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 frameshift mutagen 9-aminoacridine (9AA) causes DNA damage via a recA+-independent mechanism in Escherichia coli. In this study we have exposed E. coli cells carrying the lacZ19124 frameshift marker to 9AA in defined minimal media, washed them, and plated to score for Lac+ revertants. Our results show that 9AA-induced reversion to Lac+ occurs in the absence of any exogenous carbon source and when cells are plated on media which do not allow much, if any, cell replication prior to expression of the revertant phenotype. When glycerol (1% w/v) was added to the liquid treatment medium, the number of Lac+ E. coli revertants was similar to that obtained when no carbon source was present. By contrast the addition of glucose (1% w/v) during the mutagenesis treatment caused a significant decrease in the number of revertants. Further experiments indicate that the repressing effects of glucose may be due to a reduction in cAMP concentration, since 9AA mutagenesis was abolished in a cya strain in which no adenylate cyclase is produced. These results are consistent with (but do not prove) the notion that at least one part of the process leading to 9AA mutagenesis is subject to catabolite repression.
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PMID:RecA-independent mutagenesis in Escherichia coli may be subject to glucose repression. 304 Dec 5

The role of G proteins in mediating adrenoceptor-prostacyclin synthesis coupling was investigated using the G protein activator, sodium fluoride. Sodium fluoride (NaF) stimulated in vitro rat aortic prostacyclin (PGI2) synthesis (EC50 = 5 x 10(-3) mol.l-1), an action inhibited completely by the presence of EDTA (10(-2) mol.l-1). The NaF-PGI2 dose-response curve was moved to the left by the presence of adrenaline, phorbol 12,13-dibutyrate (PDBU) and the Ca2+ ionophore A23187 in the incubation media. NaF-stimulated (5 x 10(-3) mol.l-1) PGI2 synthesis was inhibited by the Ca2+ channel blockers, verapamil and nifedipine, the protein kinase C inhibitor, H7, and lanthanum. Prazosin and yohimbine were without effect on NaF action, but partially inhibited adrenaline-potentiated NaF-stimulated PGI2 synthesis. Cyclic adenosine-3',5'-monophosphate (cAMP) and dibutyryl cAMP were without effect on de novo or NaF-, adrenaline-, PDBU- or A23187-stimulated PGI2 synthesis. Since fluoride is known to stimulate adenyl cyclase and phospholipase C, these data suggest that: (1) NaF stimulates in vitro rat aortic PGI2 synthesis by initiating Ca2+ influx; (2) this Ca2+ influx is mediated by protein kinase C, probably through G protein activation of phospholipase C and the generation of the protein kinase C activator, diacyl glycerol; and (3) adenyl cyclase and protein kinase A are not involved in NaF-stimulated PGI2 synthesis by the rat aorta.
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PMID:Fluoride stimulates in vitro vascular prostacyclin synthesis: interrelationship of G proteins and protein kinase C. 313 Nov 47

The GTP-dependence for stimulatory and inhibitory regulation of plasma membrane adenylate cyclase activity was measured in plasma membrane fractions isolated from a variety of cell types (platelets, lymphocytes, PC12 cells, GH3 cells, NBP2 cells, and hepatocytes). This report shows that the isolation of plasma membranes for the study of GTP-dependent adenylate cyclase activity was, for some cells, enhanced by the exposure of the cells to glycerol prior to cell lysis. The isolation of plasma membranes from other cells, which did not appear to be sensitive to glycerol pretreatment, was enhanced by the removal of heavy particulate matter prior to fractionation of the cell lysate. The regulation of enzyme activity by various agents was found to be dependent upon the presence of (exogenous) GTP to varying degrees, indicating variable contamination of membrane preparations with GTP. It is concluded that (i) exposure of platelets and lymphocytes to glycerol prior to cell lysis decreases subsequent contamination of the plasma membrane preparation with GTP, and (ii) although glycerol pretreatment of other cells does not ensure the subsequent isolation of plasma membrane adenylate cyclase activity displaying high requirements for (exogenous) GTP, it is a reasonable first approach to be used during the development of procedures for the isolation of plasma membranes.
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PMID:Evaluation of methods for the isolation of plasma membranes displaying guanosine 5'-triphosphate-dependence for the regulation of adenylate cyclase activity: potential application to the study of other guanosine 5'-triphosphate-dependent transduction systems. 324 66

