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)

We have previously reported incorporation of Triton X-100-solubilized bovine calf testis membrane protein into liposomes. The resulting proteoliposomes responded to FSH by exchange of bound GDP for [3H]5'-guanylyl imidodiphosphate ([3H]Gpp(NH)p) and by activation of adenylate cyclase (AC) (Grasso, P., Dattatreyamurty, B. and Reichert, L.E., Jr. (1988) Mol. Endocrinol. 2, 420-430). This model system was utilized to study the effects of FSH on the quaternary structure of FSH receptor-associated GTP-binding protein by comparing the gel filtration profiles of proteoliposomes solubilized with Triton X-100 after exposure to [3H]Gpp(NH)p in the presence or absence of FSH. FSH caused a redistribution of radioactivity (due to bound [3H]Gpp(NH)p) from a high molecular weight fraction (Mr greater than 100,000) to a fraction of much lower molecular weight (Mr approximately 23,000). These results are interpreted to reflect an FSH-induced dissociation of [3H]Gpp(NH)p-bound G protein from its receptor-associated complex. The apparent Mr of approximately 23,000 for the FSH receptor-associated GTP-binding protein suggests that it may represent yet another member of a family of low molecular weight GTP-binding proteins, possibly a ras gene product, recently identified in various mammalian tissues.
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PMID:Follicle stimulating hormone (FSH) induces G protein dissociation from FSH receptor-G protein complexes in reconstituted proteoliposomes. 250 56

The human gastric tumoral cell line HGT-1 was previously shown to contain a membrane somatostatin receptor negatively coupled to adenylate cyclase through a pertussis toxin-sensitive inhibitory GTP-binding regulatory protein (Gi) (Reyl-Desmars, F., Laboisse, C., and Lewin, M. J. M. (1986) Regul. Pept. 16, 207-215). In this study, we have solubilized this receptor in a free unoccupied form using Triton X-100 as detergent and [125I-Tyr11]somatostatin-14 to monitor specific binding. Furthermore, we have prepared a monoclonal antibody against a chromatographically enriched soluble receptor fraction and used this antibody (30F3) to immunopurify the receptor in conjunction with Sepharose-somatostatin-14 immunopurification and steric exclusion high pressure liquid chromatography (HPLC). The purified fraction showed 18,600-fold enrichment in terms of specific binding (i.e. from 0.6 +/- 0.05 to 11,300 +/- 830 pmol/mg of protein) and a single dissociation constant (kappa D) of 76 +/- 8 nM. On HPLC, it migrated as a single and symmetric 90-kDa peak. Moreover, after 125I-protein labeling, it gave a single 90-kDa band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis autoradiography. On the other hand, the 30F3 monoclonal antibody immunoblotted with a single 90-kDa band contained in the HGT-1 cell membrane. We therefore suggest that this antibody is specific to the HGT-1 membrane somatostatin receptor, that this receptor has a molecular mass of 90 kDa, and that we have obtained a homogeneous preparation of nondenatured receptor suitable for further cloning studies.
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PMID:Solubilization and immunopurification of a somatostatin receptor from the human gastric tumoral cell line HGT-1. 257 96

Adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein phosphorylation is considered a key step in the cellular action of vasopressin (AVP) to regulate water permeability in collecting tubules. However, the proteins serving as a substrate(s) for phosphorylation in undisrupted cells have not yet been identified. In the present study, we developed a method for investigation of in situ phosphorylation of microdissected segments of medullary collecting tubules (MCT) from rat kidney. Incubation of microdissected MCT segments with low concentrations of saponin, "semipermeabilization," increased permeability of the membrane for ATP but did not allow leakage of macromolecules such as lactate dehydrogenase. This treatment also did not cause major disruption of cell structure, or impairment of AVP-sensitive adenylate cyclase. Incubation of semipermeabilized MCT with gamma-[32P]ATP resulted in incorporation of 32Pi into two major protein bands [band "A" of apparent molecular mass (Mr) approximately equal to 66 kDa, and band "B" of Mr approximately equal to 45 kDa] detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis and subsequent autoradiography. Similar incubation of tubules disrupted by hyposmotic solutions and a stronger detergent Triton X-100 resulted in 32Pi incorporation into multiple protein bands. Incubation of MCT with 1 microM AVP resulted in increased 32Pi radioactivity in band A and decreased 32Pi radioactivity in band B. These findings demonstrate a novel method for identification of endogenous protein substrate(s) for cAMP-dependent protein kinase and other protein kinases and phosphatases that are probably involved in post-cAMP steps in the cellular action of AVP in the intact cells of collecting tubules.
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PMID:In situ phosphorylation of proteins in MCTs microdissected from rat kidney: effect of AVP. 283 21

