Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:4.6.1.1 (adenylate cyclase)
19,190 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Intracellular recordings were made from rat dorsal horn neurons in the in vitro slice preparation to study the actions of cyclic adenosine 3',5'-monophosphate (cyclic AMP). In the presence of TTX, bath application of the membrane permeable analogue of cyclic AMP, 8-Br cyclic AMP (25-100 microM) caused a small depolarization of the resting membrane potential accompanied by a variable change in membrane input resistance. In addition, 8-Br cyclic AMP caused a long-lasting increase in the spontaneous synaptic activity and the amplitude of presumed monosynaptic excitatory postsynaptic potentials evoked in the substantia gelatinosa neurons by orthodromic stimulation of a lumbar dorsal root. When the fast voltage-sensitive Na conductance was blocked by TTX, 8-Br cyclic AMP enhanced in a reversible manner, the depolarizing responses of a proportion of dorsal horn neurons to N-methyl-D-aspartic acid (NMDA), alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), quisqualic acid (QA) and kainic acid (KA). The effects of 8-Br cyclic AMP on the resting membrane potential and the NMDA response of dorsal horn neurons were mimicked by reducing phosphodiesterase activity with bath application of 3-isobutyl-1-methylxanthine, but not by cyclic AMP applied extracellularly. Moreover, we have found that intracellular application of a protein inhibitor of cyclic AMP-dependent protein kinase (PKI) into dorsal horn neurons prevents the 8-Br cyclic AMP-induced potentiation of the NMDA response of these cells. These results suggest that in the rat spinal dorsal horn the activation of the adenylate cyclase-cyclic AMP-dependent protein kinase system may be involved in the enhancement of the sensitivity of postsynaptic excitatory amino acid (NMDA, AMPA, KA) receptors and modulation of primary afferent neurotransmission, including nociception.
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PMID:Cyclic adenosine 3'5'-monophosphate potentiates excitatory amino acid and synaptic responses of rat spinal dorsal horn neurons. 133 73

The effect of the putative amino acid transmitter, L-glutamate, on adenylate cyclase in crude membrane preparations of the rat tapeworm Hymenolepis diminuta was investigated to determine if glutamate effects the generation of the second messenger cAMP. Addition of glutamate at 10(-3) and 5.5 x 10(-9) M resulted in significant elevations in basal activity of adenylate cyclase, while concentrations in the 10(-5)-10(-7) M range caused significant depressions below basal activity. Assays with glutamate agonists and other acidic compounds showed glutamate to be the only amino acid, dicarboxylic acid, or acidic compound capable of this pattern of stimulation and inhibition. While the response of adenylate cyclase to glutamate agonists suggested that an N-methyl-D-aspartic acid (NMDA) type receptor may be present, glutamate agents acting as NMDA antagonists in vertebrate systems were agonists. Metabolic end products of glycolysis stimulated adenylate cyclase, suggesting that these, along with metabolic glutamate may regulate glycolytic enzymes. Only 10(-3) M L-glutamate significantly stimulated adenylate cyclase activity in tissue slices, and this response was restricted to those slices rich in nervous tissues. L-Glutamate eliminated the 5-hydroxytryptamine (5-HT) stimulated adenylate cyclase response suggesting that glutamate can modulate the 5-HT stimulated elevations in adenylate cyclase activity. The data support the hypothesis that L-glutamate is a neurotransmitter-modulator in the cestode.
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PMID:The effect of L-glutamate and related agents on adenylate cyclase in the cestode Hymenolepis diminuta. 170 17

