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)

The synthesis of the heterobifunctional cross-linking reagent 2-nitro-4-azidophenylsulfenyl chloride (NAPSCl) is described. This reagent can be used to specifically attach a photoactivatable nitrophenyl azide to tryptophan-containing polypeptides and proteins lacking sulfhydryl groups. The sulfenyl chloride group of NAPSCl reacts with the indole ring of tryptophan following second-order reaction kinetics in 50-100% acetic acid. The labeled product can be effectively photolyzed at wavelengths above 300 nm. The reaction of glucagon, a peptide hormone containing a single tryptophan residue at position 25 and no cysteine, with NAPSCl gave one major product, the photosensitive derivative glucagon-NAPS. The structure and properties of the purified derivative were established by amino acid analysis, absorption spectroscopy, and photolysis. Only the tryptophan residue of this derivative was modified. The photosensitive glucagon was shown to activate the adenylate cyclase of hepatocyte plasma membranes to the same extent as the native hormone at equimolar concentrations. Glucagon-NAPS could be radiolabeled by the lactoperoxidase-catalyzed iodination of the peptide. A glucagon-specific antibody bound both radiolabeled glucagon and glucagon-NAPS peptides. The covalent labeling of protein molecules with radiolabeled glucagon-NAPS peptide upon photolysis was demonstrated. Glucagon-NAPS can be used as an effective photoaffinity probe for labeling the glucagon receptor site in plasma membranes of target cells.
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PMID:Synthesis and characterization of a heterobifunctional photoaffinity reagent for modification of tryptophan residues and its application to the preparation of a photoreactive glucagon derivative. 742 13

The combination of hydralazine and nitrates has been shown to provide long-term benefit in congestive heart failure, despite a nitrate dosage that should induce tolerance. To assess the interactions between hydralazine and nitroglycerin, aortic rings were isolated from male Wistar rats. In rings precontracted with phenylephrine, hydralazine incubation (10 microM and 0.1 mM) potentiated the responses to nitroglycerin (p < 0.05) but not to sin-1 (a direct activator of guanylate cyclase), 8-bromocyclic guanylate monophosphate, and forskolin (an adenylate cyclase activator). In similar conditions, the incubation of isoniazid (0.1 mM, used as a pyridoxal-sequestering agent without direct vasoactive properties) also potentiated the dose-response curve to nitroglycerin (p < 0.05). In aortas isolated from rats rendered nitrate tolerant in vivo (50 mg/kg subcutaneously twice daily during 4 days), hydralazine partially attenuated tolerance (p < 0.05). Our results suggest that the observed interaction between hydralazine and nitroglycerin may involve an inhibition of pyridoxal-dependent reactions, such as the catabolism of methionine and cysteine. This may enhance the availability of sulfhydryl-containing compounds, and therefore potentiate the responses to nitroglycerin.
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PMID:Interaction between hydralazine and nitrovasodilators in vascular smooth muscle. 768 11

