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1.
NADPH-oxidase-catalyzed superoxide (O2-) formation in membranes of HL-60 leukemic cells was activated by arachidonic acid in the presence of Mg2+ and HL-60 cytosol. The GTP analogues, guanosine 5'-[gamma-thio]triphosphate (GTP[gamma S] and guanosine 5'-[beta,gamma-imido]triphosphate, being potent activators of guanine-nucleotide-binding proteins (G proteins), stimulated O2- formation up to 3.5-fold. The adenine analogue of GTP[gamma S], adenosine 5'-[gamma-thio]triphosphate (ATP[gamma S]), which can serve as donor of thiophosphoryl groups in kinase-mediated reactions, stimulated O2- formation up to 2.5-fold, whereas the non-phosphorylating adenosine 5'-[beta,gamma-imido]triphosphate was inactive. The effect of ATP[gamma S] was half-maximal at a concentration of 2 microM, was observed in the absence of added GDP and occurred with a lag period two times longer than the one with GTP[gamma S]. HL-60 membranes exhibited nucleoside-diphosphate kinase activity, catalyzing the thiophosphorylation of GDP to GTP[gamma S] by ATP[gamma S]. GTP[gamma S] formation was half-maximal at a concentration of 3-4 microM ATP[gamma S] and was suppressed by removal of GDP by creatine kinase/creatine phosphate (CK/CP). The stimulatory effect of ATP[gamma S] on O2- formation was abolished by the nucleoside-diphosphate kinase inhibitor UDP. Mg2+ chelation with EDTA and removal of endogenous GDP by CK/CP abolished NADPH oxidase activation by ATP[gamma S] and considerably diminished stimulation by GTP[gamma S]. GTP[gamma S] also served as a thiophosphoryl group donor to GDP, with an even higher efficiency than ATP[gamma S]. Transthiophosphorylation of GDP to GTP[gamma S] was only partially inhibited by CK/CP. Our results suggest that NADPH oxidase is regulated by a G protein, which may be activated either by exchange of bound GDP by guanosine triphosphate or by thiophosphoryl group transfer to endogenous GDP by nucleoside-diphosphate kinase.  相似文献   

2.
The effect of GTP analogues on catecholamine secretion and [3H]arachidonic acid release from digitonin-permeabilized adrenal chromaffin cells was examined. Several GTP analogues stimulated Ca2(+)-independent exocytosis, with the order of efficacy being XTP greater than ITP greater than guanosine 5'-[beta gamma-imido]triphosphate (p[NH]ppG) greater than guanosine 5'-[gamma-thio]triphosphate (GTP[S]). The stimulatory effect of the GTP analogues appeared to be due to activation of a conventional GTP-binding protein, as it was inhibited by guanosine 5'-[beta-thio]diphosphate (GDP[S]). In contrast, Ca2(+)-dependent exocytosis was only partially inhibited by high doses of GDP[S]. GTP did not stimulate Ca2(+)-independent exocytosis, but instead was found to inhibit secretion caused by micromolar Ca2+. Arachidonic acid (100 microM) also stimulated Ca2(+)-independent catecholamine secretion. Determination of the effect of GTP analogues on release of free [3H]arachidonic acid into the medium showed that it was stimulated by GTP[S] but inhibited by GTP, p[NH]ppG, ITP and XTP. The inhibition of [3H]arachidonic acid release by XTP was not prevented by GDP[S]. These results demonstrate that activation of a GTP-binding protein by certain GTP analogues can induce Ca2(+)-independent secretion in adrenal chromaffin cells and that the effect of GTP analogues on Ca2(+)-independent secretion can be dissociated from generation of arachidonic acid.  相似文献   

3.
The stimulation of inositol phosphate generation by bombesin and GTP analogues was studied in Swiss 3T3 cells permeabilized by electroporation. Bombesin-stimulated inositol phosphate generation is potentiated by guanosine 5'-[gamma-thio]triphosphate (GTP[S]) and inhibited by guanosine 5'-[beta-thio]diphosphate at all peptide concentrations tested, with no change in the EC50 value (concn. giving half-maximal response) for the agonist. Kinetic analysis showed that, although bombesin-stimulated [3H]InsP3 generation in [3H]inositol-labelled cells was rapid (maximal by 5-10 s), the response to GTP[S] alone displayed a distinct lag time of 20-30 s. This lag time was significantly decreased by the addition of bombesin, suggesting that in this system agonist-stimulated GTP/GDP exchange occurs. In addition, bombesin-stimulated generation of Ins(1,4,5)P3 mass at 10 s was enhanced by GTP[S] in the absence of a nucleotide response alone, a result consistent with this proposal. Pretreatment of the cells with phorbol 12-myristate 13-acetate (PMA) resulted in a dose-dependent inhibition of bombesin-, but not GTP[S]-, stimulated inositol phosphate generation. Furthermore, although PMA pretreatment did not affect the lag time for InsP3 formation in response to GTP[S] alone, the degree of synergy between bombesin and the nucleotide was severely decreased at early time points. The results therefore demonstrate that the high-affinity bombesin receptor is coupled via a G-protein to phospholipase C in a manner consistent with a general model for receptor-G-protein interactions and that this coupling is sensitive to phosphorylation by protein kinase C.  相似文献   

