共查询到20条相似文献,搜索用时 0 毫秒
1.
Chirstian Roy 《Biochimica et Biophysica Acta (BBA)/General Subjects》1979,587(3):433-445
The reversibility of adenylate cyclase activation induced by vasopressin was studied by reducing the concentration of active peptide in contact with kidney medullo-papillary membranes. Reversibility of hormonal activation was only partial. The use of antagonists failed to demonstrate the reversibility of an adenylate cyclase activation induced by high affinity agonists. When antagonist was added after the agonist to membranes, a non-competitive inhibitio was apparent. Active peptide was also eliminated from the incubation medium by treatment with agents capable of reducing the disulfide bridge of the hormonal molecule. Direct effects of reducers on adenylate cyclase activity were measured on enzyme activation induced by peptides lacking a disulfide bridge. There was no apparent correlation between the abilities of different reducers to inactivate free peptide in solution and their abilities to promote the reversibility of hormone-induced enzyme activation. Upon the addition of dithiothreitol, enzyme activity could be lowered to baseal value and adenylate cyclase was again fully stimulatable. However, when dithiothreitol addition to stimulated enzyme was combined with a 60-fold dilutionof the incubation medium, no reversibility of hormonal activation occurred. These results illustrate that the processes involved in adenylate cyclase activation are only partially reversible. 相似文献
2.
On the equilibrium of the adenylate cyclase reaction 总被引:2,自引:0,他引:2
3.
Previous work suggested that hormonal activation of adenylate cyclase involves the introduction of GTP to the regulatory site, and subsequent hydrolysis of the bound GTP terminates the activation. In many tissues the turn-off GTPase reaction cannot be readily measured because of a high background of nonspecific GTP hydrolysis. To circumvent this problem a general assay for the turn-off reaction has now been developed. The adenylate cyclase is first activated by hormone and GTP and the introduction of GTP is then stopped either by addition of an excess of guanosine 5'-O-(2-thiodiphosphate) (GDP beta S) or by addition of a receptor blocking agent. The decay of adenylate cyclase activity brought on by these inhibitors is used to calculate the rate constant of the turn-off reaction. In turkey erythrocyte and rat parotid membranes the rate constant of the decay process as determined with GDP beta S is similar to that determined with the beta-adrenergic blocker propranolol. The rate constants (min-1 at 30 degrees C) for various adenylate cyclase preparations are 10 for turkey erythrocyte, 7.5 for rat parotid, and 6.2 for the rat liver enzyme. The finding of similar rate constants in the various preparations indicates that GTP hydrolysis at the regulatory site is a general mechanism for terminating the activation of adenylate cyclase. 相似文献
4.
The size of adenylate cyclase 总被引:9,自引:0,他引:9
E J Neer 《The Journal of biological chemistry》1974,249(20):6527-6531
5.
The activation of adenylate cyclase 总被引:3,自引:0,他引:3
6.
K H Jakobs 《European journal of biochemistry》1983,132(1):125-130
Epinephrine inhibits human platelet adenylate cyclase by an alpha 2-adrenoceptor-mediated and GTP-dependent process. The turn-off reaction for this epinephrine-inhibited enzyme was studied by measuring the rate of cyclic AMP formation upon addition of the alpha2-adrenoceptor antagonist, yohimbine, or upon addition of an excess of the stable GDP analog, guanosine 5'-O-(2-thiodiphosphate) (GDP beta S), which competitively inhibited the action of GTP in the epinephrine-induced inhibition. The decay of the inhibited state of the adenylate cyclase was used to calculate the rate constant of the turn-off reaction. With both methods, almost identical koff values of 0.6-0.7 min-1 at 25 degrees C were obtained for the epinephrine-inhibited platelet enzyme. Cholera toxin, which does not inhibit the epinephrine-induced GTPase stimulation in platelet membranes, did not affect this turn-off reaction. In contrast, the turn-off rate of the prostaglandin-E1-stimulated human platelet adenylate cyclase, measured with GDP beta S, was reduced from about 9 min-1 to 2 min-1 at 25 degrees C by pretreatment of the membranes with cholera toxin, which inhibits the prostaglandin-E1-stimulated GTPase activity. The data strongly suggest that the guanine nucleotide regulatory site, mediating epinephrine-induced adenylate cyclase inhibition, is activated and inactivated by similar mechanisms as is the site mediating adenylate cyclase stimulation, and that cholera toxin affects only the stimulatory site. The findings furthermore suggest that the activity states of these two regulatory sites control the activity of the adenylate cyclase. 相似文献
7.
8.