Recent studies on the glucagon antagonist des-His1-[Glu9]glucagon amide have resulted in pure inhibitors of the hormone, suggesting that the inhibitory properties may be centered around position 9. The present study was designed to investigate the chemical characteristics of substitutions in position 9 of glucagon that determine binding affinity and biological activity. Twenty replacement analogs of position 9 of glucagon were synthesized and assessed for their ability to bind to the glucagon receptor in rat hepatocyte membranes and to activate adenylate cyclase. Any substitution of aspartic acid 9 was accompanied by a severely diminished capacity to transmit the biological signal, while retaining receptor binding affinity. These results are an indication of an uncoupling of receptor binding and biological activity at this locus and define a central role of aspartic acid 9 in glucagon activity. Single replacement or deletion of either His1 or Asp9 in glucagon caused a 20- to 50-fold decrease in cyclase activity, whereas these same changes made in tandem caused virtually complete loss of activity, with decreases of 10(4)-to 10(6)-fold. These observations have led us to speculate that, at the molecular level, the region of glucagon required for transduction of the biological response may be distinct from the binding region and is mediated by a coupled interaction between His1 and Asp9 of the hormone and a complementary functional site of the glucagon receptor.
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PMID:Position 9 replacement analogs of glucagon uncouple biological activity and receptor binding. 184 33

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.
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PMID:Functional consequences of single amino acid substitutions in calmodulin-activated adenylate cyclase of Bordetella pertussis. 205 Jan 7

In Escherichia coli there is a large increase of cAMP synthesis in crp strains, which are deficient in the catabolite gene activator protein. In this work it was shown that this increase in cAMP synthesis does not occur in crp crr strains, deficient in both the catabolite gene activator protein and enzymeIII-glucose, a component of the phosphotransferase system. It was also shown that the other components of the phosphotransferase system are required to obtain the increase of cAMP synthesis in a crp background. Adenylate cyclase mutants were obtained, by random mutagenesis, which had partial adenylate cyclase activity but which did not exhibit increased levels of cAMP in a crp background. For three mutants the mutation was identified as a single point mutation. This allowed the identification of residues arginine 188, aspartic acid 414 and glycine 463 which could be involved in the catabolite gene activator protein dependent activation process.
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PMID:Characterization of Escherichia coli adenylate cyclase mutants with modified regulation. 217 76

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.
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PMID:Identification of residues essential for catalysis and binding of calmodulin in Bordetella pertussis adenylate cyclase by site-directed mutagenesis. 254 30

Escherichia coli haemolysin (HlyA), a 107K (K = 10(3) Mr) protein, is secreted to the medium in an hlyB, hlyD-dependent process. Secretion, however, depends on neither an N-terminal signal sequence nor on SecA, which is part of the normal cellular export machinery for periplasmic and outer membrane proteins. In contrast, HlyA contains a novel C-terminal secretion signal encompassing the last 27 amino acids and possibly some additional residues immediately upstream. This region is characterized by a 16 residue 'aspartic acid box' composed largely of small amino acids which we propose constitutes an important element in recognition of the membrane translocation complex constituted by HlyB and HlyD. This feature is also found at the C-terminus of the adenyl cyclase and leukotoxin A molecules and resembles a recently identified eukaryotic C-terminal signal for targeting to glycosomes. A domain of the HlyB component of the haemolysin transport system is also similar to a domain widely distributed in nature, apparently acting as an ATP-dependent transport protein for a wide variety of molecules. Secretion of haemolysin, however, is the first example of a protein translocation system involving an HlyB-like molecule. This suggests that a major role of HlyB or at least its C-terminal domain is the coupling of energy to translocation of the haemolysin. It is more likely therefore that HlyD is more involved in the actual translocation through the membrane. On the basis of genetical and biochemical studies we propose that the haemolysin is translocated directly to the medium bypassing the periplasm. We further propose that HlyB and HlyD together constitute a membrane-bound translocator specific for molecules bearing the HlyA targeting sequence, and that the organization of this complex (conceivably involving other E. coli membrane proteins) must somehow straddle the inner and outer membranes. Finally, the HlyA C-terminal domain has been successfully used to promote the secretion to the medium of a number of heterologous polypeptides, in an HlyB,D-dependent manner.
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PMID:A novel C-terminal signal sequence targets Escherichia coli haemolysin directly to the medium. 269 60

