Heavy isotope-labeling study of the metabolism of monomeric and tetrameric acetylcholinesterase forms in the murine neuronal-like T 28 hybrid cell line

We have used the method of heavy isotope labeling to study the metabolic turnover of acetylcholinesterase forms in the neuroblastoma-derived T 28 hybrid cells in their differentiated state. These cells contain mostly G1 and G4 forms, together with a small proportion of G2, and secrete all these forms into the culture medium. The cells maintained constant and equal levels of acetylcholinesterase, with the same proportions of molecular forms, in a medium containing heavy isotope-labeled amino acids and in a control light medium of similar composition. In addition, they secreted acetylcholinesterase at the same rate in both media. After transfer of the cells into the heavy medium, heavy isotope-labeled acetylcholinesterase molecules progressively replace preexisting light molecules. We analyzed heavy and light components of acetylcholinesterase for each of the two major G1 and G4 forms, by reconstructing the pattern obtained in sucrose gradient differential sedimentation, using combinations of weighted elementary distributions. Heavy molecules were detected in cellular extracts after about 30 min for G1 and 3 h for G4. Both heavy forms also appeared in the medium after a lag of about 3 h. The cellular complement of G1 was renewed much faster than that of G4, the levels of the light forms being reduced to 50% of the original level after 3.5 and 40 h, respectively. Each of these forms appeared to consist of several metabolic pools, and we present simplified models which describe their possible relationships.     

Histidine regulation of cyclic AMP metabolism in cultured renal epithelial LLC-PK1 cells.

L-Histidine and imidazole (the histidine side chain) significantly increase cAMP accumulation in intact LLC-PK1 cells. This effect is completely inhibited by isobutylmethylxanthine (IBMX). Histidine and imidazole stimulate cAMP phosphodiesterase activity in soluble and membrane fractions of LLC-PK1 cells suggesting that the IBMX-sensitive effect of these agents to stimulate cAMP formation is not due to inhibition of cAMP phosphodiesterase. Histidine and imidazole but not alanine (the histidine core structure) increase basal, GTP-, forskolin-, and AVP-stimulated adenylate cyclase activity in LLC-PK1 membranes. Two other amino acids with charged side chains (aspartic and glutamic acids) increase AVP-stimulated but neither basal- nor forskolin-stimulated adenylate cyclase activity. This suggests that multiple amino acids with charged side chains can regulate selected aspects of adenylate cyclase activity. To better define the mechanism of histidine regulation of adenylate cyclase, membranes were detergent-solubilized which prevents histidine and imidazole potentiation of forskolin-stimulated adenylate cyclase activity and suggests that an intact plasma membrane environment is required for potentiation. Neither pertussis toxin nor indomethacin pretreatment alter imidazole potentiation of adenylate cyclase. IBMX pretreatment of LLC-PK1 membranes also prevents imidazole to potentiate adenylate cyclase activity. Since IBMX inhibits adenylate cyclase coupled adenosine receptors, LLC-PK1 cells were incubated in vitro with 5'-N-ethylcarboxyamideadenosine (NECA) which produced a homologous pattern of desensitization of NECA to stimulate adenylate cyclase activity. Despite homologous desensitization, histidine and imidazole potentiation of adenylate cyclase was unaltered. These data suggest that histidine, acting via an imidazole ring, potentiates adenylate cyclase activity and thereby increases cAMP formation in cultured LLC-PK1 epithelial cells. This potentiation requires an intact plasma membrane environment, occurs independent of a pertussis toxin-sensitive substrate and of products of cyclooxygenase, and is inhibited by IBMX. This IBMX-sensitive pathway does not involve either inhibition of cAMP phosphodiesterase activity or a stimulatory adenosine receptor coupled to adenylate cyclase.     

Effects of amino acids and glucose on adenylate cyclase and cell differentiation of Dictyostelium discoideum.

