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)

To assess whether internalization of beta-adrenergic receptor occurs in the CNS, we have isolated clathrin-coated vesicles from bovine forebrain and examined them for the presence of beta-adrenergic receptor binding and adenylate cyclase activities. A coated vesicle enriched preparation isolated by successive D2O-Ficoll density gradient centrifugations was applied to a glass bead permeation column to achieve further purification. Two major peaks of protein were eluted from the column and monitored by electron microscopy and SDS-PAGE. Peak II contained almost exclusively coated vesicles (98%), whereas peak I, which appeared in the void volume, contained larger smooth vesicles and few coated vesicles. beta-Adrenergic receptor binding to peaks I and II was measured with 125I-cyanopindolol (CYP) as ligand in Sepharose 4B column assays. 125I-CYP was found to bind specifically and saturably to both peaks I and II with a Bmax of 28 +/- 4 and 32 +/- 3 fmol/mg protein, respectively. 3H-CGP 12177, a hydrophilic beta-adrenergic receptor ligand, did not label receptors present in peak II, but it specifically bound to synaptic plasma membranes (SPM) prepared from bovine hippocampus and, to a lesser extent, to peak I. These results suggest that receptors present in coated vesicles are cryptic in nature. In the displacement of 125I-CYP binding by (-)-isoproterenol, addition of 50 microM GppNHp caused a significant "right shift" with SPM and peak I but not the peak II preparation. Adenylate cyclase activities could also be detected in both peaks I and II (specific activities, 21 +/- 0.6 and 24 +/- 0.5 pmol cAMP/mg protein/min, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Detection and characterization of beta-adrenergic receptors and adenylate cyclase in coated vesicles isolated from bovine brain. 301 96

The distribution of alpha 1-adrenergic receptors in rat liver subcellular fractions was studied using the alpha 1-adrenergic receptor ligand [3H]prazosin. The highest number of [3H]prazosin binding sites was found in a plasma membrane fraction followed by 2 Golgi and a residual microsomal fraction, the numbers of binding sites were 1145, 845, 629 and 223 fmol/mg protein, respectively. When the binding in these fractions was compared with the activity of plasma membrane 'marker' enzymes in the same fractions a relative enrichment of [3H]prazosin binding sites was found in the residual microsomes and one of the Golgi fractions. Photoaffinity labelling with 125I-arylazidoprazosin in combination with SDS-polyacrylamide gel electrophoresis revealed the specific binding to 40 and 23 kDa entities in a Golgi fraction, while in plasma membranes the binders had an apparent molecular mass of 36 and 23 kDa. When [3H]prazosin was injected in vivo into rat portal blood followed by subcellular fractionation of liver, a pattern of an initial rapid decline and thereafter a slow decline of radioactivity was noted in all fractions. Additionally, in the two Golgi fractions a transient accumulation of radioactivity occurred between 5 and 10 min after the injection. The ED50 values for displacement of [3H]prazosin with adrenaline was lowest in the plasma membrane fraction, followed by the residual microsomes and Golgi fractions, the values were 10(-6), 10(-5) and 10(-4) mol/l, respectively. On the basis of lack of correlation between distribution of alpha 1-adrenergic antagonist binding and adenylate cyclase activity, differences in the molecular mass of alpha 1-adrenergic antagonist binders, differences in the kinetics of in vivo binding and accumulation of [3H]prazosin and also differences in agonist affinity between plasma membrane and Golgi fractions, it is concluded that alpha 1-adrenergic receptors are localized to low-density intracellular membranes involved in receptor biosynthesis and endocytosis.
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PMID:Subcellular distribution of alpha 1-adrenergic receptors in rat liver. 303 84