We have developed a method to ADP-ribosylate the stimulatory guanine nucleotide-binding protein of adenylate cyclase (GS) in brain membranes by using cholera toxin. In particular, we used isonicotinic acid hydrazide and 3-acetylpyridine adenine dinucleotide to inhibit the potent NAD-glycohydrolase activity of brain membranes, and we used the detergent Triton X-100 (at 0.1%) to improve the accessibility of the toxin and guanine nucleotides used for supporting the ADP-ribosylation. This method reveals that GS is a very abundant protein in membranes derived from calf brain (approximately 30 pmol/mg of protein). In brain, GS exists in large excess over the previously reported amount of the adenylate cyclase catalytic subunit. The modification of GS with an ADP-ribosyl residue (a) elicits a four- to fivefold activation of adenylate cyclase by GTP, (b) increases the stabilization of adenylate cyclase by GTP, and (c) reduces adenylate cyclase activation by fluoride but does not change basal activity, activation by guanosine 5'-(beta, gamma-imido)triphosphate, or the sensitivity of adenylate cyclase to heat-induced denaturation. A correlation between ADP-ribosylation and the alterations in the activation of adenylate cyclase by guanine nucleotides and by fluoride is presented.
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PMID:ADP-ribosylation by cholera toxin of membranes derived from brain modifies the interaction of adenylate cyclase with guanine nucleotides and NaF. 283 59

Activation of platelet adenylate cyclase by prostaglandin E1 or prostacyclin is initiated through the interaction of the agonists with the same receptors on membrane. Prostaglandin E1/prostacyclin receptors of human platelets were solubilized in buffer, containing 0.05% Triton X-100 and protease inhibitors. The soluble membrane protein was chromatographed on a DEAE-cellulose column and assayed by a microfiber filter by equilibrium binding technique. The active fractions eluted at 0.7 M KCl were pooled, and the receptors were purified to homogeneity by Sephadex G-200 gel filtration with an overall recovery of 30%. The isolated receptor was 2,200-fold purified over the starting platelets. As evidenced by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the receptor showed a molecular mass of 190,000 daltons and is composed of two nonidentical subunits with molecular masses of 85,000 and 95,000 daltons. The interaction of prostaglandin E1 with the purified receptor was rapid, saturable, reversible, and highly specific. Among all prostaglandins tested, only prostacyclin was capable of displacing [3H]prostaglandin E1 bound to the receptor. Scatchard analysis of [3H]prostaglandin E1 binding to the purified receptor suggested the presence of a single class of high affinity binding sites (Kd = 9.8 nM) and a second population of low affinity binding sites (Kd = 0.7 microM) in the same protein molecule. Incubation of the purified receptor with platelets stripped of the receptor by washing with low concentrations of Triton X-100 efficiently restored the ability of prostaglandin E1 and prostacyclin to activate adenylate cyclase in these cells.
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PMID:Purification and properties of prostaglandin E1/prostacyclin receptor of human blood platelets. 288 71

Bordetella pertussis cells express multiple virulence-associated surface proteins, including adenylate cyclase, agglutinogens 2 and 3, filamentous hemagglutinin, pertussis toxin, and outer-membrane protein (Omp) 30/32 and Omp91. Surface proteins that are not virulence-associated include three peptidoglycan-associated Omps of apparent molecular weights 40,000, 25,000, and 18,000. Omp40 is an anion-selective porin and is the most abundant surface protein of virulent and avirulent cells. Three independent approaches--immunomicroscopy, surface radioiodination, and isolation of Triton X-100-insoluble envelope proteins--suggest that the Triton-insoluble fraction of the B. pertussis cell envelope is the outer membrane. Agglutinogens 2 and 3 and filamentous hemagglutinin lie outside the outer membrane, the first two as fimbriae and the last as a microcapsule. Adenylate cyclase and pertussis toxin are present in the outer membrane but may be present transiently or present in small amounts.
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PMID:Surface proteins of Bordetella pertussis. 290 39