In Dictyostelium, extracellular cAMP interacts specifically with cell-surface receptors to promote the accumulation of a variety of intracellular second messengers, such as 3'-5' cyclic adenosine monophosphate (cAMP) and 1,4,5 inositol trisphosphate (IP3). We and others have shown that activation of the cell-surface cAMP receptor can also modulate the expression of the Dictyostelium genome during development. In at least one instance, synthesis of intracellular cAMP is required for appropriate gene regulation. However, the induction of most cAMP-dependent gene expression can occur in the absence of receptor-mediated activation of adenylate cyclase and a consequent accumulation of intracellular cAMP. These results suggest that other intracellular second messengers produced in response to receptor activation may potentially act as signal transducers to modulate gene expression during development. In vertebrate cells, IP3 and diacylglycerol (DAG) are intracellular activators of specific protein kinases; they are produced in equimolar amounts by cleavage of phosphoinositol bisphosphate after a receptor-mediated activation of a membrane-bound phosphodiesterase. IP3 and, thus, by inference, diacyl-glycerol are synthesized in Dictyostelium as a response to cAMP interacting with its cell-surface receptor. Using defined conditions to inhibit the accumulation of extracellular cAMP, we have examined the effects of these compounds on the expression of genes that require cAMP for their maximal expression. Our results suggest that intracellular IP3 and DAG may in part mediate the action of extracellular cAMP on the expression of the Dictyostelium genome.
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PMID:Regulation of gene expression by the intracellular second messengers IP3 and diacylglycerol. 326 90

In the mammalian myocardium, an active triglyceride synthesis pathway is operating, (re)esterifying activated fatty acids from endogenous or exogenous sources, with the glycolytically derived three-carbon intermediates dihydroxyacetone-phosphate and glycerol-3-phosphate by the so-called Kennedy pathway. The seven enzymes of triglyceride synthesis are membrane bound and located at the sarcoplasmic reticulum. The first enzyme in the glycerol-3-phosphate pathway, glycerol-3-phosphate acyltransferase, is proposed to be rate limiting for triglyceride formation. This microsomal enzyme is regulated by phosphorylation (inactiycation)-dephosphorylation (activation) coupled to the beta-receptor--adenyl cyclase--protein kinase system. Additional regulatory steps in triglyceride formation are the reactions catalyzed by the microsomal phosphatidic acid phosphatase and diglyceride acyltransferase. Intracellular triglycerides occur as free floating cytosolic droplets, membrane-bound particles and lipid-filled lysosomes. No consensus exists about the metabolically active portion of myocardial triglycerides. Various lipases have been proposed to be involved in endogenous lipolysis: the lysosomal acid, microsomal and soluble neutral triglyceride, intracellular lipoprotein lipases and the microsomal di- and monoglyceridase. It has been acknowledged that the bulk of the intracellular neutral lipase represents the precursor of vascular lipoprotein lipase. The presence of a neutral lipase, as distinct from lipoprotein lipase, in the rat heart was recently advocated. Endogenous lipolysis is a hormone-sensitive process. Hormone-sensitivity may involve direct alteration of enzyme activity by protein phosphorylation-dephosphorylation but is also dependent on the removal rate of product fatty acids, since feedback inhibition is a common property of all lipases in the heart.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Synthesis, storage and degradation of myocardial triglycerides. 331 Oct 5

