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)

Guanine nucleotide-binding (G) proteins are involved in several transmembrane signaling systems. Choleragen (cholera toxin) activates adenylate cyclase by catalyzing the ADP-ribosylation of Gs alpha, the stimulatory G protein of the cyclase system. This reaction is enhanced by another guanine nucleotide-binding protein termed ADP-ribosylation factor or ARF that was purified from bovine brain membranes [R. A. Kahn and A. G. Gilman, Journal of Biological Chemistry (1986) 261, 7906-7911]. It was recently found that this ARF also increases the NAD:agmatine and NAD:protein ADP-ribosyltransferase, NAD glycohydrolase and auto-ADP-ribosylation activities of the toxin. We have purified and characterized two soluble proteins from bovine brain that act in a similar fashion to enhance choleragen activity in each of these reactions. The membrane and soluble factors are all proteins of approximately 19 kDa that require GTP or GTP analogues for activity and are ADP-ribosylated by the toxin. The ARF proteins apparently interact directly with choleragen in a GTP-dependent fashion to increase its catalytic activity and thus are part of a G protein cascade through which the toxin activates adenylate cyclase. The physiological function of the ARF proteins, as well as their possible relationships to the ras oncogene products and/or the family of G proteins that includes Gs alpha, remains to be determined.
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PMID:Participation of a guanine nucleotide-binding protein cascade in cholera toxin activation of adenylate cyclase. 249 82

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 G-regulatory proteins of adenylate cyclase, tubulin, and the ras oncogene protein product require the production of GTP from ATP in order to exert their effects within the cell. This implies that the activity of nucleoside diphosphate kinase (NDPK) plays a major role in the regulation of cellular events requiring GTP and that the level of activity of this enzyme is critical. This report presents a simple method for trapping a specific isozyme of NDPK in its high-energy phosphorylated form (NDPK approximately P) using EDTA and demonstrates that this NDPK approximately P is tenfold higher in malignant colon tumor tissue than in normal colon tissue. This autophosphorylation of the 21,000 and 24,000 Mr subunits of NDPK occurs rapidly at 0 degrees C, will use either [gamma-32P]ATP, [gamma-32P]GTP, or the corresponding 8-azidopurine photoprobes, is intramolecular, displays saturation effects, and is prevented from forming if GTP gamma S is added. Dephosphorylation in the homogenate occurs rapidly upon addition of Mg2+ or any nucleoside-5'-diphosphate. The subunits autophosphorylated in the homogenates are mostly in the soluble phase, and they comigrate with the subunits of pure NDPK from human erythrocytes. Cross-addition of normal and malignant homogenates does not decrease the level of autophosphorylation of NDPK, which indicates that the level of NDPK approximately P may be a quantitative measure of the level of this specific NDPK isozyme form. Assays for NDPK activity show correspondingly elevated levels in the malignant homogenates. Using western blot and photoaffinity labeling techniques, we distinguished the NDPK approximately P subunits from two closely migrating GTP-binding proteins. These were identified as the ras gene protein product and a 20,000 Mr protein, which comigrates identically with ADP-ribosylating factor (ARF). The ARF also comigrates in a tight band that is phosphorylated by [gamma 32P]ATP or [gamma-32P]GTP when Mg2+ is present.
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PMID:Prevalence of nucleoside diphosphate kinase autophosphorylation in human colon carcinoma versus normal colon homogenates. 255 33

The yeast Saccharomyces cerevisiae contains two ras homologues, RAS1 and RAS2, whose products have been shown to modulate the activity of adenylate cyclase encoded by the CYR1 gene. To isolate temperature-sensitive mutations in the RAS2 gene, we constructed a plasmid carrying a RAS2 gene whose expression is under the control of the galactose-inducible GAL1 promoter. A ras1 strain transformed with this plasmid was subjected to ethyl methanesulfonate mutagenesis and nystatin enrichment. Screening of approximately 13,000 mutagenized colonies for galactose-dependent growth at a high temperature (37 degrees) yielded six temperature-sensitive ras2 (ras2ts) mutations and one temperature-sensitive cyr1 (cyr1ts) mutation that can be suppressed by overexpression or increased dosage of RAS2. Some ras2ts mutations were shown to be suppressed by an extra copy of CYR1. Therefore increased dosage of either RAS2 or CYR1 can suppress the temperature sensitivity caused by a mutation in the other. ras1 ras2ts and ras1 cyr1ts mutants arrested in the G1 phase of the cell cycle at the restrictive temperature, and showed pleiotropic phenotypes to varying degrees even at a temperature permissive for growth (25 degrees), including slow growth, sporulation on rich media, increased accumulation of glycogen, impaired growth on nonfermentable carbon sources, heat-shock resistance, impaired growth on low concentrations of glucose, and lithium sensitivity. Of these, impaired growth on low concentrations of glucose and sensitivity to lithium are new phenotypes, which have not been reported for mutants defective in the cAMP pathway.
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PMID:Isolation and characterization of temperature-sensitive mutations in the RAS2 and CYR1 genes of Saccharomyces cerevisiae. 255 58