This work shows that unsaturated fatty acids enhance the epinephrine-stimulated adenylate cyclase activity in bovine retina. The modulating effect on the epinephrine-stimulated formation of cyclic AMP seems to be linked to the degree of unsaturation of the fatty acid. Treatment of the intact retina with docosahexaenoic acid in the concentration range 0.5 X 10(-6)-1 X 10(-3) M does not affect the enzyme activity measured in the absence of the hormone but markedly increases the cyclase activity when the tissue is incubated in the presence of 0.1 mM epinephrine. Docosahexaenoic acid enhances the maximal response to epinephrine without affecting the apparent ED50 value for this effector. Docosahexaenoic acid at 0.5 mM also increases the hormone-stimulated adenylate cyclase activity in retinal cell-free homogenate, whereas it has no effect on the epinephrine-sensitive enzyme solubilized from the membrane fraction with 1% Triton X-305. When docosahexaenoic acid-preincubated intact retina and cell-free homogenate are incubated in the presence of defatted albumin, both the observed activating effect of the fatty acid on the epinephrine-stimulated adenylate cyclase activity and the enhancement of the enzyme response to the hormone significantly diminish.
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PMID:Modulation by docosahexaenoic acid of the epinephrine-stimulated adenylate cyclase activity of the bovine retina. 297 May 25

The enzymatic activity of adenylate cyclase in homogenates and membrane-rich fractions prepared from rabbit iris-ciliary bodies and bovine trabecular meshwork was found to be inhibited by timolol. Treatment of iris-ciliary body homogenates with Triton X-305 resulted in abolition of the inhibitory effect of the drug on the activity of the enzyme. The stimulatory effect of salbutamol on the enzyme was also susceptible to blockade by timolol. It is suggested that the hypotensive action of timolol on intraocular pressure results from structural and functional changes induced on the plasma membranes of the iris-ciliary body and trabecular meshwork by the thiadiazole group of the molecule, and, also, from the occupation of the adrenergic receptors of the iris-ciliary body by the tert-butylamino-2-hydroxypropoxy part of the compound.
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PMID:Timolol inhibits adenylate cyclase activity in the iris-ciliary body and trabecular meshwork of the eye and blocks activation of the enzyme by salbutamol. 302 33

Gs and Gi are guanine nucleotide-binding proteins that mediate the stimulation and inhibition, respectively, of adenylate cyclase. The extent to which the beta subunit common to these proteins may be associated with the cytoskeleton of S49 mouse lymphoma cells was assessed by procedures of differential detergent extraction and immunotransfer blotting. Treatment of cells with 1% Triton X-100 results in nearly quantitative extraction of cellular protein, phospholipid, tubulin, and the catalytic component of adenylate cyclase. Approximately 65% of the beta subunit is refractory to extraction. This population of the beta subunit, along with a population of actin presumed to originate from actin filaments, is subsequently solubilized with hypotonic medium containing 0.5% sodium deoxycholate and 1% Tween-40. The pattern of distribution of the beta subunit among sequential detergent extracts is corroborated by that of generated immunoreactive tryptic fragments. These results are consistent with the interaction of guanine nucleotide-binding proteins with the cytoskeleton.
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PMID:Fractionation of the beta subunit common to guanine nucleotide-binding regulatory proteins with the cytoskeleton. 309 28

Forskolin, a diterpene that exerts several pharmacological effects, activates adenylate cyclase in brain and in some other mammalian tissues. Properties of forskolin activation of adenylate cyclase from central nervous system of the dipterous Ceratitis capitata are described. The interaction of forskolin with the insect adenylate cyclase system was studied by evaluating its effect on metal-ATP kinetics, protection against thermal inactivation, membrane fluidity and enzyme modulation by fluoride, guanine nucleotides, octopamine, and ADP-ribosylation by cholera toxin. The diterpene stimulated basal enzyme activity both in membranes and Triton X-100-solubilized preparations, apparently devoid of functional regulatory unit, this effect being rapidly reversed by washing the membranes. An increase of Vmax accounts for the activation of soluble and membrane adenylate cyclase preparations by forskolin, whereas the affinity of the enzyme for the substrate was not affected. Forskolin apparently protects the membrane enzyme from thermal inactivation, and at concentrations that promote the enzyme activity the diterpene does not alter membrane microviscosity. Forskolin does not appear to alter the sensitivity of insect adenylate cyclase to sodium fluoride, guanine nucleotide, or regulatory subunit ADP ribosylated by cholera toxin, the combined effect of these factors with the diterpene resulting in a nearly additive enzymatic activation. However, forskolin blocks the octopamine stimulatory input. Results obtained with the insect adenylate cyclase system are discussed and compared to what is known about mammalian systems to propose a mechanism of enzyme activation by forskolin.
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PMID:Regulation by forskolin of octopamine-stimulated adenylate cyclase from brain of the dipterous Ceratitis capitata. 310 70


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