Proenkephalin and other prohormones require proteolytic processing at paired basic and monobasic residues for the biosynthesis of active neuropeptides. The novel "prohormone thiol protease" (PTP) has been proposed as a candidate proenkephalin processing enzyme for the production of [Met]enkephalin in chromaffin granules (Krieger, T. J., and Hook, V. Y. H. (1991) J. Biol. Chem. 266, 88376-8383). In this study, PTP was examined during elevation of cellular [Met]enkephalin by forskolin, a direct activator of adenylate cyclase that produces cAMP. Treatment of chromaffin cells with forskolin for 72 h increased enkephalin precursor cleaving activity (measured by following the conversion of the model substrate [35S-Met]preproenkephalin to trichloroacetic acid-soluble radioactivity) in isolated chromaffin granules by 170-180% over controls (100%). The increased activity was associated with the membrane fraction, rather than the soluble fraction, of chromaffin granules. The elevated activity was inhibited by E-64c, which is a potent inhibitor of PTP and cysteine proteases; however, the activity was not inhibited by serine or aspartic protease inhibitors. The elevated activity was identified as PTP based on immunoprecipitation by anti-PTP immunoglobulins. Stimulation of PTP synthesis was involved in the forskolin-induced increase in PTP activity, as demonstrated by a 10-fold increase in [35S]PTP pulse labeling in forskolin-treated chromaffin cells. Forskolin elevation of PTP protein levels within chromaffin granules was also detected in Western blots. Importantly, the forskolin-mediated rise in cellular [Met]enkephalin levels was completely blocked when cells were preincubated with the cysteine protease inhibitor Ep453, which is known to be converted by intracellular esterases to the more effective inhibitor E-64c (Buttle, D. J., Saklatvala, J., Tamai, M., and Barrett, A. J. (1992) Biochem. J. 281, 175-177). Both E-64c and Ep453 inhibit PTP, with E-64c being more potent (Azaryan, A. V., and Hook, V. Y. H. (1994b) Arch. Biochem. Biophys. 314, 171-177). These results demonstrate a role for PTP in proenkephalin processing in chromaffin cells and indicate that [Met] enkephalin formation and PTP are both regulated by cAMP.
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PMID:Stimulation of "prohormone thiol protease" (PTP) and [Met]enkephalin by forskolin. Blockade of elevated [Met]enkephalin by a cysteine protease inhibitor of PTP. 776 28

The two cysteines C494 and C569, located in the first and second extracellular loop, respectively, of the thyrotropin (TSH) receptor, were mutated to serines to test the functional significance of the putative disulfide bond between these two cysteines. Single (C494S and C569S) and double (C494/569S) mutant receptors were generated, transiently expressed in COS cells, and compared with regard to the ability to bind ligand and to mediate stimulation of adenylate cyclase activity. The double mutant retained ligand binding capacity, in contrast to the single cysteine mutants that were essentially devoid of binding capacity. The ability of the mutated receptor variants to stimulate adenylate cyclase activity was lost or greatly reduced.
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PMID:Point mutations of the thyrotropin receptor determining structural requirements for its ability to bind thyrotropin and to stimulate adenylate cyclase activity. 813 1

We have identified the palmitoylated cysteine residues of alpha q and alpha s, alpha subunits of two heterotrimeric G proteins. Mutational substitutions of serines for cysteines 9 and 10 in alpha q and cysteine 3 in alpha s profoundly alter behavior of the subunits expressed in HEK293 cells. Neither mutant alpha subunit incorporates palmitate; both mutant proteins are found in the soluble rather than the particulate fraction; mutant alpha q or alpha s cannot couple a co-expressed receptor to stimulation of phospholipase C or adenylylcyclase, respectively; cysteine substitution prevents a mutationally activated alpha q (R183C) from stimulating phospholipase C directly, and reduces but does not abolish the ability of a similarly activated alpha s (R201C) to stimulate cAMP synthesis. Substitution of a myristoylation sequence for the palmitoylation sites leads to labeling of alpha q and alpha s by myristate, rather than by palmitate. Myristoylation restores the abilities of both nonpalmitoylated alpha q and alpha s to attach to membranes and, in the case of alpha q, restores its ability to stimulate phospholipase C, whether triggered by the R183C mutation or by receptor activation. These findings identify palmitoylation as a critical determinant of membrane attachment for alpha q and alpha s and show that this modification is required for normal signaling by these proteins.
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PMID:Palmitoylation is required for signaling functions and membrane attachment of Gq alpha and Gs alpha. 822 63

