate cyclase is present in guinea-pig enterocytes, and that stimulation of cAMP formation in the intestinal mucosa may involve a calmodulin-mediated mechanism.     

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 activation of adenylate cyclase in the mouse B16 melanoma.

Calmodulin antagonists inhibited hormone-stimulated cyclic AMP accumulation in both cultured cells and cell lysates of mouse B16 melanoma. Particulate preparations of B16 melanoma contained 34-45% of total cell calmodulin, which could not be dissociated by extensive washing irrespective of the presence of EGTA in the buffer. The adenylate cyclase activity in such preparations was unaffected by the addition of exogenous calmodulin. However, the rare-earth-metal ion La3+, which can mimic or replace Ca2+ in many systems, produced an immediate inhibition of agonist-stimulated adenylate cyclase activity and preincubation of particulate preparations was La3+ followed by washing with La3+-free buffer dissociated calmodulin (96% loss) from particulate preparations. The loss of calmodulin from particulate preparations was associated with a decrease in agonist responsiveness (74%) and a marked change in the Ca2+-sensitivity of the enzyme, low concentrations of calcium (approx. 10 nM) now failing to stimulate enzyme activity, high concentrations of calcium (greater than or equal to 100 nM) producing greater-than-normal inhibition of enzyme activity. Direct activation of adenylate cyclase by the addition of pure calmodulin was now demonstrable in such calmodulin-depleted particulate preparations. Half-maximal stimulation of agonist-responsive adenylate cyclase occurred at 80 nM-calmodulin in the presence of 10 microM free Ca2+. Maximal stimulation by calmodulin (at 300-600 nM) restored enzyme activity to 89 +/- 5% (mean +/- S.E.M., n = 7) of the activity in untreated, calmodulin-intact, preparations.     

Identification of residues essential for catalysis and binding of calmodulin in Bordetella pertussis adenylate cyclase by site-directed mutagenesis.

In order to identify molecular features of the calmodulin (CaM) activated adenylate cyclase of Bordetella pertussis, a truncated cya gene was fused after the 459th codon in frame with the alpha-lacZ' gene fragment and expressed in Escherichia coli. The recombinant, 604 residue long protein was purified to homogeneity by ion-exchange and affinity chromatography. The kinetic parameters of the recombinant protein are very similar to that of adenylate cyclase purified from B.pertussis culture supernatants, i.e. a specific activity greater than 2000 mumol/min mg of protein at 30 degrees C and pH 8, a KmATP of 0.6 mM and a Kd for its activator, CaM, of 0.2 nM. Proteolysis with trypsin in the presence of CaM converted the recombinant protein to a 43 kd protein with no loss of activity; the latter corresponds to the secreted form of B.pertussis adenylate cyclase. Site-directed mutagenesis of residue Trp-242 in the recombinant protein yielded mutants expressing full catalytic activity but having altered affinity for CaM. Thus, substitution of an aspartic acid residue for Trp-242 reduced the affinity of adenylate cyclase for CaM greater than 1000-fold. Substitution of a Gln residue for Lys-58 or Lys-65 yielded mutants with a drastically reduced catalytic activity (approximately 0.1% of that of wild-type protein) but with little alteration of CaM-binding. These results substantiated, at the molecular level, our previous genetic and biochemical studies according to which the N-terminal tryptic fragment of secreted B.pertussis adenylate cyclase (residues 1-235/237) harbours the catalytic site, whereas the C-terminal tryptic fragment (residues 235/237-399) corresponds to the main CaM-binding domain of the enzyme.     

Crayfish abdominal muscle adenylate cyclase. Studies on the stimulation by a Ca2+-binding protein.

A plasma-membrane preparation of crayfish muscle showed an adenylate cyclase activity which is inhibited to about 80% of its original activity by 100 microM-EGTA. Measurements of the enzyme activity in the presence of 100 microM-EGTA and various concentrations of Ca2+ revealed an increase in enzyme activity of about 400%, indicating an adenylate cyclase which is dependent on Ca2+ for activity. Fluphenazine (1 mM), a blocker of the Ca2+-binding protein calmodulin, decreased enzyme activity to zero. The enzyme can be re-activated by the addition of certain concentrations of calmodulin to the assay medium. This suggests that crayfish muscle adenylate cyclase is dependent on Ca2+ and calmodulin for activity.     

