Evidence that the adenylate cyclase secreted from Bordetella pertussis does not enter animal cells by receptor-mediated endocytosis

Bordetella pertussis, the pathogen responsible for whooping cough, produces a calmodulin-sensitive adenylate cyclase. Several investigators have shown that the partially purified adenylate cyclase is capable of entering animal cells and elevating intracellular cAMP levels (Confer and Eaton: Science 217:948-950, 1982; Shattuck and Storm: Biochemistry 24:6323-6328, 1985). However, the mechanism for entry of the catalytic subunit of this adenylate cyclase into animal cells is unknown. It has been reported that the B. pertussis adenylate cyclase extracted from bacterial cells with urea does not enter animal cells by receptor-mediated endocytosis. There is, in addition to the cell associated form of the B. pertussis adenylate cyclase, a cell-invasive form of the enzyme secreted into the bacterial culture media. The properties of the cell-associated and secreted enzymes are significantly different (Masure and Storm: Biochemistry 28:438-442, 1989). In this study, we report evidence that the secreted form of the B. pertussis adenylate cyclase enters animal cells by a mechanism distinct from receptor-mediated endocytosis.     

The Escherichia coli adenylate cyclase complex: Activation by phosphoenolpyruvate

A model for the regulation of the activity of Escherichia coli adenylate cyclase is presented. It is proposed that Enzyme I of the phosphoenolpyruvate:sugar phosphotransferase system (PTS) interacts in a regulatory sense with the catalytic unit of adenylate cyclase. The phosphoenolpyruvate (PEP)-dependent phosphorylation of Enzyme I is assumed to be associated with a high activity state of adenylate cyclase. The pyruvate or sugar-dependent dephosphorylation of Enzyme I is correlated with a low activity state of adenylate cyclase. Evidence in support of the proposed model involves the observation that Enzyme I mutants have low cAMP levels and that PEP increases cellular cAMP levels and, under certain conditions, activates adenylate cyclase, Kinetic studies indicate that various ligands have opposing effects on adenylate cyclase. While PEP activates the enzyme, either glucose or pyruvate inhibit it. The unique relationships of PEP and Enzyme I to adenylate cyclase activity are discussed.     

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.     

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)     

Calmodulin inhibits entry of Bordetella pertussis adenylate cyclase into animal cells

Bordetella pertussis, the pathogen responsible for whooping cough, releases a soluble calmodulin-sensitive adenylate cyclase into its culture medium. Recently, Confer and Eaton [Confer, D., & Eaton, J. (1982) Science (Washington, D.C.) 217, 948-950], as well as Hanski and Farfel [Hanski, E., & Farfel, Z. (1985) J. Biol. Chem. 290, 5526-5536], have shown that crude extracts from B. pertussis containing adenylate cyclase activity cause elevations in intracellular cAMP when incubated with human neutrophils or lymphocytes. These investigators proposed that the bacterial enzyme enters animal cells and catalyzes the formation of cAMP from intracellular ATP. In this study, B. pertussis adenylate cyclase was purified to remove contaminating islet activating protein and examined for its effects on intracellular cAMP levels of human erythrocytes and N1E-115 mouse neuroblastoma cells. In both cases, the enzyme catalyzed the formation of intracellular cAMP. Addition of calmodulin to the adenylate cyclase preparations completely inhibited formation of intracellular cAMP catalyzed by the bacterial enzyme, indicating that cAMP was not synthesized extracellularly and then taken up by the cells. These experiments illustrate that the bacterial enzyme does enter animal cells and that the enzyme-calmodulin complex does not.     

The Escherichia coli adenylate cyclase complex. Regulation by enzyme I of the phosphoenolpyruvate:sugar phosphotransferase system.