By using oligonucleotide-directed mutagenesis, we have produced a point mutation (guanine to adenine) at nucleotide 388 of the gene for human beta-adrenergic receptor (beta AR) that results in a substitution of asparagine for the highly conserved aspartic acid at position 130 in the putative third transmembrane domain of the human beta AR ([Asn130]beta AR). We have examined the functional significance of this mutation in B-82 cells continuously expressing the mutant [Asn130]beta AR. The mutant [Asn130]beta AR displayed normal antagonist binding but unusually high-affinity agonist binding (5- to 10-fold higher than wild-type beta AR), consistent with a single class of high-affinity binding sites. The mutant beta AR displayed guanine nucleotide-sensitive changes in agonist affinity (3- to 5-fold shift) implying an interaction between the beta AR and the stimulatory guanine nucleotide-binding regulatory protein; however, the ability of guanine nucleotides to alter agonist affinity was attenuated. Addition of saturating concentrations of isoproterenol to cell cultures expressing mutant [Asn130]-beta ARs had no effect on intracellular levels of cAMP, indicating that the mutant beta AR is unable to affect stimulation of adenylate cyclase. These results indicate that substitution of the aspartic acid with asparagine at residue 130 of the human beta AR dissociates the well-characterized guanine nucleotide effects on agonist affinity from those on activation of the stimulatory guanine nucleotide-binding regulatory protein and adenylate cyclase and suggests the existence of two distinct counterions for the amine portion of catecholamines that are associated with high- and low-affinity agonist binding states of beta AR.
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PMID:Site-directed mutagenesis of human beta-adrenergic receptors: substitution of aspartic acid-130 by asparagine produces a receptor with high-affinity agonist binding that is uncoupled from adenylate cyclase. 284 Jun 63

The authors studied the effect of native ACTH on dehydrogenase activity of isolated strips of rat diaphragm and suspension of E. coli cells, serotype O III:B4, grown on beef extract agar in a medium with different dehydrogenation substrates. ACTH activated dehydrogenase of rat diaphragm in a medium containing pyruvate, alpha-ketoglutarate, malate, beta-hydroxybutyrate, D-aspartic acid and did not alter it in a medium containing succinate. In contradistinction to rat diaphragm, ACTH activated dehydrogenase of E. coli cells whatever the substrates used (oxaloacetate, isocitrate, alpha-ketoglutarate, succinate, fumarate, malate, pyruvate, lactate, beta-hydroxybutyrate, glucose, D-aspartic acid. Synacthen (ACTH1-24) exerted a similar effect. It is suggested that the effects of ACTH are mediated via its influence on adenylate cyclase in the absence of receptors.
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PMID:[Effect of corticotropin on the dehydrogenase activity of cells and tissues]. 298 79

Synthetic 1-84 human PTH (hPTH) peptides (with either asparagine) or aspartic acid at position 76) were compared with natural bovine PTH (bPTH) in three in vivo bioassays. Surprisingly, in the chick hypercalcemia bioassay, the human 1-84 peptides were approximately 3 times more potent on a molar basis than bPTH. In contrast, in an in vivo mouse kidney cAMP accumulation bioassay, these human peptides were 3-6 times less potent than bPTH. This low potency of synthetic hPTH relative to bPTH in the renal cAMP assay is in accordance with published relative potency estimates for natural extracted hPTH in in vitro rat renal membrane adenylate cyclase assays. The human and bovine 1-84 peptides were weakly active in an in vivo mouse calvaria cAMP accumulation system, producing a shallow dose-response curve which was not suitable for any quantitative estimates of potency. In contrast, both human and bovine 1-34 fragments were highly active in stimulating accumulation of cAMP in calvaria thus emphasizing the qualitative differences between 1-84 PTH and the 1-34 fragment of both species of PTH. Despite the homology between human and bovine 1-84 PTH, they have markedly different quantitative biological effects on hypercalcemia in chicks and in vivo renal cAMP accumulation in mice. Any estimate of the biological potency of human 1-84 PTH, relative to bovine 1-84 PTH, will need to be defined in terms of the nature and species of the biological test system.
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PMID:Biological activities of synthetic human parathyroid hormone (PTH) 1-84 relative to natural bovine 1-84 PTH in two different in vivo bioassay systems. 299 3


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