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cAMP generation in bacteria is often stimulated by sudden, but lasting, changes in extracellular conditions, whereas intracellular cAMP concentrations quickly settle at new levels. As bacteria lack G-proteins, it is unknown how bacterial adenylate cyclase (AC) activities are modulated. Mycobacterium tuberculosis has 15 class III AC genes; therefore, we examined whether mycobacteria contain a factor that may regulate AC activities. We identified mycobacterial polyphosphates with a mean chain length of 72 residues as highly potent inhibitors of dimeric class IIIa, class IIIb and class IIIc ACs from M. tuberculosis and other bacteria. The identity of the inhibitor was established by phosphatase degradation, 31P-NMR, acid or base hydrolysis, PAGE and comparisons with commercial standards, and functional substitution by several polyphosphates. The data indicate that each AC dimer occupies 8–15 phosphate residues on a polyphosphate strand. Other polyionic polymers such as polyglutamate, polylysine and hyaluronic acid do not affect cyclase activity. Notably, the structurally unrelated class I AC Cya from Escherichia coli is unaffected. Bacterial polyphosphate metabolism is generally viewed in the context of stress-related regulatory networks. Thus, regulation of bacterial class III ACs by polyphosphates could be a component of the bacterial stress response. [ABSTRACT FROM AUTHOR]

The adenylyl cyclase Rv1625c from Mycobacterium tuberculosis codes for a protein with six transmembrane spans and a catalytic domain, i.e. it corresponds to one half of the pseudoheterodimeric mammalian adenylyl cyclases (ACs). Rv1625c is active as a homodimer. We investigated the role of the Rv1625c membrane domain and demonstrate that it efficiently dimerizes the protein resulting in a 7.5-fold drop in Km for ATP. Next, we generated a duplicated Rv1625c AC dimer by a head-to-tail concatenation. This produced an AC with a domain order exactly as the mammalian pseudoheterodimers. It displayed positive cooperativity and a 60% increase of vmax compared with the Rv1625c monomer. Further, we probed the compatibility of mycobacterial and mammalian membrane domains. The second membrane anchor in the Rv1625c concatamer was replaced with membrane domain I or II of rabbit type V AC. The mycobacterial and either mammalian membrane domains are compatible with each other and both recombinant proteins are active. A M. tuberculosis Rv1625c knockout strain was assayed in a mouse infection model. In vitro growth characteristics and in vivo organ infection and mortality were unaltered in the knockout strain indicating that AC Rv1625c alone is not a virulence factor. [ABSTRACT FROM AUTHOR]

The genes Rv1318c, Rv1319c, Rv1320c and Rv3645 of Mycobacterium tuberculosis are predicted to code for four out of 15 adenylyl cyclases in this pathogen. The proteins consist of a membrane anchor, a HAMP region and a class IIIb adenylyl cyclase catalytic domain. Expression and purification of the isolated catalytic domains yielded adenylyl cyclase activity for all four recombinant proteins. Expression of the HAMP region fused to the catalytic domain increased activity in Rv3645 21-fold and slightly reduced activity in Rv1319c by 70%, demonstrating isoform-specific effects of the HAMP domains. Point mutations were generated to remove predicted hydrophobic protein surfaces in the HAMP domains. The mutations further stimulated activity in Rv3645 eight-fold, whereas the effect on Rv1319c was marginal. Thus HAMP domains can act directly as modulators of adenylyl cyclase activity. The modulatory properties of the HAMP domains were confirmed by swapping them between Rv1319c and Rv3645. The data indicate that in the mycobacterial adenylyl cyclases the HAMP domains do not display a uniform regulatory input but instead each form a distinct signaling unit with its adjoining catalytic domain. [ABSTRACT FROM AUTHOR]

We describe a single aquaporin gene in Toxoplasma gondii which, surprisingly, has only 28% sequence similarity to the aquaglyceroporin of another apicomplexan parasite, Plasmodium falciparum. Sequence comparisons showed 47% similarity to water-specific plant aquaporins and the conservation of typical pore-forming residues. We established that the Toxoplasma aquaporin protein is a bifunctional membrane pore with intermediate water and high glycerol permeability. Furthermore, we identified hydroxyurea, an antineoplastic agent with inhibitory effects on parasite proliferation, as a permeant of this channel. [Copyright &y& Elsevier]