4.
Signal-transducing guanine-nucleotide-binding regulatory proteins (G proteins) are heterotrimers, composed of the nucleotide-binding alpha subunit and a beta gamma dimer. The influence of beta gamma dimer preparations of the retinal G protein transducin (TD) was studied on formylpeptide-receptor--G-protein interactions in membranes of differentiated HL 60 cells. For this, TD was prepared from bovine rod outer segment (ROS) membranes with either GTP or its analogs, guanosine 5'-[gamma-thio]triphosphate (GTP[S]) and guanosine 5'-[beta gamma-imino]triphosphate (Gpp[NH]p). After removal of free nucleotides, TD beta gamma was separated from TD alpha and its function analyzed. Addition of TD beta gamma isolated from TD prepared with GTP[S] (TD beta gamma GTP[S]) to HL 60 membranes abolished high-affinity binding of fMet-Leu-[3H]Phe (fMet, N-formylmethionine) to its receptor. In contrast, TD beta gamma isolated from TD prepared with GTP (TD beta gamma GTP), boiled TD beta gamma GTP[S] and TD alpha prepared with GTP[S] had no or only slight effects. The inhibitory effect of TD beta gamma GTP[S] on fMet-Leu-[3H]Phe receptor binding was potentiated by GDP at low concentrations but not by GTP[S]. Furthermore, TD beta gamma GTP[S], but not TD beta gamma GTP or TD beta gamma isolated from TD prepared with Gpp[NH]p (TD beta gamma Gpp[NH]p), prevented fMet-Leu-Phe-stimulated binding of [35S]GTP[S] to G proteins in HL 60 membranes, measured in the presence of GDP. When TD beta gamma GTP was incubated with GTP [S] and TD-depleted illuminated ROS membranes, and subsequently separated from the membranes and free GTP[S], this TD beta gamma GTP, similar to TD beta gamma GTP[S], abolished high-affinity binding of fMet-Leu-[3H]Phe to its receptor, fMet-Leu-Phe-stimulated binding of [35S]GTP[S], and fMet-Leu-Phe-stimulated GTP hydrolysis in HL 60 membranes. Inhibition of [35S]GTP[S] binding by TD beta gamma was not seen in the presence of the metabolically stable GDP analog, guanosine 5'-[beta-thio]diphosphate. In order to obtain an insight into the modification of TD beta gamma apparently caused by GTP[S], and into its mechanism of action in HL 60 membranes, TD, TD alpha and TD beta gamma, all prepared in the presence of GTP, were incubated with [35S]GTP[S] and TD-depleted illuminated ROS membranes. Fluorographic analysis of the supernatant proteins revealed 35S labelling of the beta band of the G protein. When apparently thiophosphorylated TD beta gamma was incubated with [3H]GDP in the presence of HL 60 membranes, [3H]GTP[S] was rapidly formed.(ABSTRACT TRUNCATED AT 400 WORDS)  相似文献   

5.
We investigated the mechanism(s) whereby activation of a growth-factor receptor typically endowed with tyrosine kinase activity, such as the platelet-derived growth factor (PDGF) receptor, triggers phosphoinositide hydrolysis. In Swiss 3T3 cells permeabilized with streptolysin O, an analogue of GTP, guanosine 5'-[gamma-thio]triphosphate, was found to potentiate the coupling of the bombesin receptor to phospholipase C. In contrast, the activation of the enzyme by PDGF occurred in a GTP-independent manner. Moreover, the inactive analogue of GTP, guanosine 5'-[beta-thio]diphosphate, significantly inhibited the bombesin-induced InsP3 generation, whereas it did not decrease the same effect when stimulated by PDGF.  相似文献   