The activity of adenylate cyclase of Escherichia coli measured in toluene-treated cells under standard conditions is subject to control by the phosphoenolpyruvate:sugar phosphotransferase system (PTS). Sugars such as glucose, which are transported by the PTS, will inhibit adenylate cyclase provided the PTS is functional. An analysis was made of the properties of E. coli strains carrying mutations in PTS proteins. Leaky mutants in the PTS protein HPr are similar to wild-type strains with respect to cAMp regulation; adenylate cyclase activity in toluene-treated cells and intracellular cAMP levels are in the normal range. Furthermore, adenylate cyclase in toluene-treated cells of leaky HPr mutants is inhibited by glucose. In contrast, mutations in the PTS protein Enzyme I result in abnormalities in cAMP regulation. Enzyme I mutants generally have low intracellular cAMP levels. Leaky Enzyme I mutants show an unusual phosphoenolpyruvate-dependent activation of adenylate cyclase that is not seen in Enzyme I⁺ revertants or in Enzyme I deletions. A leaky Enzyme I mutant exhibits changes in the temperature-activity profile for adenylate cyclase, indicating that adenylate cyclase activity is controlled by Enzyme I. Temperature-shift studies suggest a functional complex between adenylate cyclase and a regulator protein at 30 °C that can be reversibly dissociated at 40 °C. These studies further support the model for adenylate cyclase activation that involves phosphoenolpyruvate-dependent phosphorylation of a PTS protein complexed to 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.     

The effect of growth conditions on adenylate cyclase activity and virulence-related properties of Bordetella pertussis

Growth of Bordetella pertussis in Stainer & Scholte medium in which the NaCl had been replaced by one of several inorganic or organic salts resulted in a large decrease in adenylate cyclase activity, histamine-sensitizing activity and in the amounts of two cell-envelope polypeptides of Mr 28000 and 30000. Although some variation between strains was observed, there was never a case where one of these properties was lost independently of the others. Cultures in which these properties were lost had decreased amounts of extracellular cAMP when compared to NaCl-grown cultures. Adenylate cyclase activity was detected in three locations of B. pertussis cultures (extracellular, extracytoplasmic but cell-associated, and cytoplasmic). After growth in medium containing high concentrations of MgSO4, enzyme activity was decreased to a similar extent in all three locations.     

Alteration in the protein components of catecholamine-sensitive adenylate cyclase during maturation of rat reticulocytes

The maturing rat reticulocyte was used as a model system in which to study developmental changes in the protein components of hormone-sensitive adenylate cyclase. Plasma membranes from rat erythrocytes display 10 to 20% of the adenylate cyclase activity and 30 to 50% of the beta-adrenergic receptors which are measured in membranes from rat reticulocytes, as noted by others. Reticulocyte membranes also display equal activities in response to (-)-isoproterenol in the presence of either GTP or GTP gamma S, whereas erythrocyte membrane adenylate cyclase is twice as active in the presence of isoproterenol plus GTP gamma S as in the presence of isoproterenol plus GTP. We have studied this system in greater detail by developing or applying independent assays for the catalytic protein (C) and the guanine nucleotide-binding regulatory protein (G/F) of adenylate cyclase. C was assayed in membranes by its intrinsic Mn2+-stimulated activity. It was also measured by reconstituting membranes with saturating amounts of GTP gamma S-activated G/F, yielding an operationally defined Vmax for the catalyst. By either assay, reticulocytes display about 3-fold greater C activity than do erythrocytes. G/F was assayed by its ability to confer GTP gamma S-stimulated activity upon C (which was supplied by membranes of cyc- S49 lymphoma cells). This assay indicates that reticulocyte membranes contain about 3 times as much G/F as do erythrocyte membranes. Cholera toxin and [32P]NAD were used to [32P]ADP-ribosylate the 45,000- and 52,000-dalton subunits of G/F. Total incorporation of 32P into these subunits decreased 3- to 4-fold with reticulocyte maturation. The ratio of label in the 52,000-dalton peptide to that in the 45,000-dalton peptide decreased from 0.29 in reticulocyte membranes to 0.14 in erythrocyte membranes. The apparently coordinate decrease in the amounts of C, G/F, and beta-adrenergic receptors suggest that the stoichiometry between these components is maintained during maturation, and may account for the decrease in adenylate cyclase in the membranes. However, the qualitative changes in responsiveness to hormones in the presence of GTP or GTP gamma S may be related to loss or proteolysis of the 52,000-dalton subunit of G/F.     