The gene cyaB1 from the cyanobacterium Anabaena sp. PCC 7120 codes for a protein consisting of two N-terminal GAF domains (GAF-A and GAF-B), a PAS domain and a class III adenylyl cyclase catalytic domain. The catalytic domain is active as a homodimer, as demonstrated by reconstitution from complementary inactive point mutants. The specific activity of the holoenyzme increased exponentially with time because the product cAMP activated dose dependently and nucleotide specifically (half-maximally at 1 μM), identifying cAMP as a novel GAF domain ligand. Using point mutants of either the GAF-A or GAF-B domain revealed that cAMP activated via the GAF-B domain. We replaced the cyanobacterial GAF domain ensemble in cyaB1 with the tandem GAF-A/GAF-B assemblage from the rat cGMP-stimulated phosphodiesterase type 2, and converted cyaB1 to a cGMP-stimulated adenylyl cyclase. This demonstrated the functional conservation of the GAF domain ensemble since the divergence of bacterial and eukaryotic lineages >2 billion years ago. In cyanobacteria, cyaB1 may act as a cAMP switch to stabilize committed developmental decisions. [ABSTRACT FROM AUTHOR]

Guanylyl cyclases in eukaryotic unicells were biochemically investigated in the ciliates Paramecium and Tetrahymena, in the malaria parasite Plasmodium and in the ameboid Dictyostelium. In ciliates guanylyl cyclase activity is calcium-regulated suggesting a structural kinship to similarly regulated membrane-bound guanylyl cyclases in vertebrates. Yet, cloning of ciliate guanylyl cyclases revealed a novel combination of known modular building blocks. Two cyclase homology domains are inversely arranged in a topology of mammalian adenylyl cyclases, containing two cassettes of six transmembrane spans. In addition the protozoan guanylyl cyclases contain an N-terminal P-type ATPase-like domain. Sequence comparisons indicate a compromised ATPase function. The adopted novel function remains enigmatic to date. The topology of the guanylyl cyclase domain in all protozoans investigated is identical. A recently identified Dictyostelium guanylyl cyclase lacks the N-terminal P-type ATPase domain. The close functional relation of Paramecium guanylyl cyclases to mammalian adenylyl cyclases has been established by heterologous expression, respective point mutations and a series of active mammalian adenylyl cyclase/ Paramecium guanylyl cyclase chimeras. The unique structure of protozoan guanylyl cyclases suggests that unexpectedly they do not share a common guanylyl cyclase ancestor with their vertebrate congeners but probably originated from an ancestral mammalian-type adenylyl cyclase. [ABSTRACT FROM AUTHOR]

The gene Rv1625c from Mycobacterium tuberculosis encodes a membrane‐anchored adenylyl cyclase corresponding to exactly one‐half of a mammalian adenylyl cyclase. An engineered, soluble form of Rv1625c was expressed in Escherichia coli. It formed a homodimeric cyclase with two catalytic centers. Amino acid mutations predicted to affect catalysis resulted in inactive monomers. A single catalytic center with wild‐type activity could be reconstituted from mutated monomers in stringent analogy to the mammalian heterodimeric cyclase structure. The proposed existence of supramolecular adenylyl cyclase complexes was established by reconstitution from peptide‐linked, mutation‐inactivated homodimers resulting in pseudo‐trimeric and ‐tetrameric complexes. The mycobacterial holoenzyme was expressed successfully in E.coli and mammalian HEK293 cells, i.e. its membrane targeting sequence was compatible with the bacterial and eukaryotic machinery for processing and membrane insertion. The membrane‐anchored mycobacterial cyclase expressed in E.coli was purified to homogeneity as a first step toward the complete structural elucidation of this important protein. As the closest progenitor of the mammalian adenylyl cyclase family to date, the mycobacterial cyclase probably was spread by horizontal gene transfer. [ABSTRACT FROM AUTHOR]

The gene Rv1625c from Mycobacterium tuberculosis encodes a membrane-anchored adenylyl cyclase corresponding to exactly one-half of a mammalian adenylyl cyclase. An engineered, soluble form of Rv1625c was expressed in Escherichia coli. It formed a homodimeric cyclase with two catalytic centers. Amino acid mutations predicted to affect catalysis resulted in inactive monomers. A single catalytic center with wild-type activity could be reconstituted from mutated monomers in stringent analogy to the mammalian heterodimeric cyclase structure. The proposed existence of supramolecular adenylyl cyclase complexes was established by reconstitution from peptide- linked, mutation-inactivated homodimers resulting in pseudo-trimeric and -tetrameric complexes. The mycobacterial holoenzyme was expressed successfully in E.coli and mammalian HEK293 cells, i.e. its membrane targeting sequence was compatible with the bacterial and eukaryotic machinery for processing and membrane insertion. The membrane-anchored mycobacterial cyclase expressed in E.coli was purified to homogeneity as a first step toward the complete structural elucidation of this important protein. As the closest progenitor of the mammalian adenylyl cyclase family to date, the mycobacterial cyclase probably was spread by horizontal gene transfer. [ABSTRACT FROM AUTHOR]