Microtubule-associated adenylate cyclase 总被引:1,自引:0,他引:1
Twice-cycled bovine brain or rat brain microtubule protein contains an adenylate cyclase activity that passes 0.2 micron filters, is activated 2-7-fold by 30 microM forskolin, shows modest stimulation by fluoride (especially in the presence of added AI3+), but is virtually insensitive to added guanine nucleotides. The activity is insensitive to various hormones or Ca2+/calmodulin. The adenylate cyclase is active with both Mg2+ and Mn2+ but activity is less in the presence of Mg2+ than with Mn2+. The cyclase is inhibited by agonists of the adenosine P site. It is proposed that the catalytic unit of adenylate cyclase and probably small quantities of the guanine nucleotide regulatory protein, Ns, are cycled along with microtubules. 相似文献
9.
W. Schulze 《Histochemistry and cell biology》1982,75(1):133-143
10.
Mechanism of the adenylate cyclase reaction. Stereochemistry of the reaction catalyzed by the enzyme from Brevibacterium liquefaciens 总被引:2,自引:0,他引:2
Adenylate cyclase from Brevibacterium liquefaciens (ATCC 14929) catalyzes the formation of the RP-diastereomer of adenosine 3':5'-cyclic monophosphorothioate from the SP-diastereomer of adenosine-5'-(1-thiotriphosphate). The reaction catalyzed by this adenylate cyclase proceeds with inversion of configuration at phosphorus, indicating that the cyclization reaction is direct and does not involve formation of an adenylated enzyme intermediate. 相似文献
11.
Eva J. Neer 《Journal of cellular biochemistry》1976,4(1):51-61
The size distribution of adenylate cyclase from the rate renal medulla solubilized with the nonionic detergents Triton X-100 and Lubrol PX was determined by gel filtration and by centrifugation in sucrose density gradients made up in H2O or D2O. The physical parameters of the predominant from in Triton X-100 are 220,w, 5.9 S; Stokes radius, 62 A; partial specific volume (v ), 0.74 ml/g; mass, 159,000 daltons; f/f0, 1.6; axial ratio (prolate ellipsoid), 11. For the minor form the values are : 220,w, 3.0; Stokes radius, 28 A; mass, 38,000 daltons; f/f0, 1.2. The corresponding values determined in Lubrol PX are similar. The value of v for the enzyme indicates that it binds less than 0.2 mg detergent/mg protein. Since interactions with detergents probably substitute for interactions with lipids and hydrophobic amino acid side chains, these findings suggest that no more than 5% of the surface of adenylate cyclase is involved in hydrophobic interactions with other membrance components. Thus, most of the mass of the enzyme is not deeply embedded in the lipid bilayer of the plasma membrance. Similar studies have been performed on the soluble guanylate cyclase of the rate renal medulla. In the absence of detergent, the molecular properties of this enzyme are: s20,w, 6.3 S; Stokes radius, 54 A, v , 0.75 ml/g; mass, 154,000 daltons f/f0, 1.4; axial ratio, 7. The addition of 0.1% Lubrol PX to this soluble enzyme increases its activity two- to fourfold and changes the physical properties to : s20,w, 5.5 S; Stokes radius, 62 A; v , 0.74 ml/g; mass, 148,000 daltons; f/f0, 1.6; axial ratio, 11. These results show that Lubrol PX activates the enzyme by causing a conformational change with unfolding on the polypeptide chain. Guanylate cyclase from the particulate cell fraction can be solubilized with Lubrol PX but has properties quite different from those of the enzyme in the soluble cell fraction. It is a heterogeneous aggregrate with s20,w, 10 S; Stokes radius, 65 A; mass about 300,000 daltons. The conditions which solubilize guanylate cyclase also solubilize adenylate cyclase and the two activities can be separated on the same sucrose gradient. 相似文献
12.
E J Neer 《The Journal of biological chemistry》1973,248(13):4775-4781
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14.
K L Olgiati D G Toscano W M Atkins W A Toscano 《Archives of biochemistry and biophysics》1984,231(2):411-415
Gossypol, a polyphenolic binaphthalene -dialdehyde reputed to exert contraceptive action in males, reversibly inhibits adenylate cyclase [ATP pyrophosphate lyase (cyclizing), EC 4.6.1.1] in a concentration-dependent manner. In membranes prepared from a variety of organs, the half-maximal inhibitory concentration (IC50) ranges from 75 microM (rat Leydig tumor cells) to 250 microM (rat liver membranes). Kinetic studies using partially purified catalytic subunit isolated from bovine testis show that gossypol is competitive with ATP with an apparent Ki of 110 microM. These data suggest that gossypol inhibition of adenylate cyclase is due to direct interaction at the nucleotide-binding domain of the catalytic subunit of the enzyme. 相似文献
15.