Activators of protein kinase C, a calcium- and phospholipid-dependent protein kinase, inhibit vasopressin-stimulated water flow in toad bladder. To determine the biochemical mechanisms of this inhibition, we examined the effects of activators of protein kinase C on arginine vasopressin (AVP)-stimulated adenylate cyclase activity in cultured rabbit cortical collecting tubular cells. The phorbol ester, 4 beta-phorbol 12-myristate 13-acetate (PMA), the diacylglycerol, 1-oleyl-2-acetyl glycerol (OAG), and the diacylglycerol kinase inhibitor, R59022, all rapidly activate protein kinase C in collecting tubular cells. Pretreatment with PMA produces a delayed inhibition (greater than or equal to 4 h) of AVP-stimulated adenylate cyclase activity. The 4-h time lag suggests that the effects of protein kinase C are mediated indirectly, possibly as a consequence of stimulating cell proliferation. PMA does not inhibit cholera toxin- or forskolin-stimulated adenylate cyclase activity, suggesting an effect on the vasopressin receptor or coupling of the receptor to the stimulatory guanine nucleotide regulatory protein. Neither prostaglandins nor the inhibitory guanine nucleotide regulatory protein appear to mediate this effect. In contrast, treatment with either OAG or R59022 produces a rapid inhibition of both AVP- and forskolin-stimulated adenylate cyclase activity suggesting a prominent distal site of action, presumably at the catalytic subunit of adenylate cyclase. The results demonstrate that different activators of protein kinase C inhibit AVP-stimulated adenylate cyclase activity by distinctly different mechanisms possibly by altering the substrate specificity or activating multiple forms of the kinase. These results have important implications when using different activators to study the biological effects of protein kinase C.
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PMID:Phorbol esters inhibit adenylate cyclase activity in cultured collecting tubular cells. 333 16

The mechanism of the inhibitory action of forskolin, a plant-derived stimulator of adenylate cyclase, on glucose transport in rat adipose cells was studied. Lipolysis (glycerol release) and glucose transport activity (initial 3-O-methylglucose uptake rate) were measured after treatment of intact cells. In isolated plasma membranes, D-glucose transport and glucose-inhibitable binding of cytochalasin B, a specific labeling agent for the glucose transporter, were assayed. Forskolin inhibited insulin-stimulated glucose transport in intact cells at low concentrations which failed to stimulate lipolysis. Furthermore, the adenylate cyclase inhibitor prostaglandin E2 reduced forskolin-stimulated lipolysis but failed to reverse the transport inhibition. Therefore, the effects of the agent on lipolysis appeared to be dissociable from those on glucose transport. In plasma membrane vesicles, forskolin inhibited D-glucose transport in a competitive manner by an increase in the apparent transport Km without any detectable change in Vmax. In parallel to the transport inhibition, the agent inhibited the specific binding of cytochalasin B in both plasma membranes and low density microsomes, which contain the intracellular pool of glucose transporters in insulin-sensitive cells. The Kl of this inhibition (205 nM) was very similar to that of the inhibition of glucose transport in the membrane vesicles (203 nM). It is concluded that forskolin inhibits glucose transport by a direct interaction with the transporter (or a closely related protein) rather than through activation of adenylate cyclase.
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PMID:Forskolin inhibits insulin-stimulated glucose transport in rat adipose cells by a direct interaction with the glucose transporter. 347 May 98

The kinetics of the ionic regulation of an adenylate cyclase associated with the excitable ciliary membrane from Paramecium tetraurelia was examined. Glycerol (30%, v/v) stabilized the enzyme, and activated by an increase in Vmax. (3-fold) and a decrease in the apparent Km for MgATP (6-fold). Kinetic analysis of Mg2+ effects showed a stimulation via a single metal-binding site separate from the substrate site, with a dissociation constant, Ks, of 0.27 mM. Analysis of Ca2+ effects showed (i) an uncompetitive inhibition with respect to substrate MgATP, and (ii) dependence of the extent of inhibition on the free Mg2+ concentration. Ki values ranged from 4 to 130 microM-Ca2+ in the presence of 0.55-2 mM-Mg2+ respectively. This indicates competition between Mg2+ and Ca2+ at the metal-binding site. The Ca2+ effect was specific; Sr2+ and Ba2+ were almost without effect, and 100 microM-Ba2+ did not interfere with the Ca2+ inhibition. The actions of Ca2+ were readily reversible after addition of EGTA. K+ activated the adenylate cyclase at concentrations around 20 mM. The stimulatory potency of K+ was dependent on the free Mg2+ concentration. At 1 mM free Mg2+, 20 mM-K+ doubled the adenylate cyclase activity. The inhibitory Ca2+ and stimulatory K+ inputs were independent of each other.
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PMID:Ionic regulation of adenylate cyclase from the cilia of Paramecium tetraurelia. 349 99

