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1.
Changes in the form of Arrhenius plots of the activity of glucagon-stimulated adenylate cyclase and other hamster liver plasma-membrane enzymes occurring on hibernation.
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1. Arrhenius plots of the glucagon-stimulated adenylate cyclase, 5'-nucleotidase, (Na+ + K+)-stimulated adenosine triphosphatase and Mg2+-dependent adenosine triphosphatase activities of control hamster liver plasma membranes exhibited two break points at around 25 and 13 degrees C, whereas Arrhenius plots of their activities in hibernating hamster liver plasma membranes exhibited two break points at around 25 and 4 degrees C. 2. A single break occurring between 25 and 26 degrees C was observed in Arrhenius plots of the activities of fluoride-stimulated adenylate cyclase, basal adenylate cyclase and cyclic AMP phosphodiesterase of liver plasma membranes from both control and hibernating animals. 3. Arrhenius plots of phosphodiesterase I activity showed a single break at 13 degrees C for membranes from control animals, and a single break at around 4 degrees C for liver plasma membranes from hibernating animals. 4. The temperature at which break points occurred in Arrhenius plots of glucagon- and fluoride-stimulated adenylate cyclase activity were decreased by about 7--8 degrees C by addition of 40 mm-benzyl alcohol to the assays. 5. Discontinuities in the Arrhenius plots of 4-anilinonaphthalene-1-sulphonic acid fluorescence occurred at around 24 and 13 degrees C for liver plasma membranes from control animals, and at around 25 and 4 degrees C for membranes from hibernating animals. 6. We suggest that in hamster liver plasma membranes from control animals a lipid phase separation occurs at around 25 degrees C in the inner half of the bilayer and at around 13 degrees C in the outer half of the bilayer. On hibernation a change in bilayer asymmetry occurs, which is expressed by a decrease in the temperature at which the lipid phase separation occurs in the outer half of the bilayer to around 4 degrees C. The assumption made is that enzymes expressing both lipid phase separations penetrate both halves of the bilayer, whereas those experiencing a single break penetrate one half of the bilayer only. 相似文献
2.
Lysophosphatidylcholines can modulate the activity of the glucagon-stimulated adenylate cyclase from rat liver plasma membranes.
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1. Synthetic lysophosphatidylcholines inhibit the glucagon-stimulated adenylate cyclase activity of rat liver plasma membranes at concentrations two to five times lower than those needed to inhibit the fluoride-stimulated activity. 2. Specific 125I-labelled glucagon binding to hormone receptors is inhibited at concentrations similar to those inhibiting the fluoride-stimulated activity. 3. At concentrations of lysophosphatidylcholines immediately below those causing inhibition, an activation of adenylate cyclase activity or hormone binding was observed. 4 These effects are essentially reversible. 5. We conclude that the increased sensitivity of glucagon-stimulated adenylate cyclase to inhibition may be due to the lysophosphatidylcholines interfering with the physical coupling between the hormone receptor and catalytic unit of adenylate cyclase. 6. We suggest that, in vivo, it is possible that lysophosphatidylcholines may modulate the activity of adenylate cyclase only when it is in the hormone-stimulated state. 相似文献
3.
The activity of glucagon-stimulated adenylate cyclase from rat liver plasma membranes is modulated by the fluidity of its lipid environment. 总被引:2,自引:7,他引:2
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1. The local anaesthetic benzyl alcohol progressively activated glucagon-stimulated adenylate cyclase activity up to a maximum at 50 mM-benzyl alcohol. Further increases in benzyl alcohol concentration inhibited the activity. The fluoride-stimulated adenylate cyclase activity was similarly affected except for an inhibition of activity occurring at low benzyl alcohol concentrations (approx. 10 mM. 2. The fluoride-stimulated adenylate cyclase activity of a solubilized enzyme preparation was unaffected by any of the benzyl alcohol concentrations tested. 3. Increases in 3-phenylpropan-1-ol and 5-phenylpentan-1-ol concentrations progressively activated both the fluoride- and glucagon-stimulated adenylate cyclase activities up to a maximum, above which further increases in alcohol concentration inhibited the activities. 4. The 'break' points in Arrhenius plots of glucagon-stimulated adenylate cyclase activity in native plasma membranes, and in plasma membranes fused with synthetic dimyristoyl phosphatidylcholine so as to constitute 60% of the total lipid pool, were decreased by approx. 6 degrees C by addition of 40 mM-benzyl alcohol. This was accompanied by a fall in the associated activation energies. 6. Arrhenius plots of fluoride-stimulated adenylate cyclase activity in the presence and absence of 40 mM-benzyl alcohol were linear, although addition of benzyl alcohol caused a dramatic decrease in the associated activation energy of the reaction. 7. 5'-Nucleotidase activity was stimulated by benzyl alcohol, and the 'break' point in the Arrhenius plot of its activity was decreased by about 6 degrees C by addition of 40 mM-benzyl alcohol to the assay. 8. It is suggested that benzyl alcohol effects a fluidization of the bilayer, which is clearly demonstrated by its ability to lower the temperature of a lipid phase separation occurring at 28 degrees C in the outer half of the bilayer to around 22 degrees C. The increase in bilayer fluidity relieves a physical constraint on the membrane-bound adenylate cyclase, activating the enzyme. 9. The various inhibition phenomena are discussed in detail, together with the suggestion that the interaction between the uncoupled catalytic unit of adenylate cyclase and the lipids of the bilayer is altered on its physical coupling to the glucagon receptor. 相似文献
4.