Starvation triggers the differentiation of Dictyostelium discoideum amoebas to aggregation competence. To determine more precisely the nature of the starvation signal, the ability of various components of the growth medium to inhibit differentiation was examined. Changes in adenylate cyclase (the enzyme which generates the cAMP pulses basic to the differentiation process), various physiological and biochemical markers of developing cells, and the ability of amoebas to form specific intercellular contacts were monitored. We show that amino acid mixtures inhibit cell differentiation by preventing the increase of adenylate cyclase activity which normally occurs during the early hours of starvation. High concentrations of glucose also inhibit the differentiation process but at a later stage: The rise in adenylate cyclase still occurs when cells are starved in the presence of sugar, but the enzyme does not appear to function in vivo. Exogenously generated cAMP pulses are not able to bypass the block exerted by amino acids but can bypass the block exerted by glucose. Results support the hypothesis that the presence of amino acids inhibits adenylate cyclase synthesis, while the presence of 3% glucose blocks endogenous activation of adenylate cyclase, perhaps as a consequence of high osmotic pressure.     

Prolonged activation of inhibitory somatostatin receptors increases adenylate cyclase activity in wild-type and Gs alpha-deficient (cyc-) S49 mouse lymphoma cells.

Many cells develop enhanced adenylate cyclase activity after prolonged exposure to drugs that acutely inhibit the enzyme and it has been suggested that this adaptation may be due to an increase in Gs alpha. We have treated wild-type and Gs alpha-deficient cyc- S49 mouse lymphoma cells with a stable analogue (SMS 201-995) of the inhibitory agonist somatostatin. After incubation with SMS for 24 h, the forskolin-stimulated cAMP synthetic rate in intact cyc- cells was increased by 76%, similar to the increase found in the wild-type cells. Forskolin-stimulated adenylate cyclase activity in the presence of Mn2+ was also increased in membranes prepared from SMS-treated cyc- cells; however, guanine nucleotide-mediated inhibition of adenylate cyclase activity was not changed despite a small decrease in inhibitory Gi alpha subunits detected by immunoblotting. Pretreatment of cyc- cells with pertussis toxin prevented SMS from inducing the enhancement of forskolin-stimulated cAMP accumulation in intact cells. After chronic incubation of cyc- cells with SMS, exposure to N-ethylmaleimide, which abolished receptor-mediated inhibition of cAMP accumulation, did not attenuate the enhanced rate of forskolin-stimulated cAMP synthesis compared to N-ethylmaleimide-treated controls. These results with cyc- cells demonstrate that an adaptive increase in adenylate cyclase activity induced by chronic treatment with an inhibitory drug can occur in the absence of expression of Gs alpha.     

Picomolar-affinity binding and inhibition of adenylate cyclase activity by melatonin in syrian hamster hypothalamus

1. The effect of melatonin on forskolin-stimulated adenylate cyclase activity was measured in homogenates of Syrian hamster hypothalamus. In addition, the saturation binding characteristics of the melatonin receptor ligand, [125I]iodomelatonin, was examined using an incubation temperature (30 degrees C) similar to that used in enzyme assays. 2. At concentrations ranging from 10 pM to 1 nM, melatonin caused a significant decrease in stimulated adenylate cyclase activity with a maximum inhibition of approximately 22%. 3. Binding experiments utilizing [125I]iodomelatonin in a range of approximately 5-80 pM indicated a single class of high-affinity sites: Kd = 55 +/- 9 pM, Bmax = 1.1 +/- 0.3 fmol/mg protein. 4. The ability of picomolar concentrations of melatonin to inhibit forskolin-stimulated adenylate cyclase activity suggests that this affect is mediated by picomolar-affinity receptor binding sites for this hormone in the hypothalamus.     

Epidermal growth factor, a modulator of luteal adenylate cyclase. Characterization of epidermal growth factor receptors and its interaction with adenylate cyclase system in bovine luteal cell membrane.

The epidermal growth factor (EGF) binding sites on bovine luteal cell membrane have been characterized in detail, and evidence has been obtained for a direct stimulatory effect of EGF on membrane-associated adenylate cyclase activity. The membrane fraction prepared showed the presence of high affinity (Ka = 1.2 +/- 0.7 x 10(-11) M-1), specific, and saturable EGF receptors of Mr = 170,000. The EGF receptors underwent rapid autophosphorylation and down-regulation following treatment of the cells with EGF. Treatment of the cells with 4 beta-phorbol 12-myristate 13-acetate resulted in a diminished binding of 125I-EGF to the receptors. When luteal cells were preincubated with EGF, both basal and forskolin-stimulated adenylate cyclase activity was increased severalfold. This enhancement of the adenylate cyclase activity was dependent upon the duration of the exposure to EGF and on the concentration of the growth factor. An optimal enhancement was observed when the cells were preincubated with 10 ng/ml EGF for 10-15 min. Furthermore, when the membrane fraction prepared from luteal cells was preincubated in vitro with EGF, a similar dose-related and time-dependent increase in basal, as well as forskolin-stimulated, adenylate cyclase activity was observed. These results demonstrate that luteal cell adenylate cyclase activity is finely regulated by EGF. Such a direct interaction between EGF and membrane-associated adenylate cyclase has not been previously recognized.     