Characterization of a calmodulin-stimulated adenylate cyclase from abalone spermatozoa

Abalone sperm adenylate cyclase activity is particulate in nature and displays a high Mg2+-supported activity (Mg2+/Mn2+ = 0.8) as compared to other sperm adenylate cyclases. Approximately 90% of the enzyme activity in crude homogenates is inhibited by EGTA in a concentration-dependent manner which is overcome by added micromolar free Ca2+. The EGTA-inhibited Ca2+-stimulated enzyme activity is also inhibited by phenothiazines. Added calmodulin, however, has no effect on enzyme activity prepared from crude homogenates. Preparation of a twice EGTA-extracted 48,000 X g pellet fraction yields a particulate enzyme activity that can be stimulated 10-65% by added calmodulin in the presence of micromolar free Ca2+. Detergent extraction (1% Lubrol PX) of the EGTA-washed 48,000 X g pellet solubilizes 2-5% of the total particulate adenylate cyclase activity, and this solubilized enzyme is activated up to 125% by calmodulin. The ability of the different enzyme preparations to be stimulated by calmodulin is inversely proportional to the endogenous calmodulin concentration. Calmodulin stimulation of the Lubrol PX-solubilized enzyme is specific to this Ca2+-binding protein and is mediated as an effect on the velocity of the enzyme. This stimulation is completely Ca2+ dependent and is fully reversible. These data suggest that the control of sperm cAMP synthesis by changes in Ca2+ conductance may be mediated via this Ca2+-binding protein.     

Calcium and calmodulin in the regulation of human thyroid adenylate cyclase activity.

TSH (thyrotropin)-stimulated human thyroid adenylate cyclase has a biphasic response to Ca2+, being activated by submicromolar Ca2+ (optimum 22nM), with inhibition at higher concentrations. Calmodulin antagonists caused an inhibition of TSH-stimulated adenylate cyclase in a dose-dependent manner. Inhibition of TSH-and TSIg-(thyroid-stimulating immunoglobulins)-stimulated activity was more marked than that of basal, NaF- or forskolin-stimulated activity. This inhibition was not due to a decreased binding of TSH to its receptor. Addition of pure calmodulin to particulate preparations of human non-toxic goitre which had not been calmodulin-depleted had no effect on adenylate cyclase activity. EGTA was ineffective in removing calmodulin from particulate preparations, but treatment with the tervalent metal ion La3+ resulted in a loss of up to 98% of calmodulin activity from these preparations. Addition of La3+ directly to the adenylate cyclase assay resulted in a partial inhibition of TSH- and NaF-stimulated activity, with 50% inhibition produced by 5.1 microM and 4.0 microM-La3+ respectively. Particulate preparations with La3+ showed a decrease of TSH- and NaF-stimulated adenylate cyclase activity (approx. 40-60%). In La3+-treated preparations there was a decrease in sensitivity of TSH-stimulated adenylate cyclase to Ca2+ over a wide range of Ca2+ concentrations, but most markedly in the region of the optimal stimulatory Ca2+ concentration. In particulate preparations from which endogenous calmodulin had been removed by La3+ treatment, the addition of pure calmodulin caused an increase (73 +/- 22%; mean +/- S.E.M., n = 8) in TSH-stimulated thyroid adenylate cyclase activity. This was seen in 8 out of 13 experiments.     

Bordetella pertussis adenylate cyclase. Purification, characterization, and radioimmunoassay

The extracellular adenylate cyclase of Bordetella pertussis was purified either as a free enzyme or as a complex with calmodulin. The purified enzyme has a specific activity of 1600 mumol of cAMP min-1 X mg-1 and exists under two molecular forms of 45 and 43 kDa which are apparently structurally related. Calmodulin increased considerably the resistance of adenylate cyclase to inactivation by trypsin. Although trypsin cleaved the adenylate cyclase-calmodulin complex, the digested fragments remained associated by noncovalent interactions in an active conformation. Specific mouse anti-adenylate cyclase antibodies inhibit adenylate cyclase activity and were used to develop a specific radioimmunoassay that allows detection of as little as 5 ng of adenylate cyclase in culture supernatants.     