5.
Elevated membrane cholesterol concentrations inhibit glucagon-stimulated adenylate cyclase. 总被引:2,自引:2,他引:2
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A method was devised which increases the cholesterol concentration of rat liver plasma membranes by exchange from cholesterol-rich liposomes at low temperature (4 degrees C). When the cholesterol concentration of liver plasma membranes is increased, there is an increase in lipid order as detected by a decrease in mobility of an incorporated fatty acid spin probe. This is accompanied by an inhibition of adenylate cyclase activity. The various ligand-stimulated adenylate cyclase activities exhibit different sensitivities to inhibition by cholesterol, with inhibition of glucagon-stimulated greater than fluoride-stimulated greater than basal activity. The bilayer-fluidizing agent benzyl alcohol is able to reverse the inhibitory effect of cholesterol on adenylate cyclase activity in full. The thermostability of fluoride-stimulated cyclase is increased in the cholesterol-rich membranes. Elevated cholesterol concentrations abolish the lipid-phase separation occurring at 28 degrees C in native membranes as detected by an incorporated fatty acid spin probe. This causes Arrhenius plots of glucagon-stimulated adenylate cyclase activity to become linear, rather than exhibiting a break at 28 degrees C. It is suggested that the cholesterol contents of both halves of the bilayer are increased by the method used and that inhibition of adenylate cyclase ensues, owing to the increase in lipid order and promotion of protein-protein and specific cholesterol-phospholipid interactions. 相似文献
6.
A D Whetton L Needham G P Margison N J Dodd M D Houslay 《Biochimica et biophysica acta》1984,773(1):106-112
The effect of the hepatocarcinogen dimethylnitrosamine on rat liver plasma membrane adenylate cyclase activity and lipid fluidity was assessed. Glucagon-stimulated adenylate cyclase activity exhibited a complex response to increasing concentrations of dimethylnitrosamine, whereas fluoride-stimulated adenylate cyclase activity was progressively inhibited. Maximal inhibitory effects were observed at a concentration of 15 mM in both cases. The activity of detergent-solubilized adenylate cyclase was unaffected by dimethylnitrosamine. ESR analysis using a fatty acid spin probe showed that dimethylnitrosamine produced a marked, dose-dependent reduction in the fluidity of the plasma membrane with a maximal effect occurring at 20 mM. Dimethylnitrosamine also elevated the temperature at which the lipid phase separation occurred in rat liver plasma membranes, from 28 degrees C to 31 degrees C. The non-carcinogenic but structurally similar compound, dimethylamine hydrochloride neither inhibited adenylate cyclase nor decreased plasma membrane fluidity. It is suggested that the decrease in membrane fluidity, induced by dimethylnitrosamine, via its effects on membrane fluidity, could influence plasma membrane function and cellular regulation. 相似文献
7.
P J Bilan J B Weitzman T Pflieger C R Lancaster A Stafford R M Epand 《Biochimie et biologie cellulaire》1989,67(11-12):759-762
Lipoprotein fractions from some individuals have inhibitory effects on rat liver adenylate cyclase. Precipitation of the lipoprotein fractions with acetone released an inhibitory factor, which was soluble in acetone-H2O (3:1, v/v). The inhibition was greater against glucagon-stimulated activity than against basal activity. Acetone extraction increased the potency of inhibition. All three lipoprotein fractions, i.e., very low, low, and high density lipoproteins, released some inhibitory component after acetone extraction. The inhibitor was concentrated in the lipoprotein fractions, since acetone extraction of plasma did not release an inhibitor. The acetone extract from the very low density lipoprotein was the most inhibitory. This material was further purified and partially characterized. The inhibitor had a molecular mass of about 500. It was inhibitory at micromolar concentrations. The material was sufficiently hydrophobic to migrate in normal-phase thin-layer chromatography (TLC). Nuclear magnetic resonance results indicated that it was not a polar lipid. There were several different inhibitory factors that were separable by TLC. The sequestration of these inhibitors into lipoproteins reduced their effectiveness in inhibiting the action of counter-regulatory hormones, such as glucagon. 相似文献
8.