We cloned a guanylyl cyclase of 280 kDa from the ciliate Paramecium which has an N-terminus similar to that of a P-type ATPase and a C-terminus with a topology identical to mammalian adenylyl cyclases. Respective signature sequence motifs are conserved in both domains. The cytosolic catalytic C1a and C2a segments of the cyclase are inverted. Genes coding for topologically identical proteins with substantial sequence similarities have been cloned from Tetrahymena and were detected in sequences from Plasmodium deposited by the Malaria Genome Project. After 99 point mutations to convert the Paramecium TAA/TAG-Gln triplets to CAA/CAG, together with partial gene synthesis, the gene from Paramecium was heterologously expressed. In Sf9 cells, the holoenzyme is proteolytically processed into the two domains. Immunocytochemistry demonstrates expression of the protein in Paramecium and localizes it to cell surface membranes. The data provide a novel structural link between class III adenylyl and guanylyl cyclases and imply that the protozoan guanylyl cyclases evolved from an ancestral adenylyl cyclase independently of the mammalian guanylyl cyclase isoforms. Further, signal transmission in Ciliophora (Paramecium, Tetrahymena) and in the most important endoparasitic phylum Apicomplexa (Plasmodium) is, quite unexpectedly, closely related. [ABSTRACT FROM AUTHOR]

A guanylate cyclase of high specific activity was localized in the ciliary membrane from Tetrahymena pyriformis. Purity of cilia was checked by electron microscopy and purity of membrane fractions isolated by a sucrose density gradient by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. Enzyme activity was due to the presence of endogenous calmodulin as evidenced by the inhibition of guanylate cyclase by addition of antiserum against calmodulin from Tetrahymena or soybean. Removal of endogenous calmodulin by La3+-treatment of ciliary membranes resulted in loss of guanylate cyclase activity. In addition to protozoan calmodulins, the original activity could also be restored by the nonhomologous calmodulins from soybean and pig brain but not by calcium-binding proteins like Dictyostelium calmodulin, parvalbumin, and troponin C, lacking the trimethyllysine characteristic for mammalian calmodulins. However, only calmodulins from the protozoans Tetrahymena and Paramecium stimulated guanylate cyclase activity in excess of the initial activity. This indicates that the guanylate cyclase either contains t binding sites for calmodulin with different specificities or that a single, but only partially occupied binding site is dified possibly by hydrolytic exo-proteases during membrane preparation. The ciliary membrane m trahymena contains a discrete calcium-permeability as demonstrated by calcium-flux measurements using the calcium indicator dye arsenazo III. In analogy to the excitable ciliary membrane of the larger relative Paramecium, the ciliary membrane of Tetrahymena may thus carry the voltage-sensitive calcium-channels known from electrophysiological studies. [ABSTRACT FROM AUTHOR]

The membraneous guanylute cyclase of cilia from Paramecium tetraurelia used MgGTP and MnGTP as substrate with Michaelis constants for GTP of 71.5 μM and 36 μM, respectively. A linear Arrhenius plot indicated that a single enzyme entity exists not sensitive to possible phase transitions of membrane lipids. Guanylate cyclase is activated by low concentrations (< 100 μM)and inhibited by high concentrations (> 100 μM) of calcium, half-maximal effects were obtained with 8 μM and 500 μM Ca2+, respectively. Only strontium ions displayed partial activating and inhibiting potency, all other divalent cations tested, Ba2+, Fe2+, Co2+, Mn2+, Sn2+ and Ni2+ had no effect on guanylate cyclase activity. Ca2+ activation increased V; Km remained identical. The Ca2+ stimulated activity was not inhibited by trifluoperazine, tentatively suggesting that the stimulation may not be mediated by calmodulin. Ca2+ inhibition was due to a single binding site of Ca2+ at the guanylate cyclase as evidence by a Hill coefficient h = -1 and was noncompetitive. The lanthanides La3+, Ce3+ and Tb3+ were powerful inhibitors of guanylate cyclase, with La3+ the half-maximal effect was obtained with 0.6 μM, it was kinetically a mixed-type inhibition. La3+ and Ca3+ competed for the same binding site on the guanylate cyclase as determined by detailed kinetic analysis. Addition of EGTA reversed the activation and inhibition by Ca-2+ and the inhibition by La3+. It is discussed that guanylate cyclase may be the initial target enzyme in the cilia for the calcium transient of the calcium/potassium action potential of Parcunecium. [ABSTRACT FROM AUTHOR]