Numerous gram-negative and gram-positive bacteria take up carbohydrates through the phosphoenolpyruvate (PEP):carbohydrate phosphotransferase system (PTS). This system transports and phosphorylates carbohydrates at the expense of PEP and is the subject of this review. The PTS consists of two general proteins, enzyme I and HPr, and a number of carbohydrate-specific enzymes, the enzymes II. PTS proteins are phosphoproteins in which the phospho group is attached to either a histidine residue or, in a number of cases, a cysteine residue. After phosphorylation of enzyme I by PEP, the phospho group is transferred to HPr. The enzymes II are required for the transport of the carbohydrates across the membrane and the transfer of the phospho group from phospho-HPr to the carbohydrates. Biochemical, structural, and molecular genetic studies have shown that the various enzymes II have the same basic structure. Each enzyme II consists of domains for specific functions, e.g., binding of the carbohydrate or phosphorylation. Each enzyme II complex can consist of one to four different polypeptides. The enzymes II can be placed into at least four classes on the basis of sequence similarity. The genetics of the PTS is complex, and the expression of PTS proteins is intricately regulated because of the central roles of these proteins in nutrient acquisition. In addition to classical induction-repression mechanisms involving repressor and activator proteins, other types of regulation, such as antitermination, have been observed in some PTSs. Apart from their role in carbohydrate transport, PTS proteins are involved in chemotaxis toward PTS carbohydrates. Furthermore, the IIAGlc protein, part of the glucose-specific PTS, is a central regulatory protein which in its nonphosphorylated form can bind to and inhibit several non-PTS uptake systems and thus prevent entry of inducers. In its phosphorylated form, P-IIAGlc is involved in the activation of adenylate cyclase and thus in the regulation of gene expression. By sensing the presence of PTS carbohydrates in the medium and adjusting the phosphorylation state of IIAGlc, cells can adapt quickly to changing conditions in the environment. In gram-positive bacteria, it has been demonstrated that HPr can be phosphorylated by ATP on a serine residue and this modification may perform a regulatory function.
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PMID:Phosphoenolpyruvate:carbohydrate phosphotransferase systems of bacteria. 824 40

PEC-60, a sixty-residue novel regulatory peptide with N-terminal glutamic acid and C-terminal cysteine is abundant in intestinal tissue. PEC-60-like immunoreactivity is found in catecholamine neurons and intracerebroventricular injections of PEC-60 reduce dopamine utilization within the caudate nucleus indicating a possible role of this peptide in the central nervous system. We have investigated the effect of PEC-60 on cAMP formation in membrane preparations from rat caudate nucleus. We have found that PEC-60 significantly and dose-dependently reduces basal and forskolin stimulated cAMP production. The results demonstrate for the first time the interaction of PEC-60 with specific binding sites that regulate adenylate cyclase in inhibitory fashion in rat caudate nucleus.
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PMID:PEC-60, a novel regulatory peptide reduces cyclic AMP formation in rat caudate nucleus. 826 23

Most G-protein-coupled receptors have conserved cysteine residues in their C-terminal cytoplasmic domain that appear to be generally palmitoylated. An example is the human arginine vasopressin V2 receptor with cysteine residues at positions 341 and 342. Site-directed mutagenesis of the putative palmitoylation site was used to study the significance of palmitoylation for the V2 receptor. A multifunctional expression plasmid was constructed by cloning the V2 receptor cDNA into the vector pCDNAI.Neo. The resulting plasmid allowed site-directed mutagenesis experiments without subcloning, and stable and transient expression of the V2 receptor in Ltk- and COS.M6 cells respectively. The conserved cysteine residues Cys-341 and Cys-342 were placed by serine residues, yielding the single mutants C-341S and C-342S and the double mutant C-341S/C-342S. Functional expression in stably transfected Ltk- cells showed that the affinity of the three mutant receptors for arginine vasopressin was not altered. In contrast with the activation of adenylate cyclase through beta 2 adrenergic receptors, arginine vasopressin stimulated adenylate cyclase to the same extent and with similar EC50 values in both wild-type and mutant receptors. Transient expression of the C-341S/C-342S mutant receptor in COS.M6 cells confirmed an unaltered affinity of the mutant receptor for arginine vasopressin. However, the number of arginine vasopressin-binding sites on the cell surface was reduced by 30%, suggesting that the transport of the mutant receptor to the cell surface was impaired. In addition, the decrease in detectable arginine vasopressin-binding sites on the cell surface following pre-exposure to hormone was reduced, indicating that the sequestration/internalization of the mutant receptor on the cell surface was affected. The present data indicate that palmitoylation of the V2 receptor is important for intracellular trafficking and/or sequestration/internalization but not for agonist binding or activation of the Gs/adenylate cyclase system.
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PMID:Properties of the human arginine vasopressin V2 receptor after site-directed mutagenesis of its putative palmitoylation site. 857