Adrenergic agonists induce heterologous sensitization of adenylate cyclase in NS20Y-D(2L) cells

Adenylate cyclase activity in NS20Y cells expressing D2L dopamine receptors was examined following chronic treatment with norepinephrine and epinephrine. Initial acute experiments revealed that both norepinephrine and epinephrine inhibited forskolin-stimulated cyclic AMP accumulation via D2 receptors. Furthermore, chronic 18 h activation of D2 dopamine receptors by norepinephrine or epinephrine induced a marked increase (>10-fold) in subsequent forskolin-stimulated cyclic AMP accumulation. This heterologous sensitization of adenylate cyclase activity was blocked by D2 dopamine receptor antagonists and by pertussis toxin pretreatment. In contrast, concurrent activation of Galpha(s) or adenylate cyclase did not appear to alter noradrenergic agonist-induced sensitization.     

Adenylate cyclase in membrane fractions of RIN-A2-cells: studies with forskolin, NaF, GppNHp and NEM

In crude membrane fractions of rat pancreatic islets and of RIN-A2-cells, forskolin and NaF stimulated adenylate cyclase activity. Basal and stimulated enzyme activity was approximately 3 to 6 fold higher in membranes of RIN-A2-cells than in membranes of islet cells. In RIN-A2-cells GppNHp and NEM inhibited forskolin-stimulated enzyme activity. The inhibitory effect of GppNHp could be reduced by NEM. It is suggested that the adenylate cyclase system of RIN-A2-cells contains inhibitory and stimulatory N-proteins and that there are critical thiols related to Ni, Ns and/or the catalytic unit. Thus, membrane fractions of RIN-A2-cells may be an appropriate model for studies on the adenylate cyclase system of insulin-producing cells.     

Guanine nucleotide-independent inhibition of adenylate cyclase by a stimulatory hormone.

Stimulation of basal adenylate cyclase activity in membranes of neuroblastoma x glioma hybrid cells by prostaglandin E1 (PGE1) is half-maximal and maximal (about 8-fold) at 0.1 and 10 microM respectively. This hormonal effect requires GTP, being maximally effective at 10 microM. However, at the same concentrations that stimulate adenylate cyclase in the presence of GTP, PGE1 inhibited basal adenylate cyclase activity when studied in the absence of GTP, by maximally 60%. A similar dual action of PGE1 was observed with the forskolin-stimulated adenylate cyclase, although the potency of PGE1 in both stimulating and inhibiting adenylate cyclase was increased and the extent of stimulation and inhibition of the enzyme by PGE1 was decreased by the presence of forskolin. The inhibition of forskolin-stimulated adenylate cyclase by PGE1 occurred without apparent lag phase and was reversed by GTP and its analogue guanosine 5'-[gamma-thio]triphosphate at low concentrations. Treatment of neuroblastoma x glioma hybrid cells or membranes with agents known to eliminate the function of the inhibitory GTP-binding protein were without effect on PGE1-induced inhibition of adenylate cyclase. The data suggest that stimulatory hormone agonist, apparently by activating one receptor type, can cause both stimulation and inhibition of adenylate cyclase, and that the final result depends only on the activity state of the stimulatory GTP-binding protein, Gs. Possible mechanisms responsible for the observed adenylate cyclase inhibition by the stimulatory hormone PGE1 are discussed.     

Phorbol esters induce two distinct changes in GH3 pituitary cell adenylate cyclase activity

Phorbol esters alter cyclic AMP levels in a number of tissues, including the anterior pituitary. We report that membrane preparations from GH3 cells exposed to phorbol esters exhibit decreased vasoactive intestinal peptide (VIP)-stimulated and enhanced forskolin-stimulated adenylate cyclase activity. The responsiveness of adenylate cyclase activity to NaF, guanylyl-imidodiphosphate, and Mn2+ was also reduced by phorbol ester treatment. The ability of somatostatin to inhibit forskolin-stimulated adenylate cyclase activity was reduced while phorbol ester exposure had no apparent effect on somatostatin inhibition of VIP-stimulated adenylate cyclase activity. We suggest that protein kinase C alters at least two distinct components of the adenylate cyclase system. One modification disrupts hormone receptor-Gs interaction (lowering VIP efficacy) and the second perturbation augments the activity of the adenylate cyclase catalytic subunit.     