C C McOsker G A Weiland D B Zilversmit 《The Journal of biological chemistry》1983,258(21):13017-13026
The nonspecific lipid transfer protein from beef liver was used to modify the phospholipid composition of intact turkey erythrocytes in order to study the dependence of isoproterenol-stimulated adenylate cyclase activity on membrane phospholipid composition. Incorporation of phosphatidylinositol into turkey erythrocytes inhibited isoproterenol-stimulated cyclic AMP accumulation in a linear, concentration-dependent manner. Inhibition was relatively specific for phosphatidylinositol; phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylglycerol and phosphatidic acid were from 3 to 7 times less effective as inhibitors of hormone-stimulated cyclase activity. Inhibition by phosphatidylinositol was not reversible when up to 90% of the incorporated phosphatidylinositol was removed, either by incubation with phosphatidylinositol-specific phospholipase C or a second incubation with transfer protein; possibly adenylate cyclase activity depends on a small pool of phosphatidylinositol that is inaccessible to either phospholipase C hydrolysis or removal by lipid transfer protein. Phosphatidylinositol incorporation inhibits adenylate cyclase activity by uncoupling beta-adrenergic receptors from the remainder of the cyclase complex. Phosphatidylinositol incorporation had no effect on stimulation of cAMP accumulation by either cholera toxin or forskolin, indicating that inhibition occurs only at the level of receptor. Phosphodiesterase activity was not altered in phosphatidylinositol-modified cells. Inhibition of cAMP accumulation was not the result of changes in either membrane fluidity or in cAMP transport out of modified turkey erythrocytes. Phosphatidylinositol inhibition of isoproterenol-stimulated cyclase activity may serve as a useful model system for hormone-induced desensitization. 相似文献
9.
M D Houslay T R Hesketh G A Smith G B Warren J C Metcalfe 《Biochimica et biophysica acta》1976,436(2):495-504
1. The lipids composition of rat liver plasma membranes was substantially altered by introducing synthetic phosphatidylcholines into the membrane by the techniques of lipid substitution or lipid fusion. 40-60% of the total lipid pool in the modified membranes consisted of a synthetic phosphatidylcholine. 2. Lipid substitution, using cholate to equilibrate the lipid pools, resulted in the irreversible loss of a major part of the adenylate cyclase activity stimulated by F-, GMP-P(NH)P or glucagon. However, fusion with presonicated vesicles of the synethic phosphatidylcholines causes only small losses in adenylate cyclase activity stimulated by the same ligands. 3. The linear form of the Arrhenius plots of adenylate cyclase activity stimulated by F- or GMP-(NH)P was unaltered in all of the membrane preparations modified by substitution or fusion, with very similar activation energies to those observed with the native membrane. The activity of the enzyme therefore appears to be very insensitive to its lipid environment when stimulated by F- or gmp-p(nh)p. 4. in contrast, the break at 28.5 degrees C in the Arrhenius plot of adenylate cyclase activity stimulated by glucagon in the native membrane, was shifted upwards by dipalmitoyl phosphatidylcholine, downwards by dimyristoyl phosphatidylcholine, and was abolished by dioleoyl phosphatidylcholine. Very similar shifts in the break point were observed for stimulation by glucagon or des-His-glucagon in combination with F- or GMP-P(NH)P. The break temperatures and activation energies for adenylate cyclase activity were the same in complexes prepared with a phosphatidylcholine by fusion or substitution. 5. The breaks in the Arrhenius plots of adenylate cyclase activity are attributed to lipid phase separations which are shifted in the modified membranes according to the transition temperature of the synthetic phosphatidylcholine. Coupling the receptor to the enzyme by glucagon or des-His-glucagon renders the enzyme sensitive to the lipid environment of the receptor. Spin-label experiments support this interpretation and suggest that the lipid phase separation at 28.5 degrees C in the native membrane may only occur in one half of the bilayer. 相似文献
10.