Regulation of ciliary adenylate cyclase by Ca2+ in Paramecium.

In the ciliated protozoan Paramecium, Ca2+ and cyclic nucleotides are believed to act as second messengers in the regulation of the ciliary beat. Ciliary adenylate cyclase was activated 20-30-fold (half-maximal at 0.8 microM) and inhibited by higher concentrations (10-20 microM) of free Ca2+ ion. Ca2+ activation was the result of an increase in Vmax., not a change in Km for ATP. The activation by Ca2+ was seen only with Mg2+ATP as substrate; with Mn2+ATP the basal adenylate cyclase activity was 10-20-fold above that with Mg2+ATP, and there was no further activation by Ca2+. The stimulation by Ca2+ of the enzyme in cilia and ciliary membranes was blocked by the calmodulin antagonists calmidazolium (half-inhibition at 5 microM), trifluoperazine (70 microM) and W-7 (50-100 microM). When ciliary membranes (which contained most of the ciliary adenylate cyclase) were prepared in the presence of Ca2+, their adenylate cyclase was insensitive to Ca2+ in the assay. However, the inclusion of EGTA in buffers used for fractionation of cilia resulted in full retention of Ca2+-sensitivity by the ciliary membrane adenylate cyclase. The membrane-active agent saponin specifically suppressed the Ca2+-dependent adenylate cyclase without inhibiting basal activity with Mg2+ATP or Mn2+ATP. The ciliary adenylate cyclase was shown to be distinct from the Ca2+-dependent guanylate cyclase; the two activities had different kinetic parameters and different responses to added calmodulin and calmodulin antagonists. Our results suggest that Ca2+ influx through the voltage-sensitive Ca2+ channels in the ciliary membrane may influence intraciliary cyclic AMP concentrations by regulating adenylate cyclase.     

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.     

Inhibition of a Low Km GTPase Activity in Rat Striatum by Calmodulin

In rat striatum, the activation of adenylate cyclase by the endogenous Ca2+-binding protein, calmodulin, is additive with that of GTP but is not additive with that of the nonhydrolyzable GTP analog, guanosine-5'-(beta, gamma-imido)triphosphate (GppNHp). One possible mechanism for this difference could be an effect of calmodulin on GTPase activity which has been demonstrated to "turn-off" adenylate cyclase activity. We examined the effects of Ca2+ and calmodulin on GTPase activity in EGTA-washed rat striatal particulate fractions depleted of Ca2+ and calmodulin. Calmodulin inhibited GTP hydrolysis at concentrations of 10(-9)-10(-6) M but had no effect on the hydrolysis of 10(-5) and 10(-6) M GTP, suggesting that calmodulin inhibited a low Km GTPase activity. The inhibition of GTPase activity by calmodulin was Ca2+-dependent and was maximal at 0.12 microM free Ca2+. Maximal inhibition by calmodulin was 40% in the presence of 10(-7) M GTP. The IC50 for calmodulin was 100 nM. In five tissues tested, calmodulin inhibited GTP hydrolysis only in those tissues where it could also activate adenylate cyclase. Calmodulin could affect the activation of adenylate cyclase by GTP in the presence of 3,4-dihydroxyphenylethylamine (DA, dopamine). Calmodulin decreased by nearly 10-fold the concentration of GTP required to provide maximal stimulation of adenylate cyclase activity by DA in the striatal membranes. The characteristics of the effect of calmodulin on GTPase activity with respect to Ca2+ and calmodulin dependence and tissue specificity parallel those of the activation of adenylate cyclase by calmodulin, suggesting that the two activities are closely related.(ABSTRACT TRUNCATED AT 250 WORDS)