Cell-specific localization of prostaglandin E2-sensitive adenylate cyclase in rabbit endometrium

Epithelial and stromal cells were isolated from endometrium of Day 1 pseudopregnant rabbits by enzymatic digestion with trypsin or trypsin:collagenase:deoxyribonuclease. Dispersed cells were grown in RPMI 1640 supplemented with 10% whole or steroid-depleted fetal bovine serum (FBS). Epithelial and stromal cells reached confluency after 6 to 7 days in culture and showed specific characteristics. Cells could be differentiated according to morphology, growth patterns, electrophoretic patterns, and response to estrogen or progesterone. Hormonal stimulation of adenylate cyclase activity was measured in broken cell preparations by catalytic transformation of alpha-32P-adenosine triphosphate into 32P-adenosine 3'-5' cyclic monophosphate (cAMP). Adenylate cyclase activity was present in fresh endometrial tissue and in dispersed cells after 7 days in culture. The enzyme activity was significantly higher in stromal than in epithelial cells at all stimulation levels: basal (9.2 +/- 1.0 vs. 2.3 +/- 0.6, p less than 0.001) and guanosine triphosphate (GTP, 300 microM) (25.4 +/- 2.9 vs. 7.0 +/- 1.6, p less than 0.001). Net response to prostaglandin E2 (PGE2, 10 microM) was three times higher (p less than 0.001) in stromal (17 +/- 2) than in epithelial (5.0 +/- 1) cells. These results suggest that PGE2 can stimulate adenylate cyclase in rabbit endometrium and that the enzyme is preferentially localized in the stroma. Our results are in agreement with the hypothesis that cAMP formed in endometrium in response to PGE2 might be involved in the decidual reaction.     

Adenylate cyclase activity in a higher plant, alfalfa (Medicago sativa).

An adenylate cyclase activity in Medicago sativa L. (alfalfa) roots was partially characterized. The enzyme activity remains in the supernatant fluid after centrifugation at 105,000 g and shows in crude extracts an apparent Mr of about 84,000. The enzyme is active with Mg2+ and Ca2+ as bivalent cations, and is inhibited by EGTA and by chlorpromazine. Calmodulin from bovine brain or spinach leaves activates this adenylate cyclase.     

Adenylate cyclase in Saccharomyces cerevisiae is a peripheral membrane protein.

The adenylate cyclase system of the yeast Saccharomyces cerevisiae contains the CYR1 polypeptide, responsible for catalyzing formation of cyclic AMP (cAMP) from ATP, and two RAS polypeptides, which mediate stimulation of cAMP synthesis of guanine nucleotides. By analogy to the mammalian enzyme, models of yeast adenylate cyclase have depicted the enzyme as a membrane protein. We have concluded that adenylate cyclase is only peripherally bound to the yeast membrane, based on the following criteria: (i) substantial activity was found in cytoplasmic fractions; (ii) activity was released from membranes by the addition of 0.5 M NaCl; (iii) in the presence of 0.5 M NaCl, activity in detergent extracts had hydrodynamic properties identical to those of cytosolic or NaCl-extracted enzyme; (iv) antibodies to yeast adenylate cyclase identified a full-length adenylate cyclase in both membrane and cytosol fractions; and (v) activity from both cytosolic fractions and NaCl extracts could be functionally reconstituted into membranes lacking adenylate cyclase activity. The binding of adenylate cyclase to the membrane may have regulatory significance; the fraction of activity associated with the membrane increased as cultures approached stationary phase. In addition, binding of adenylate cyclase to membranes appeared to be inhibited by cAMP. These results indicate the existence of a protein anchoring adenylate cyclase to the membrane. The identity of this protein remains unknown.     

Isolation of a protein fraction from Bordetella pertussis that facilitates entry of the calmodulin-sensitive adenylate cyclase into animal cells

Bordetella pertussis, the pathogen responsible for whooping cough, releases a soluble calmodulin-sensitive adenylate cyclase into its culture medium. Several investigators have shown that the partially purified adenylate cyclase is capable of entering animal cells and elevating intracellular cAMP levels [Confer, D. L., & Eaton, J. W. (1982) Science 217, 948-950; Shattuck, R. L., & Storm, D. R. (1985) Biochemistry 24,6323-6328]. However, the mechanism for entry of the catalytic subunit of the adenylate cyclase into animal cells is unknown. Recently, it was determined that the purified catalytic subunit of the enzyme is unable to enter animal cells [Masure, H. R., Oldenburg, D. J., Donovan, M. G., Shattuck, R. L., & Storm, D. R. (1988) J. Biol. Chem. 263, 6933-6940]. On the basis of these data and other observations, we hypothesized that the culture medium of B. pertussis contains one or more additional polypeptides which facilitate entry of the adenylate cyclase catalytic subunit into animal cells. In this study, we report that a cell-invasive preparation of B. pertussis adenylate cyclase was rendered noninvasive after passage through a wheat germ lectin-agarose column. A fraction was eluted from the wheat germ lectin-agarose column with N-acetyl-D-glucosamine. This fraction, when combined with the noninvasive adenylate cyclase, was able to restore the ability of the adenylate cyclase preparation to enter neuroblastoma cells and increase intracellular cAMP levels. Furthermore, the fraction eluted from the wheat germ lectin-agarose column was found to be trypsin and chymotrypsin sensitive, suggesting that this material was proteinaceous.(ABSTRACT TRUNCATED AT 250 WORDS)     