The role of a guanine nucleotide-binding protein in the activation of rat liver plasma-membrane adenylate cyclase by forskolin. 总被引:3,自引:4,他引:3
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The effects of the photoreactive GTP analogue GTP-gamma-azidoanilide on rat liver plasma-membrane adenylate cyclase are described. U.v. irradiation in the presence of the analogue abolished activation by any effector or combination of effectors that function via the activatory G protein. Partial protection against this inhibition was given by F- and guanosine 5'-[gamma-thio]triphosphate. It is concluded that GTP-gamma-azidoanilide acts by a light-induced covalent reaction with the G protein. In the dark the effects of the analogue were similar to those of GTP. Irradiation in the presence of GTP-gamma-azidoanilide was found to reduce but not to abolish activation of rat liver plasma membrane adenylate cyclase by forskolin. The activation by forskolin and GTP together were greater than the sum of the individual activations. Forskolin doubled adenylate cyclase activity in the presence of glucagon and guanosine 5'-[beta, gamma-imido]triphosphate, which might be expected to activate to the maximum possible extent via the G protein. It is concluded that there are two components to the forskolin activation, a guanine nucleotide-dependent and a guanine nucleotide-independent component. 相似文献
11.
Endocytosis of beta-adrenergic ligands by rat liver. Comparison of beta-adrenergic receptor and adenylate cyclase distribution in endosome and plasma-membrane fractions.
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The internalization of beta-adrenergic receptors was investigated in rat livers perfused with an agonist ([3H]isoprenaline) or an antagonist ([125I]iodocyanopindolol). Analytical centrifugation of liver homogenates indicated that the ligands were transferred rapidly to endosomal and lysosomal positions in sucrose gradients. Endosome fractions contained beta-adrenergic binding sites, but adenylate cyclase activity was low and poorly activated by isoprenaline. The results indicate that the receptor-regulatory-protein-adenylate cyclase complex was disassembled during uptake of beta-adrenergic ligands, with the adenylate cyclase being retained at the plasma membrane. 相似文献
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13.
The effects of the alpha 1-adrenergic agonist methoxamine and the alpha 2-adrenergic agonist clonidine on isoproterenol stimulated adenylate cyclase activity were examined in plasma membranes prepared from female human subcutaneous adipose tissue. It was found that in the presence of 10 microM GTP and 100 mM NaCl increasing concentrations of both agonists inhibited basal and isoproterenol-stimulated adenylate cyclase activity. The inhibitory action of 5 x 10(-7) M clonidine could not be overcome by increasing concentrations of isoproterenol. These results suggest both alpha 1- and alpha 2-adrenergic agonists inhibit beta-agonist-stimulated adenylate cyclase activity in human adipose tissue. 相似文献
14.
1. Intact mouse neuroblastoma NS20 cells, in the presence of cyclic adenosine 3':5'-monophosphate (cAMP) phosphodiesterase inhibitor, responded to adenosine (200 muM) and 2-chloroadenosine (200 muM) with a 20-fold increase in intracellular cAMP levels. AMP (200 muM) additions caused only a 3.5-fold cAMP level elevation. ATP, ADP, guanosine, cytidine, uridine, and guanine, all at 200 muM, had no effect on the cAMP level of these cells. 2. Homogenate NS20 adenylate cyclase activity was increased 2.5- to 4-fold by addition of 200 muM adenosine, 2-chloroadenosine, 2-hydroxyadenosine, or 8-methylaminoadenosine. Prostaglandin E1 additions (1.4 muM) produced about an 8-fold stimulation of homogenate cyclase activity. The Km of homogenate cyclase activation by adenosine and 2-chloroadenosine was 67.6 and 6.7 muM, respectively. Addition of 7-deazaadenosine, tolazoline, yohimbine, guanosine, cytosine, guanine, 2-deoxy-AMP, and adenine 9-beta-D-xylopyranoside, all at 200 muM were found to be without effect on homogenate NS20 adenylate cyclase. Two classes of inhibitors of homogenate NS20 adenylate cyclase activity were observed. One class, which included AMP, adenine, and theophylline, blocked 2-chloroadenosine but not prostaglandin E1 stimulation of cyclase. Theophylline was shown to be a competitive inhibitor of 2-chloroadenosine, with a Ki of 35 muM. The second class of inhibitors, which included 2'- and 5'-deoxyadenosine, inhibited unstimulated, 2-chloroadenosine and prostaglandin E1-stimulated homogenate cyclase activity to about the same degree. 3. Activation of NS20 homogenate adenylate cyclase by adenosine appears to be noncooperative. 4. The inhibitory action of putative "purinergic" neurotransmitters is postulated to be due to their effects on adenylate cyclase activity. 相似文献
15.
16.
Acidic phospholipid species inhibit adenylate cyclase activity in rat liver plasma membranes.