Veratridine opens voltage-dependent Na channels in many metazoans. In Paramecium, which has voltage-dependent Ca channels and a Ca/K action potential, no such Na channels are known. A Ca-inward current is correlated to an intracellular increase in cGMP. The addition of veratridine to Paramecium wildtype and to pawn mutant cells, which lack the Ca-inward current, transiently increased intracellular levels of cGMP about sevenfold to 40 pmol/mg protein. A half-maximal effect was obtained with 250 μ m veratridine. The increase in cGMP was maximal about 15 sec after the addition of veratridine and declined rapidly afterwards. Intracellular cAMP levels were not affected. The effect of veratridine on cGMP was dependent on the presence of extracellular Ca. The time dependence and extent of stimulation closely resembled the effects observed after stimulation by Ba, which causes the repetitive firing of action potentials, Ca-dependent ciliary reversal, and cGMP formation. The effects of Ba and veratridine were not additive. Wildtype cells and, surprisingly, also pawn mutant cells showed avoiding reactions upon addition of veratridine indicating that it induced a Ca influx into the cilia, which causes ciliary reversal. The potency of veratridine to stimulate cGMP formation was little affected by Na in wildtype cells, three pawn mutant strains, and in the cell line fast-2, which is defective in a Ca-dependent Na-inward current. Divalent cations (Ca, Mg, and Ba) inhibited the effects the veratridine similar to metazoan cells. The results indicate that veratridine can open the voltage-operated Ca channels in Paramecium wildtype and, most interestingly, in pawn mutant cells. The pawn mutation is suggested to represent a defect in the activation of the Ca channel. This explains the lack of differences in ciliary proteins between wildtype and pawn cells reported earlier. [ABSTRACT FROM AUTHOR]

The Ca-inward current of Paramecium is related to cGMP production by a Ca-dependent guanylate cyclase. Excitation with Ba increases cGMP levels about ninefold to 45 pmol/ mg within 15 sec. Inhibition of cGMP hydrolysis reveals a large rate of synthesis of up to 25 pmol cGMP/mg·sec, or about 1.2 ·10 molecules/cell·sec. Because no other factors than the Ca-inward current were found to affect cGMP formation in Paramecium, we used it as a quantitative measure of Ca channel activity. After a transient stimulation of cGMP formation by 1 mm Ba, an additional increase of Ba to 5 mm did not result in a renewed elevation of cGMP levels. The extent of desensitization towards a second stimulus was graded with the strength of the first stimulus. Termination of the first stimulus after various time intervals and restimulation after 3 min with 1 mm Ba revealed a time-dependent inactivation of the Ca channel, which could be fitted by a single exponential. The inactivated form of the channel was stable for a few minutes at room temperature. The partial desensitization of Paramecium reduced the maximal response, but did not shift the dose-response curve for Ba. Veratridine, which activates the Ca channel, was also used as a first stimulus. It effectively and transiently inactivated the channel resulting in a complete loss of both a behavioral response of Paramecium and cGMP elevation towards a second stimulus. The time course of reactivation of channel excitability was studied at different temperatures. Half times of recovery were 51 and 7.5 min at 12 and 25°C, respectively. Reactivation curves can be described by a single exponential, indicating a first order reaction. The activation energy was 100 kJ/mol. The extremely high rate of cGMP turnover in Paramecium is reminiscent of findings in visual cells. A model for regulation of the voltage-dependent Ca channel of Paramecium is proposed. [ABSTRACT FROM AUTHOR]

A stopped-flow spectrophotometric technique was used to study the kinetics of Ca flux into ciliary membrane vesicles from Paramecium tetraurelia wild-type and several 'pawn' mutants with defective Ca conductances. 15 mm Arsenazo III was used as metallochromic indicator and as intravesicular Ca trap. The absolute amount of Ca-permeable vesicles was significantly reduced in preparations from the 'pawn' mutants compared to wild-type. However, influx kinetics were identical for vesicles from wild-type and 'pawn' mutant Paramecia when the fraction of Ca-permeable vesicles was taken into account. Ca influx was rapid with a time constant of about 1.5 sec and an initial saturation rate of arsenazo III of about 50%/single vesicle ×sec. Ca influx rates were half-maximal at approximately 20 μ m Ca. Comparisons of Ba toxicity tested with a behavioral assay, Ca inward conductances under voltage-clamp conditions and Ca influx kinetics between wild-type and the 'pawn' mutants pwA (d4-94), leaky pwB (d4-96) and the double mutant pwA/pwB indicated that Ca transport in all types of ciliary membrane vesicles occurred through similar Ca gates. [ABSTRACT FROM AUTHOR]