cAMP imaging of cells treated with pertussis toxin, cholera toxin, and anthrax edema toxin

The enzymatic activity of the three most studied bacterial toxins that increase the cytosolic cAMP level: pertussis toxin (PT), cholera toxin (CT), and anthrax edema toxin (ET), was imaged by fluorescence videomicroscopy. Three different cell lines were transfected with a fluorescence resonance energy transfer biosensor based on the PKA regulatory and catalytic subunits fused to CFP and YFP, respectively. Real-time imaging of cells expressing this cAMP biosensor provided time and space resolved pictures of the toxins action. The time course of the PT-induced cAMP increase suggests that its active subunit enters the cytosol more rapidly than that deduced by biochemical experiments. ET generated cAMP concentration gradients decreasing from the nucleus to the cell periphery. On the contrary, CT, which acts on the plasma membrane adenylate cyclase, did not. The potential of imaging methods in studying the mode of entry and the intracellular action of bacterial toxins is discussed.     

Nature of the Cytoplasmic Factor(s) Modulating Adenylate Cyclase in the Developing Rat Brain

Abstract: Adenylate cyclase activity in the particulate fraction from rat brain was markedly enhanced by the cytoplasmic fraction, which itself contained negligible enzyme activity, indicating the presence of some stimulatory factor(s) in the supernatant. Activation of adenylate cyclase was dependent on the supernatant concentration up to 1 mgiml, but higher concentration of the supernatant did not produce further activation of the enzyme. The supernatant retained its stimulatory activity after boiling for 5 min, extensive dialysis, and phospholipase A and DNAase treatments, but was completely inactivated by digestion with trypsin. Ability of the supernatant to activate adenylate cyclase was low during fetal life, increased severalfold neonatally, and declined somewhat thereafter to an adult level. Adenylate cyclase in the particulate fraction from 2-day-old rat brain was also activated by GTP, calcium-dependent regulator (CDR) of cyclic AMP phosphodiesterase in the presence of 100 pM-Ca¹, and by NaF. The supernatant produced additive activation of the enzyme with NaF but not with GTP or CDR, suggesting a common site of action of the supernatant factor(s) and the latter two agents. DEAE-cellulose chromatography of the boiled supernatant resolved the heat-stable proteins into several peaks. Adenylate cyclase activator eluted in two distinct peaks, one of which also contained CDR activity. It is concluded that rat brain supernatant contains some factor in addition to CDR which activates particulate adenylate cyclase.     

Evidence for reversability of age-related decrease in human lymphocyte adenylate cyclase activity

Age-related loss of adenylate cyclase responsiveness to guanyl nucleotide was demonstrated in lymphocytes freshly isolated from human subjects. Enzyme activity of cells from young (<40 years) and elderly (>65 years) subjects were markedly sensitive to inhibition by non-ionic detergents. When enzyme activity in the presence of guanyl nucleotide and low concentrations of Triton X-100 was determined in a mixture of cells from the young and aged donors, the activity was 40±17 percent (mean ± S.D.) greater than anticipated from the activity of the cells of the two age groups assayed separately. The detergent range which facilitated the enhanced enzyme activity was too low to extract the catalytic subunit of adenylate cyclase from the cells. These results further suggest that in man, changes distal to receptors contribute to diminished responsiveness of lymphocyte adenylate cyclase as a function of age. In addition, these age-related changes may be partially reversible by reconstitution with factors from cells from younger subjects.     