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Incubation of rat liver plasma membranes with liposomes of dioleoyl phosphatidic acid (dioleoyl-PA) led to an inhibition of adenylate cyclase activity which was more pronounced when fluoride-stimulated activity was followed than when glucagon-stimulated activity was followed. If Mn2+ (5 mM) replaced low (5 mM) [Mg2+] in adenylate cyclase assays, or if high (20 mM) [Mg2+] were employed, then the perceived inhibitory effect of phosphatidic acid was markedly reduced when the fluoride-stimulated activity was followed but was enhanced for the glucagon-stimulated activity. The inhibition of adenylate cyclase activity observed correlated with the association of dioleoyl-PA with the plasma membranes. Adenylate cyclase activity in dioleoyl-PA-treated membranes, however, responded differently to changes in [Mg2+] than did the enzyme in native liver plasma membranes. Benzyl alcohol, which increases membrane fluidity, had similar stimulatory effects on the fluoride- and glucagon-stimulated adenylate cyclase activities in both native and dioleoyl-PA-treated membranes. Incubation of the plasma membranes with phosphatidylserine also led to similar inhibitory effects on adenylate cyclase and responses to Mg2+. Arrhenius plots of both glucagon- and fluoride-stimulated adenylate cyclase activity were different in dioleoyl-PA-treated plasma membranes, compared with native membranes, with a new 'break' occurring at around 16 degrees C, indicating that dioleoyl-PA had become incorporated into the bilayer. E.s.r. analysis of dioleoyl-PA-treated plasma membranes with a nitroxide-labelled fatty acid spin probe identified a new lipid phase separation occurring at around 16 degrees C with also a lipid phase separation occurring at around 28 degrees C as in native liver plasma membranes. It is suggested that acidic phospholipids inhibit adenylate cyclase by virtue of a direct headgroup specific interaction and that this perturbation may be centred at the level of regulation of this enzyme by the stimulatory guanine nucleotide regulatory protein NS. 相似文献
17.
Epinephrine-sensitive adenylate cyclase of human fat cell ghosts. Modulation of enzyme activity by GTP 总被引:1,自引:0,他引:1
Some of the effects of GTP on human fat cell adenylate cyclase activity were studied. This nucleotide caused a dose-dependent inhibition of basal activity without affecting the hormone-activated rate of cAMP formation. Maximal effects were observed at GTP-concentrations of about 1 x 10(-4) M. The relative extent of hormonal stimulation was about 1.5-fold increased in the presence of 0.1 mM GTP. 相似文献
18.
19.
Vasoactive intestinal polypeptide and glucagon: stimulation of adenylate cyclase activity via distinct receptors in liver and fat cell membranes 总被引:8,自引:0,他引:8
B Desbuguois M H Laudat P Laudat 《Biochemical and biophysical research communications》1973,53(4):1187-1194
Vasoactive intestinal polypeptide (VIP), a peptide hormone that is chemically and biologically related to glucagon and secretin, stimulates the activity of adenylate cyclase in liver and fat cell membranes. Effects of combinations of VIP with glucagon and secretin at concentrations that maximally activate adenylate cyclase suggest that in adipose tissue, the three hormones act on the same enzyme, whereas in liver, VIP and secretin activate a common enzyme that is distinct from that responding to glucagon. Studies with radioiodinated derivatives of VIP and glucagon indicate that these hormones interact with separate receptors. Secretin, which gives a maximal stimulation of adenylate cyclase activity virtually identical to that elicited by VIP, inhibits the binding of the latter to its receptor. However, the apparent affinity of secretin for adenylate cyclase and for the VIP receptor is about two order of magnitude lower than that of VIP. It is suggested that VIP and secretin may activate adenylate cyclase via a common receptor. 相似文献
20.
A soluble form of adenylate cyclase was extracted from mycelia of Neurospora crassa wild-type strains. This enzyme activity was purified by chromatography on hexyl-amino-Sepharose, agarose and Blue Sepharose and preparative polyacrylamide-gel electrophoresis. On sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, peak fractions from the later purification steps showed a main polypeptide band with an apparent molecular weight of about 66 000. The following hydrodynamic and molecular parameters were established for the Neurospora soluble adenylate cyclase activity: sedimentation coefficient, 6.25 S; Stokes radius, 7.3 nm; partial specific volume, 0.74 ml/g; molecular weight, 202 000; frictional ratio, 1.65. The isoelectric point of this enzyme activity was 4.65. The enzyme was not activated by GTP, [beta gamma-imido]GTP, fluoride or cholera toxin. 相似文献