A conserved arginine in the distal third intracellular loop of the mu-opioid receptor is required for G protein activation

In the present study, the functional significance of the intracellular C-terminal loop of the mu-opioid receptor in activating Gi proteins was determined by constructing a C-terminal deletion mutant mu(C delta 45) receptor, which lacks the carboxyl 45 amino acids. When the truncated mu(C delta 45) receptor was stably expressed in human embryonic kidney (HEK) 293 cells, the efficacy and the potency of [D-Ala2,N-Me-Phe4,Gly-ol5]enkephalin (DAMGO), a specific mu-opioid receptor agonist, to inhibit forskolin-stimulated adenylate cyclase activity were not significantly affected. Similar to other G-coupled receptors, the third cytoplasmic loop of the mu-opioid receptor contains conserved basic residues (R276/R277/R280) at the C-terminal segment. Mutating these basic residues to neutral amino acids (L276/M277/L280) greatly impaired the ability of DAMGO to inhibit forskolin-stimulated cyclic AMP formation. Replacing R276/R277 with L276/M277 did not affect the efficacy and potency by which DAMGO inhibits the adenylate cyclase activity. In HEK 293 cells stably expressing mutant (R280L) mu-opioid receptors, the ability of DAMGO to inhibit forskolin-stimulated cyclic AMP production was greatly reduced. These results suggest that the intracellular carboxyl tail of the mu-opioid receptor does not play a significant role in activating Gi proteins and that the arginine residue (R280) at the distal third cytoplasmic loop is required for Gi activation by the mu-opioid receptor.     

Increased sensitivity of fat cell adenylate cyclase to stimulatory agonists during fasting is not related to impaired inhibitory coupling system

In rat adipocytes, inhibition of the forskolin-stimulated cyclic AMP response by nicotinic acid and N6-phenylisopropyladenosine was unaltered by a 72 h fasting. Under assay conditions favouring inhibition, basal and forskolin-stimulated adenylate cyclase responses to inhibition by GTP and nicotinic acid were also unimpaired by fasting. Under the same conditions, however, low GTP concentrations elicited a clear activatory effect in membranes from fasted but not from fed rats. Fasting failed to alter the incorporation of [32P]ADP ribose into the alpha i-subunit of Ni and the attenuation of nicotinic acid inhibitory action that are both induced by pertussis toxin. These results, demonstrating unimpaired inhibitory control of adenylate cyclase in starved rat adipocytes, suggest that the permissive effect of fasting on the action of stimulatory receptor agonists in fat cells reflects a specific increase in the activity of the adenylate cyclase stimulatory coupling system.     

Galanin inhibits adenylate cyclase of rat brain membranes.

The ubiquitous neuropeptide, galanin, strongly inhibits adenylate cyclase in rat brain membranes. While basal enzyme activity was not altered, galanin from 10(-11) M to 5 x 10(-7) M decreased forskolin- and VIP-stimulated adenylate cyclase with a half-maximal effect being elicited by 0.7 nM neuropeptide and a maximal 80% inhibition of the enzyme activity. The galanin fragments (2-29) and (1-15) dose-dependently inhibited the forskolin-stimulated adenylate cyclase, while the fragments (3-29) and (10-29) were found inactive. These results indicate that the regulatory action of galanin in the central nervous system involves the coupling of galanin receptors to the inhibition of the adenylate cyclase system.     

Inhibition of adenylate cyclase activity by polyamines in human erythrocyte plasma membranes

Polyamines (spermidine, spermine and putrescine) inhibited the adenylate cyclase activity in a concentration dependent manner in human erythrocyte plasma membranes. Spermidine (Spd) exhibited more inhibitory effect than spermine (Spm) and putrescine (Put). On the contrary, the addition of amino acids (arginine, glutamine and lysine) did not influence the basal enzyme activity. Other cations (polylysine, polyarginine and polyglutamine) also did not affect the enzyme activity. Addition of all the three polyamines (Spd, Spm and Put) in the reaction mixture exhibited moderate inhibitory effect on the adenylate cyclase activity whether it was basal or activated with sodium fluoride or with forskolin. Since the three polyamines exhibited maximum inhibitory effect at 10 microM concentration which is within physiological limit for mammalian tissues, we suggest that there may be a regulatory function of these molecules on adenylate cyclase activity in human erythrocytes.     