Muscarinic receptors have been purified from calf forebrain plasma cell membranes by affinity chromatography on a dexetimide-agarose gel. SDS-PAGE analysis showed a single 70 kDa band. Monoclonal antibodies have been prepared against these affinity purified 70 kDa protein(s). One antibody, M-35, immunoprecipitated up to 80% of digitonin-solubilized muscarinic receptors. M-35 had agonist-like effects on guinea-pig myometrium: it increased the intracellular cyclic GMP content, decreased prostaglandin-induced cyclic AMP accumulation and caused muscle contractions. The two first effects were inhibited by atropine. M-35 was used to visualize muscarinic receptors at the surface of human fibroblastic cells. In the particular cell line used, the receptors have a low affinity for pirenzepine, were negatively coupled to adenylate cyclase and mediated increase in the phosphatidyl-inositol breakdown.
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PMID:Immunochemical studies of the muscarinic acetylcholine receptor. 304 Sep 87

The abalone sperm adenylate cyclase does not appear to be regulated by guanine nucleotides, but has a Mg2+-supported catalytic activity similar to other hormone- and guanine nucleotide-regulated enzymes (Kopf, G. S., and Vacquier, V. D. (1984) J. Biol. Chem. 259, 7590-7596; Kopf, G. S., and Vacquier, V. D. (1985) Biol. Reprod. 33, 1094-1104). The present studies were undertaken to ascertain whether the abalone enzyme has associated guanine nucleotide-binding regulatory proteins. Membrane fractions were incubated with either islet-activating protein (IAP) or cholera toxin and analyzed by sodium dodecyl sulfate SDS-polyacrylamide gel electrophoresis for the presence of toxin-catalyzed ADP-ribosylated proteins. The supernatant from a Lubrol PX-extracted 48,000 X g pellet fraction contained a Mr = 41,000 IAP substrate. This substrate could not be ADP-ribosylated prior to detergent extraction. Lubrol PX-solubilized fractions of membrane preparations from mouse, bovine, and human sperm also contained a Mr = 41,000 IAP substrate. These proteins co-migrated on sodium dodecyl sulfate-polyacrylamide gels with the Mr = 41,000 alpha i-subunit of the inhibitory guanine nucleotide-binding regulatory protein (Gi) from transformed chicken embryo fibroblast and mouse S-49 lymphoma membrane extracts. The sperm IAP substrates displayed similar protease digest patterns to alpha i of mouse S-49 lymphoma cells. Sea urchin sperm analyzed in a similar manner contained a Mr = 39,000 IAP substrate. Cholera toxin-catalyzed ADP-ribosylation of specific sperm membrane proteins was not observed in any of the sperm preparations tested. The presence of the beta-subunit common to both the stimulatory and inhibitory guanine nucleotide-binding regulatory heterotrimers was confirmed in sperm using an antiserum directed against the purified beta-subunit of the guanine nucleotide-binding regulatory proteins from bovine brain. It is concluded that all of the sperm tested, with the possible exception of sea urchin sperm, contain a Gi-like protein. Additional properties of these proteins and their role(s) in sperm function are currently being examined.
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PMID:Evidence for a guanine nucleotide-binding regulatory protein in invertebrate and mammalian sperm. Identification by islet-activating protein-catalyzed ADP-ribosylation and immunochemical methods. 308 10

The existence of a GTP-binding protein of the Ns type in Trypanosoma cruzi was explored. Epimastigote membranes were labelled by cholera toxin in the presence of [adenine-14C]NAD+. After SDS/polyacrylamide-gel electrophoresis of extracted membrane proteins, a single labelled polypeptide band of apparent Mr approx. 45,000 was detected. Epimastigote cells were treated with N-ethylmaleimide and electrofused to lymphoma S49 cells lacking the Ns protein. Evidence indicates that in such electrofusion-generated cell hybrids a heterologous adenylate cyclase system was reconstituted with the Ns protein provided by T. cruzi epimastigotes.
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PMID:Evidence for the existence of an Ns-type regulatory protein in Trypanosoma cruzi membranes. 309 61