It is well known that many of thyroid carcinoma are capable of responding to TSH, but our studies shown that there are some alteration in this responsiveness. The adenylate cyclase responsiveness to TSH was usually greater in thyroid carcinoma than in adjacent histologically normal thyroid tissue. The level of increased response of adenylate cyclase were correlated with the level of enhanced expression of ras oncogene product p21 assessed by Western blotting analysis. The TSH induced desensitization of adenylate cyclase was not observed in some differentiated carcinoma. This loss of desensitization may be reflect the change in ADP-ribosylable Gi protein. In the differentiated carcinoma, the capacity of EGF receptor was higher than that in normal thyroid. The EGF binding to cultured carcinoma cells did not increase in response to TSH. These altered properties of transmembrane control in human thyroid carcinoma may be related to the neoplastic growth.
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PMID:[Transmembrane controls in cultured human thyroid carcinoma]. 256 2

The author deals with mechanisms of paraneoblastic hormone production. Paraneoblastic production of protein hormones is closely linked with processes of malignant transformation of cells which takes place as a result of dysregulation of hyperactive c-oncogens. Products of these oncogens either directly code growth factors or form their receptors or the receptors of dijerent hormones. The product of the family of c-ras-oncogens creates by excessive formation of G-proteins an excessively long activation of adenyl cyclase by hormones which act via cAMP. Under the influence of c-oncogens proliferating and differentiating stem cells reach in larger amounts the APUD stage of differentiation, where they express their genes for the formation of the entire range of protein hormones. Their ectopic formation may acquire biological or clinical significance, as relatively many cells remain in the APUD stage of differentiation, if the process of postscriptional adjustment of the formed hormones is not impaired.
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PMID:[Paraneoblastic endocrinne syndromes--mechanisms of paraneoblastic hormone production]. 273 96

Adenylate cyclase in permeabilized cells of Saccharomyces cerevisiae was examined. Among various permeabilization procedures, including organic solvents, detergents and other reagents, dimethylsulfoxide (DMSO) and digitonin treatments resulted in the highest recovery of adenylate cyclase activity. Incubation of cells at 30 degrees C with digitonin at 0.01% to 0.1%, or DMSO at 20% to 40% for 15 to 30 min gave optimal adenylate cyclase activity. The enzyme activity in digitonin-permeabilized cells could be supported only by Mn2+, whereas Mg2+ with or without guanine nucleotides did not support cyclase activity. DMSO-permeabilized cells exhibit efficient Mn2+- and Mg2+/Gpp[NH]p-dependent stimulation. Furthermore, digitonin added to yeast membranes at a 1:50 detergent to protein ratio (w/w) abolishes guanyl nucleotide regulation without significantly affecting the Mn2+-supported cyclase activity. The superiority of DMSO is further supported by the fact that recovery of adenylate cyclase activity is better in the DMSO-treated cells than in the digitonin-treated cells. DMSO most probably causes less disturbance of the fabric of the native cell. We conclude that digitonin, but not DMSO, uncouples the catalytic unit of adenylate cyclase from the regulatory GTP binding (ras) proteins.
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PMID:Guanine nucleotide regulation of adenylate cyclase in permeabilized cells of Saccharomyces cerevisiae. 283 Sep 10

The plasma membrane-bound mammalian ras proteins of relative molecular mass 21,000 (ras p21) share biochemical and structural properties with other guanine nucleotide-binding regulatory proteins (G-proteins). Oncogenic ras p21 variants result from amino acid substitutions at specific positions that cause p21 to occur predominantly complexed to GTP in vivo. Recently, a GTPase activating protein (GAP) with cytosolic activity has been discovered that stimulates the GTPase activity of normal but not of oncogenic ras p21. GAP might be either a negative regulatory agent which acts further upstream in the regulatory pathway or the downstream target of ras p21. We have identified a protein from bovine brain with apparent relative molecular mass 125,000 that has GAP activity. Here, using pure GAP in a kinetic competition assay, we show that GAP interacts preferentially with the active GTP complexes of both normal and oncogenic Harvey (Ha) ras p21 compared with the inactive GDP complexes. We also report the cloning and sequencing of the complementary DNA for bovine GAP. Regions of GAP share amino acid similarity with the noncatalytic domain of adenylate cyclase from the yeast Saccharomyces cerevisiae and with regions conserved between phospholipase C-148, the crk oncogene product and the nonreceptor tyrosine kinases.
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PMID:Cloning of bovine GAP and its interaction with oncogenic ras p21. 284 90