Somatostatin induces translocation of the beta-adrenergic receptor kinase and desensitizes somatostatin receptors in S49 lymphoma cells

The beta-adrenergic receptor kinase is a cytosolic enzyme that specifically phosphorylates the agonist-occupied form of the beta-adrenergic receptor (beta AR). Beta AR kinase appears to be translocated from the cytosol to the plasma membrane when kin- S49 lymphoma cells are incubated with either beta-adrenergic agonists or prostaglandin E1, both of which act through receptors which stimulate adenylate cyclase. We report here that brief (approximately 20 min) exposure of wild type S49 lymphoma cells to somatostatin (which inhibits adenylate cyclase) promotes the translocation of beta AR kinase to an extent comparable to that observed in the presence of the beta agonist isoproterenol or prostaglandin E1. Beta AR kinase activity can be measured using either beta AR or rhodopsin, the retinal receptor for light, as a substrate. The translocation process triggered by somatostatin is rapid, reversible, and is associated with somatostatin receptor desensitization. The latter is apparent as an attenuation of the inhibition by somatostatin of forskolin-stimulated adenylate cyclase activity in membranes of S49 cells preincubated in the presence of the peptide. These results strongly suggest that beta AR kinase is able to phosphorylate and desensitize both stimulatory and inhibitory adenylate cyclase-coupled receptors, thus emerging as a general kinase that regulates the function of different receptors in an agonist-specific fashion.     

Isolation of adenylate cyclase-enriched membranes from mammalian cells using concanavalin A.

Plasma membrane vesicles containing adenylate cyclase and beta-adrenergic receptors were prepared from 1321N1 human astrocytoma cells by a procedure involving the use of concanavalin A to stabilize the plasma membrane to fragmentation and vesiculation upon cell lysis. Treatment of cells with concanavalin A causes these plasma membrane markers to sediment to a higher density of sucrose and in a narrower band than observed with untreated cells. Upon treatment of the heavy membrane fragments with alpha-methylmannoside to remove bound concanavalin A, the enzyme markers again sediment a lower densities of sucrose. This reversible change in sedimentation behavior has been used to obtain preparations of plasma membranes enriched 14- to 21-fold (recovery 25%) in adenylate cyclase activity and about 12-fold (recovery 16%) in beta-adrenergic receptor density, as compared to lysates. The adenylate cyclase of purified membranes responded normally to isoproterenol and prostaglandin E1. Experiments with S49 and YAC mouse lymphoma cells and human skin fibroblasts indicate that this procedure may be adaptable to the isolation of plasma membranes from a variety of cultured cell lines.     

Calcium inhibition of adenylate cyclase: Studies in turkey erythrocyte and S49 CYC− cell membranes

We studied the mechanism of calcium inhibition of adenylate cyclase using partially purified components of the enzyme complex and computer analysis of free metal and substrate concentrations. A sigmoidal relationship was observed between percentage maximal adenylate cyclase activity with 1-isoproterenol/guanylyl-β,γ-imidodiphosphate and the calculated free calcium. Fifty percent inhibition occurred at 2.5 × 10^?4m free calcium. This inhibition appeared to be independent of calmodulin. Calcium inhibited the holocatalytic enzyme in a manner indentical to that of the native enzyme, but did not affect the ability of 1-isoproterenol and guanylyl-β,γ-imidodiphosphate to promote the formation of the holocatalytic state. There was no effect of calcium on the conformation of the activated G unit nor on the holocatalytic enzyme as determined by sedimentation velocity analysis. Calcium did not cause detectable dissociation of the activated G unit from the catalytic unit, nor convert activated G unit to an inactive form. Calcium inhibition of the catalytic unit of adenylate cyclase was studied in S49 CYC^? lymphoma cell membranes. High concentrations of calcium inhibited manganese-stimulated CYC^? enzyme, but this could be explained by competition between calcium and manganese for ATP. With addition of forskolin, CYC^? adenylate cyclase utilized MgATP^2? as substrate and was shown to have a separate binding site for free magnesium. Calcium inhibited forskolin-stimulated CYC^? enzyme by competing with free magnesium for its regulatory site. Calcium inhibition was noncompetitive with respect to MgATP^2?. We conclude that calcium inhibits adenylate cyclase by direct competition with magnesium for a regulatory site on the catalytic unit.     