Hormonal stimulation of adenylate cyclase from bovine cerebral cortex is mediated by a guanine-nucleotide regulatory protein (Gs). This protein contains at least three polypeptides: a guanine nucleotide-binding alpha s component and a beta X gamma component, which modulates the function of alpha s. The alpha s component from many tissues can be ADP-ribosylated with cholera toxin, but has been unusually difficult to modify in brain. We have improved incorporation of ADP-ribose by including isonicotinic acid hydrazide to inhibit the potent NAD glycohydrolase activity of brain. ADP-ribosylation is further improved by addition of detergent to render the substrates accessible and 20 mM-EDTA to chelate metal ions. Although Mg2+ is absolutely required for activation of adenylate cyclase by the GTP analogue guanosine 5'-[beta gamma-imido]triphosphate (p[NH]ppG), it is not obligatory for p[NH]ppG-stimulated ADP-ribosylation by cholera toxin. Under these conditions, the ADP-ribosylation of brain membranes is not enhanced by a cytosolic protein. We find that there are two major sizes of brain alpha s, which we have named 'alpha sL', with an apparent Mr of 42,000-45,000, and 'alpha sH' with an apparent Mr of 46,000-51,000 depending on the gel-electrophoretic system used. The alpha sL and alpha sH components can incorporate different amounts of ADP-ribose depending on the reaction conditions, so that one or the other may appear to predominate. Thus we show that incomplete ADP-ribosylation by cholera toxin is not a good indication of the relative amounts of alpha s units. Functionally, however, both forms of alpha s appear to be similar. Both forms associate with the catalytic unit of adenylate cyclase, but neither of them does so preferentially. There is an excess of each of them over the amount associated with catalytic unit. We have now substantially purified Gs from brain by a modification of the method of Sternweis et al. [(1981) J. Biol. Chem. 256, 11517-11526] as well as by a new, simplified, procedure. On SDS/polyacrylamide-gel electrophoresis, the purified brain Gs contains both the 45 and 51 kDa alpha s polypeptides revealed by ADP-ribosylation and a beta X gamma component. Activation of purified alpha s by guanine nucleotides or fluoride can be reversed by addition of purified beta X gamma component. The activated form of purified brain Gs has an Mr of 49,000 as determined by hydrodynamic measurements, which is consistent with the idea that the active form of brain Gs is the dissociated one.
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PMID:The stimulatory guanine-nucleotide regulatory unit of adenylate cyclase from bovine cerebral cortex. ADP-ribosylation and purification. 310 73

Information available at present documents the existence of three well-defined classes of guanine nucleotide binding proteins functioning as signal transducers: Gs and Gi which stimulate and inhibit adenylate cyclase, respectively, and transducin which transmits and amplifies the signal from light-activated rhodopsin to cGMP-dependent phosphodiesterase in ROS membranes. Go is a fourth member of this family. Its function is the least known among GTP binding signal transducing proteins. The family of G proteins has a number of properties in common. All are heterotrimers consisting of three subunits, alpha, beta, and gamma. Each of the subunits may be heterogeneous depending on species and tissue of origin and may be posttranslationally modified covalently. The alpha subunits vary in size from 39 to 52 kDa. The sequences for Gs alpha and transducin alpha have 42% overall homology and those of Gi alpha and Gs alpha 43%, whereas those of Gi alpha and transducin alpha have a higher degree (68%) of homology. All alpha subunits bind guanine nucleotides and are ADP-ribosylated by either pertussis toxin (Gi, transducin, Go) or cholera toxin (Gs, Gi, transducin). Thus, transducin and Gi, which have the highest degree of sequence homology, are also ADP-ribosylated by both toxins. The beta subunits have molecular weights of 36 and 35 kDa, respectively. While Gs, Gi, and Go contain a mixture of both, transducin contains only the larger (36-kDa) beta-polypeptide. The relationship of the 36- and the 35-kDa beta subunits is not defined. Although the complete sequence of the 36-kDa beta subunit of transducin has been deduced from the cDNA sequence, complete sequences of other beta subunits are not yet available so that detailed comparisons cannot be made at present. However, the proteolytic profiles of each class of the beta subunits of different G proteins are indistinguishable. The gamma subunit of bovine transducin has been completely sequenced. It has a Mr of 8400. Again complete sequences of other gamma subunits are not yet available. While the gamma subunits of Gs, Gi, and Go have identical electrophoretic mobility in SDS gels, they differ significantly in this respect from the gamma subunit of transducin. Moreover, crossover experiments point to functional differences between gamma subunits from G protein and transducin complexes. In addition, a role for beta, gamma in anchoring guanine nucleotide binding proteins to membranes has been postulated.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Structural and functional relationships of guanosine triphosphate binding proteins. 313 54