Isolation of adenylate cyclase-enriched membranes from human platelets was attempted using glycerol lysis technique followed by ultracentrifugation on discontinuous sucrose gradients composed of 24, 30, 34, 37, and 41% (w/w). Adenylate cyclase activity was enriched 4-fold in sample/24% sucrose interface, 7-fold in 24%/30% sucrose interface, and 4-fold in 30%/34% sucrose interface fractions with the recovery of 15-20% of the total activity. The enrichment and subcellular distribution of adenylate cyclase resembled in general those of phosphodiesterase and acid phosphatase with slight differences in each other. Protein profiles from SDS-polyacrylamide gel electrophoresis showed that the heavy chain of myosin (Mr = 200,000) was enriched in sample/24% sucrose interface and lower molecular weight proteins in 34%/37% sucrose interface and pellet. The interface fractions between 24 and 34% sucrose were, therefore, collected as adenylate cyclase-enriched membranes. Adenylate cyclase associated with the membranes displayed high specific activity (0.1 and 1-2 nmol/min/mg protein in the absence and presence of stimulants, respectively), and possessed sensitivities to prostaglandins (E1, I2, and D2) as well as cholera toxin. Activation of adenylate cyclase by these compounds required added GTP, indicating that the contamination of the membrane preparations with GTP-like substance (s) was minimal, if at all present.
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PMID:Isolation and partial characterization of adenylate cyclase-enriched membranes from human platelets. 371 86

Rapid destabilization of FSH receptor after solubilization by detergents is a serious problem complicating its purification and further study. We have developed a procedure for the solubilization of stable and functional FSH receptors with Triton X-100. The new protocol selectively utilizes pure lighter membranes isolated from bovine calf testes by preparative sucrose density gradient centrifugation as the source of receptor. The conditions of detergent solubilization were optimized to reduce the required ratio of Triton X-100 to membrane protein to a minimum. In addition, during detergent extraction the membranes were treated with petroleum ether to remove interfering neutral lipids, thus facilitating solubilization of FSH receptors by the detergent. FSH receptors so obtained appeared to be soluble by criteria such as failure to sediment at 145,000 X g after 90 min, passage through 0.22-micron Millipore filters, and retardation upon chromatography on Sepharose 6B column. Approximately 86% of receptors originally present in the light membranes were recovered after solubilization, with a 24-fold increase in specific activity. The detergent-soluble fraction has several interesting properties not previously reported. It contains only high affinity receptors for FSH (Ka = 1.02 X 10(10) M-1), which are stable in the absence of glycerol for 4 days at 1 degree C or 6 months at -80 degrees C. Luteinizing hormone and human chorionic gonadotropin receptor activity usually associated with detergent-solubilized extracts of testes is low due to incomplete solubility of these receptors under the conditions utilized for solubilization of FSH receptors. Of particular interest is the ability of the receptor in the detergent extract to respond to added FSH with stimulation of adenylate cyclase activity. Adenylate cyclase activity also responds to F- stimulation and the detergent extract retains full guanosine 5'-imidotriphosphate-binding activity. This suggests that under the extraction conditions employed, a high proportion of soluble receptors are associated with related components of the adenylate cyclase system. Our data are consistent with the notion that the solubilized hormone-binding sites represent the physiologically relevant and functional receptors originally present in the light membrane fraction of calf testis. The availability of this detergent-soluble, stable and functional receptor fraction in larger amounts (2.2 g of protein from each batch of 11.5 kg bovine calf testes) than heretofore possible should facilitate further studies on FSH receptor purification and its mechanism of action.
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PMID:Solubilization of functional and stable follitropin receptors from light membranes of bovine calf testis. 375 49


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