Mechanism of galanin-inhibited insulin release. Occurrence of a pertussis-toxin-sensitive inhibition of adenylate cyclase

In the insulin-secreting beta cell line Rin m 5F, galanin, a newly discovered ubiquitous neuropeptide, inhibited, by 50%, the stimulation of insulin release induced by gastric inhibitory polypeptide (GIP) or forskolin, i.e. two cAMP-generating effectors. In contrast, it failed to decrease the stimulation of insulin release elicited by either the Ca2+-mobilizing agent, carbamoylcholine, or by dibutyryl-cAMP. Concomitantly, galanin inhibited the GIP- and forskolin-stimulated cAMP production. Furthermore, adenylate cyclase in membranes from Rin m 5F cells was highly sensitive to galanin, which exerted a marked inhibitory effect on the forskolin-stimulated enzyme activity. All these galanin effects were observed at low physiological doses, in the nanomolar range. Overnight treatment of the Rin m 5F cells with pertussis toxin completely abolished the inhibitory effect of galanin on insulin release, cAMP production and adenylate cyclase activity. Moreover, pertussis toxin specifically ADP-ribosylated a 39-kDa protein present in membranes from those cells. Taken together, these data show that the galanin inhibition of insulin release most likely occurs through the inhibition of adenylate cyclase, involving a petussis-toxin-sensitive inhibitory GTP-binding regulatory protein.     

Analysis by cell hybridization of mechanisms that regulate beta-adrenergic responses in reticulocytes and in differentiating erythroid cells.

In intact reticulocytes, but not in fragmented membranes, the loss of adenylate cyclase activity during cell maturation followed a biphasic time course. A rapid phase (t1/2 approximately 2 h) during which the initial activity was reduced by 40-50% was followed by a slow phase with t1/2 close to 3 days. The fast decay seemed to occur on the adenylate cyclase level since (-)isoprenaline- or forskolin-stimulated activities behaved similarly and bacterial toxin-monitored Gs and Gi proteins remained stable. The mechanism of the initial decrease in hormonal responsiveness was further analysed in hybrid cells prepared by fusing reticulocytes with Friend erythroleukemia (MEL) cells. The hybrids contained reticulocyte-derived beta-adrenoceptors and MEL cell-derived adenylate cyclase and G proteins. Fusion of reticulocytes to native MEL cells caused adenylate cyclase activity to drop by 30% at 2 h and 45% at 18 h after fusion. By contrast, hybrids prepared after dimethylsulfoxide-induced differentiation of MEL cells showed stable or increasing rates of receptor-coupled cAMP formation between 2 and 18 h after fusion, concomitant with the enhanced activity of the Gs protein in these cells. A cyclase-stimulating factor present in the cytosol of MEL cells and of reticulocytes appeared not to be involved in short-term regulation of hormonal responsiveness. We conclude that the strength of beta-adrenergic responses in erythroid progenitor cells is primarily regulated by modulating G protein-mediated receptor cyclase coupling while reticulocytes, during early maturation, seem to rely on direct inactivation of adenylate cyclase, probably via a cytosolic proteolytic pathway.     

The calmodulin-sensitive adenylate cyclase of Bordetella pertussis: cloning and expression in Escherichia col

The adenylate cyclase toxin of the prokaryote Bordetella pertussis is stimulated by the eukaryotic regulatory protein, calmodulin. A general strategy, using the adenylate-cyclase-calmodulin interaction as a tool, has permitted cloning and expression of the toxin in Escherichia coli in the absence of any B. pertussis trans-activating factor. We show that the protein is synthesized in a large precursor form composed of 1706 amino acids. The calmodulin-stimulated catalytic activity resides in the amino-terminal 450 amino acids of the adenylate cyclase. The enzyme expressed in E. coli is recognized in Western blots by antibodies directed against purified B. pertussis adenylate cyclase, and its activity is inhibited by these antibodies.