The study of the five somatostatin receptor subtypes (SSTx, where x is the subtype number) has been hampered by the lack of high affinity antagonists. Potent and selective antagonists would increase our understanding of SST structure, function, and regulation. In this study, the identification of novel disulfide-linked cyclic octapeptide antagonists of somatostatin is described. The antagonists contain a core structure of a DL-cysteine pair at positions 2 and 7 of the peptides. Substitution of a D-cysteine at position 2 with an L-cysteine converts the full antagonist into a full agonist. All somatostatin receptor subtypes are coupled to inhibition of adenylate cyclase. The functional properties of these peptides have been determined in radioligand binding assays, in functional coupling of the SST2 subtype to yeast pheromone response pathway, and in cAMP accumulations. One peptide antagonist [Ac-4-NO2-Phe-c(D-Cys-Tyr-D-Trp-Lys-Thr-Cys)-D-Tyr-NH2] displays a binding affinity to SST2 comparable with that observed for the native hormone (Ki = 0.2 nM) and reverses somatostatin-mediated inhibition of cAMP accumulation in rat somatomammotroph GH4C1 cells, cells transfected with the SST2 and SST5 subtypes, as well as somatostatin-stimulated growth of yeast cells expressing the SST2 subtype. This class of somatostatin antagonists, which are the first to be described, should be useful for determination of somatostatin's diverse functions in vivo and in vitro.
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PMID:Identification and characterization of novel somatostatin antagonists. 886 14

The alpha2A-adrenoceptor is the prototypic example of the family of G-protein-coupled receptors which function by activation of 'Gi-like' pertussis toxin-sensitive G-proteins. A number of members of this subfamily of G-proteins are often co-expressed in a single cell type. To examine the interaction of this receptor with individual Gi-family G-proteins the porcine alpha2A-adrenoceptor was transiently transfected into COS-7 cells either alone or with each of wild-type Gi1alpha, Gi2alpha and Gi3alpha or mutations of each of these G-proteins in which the cysteine residue which is the target for pertussis toxin-catalysed ADP-ribosylation was exchanged for a glycine residue. The alpha2-adrenoceptor agonist UK14304 stimulated both high-affinity GTPase activity and the binding of guanosine 5'-[gamma-35thio]-triphosphate (GTP[35S]), when expressed without any additional G-protein. These effects were greatly reduced by pretreatment of the cells with pertussis toxin. Co-expression of each of the wild-type Gi-like G-protein alpha-subunits resulted in enhanced agonist activation of the cellular G-protein population which was fully prevented by pretreatment with pertussis toxin. Co-expression of the receptor along with the cysteine-to-glycine mutations of Gi1alpha, Gi2alpha and Gi3alpha resulted in agonist stimulation of these G-proteins, which was as great as that of the wild type proteins, but now the agonist stimulation produced over that due to the activation of endogenously expressed Gi-like G-proteins was resistant to pertussis toxin treatment. The Cys --> Gly mutations of Gi1alpha, Gi2alpha and Gi3alpha were each also able to limit agonist-mediated stimulation of adenylate cyclase activity. The degree of agonist-mediated activation of the pertussis toxin-resistant mutant of Gi1alpha was correlated highly both with the level of expression of this G-protein and with the level of expression of the alpha2A-adrenoceptor. Half-maximal stimulation of high-affinity GTPase activity of the Cys --> Gly mutants of Gi1alpha, Gi2alpha and Gi3alpha required 10-15-fold higher concentrations of agonist than did stimulation of their wild-type counterparts, consistent with a model in which the affinity of functional interactions of the alpha2A-adrenoceptor with the wild-type G-protein is greater than with the pertussis toxin-resistant mutant G-protein.
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PMID:Interactions of the alpha2A-adrenoceptor with multiple Gi-family G-proteins: studies with pertussis toxin-resistant G-protein mutants. 903 59


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