A type 1 serine/threonine protein phosphatase (PP1) which is mostly localized in the excitable ciliary membranes from the protozoan Paramecium, was purified to homogeneity. Approximately 4 μg enzyme of 37 kDa was isolated from 1001 axenic culture. The enzymic properties were charac- terized using phosphorylase a from rabbit skeletal muscle as a substrate and several known effectors of mammalian PP1. The protozoan PP1 was enzymically indistinguishable from its mammalian congener. The amino acid sequence of the Paramecium PP1 was deduced from its cDNA. The full-length clone was obtained in several steps starting with a pair of degenerate primers made according to the two most conserved peptides of rabbit PP1 and PP2A. The gene encodes a protein of 36392 Da. The identity of the cloned gene and the isolated ciliary PP1 was unequivocally established by microsequencing of four tryptic and cyanogen-bromide peptides which were generated from the purified protein. Paramecium PP1 shows 75% amino-acid-sequence identity with rabbit PP1α. Areas of major differences are the C-termini and N-termini and a sequence between residues 219–242. [ABSTRACT FROM AUTHOR]

Basal and stimulated levels of inositol phosphates were determined in the protozoan Paramecium labelled with myo-[³H]inositol. Under resting conditions, intracellular InsP6 (phytic acid), InsP5 and InsP4 concentrations were 140, 10 and 2 µM, respectively. InsP5 was comprised of 56% Ins(l,2,3,4,5)P5 and/or Ins(1,2,3,5,6)P5, 40% Ins(1,2,4,5,6)P5 and/or Ins(2,3,4,5,6)P5 and small amounts of lns(l,3,4,5,6)P5 and lns(1,2,3,4,6)P5. InsP4 was mainly ins(l, 4, 5, 6)P4 and/or Ins(3, 4, 5, 6)P4. Other inositol phosphates were not detected at a detection limit of 50- 85 nM. Using various depolarizing and hyperpolarizing stimuli, no significant changes in level of inositol phosphates were observed in vivo, indicating that in the ciliate a contribution of inositol phosphates to signal transduction mechanisms is unlikely. In homogenates prepared from rnyo-[³H]inositol-labelled cells, a marked relative increase in InsP3 and InsP4 over the concentrations in vivo was observed. These inositol phosphates were identified as degradation products of endogenous InsP6. A novel separation methodology for inositol phosphates was established to allow unequivocal assignment of phosphate locations of all dephosphorylated lnsP6-derived products. The dephosphorylation was catalyzed by a phytase-like enzyme with a molecular mass of 240 kDa, most likely of a hexameric structure. The enzyme had a pH optimum of 7.0 and did not require divalent cations for activity. Substrate concentrations above 300 µM were inhibitory. Dephosphorylation of InsP6 by the Pararnecium enzyme differs from that of phytases from plants in that it proceeds via a sequential release of phosphate groups from positions 6, 5, 4 and 3 of the myo-inositol ring or/and positions 4, 5, 6 and 1. [ABSTRACT FROM AUTHOR]

Prolonged pretreatment of rats with the atypical antidepressant rolipram attenuates noradrenaline (NA) sensitivity of the cerebral cortical cAMP generating system. The development of this down-regulation is time (7 d treatment required) and dose dependent (EC=0.35 mg/kg). Density of β-adrenoceptor as measured by (−)-H-dihydroalprenolol [(−)-H-DHA] binding is also reduced by rolipram pretreatment. The effect of rolipram is absolutely stereospecific for the (−)-enantiomer (ED=0.18 mg/kg). In addition, only with this isomer, a reduction in daily weight gain was found compared to sham treated controls. Presynaptic denervation using intracerebroventricular (i.c.v.) injections of 6-hydroxydopamine (6-OHDA) prior to or during rolipram treatment did not completely block the effect of a rolipram treatment on down-regulation of cerebral cortical β-adrenoceptors. The data favor a prea- and postsynaptic action of rolipram different from all other antidepressants studied so far in this experimental setting. Rolipram is known as inhibitor of brain phosphodiesterase. Using partially purified calmodulin-independent phosphodiesterase from brain it is shown that exclusively the (−)-enantiomer of rolipram inhibits phosphodiesterase with an IC of 1.25 μmol/l whereas the (+)-isomer possesses little potency. Since a marked stereospecificity for the (−)-isomer of rolipram was displayed in all pharmacological parameters tested so far with (+)- and (−)-rolipram, it is suggested that stereospecific and isozyme specific inhibition of cAMP-phosphodiesterase is, at least in part, related to the mechanism of action of the potential antidepressant drug rolipram and possibly of other antidepressants as well. [ABSTRACT FROM AUTHOR]