The acute in vitro effects of ethanol on cerebral cortical adenylate cyclase activity and beta-adrenergic receptor characteristics suggested a site of action of ethanol at Gs, the stimulatory guanine nucleotide binding protein. After chronic ethanol ingestion, the beta-adrenergic receptor appeared to be uncoupled (i.e., the form of the receptor with high affinity for agonist was undetectable), and stimulation of adenylate cyclase activity by isoproterenol or guanine nucleotides was reduced, suggesting an alteration in the properties of Gs. To further characterize this change, cholera and pertussis toxin-mediated 32P-ADP-ribosylation of mouse cortical membranes was assessed in mice that had chronically ingested ethanol in a liquid diet. 32P-labeled proteins were separated by SDS-PAGE and quantitated by autoradiography. There was a selective 30-50% decrease in cholera toxin-induced labeling of 46 kDa protein band in membranes of ethanol-fed mice, with no apparent change in pertussis toxin-induced labeling. The 46 kDa protein has a molecular weight similar to that of the alpha subunit of Gs, suggesting a reduced amount of this protein or a change in its characteristics as a substrate for cholera toxin-induced ADP-ribosylation in cortical membranes of ethanol-fed mice.
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PMID:Cholera toxin-induced ADP-ribosylation of a 46 kDa protein is decreased in brains of ethanol-fed mice. 314 19

The adult rat lung cytoplasm contains some factors which markedly stimulate adenylate cyclase activity in plasma membranes (Nijjar, M.S. Biochim. Biophys. Acta 584:43-50, 1979). Adenylate cyclase activator (ACA) was purified from rat lungs by DEAE-cellulose chromatography, preparative isoelectric focusing and by repeated high-performance liquid chromatography on a Sepharogel TSK 2000SW column. The final preparation showed about 200 fold purification in ACA activity over the original lung supernatant, and appeared to be homogeneous on the basis of its migration into a single band on SDS-polyacrylamide gel electrophoresis, and co-elution of ACA activity with protein from a gel exclusion column. ACA is an acidic (pI 4.8 +/- 0.1), heat labile, monomeric protein of 40,000 +/- 2,000 dalton molecular weight, and does not resemble calmodulin.
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PMID:Further purification and partial characterization of the rat lung cytoplasmic factors modulating adenylate cyclase activity in plasma membrane. 317 46

Photoaffinity labeling analogs of the adenylate cyclase activator forskolin (PF) have been synthesized, purified and tested for their effect on preparations of membrane-bound, Lubrol solubilized and forskolin affinity-purified adenylate cyclase (AC). All analogs of forskolin significantly activated AC. However, in the presence of 0.1 to 0.3 microM forskolin, the less active forskolin photoaffinity probes at 100 microM caused inhibition. This inhibition was dose-dependent for PF, suggesting that PF may complete with F for the same binding site(s). After cross-linking [125I]PF-M (see Figure 1 for structure) to either membrane or Lubrol-solubilized AC preparations by photolysis, a radiolabeled 100-110 kDa protein band was observed after autoradiography following SDS-PAGE. F at 100 microM blocked the photoradiolabeling of this protein. Radioiodination of forskolin-affinity purified AC showed several protein bands on autoradiogram, however, only one band (Mr = 100-110 kDa) was specifically labeled by [125I]PF-M following photolysis. The photoaffinity-labeled protein of 100-110 kDa of AC preparation of rat adipocyte may be the catalytic unit of adenylate cyclase of rat adipocyte itself as supported by the facts that [a] no other AC-regulatory proteins are known to be of this size, [b] the catalytic unit of bovine brain enzyme is in the same range and [c] this PF specifically stimulates AC activity when assayed alone, and weekly inhibits forskolin-activation of cyclase. These studies indicate that radiolabeled PF probes may be useful for photolabeling and detecting the catalytic unit of adenylate cyclase.
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PMID:Modification of adenylate cyclase by photoaffinity analogs of forskolin. 327 97


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