Adenylate cyclase activity and cyclic nucleotide content in human adrenal tumors under Icenko-Cushing syndrome

The adenylate cyclase activity and cyclic nucleotide content in excised human adrenal tumours (Icenko-Cushing syndrome) were determined. The experimental data were compared to those obtained for hyperplastic adrenals. All adrenal tumours under study revealed a decreased cAMP level, an increased cGMP level and a resulting decrease of the cAMP/cGMP ratio. In malignant adrenal tumours the adenylate cyclase activity was sharply increased in comparison with that in hyperplastic adrenals. In the majority of malignant tumours the adenylate cyclase response to ACTH was either altogether absent or sharply decreased. In benign adrenal tumours the basal activity of the enzyme was unchanged and the enzyme response to ACTH was essentially normal. The decrease of adenylate cyclase response to ACTH in malignant tumours is apparently not due to the impaired catalytic activity of the enzyme, since its response to stimulation by sodium fluoride remains unaffected. In some tumours (one malignant and two benign ones) a non-specific stimulation of adenylate cyclase by hormones, which are not natural activators of the enzyme was observed. It was assumed that these changes are due to the damage of hormonal receptors in adrenal tumours.     

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.     

The regulatory component of adenylate cyclase. Purification and properties

The regulatory component (G/F) of adenylate cyclase, which has been purified previously, contains three putative subunits with molecular weights of 52,000, 45,000, and 35,000 (Northup, J. K., Sternweis, P. C., Smigel, M. D., Schleifer, L. S., Ross, E. M., and Gilman, A. G. (1980) Proc. Natl. Acad. Sci. U. S. A. 77, 6516-6520). The published procedure has been modified to reduce the time required for preparation and to increase the yield. Application of the improved procedure allows purification of .5 to 1.0 mg of purified G/F from 1.5 kg of frozen rabbit liver. Greater than 95% of the protein observed on sodium dodecyl sulfate polyacrylamide gels is found in the three bands mentioned above. Purified G/F has the following properties: 1. Hydrodynamic measurements in cholate indicate that purified hepatic G/F has a molecular weight of about 70,000. If G/F is activated with either fluoride or GTP analogs, its apparent molecular weight is reduced to 50,000. 2. The measurement of G/F by reconstitution with the catalytic moiety of adenylate cyclase is dependent on the concentrations of both G/F and catalytic moiety. This interaction is consistent with a model derived from a simple bimolecular binding equilibrium. 3. Purified G/F can be activated by fluoride and guanine nucleotide analogs in a Mg2+-dependent reaction. The rate of activation by guanine nucleotides is markedly stimulated by high concentrations of Mg2+, indicating a site of action of divalent metallic cations on G/F. 4. The 52,000- and 45,000-dalton polypeptides can be partially resolved by heptylamine-Sepharose chromatography. G/F fractions that are enriched in the 52,000-dalton protein reconstitute hormone-stimulated adenylate cyclase activity more efficiently and are activated by GTP analogs more rapidly than are fractions that are essentially free of this polypeptide. The 35,000-dalton protein is present in all cases.     

Adenylate cyclase activity during phenotypic variation of Bordetella pertussis

During MgSO4-induced modulation of Bordetella pertussis, adenylate cyclase activity, histamine-sensitizing activity (HSA) and the major cell-envelope polypeptides with Mr 28000 and 30000 (X polypeptides) were lost synchronously at a rate which could be accounted for by a simple growth-dilution effect. MgSO4 and other compounds which induced the above phenotypic change caused little inhibition of adenylate cyclase activity. Nicotinic acid was the sole exception and at 4.1 mM-caused 60% inhibition of activity. Lysates of modulated cells, mixed with lysates of unmodulated cells, had no effect on either adenylate cyclase activity or HSA. Protein synthesis was a prerequisite for MgSO4-induced modulation and also for the reversal of this process. Exogenous cAMP and dibutyryl cAMP (5 mM) had no counteracting effect on MgSO4- or nicotinic acid-induced modulation. The concentration of MgSO4 required to induce loss of the X polypeptides (10 to 11 mM) was not altered by promoting adenylate cyclase activity by including an activator in the growth medium. In one culture containing 10 mM-MgSO4 and activator, partial loss of the X polypeptides occurred and yet the extracellular cAMP concentration was twice that of cultures without activator and where full expression of the X polypeptides occurred. [3H]cAMP-binding activity was detected in cell extracts of several strains of B. pertussis, but antiserum against purified Escherichia coli catabolite repressor protein gave no reaction with B. pertussis cell extracts. Respiration rates with amino acids were similar for modulated and unmodulated variants and an avirulent strain of B. pertussis. These results are discussed in relation to a possible causal role for adenylate cyclase in modulation of B. pertussis.