Experiments using a cytochemical method showed the presence of a specific precipitate of the adenylate cyclase (AC) reaction on the sarcolemma and in the subsarcolemmal cisternae and junctional sarcoplasmic reticulum in rat cardiomyocytes. The localization of AC in the given organelles draws attention to the mutual association between Ca2+ ions and cAMP in the modulation of cardiac contractions. Trifluoperazine (TFP) and chlorpromazine (CHP) are known as phenothiazine derivatives inhibiting cellular enzymatic processes dependent on calmodulin and Ca2+. AC is one of these enzyme systems. The administration of TFP and CHP (both in a dose of 0.1 and 3 mmol.1(-1)) did not affect the cytochemical localization of the enzyme. Quantitative determination of 125I-cAMP by RIA showed that CHP inhibited AC activity in both concentrations. TFP, on the other hand, did not inhibit AC activity in 0.1 mmol.1(-1) concentration and actually stimulated its activation in 3 mmol.1(-1) concentration. The different action of the phenothiazine derivatives on AC activity can be attributed partly to the different affinity of TFP and CHP for calmodulin and partly to interaction of the inhibitor-calmodulin complex with the phosphodiesterase (PDE) system. 相似文献
16.
Cell-free extract of E. coli possessed an inhibited adenylate cyclase activity after a previous anaerobic incubation of cells with glucose which is transported and metabolized. The degree of the inhibition depends on incubation conditions. Glucose analogues that are only transported but not metabolized, are not inhibitory. To restore the adenylate cyclase activity, the cells have to be cultivated aerobically prior to disintegration for a defined period of time without glucose. 相似文献
17.
In this study, we have observed that cells increase their intracellular cAMP to relatively high levels during receptor capping induced by either ligand-dependent (anti-Thy-1 antibody) or ligand-independent (colchicine) treatment. In addition, we have found that under capping conditions, membrane-bound adenylate cyclase is induced to co-cap with independent membrane molecules such as Thy-1 antigens. These findings suggest that the binding of anti-Thy-1 to its receptors or treatment with colchicine induces the molecular reorganization of membrane-bound adenylate cyclase which may be responsible for activating the contractile machinery required for the collection of surface receptors into a cap structure. 相似文献
18.
Purification of the catalyst of adenylate cyclase 总被引:12,自引:0,他引:12
M D Smigel 《The Journal of biological chemistry》1986,261(4):1976-1982
The catalytic moiety of hormone-sensitive adenylate cyclase has been purified from bovine brain. It is isolated largely without its guanine nucleotide-binding regulatory protein, Gs, by affinity chromatography on 7-O-hemisuccinyldeacetylforskolin-agarose. It appears to be a single polypeptide which migrates on sodium dodecyl sulfate-polyacrylamide gels with an apparent Mr of approximately 120,000. When subjected to electrophoresis on gradient (5-10%) sodium dodecyl sulfate-polyacrylamide gels, it displays a larger apparent Mr of 150,000. The adenylate cyclase activity of the preparation can be stimulated by the addition of Gs, forskolin, or calcium-calmodulin. The preparation has been reconstituted with purified beta-adrenergic receptors and Gs to form a hormone-stimulated adenylate cyclase system (May, D., Ross, E.M., Gilman, A.G., and Smigel, M.D. (1985) J. Biol. Chem. 260, 15829-15833). In contrast to its stimulation by Gs, inhibition by the alpha subunits of Gi and Go, G proteins known to be coupled to inhibitory receptors (Sternweis, P., and Florio, V. (1985) J. Biol. Chem. 260, 3477-3483), is not seen. Preparations of adenylate cyclase show varying degrees of inhibition by added G protein beta . gamma subunit. This inhibition can be explained as reflecting a variable, small (under 5%) contamination of the preparation by Gs alpha which would be deactivated by complexing with the added beta . gamma subunit. 相似文献
19.
Bimodal regulation of adenylate cyclase 总被引:19,自引:0,他引:19
D M Cooper 《FEBS letters》1982,138(2):157-163
20.
ATP-dependent activation of adenylate cyclase 总被引:3,自引:0,他引:3
Incubation of rat liver plasma membranes with MgCl2, ATP, and an ATP-regenerating system at 4 degrees C provides a 4-7-fold persistent activation of adenylate cyclase. Enzyme activation is time-dependent and 48 h of incubation is usually required to achieve maximal stimulation of adenylate cyclase activity. The activation described is not affected by GTP, cAMP, or cGMP, and does not occur when ATP is replaced by a nonphosphorylating analogue, adenyl-5'-imidodiphosphate. In addition to ATP, the activation requires Mg2+ and an ATP-regenerating system. The activation described is not additive with that produced by fluoride and analysis of basal and fluoride activities following extended incubation for 48 h reveals identical activities which decay at the same rate. These results are consistent with our model (11) which invokes phosphorylation-dephosphorylation mechanisms in regulating adenylate cyclase activity. 相似文献