Reconstitution of purified mu opioid receptors with purified guanine nucleotide-binding regulatory proteins (G proteins) was investigated. mu opioid receptors were purified by 6-succinylmorphine AF-AminoTOYOPEARL 650M affinity chromatography and by PBE isoelectric chromatography. The purified mu opioid receptor (pI 5.6) migrated as a single Mr 58,000 polypeptide by NaDodSO4/PAGE, a value identical to that obtained by affinity cross-linking purified mu receptors. When purified mu receptors were reconstituted with purified Gi, the G protein that mediates the inhibition of adenylate cyclase, the displacement of [3H]naloxone (a mu opioid antagonist) binding by [D-Ala2,MePhe4,Gly-ol5]enkephalin (a mu opioid agonist) was increased 215-fold; this increase was abolished by adding 100 microM (guanosine 5'-[gamma-thio]triphosphate. Similar increases in agonist displacement of [3H]naloxone binding (33-fold) and its abolition by guanosine 5'-[gamma-thio]triphosphate were observed with Go, the G protein of unknown function, but not with the v-Ki-ras protein p21. In reconstituted preparations with Gi or Go, neither [D-Pen2,D-Pen5]enkephalin (a delta opioid agonist; where Pen is penicillamine) nor U-69,593 (a kappa opioid agonist) showed displacement of the [3H]naloxone binding. In addition, the mu agonist stimulated both [3H]guanosine 5'-[beta,gamma-imido]triphosphate binding (in exchange for GDP) and the low-Km GTPase in such reconstituted preparations, with Gi and Go but not with the v-Ki-ras protein p21, in a naloxone-reversible manner. The stoichiometry was such that the stimulation of 1 mol of mu receptor led to the binding of [3H]guanosine 5'-[beta,gamma-imido]triphosphate to 2.5 mol of Gi or to 1.37 mol of Go. These results suggest that the purified mu opioid receptor is functionally coupled to Gi and Go in the reconstituted phospholipid vesicles.
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PMID:Reconstitution of rat brain mu opioid receptors with purified guanine nucleotide-binding regulatory proteins, Gi and Go. 284 1

The c-Ha-ras oncogene has been implicated as a causative agent in the development of tumors in humans as well as mice. The molecular nature of the ras-induced tumorigenic process remains unclear, however. To address this question directly we have constructed a cell line which carries a zinc-inducible metallothionein-ras hybrid oncogene, transformant 212. Upon exposure to zinc for 24-48 hr, 212 cells assume a highly transformed morphology, concomitant with the induction of ras-expression. Natural killer cells constitute a subpopulation of lymphoid effector cells which have for a long time been hypothesized to be involved in the earliest stages of antitumor surveillance. Central to this hypothesis is the prediction that NK sensitivity arises during cellular transformation. By carrying out cytotoxicity assays against the 212 transformant, we showed that, indeed, increased sensitivity to NK-mediated lysis correlated with expression of the ras oncogene, which is consistent with the above hypothesis. We then addressed the question of the biochemical mechanism of ras-induced transformation. Owing to their similarity to G proteins, regulatory elements interposed between cell-surface receptors and their effector enzymes, it has been postulated that p21, the ras oncogene protein, mediates its transforming effects by constitutive activation of proliferative signal transduction pathways. We studied the effect of ras expression on the regulation of adenylate cyclase (A.C.), key enzyme of one such major pathway. We found that ras expression correlated with a dampening of responsiveness of A.C. to several stimuli, including hormones such as isoproterenol and other agents such as GMP-PNP, forskolin and fluoride-ion. Accumulation of cAMP as measured by RIA in intact cells, as basal or in response to stimulation of A.C. activity with forskolin, was also decreased (approximately 10-fold) with ras expression. Because the regulation of calcium, another important second messenger is dependent, in part, upon cAMP and GTP-binding proteins, we investigated the possible influence of ras expression on the intracellular concentration of calcium. Steady-state intracellular free Ca2+ concentration, as measured by fluorimetry, was indeed increased by approximately 50-125% in association with ras expression. Finally, we studied the possible influence of p21ras on protein kinase C (PKC), which is a key enzyme in the important signal transduction pathway of phosphatidylinositol lipid turnover. We assessed PKC activity directly, in a cell-free system, by measuring the ability of the enzyme to transfer radiolabelled phosphate from gamma-32P-ATP to histone, and exogenous substrate.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:The cell biology of ras-induced transformation: insights from studies utilizing an inducible hybrid oncogene system. 284 54


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