6.
1. Guanosine 5'-[gamma-thio]triphosphate (GTP[S]) stimulated by 50% the rate of release of [3H]choline and [3H]phosphorylcholine in rat liver plasma membranes labelled with [3H]choline. About 70% of the radioactivity released in the presence of GTP[S] was [3H]choline and 30% was [3H]phosphorylcholine. 2. The hydrolysis of phosphorylcholine to choline and the conversion of choline to phosphorylcholine did not contribute to the formation of [3H]choline and [3H]phosphorylcholine respectively. 3. The release of [3H]choline from membranes was inhibited by low concentrations of SDS or Triton X-100. Considerably higher concentrations of the detergents were required to inhibit the release of [3H]phosphorylcholine. 4. Guanosine 5'-[beta gamma-imido]triphosphate and guanosine 5'-[alpha beta-methylene]triphosphate, but not adenosine 5'-[gamma-thio]-triphosphate, stimulated [3H]choline release to the same extent as did GTP[S]. The GTP[S]-stimulated [3H]choline release was inhibited by guanosine 5'-[beta-thio]diphosphate, GDP and GTP but not by GMP. 5. It is concluded that, in rat liver plasma membranes, (a) GTP[S]-stimulated hydrolysis of phosphatidylcholine is catalysed predominantly by phospholipase D with some contribution from phospholipase C, and (b) the stimulation of phosphatidylcholine hydrolysis by GTP[s] occurs via a GTP-binding regulatory protein.  相似文献   

7.
The regulation of Cl- conductance by cytoplasmic nucleotides was investigated in pancreatic and parotid zymogen granules. Cl- conductance was assayed by measuring the rate of cation-ionophore-induced osmotic lysis of granules suspended in iso-osmotic salt solutions. Both inhibition and stimulation were observed, depending on the type and concentration of nucleotide. Under optimal conditions, the average inhibition measured in different preparations was 1.6-fold, whereas the average stimulation was 4.4-fold. ATP was inhibitory at 1-10 microM but stimulated Cl- conductance above 50 microM. Stimulation by ATP was more pronounced in granules with low endogenous Cl- conductance. The potency of nucleotides in terms of inhibition was ATP greater than adenosine 5'-[gamma-thio]triphosphate (ATP[S]) greater than UTP much greater than or equal to CTP much greater than or equal to GTP much greater than or equal to guanosine 5'-[gamma-thio]triphosphate (GTP[S]) much greater than or equal to ITP. The potency with respect to stimulation had the following order: adenosine 5'-[beta gamma-methylene]triphosphate (App[CH2]p) greater than ATP greater than guanosine 5'-[beta-thio]diphosphate (GDP[S]). Adenosine 5'-[beta gamma-imido]triphosphate (App[NH]p) was also stimulatory, and was more potent than ATP in the parotid granules, but less potent in the pancreatic granules. Aluminium fluoride stimulated Cl- conductance maximally at 15-30 microM-Al3+ and 10-15 mM-F. F was less effective at higher concentrations. Protein phosphorylation by kinases was apparently not involved, since the nucleotide effects (1) could be mimicked by non-hydrolysable analogues of ATP and GTP, (2) showed reversibility, and (3) were not abolished by the protein kinase inhibitors 1-(5-isoquinolinesulphonyl)-2-methylpiperazine (H-7) or staurosporine. The data suggest the presence of at least two binding sites for nucleotides, whereby occupancy of one induces inhibition and occupancy of the other induces stimulation.  相似文献   

8.
The non-differentiated HL60 cell can be stimulated to secrete when Ca2+ and guanosine 5'-[gamma-thio]-triphosphate (GTP gamma S) are introduced into streptolysin-O-permeabilized cells. Secretion is accompanied by activation of polyphosphoinositide phosphodiesterase (PPI-pde). Both responses show a concentration-dependence on Ca2+ between pCa 8 and pCa 5. The half-maximal requirements for Ca2+ for PPI-pde activation and secretion are pCa 6.4 +/- 0.1 and pCa 6.2 +/- 0.2 respectively. The rank order of potency of the GTP analogues to stimulate PPI-pde activation and secretion is similar; GTP gamma S greater than guanosine 5'-[beta gamma-imido]-triphosphate greater than guanosine 5'-[beta gamma-methylene]triphosphate greater than XTP approximately equal to ITP, but the maximal response achieved by each compound compared with GTP gamma S is much greater for secretion than for PPI-pde activation. A dissociation of the two responses is obtained with 10 mM-XTP and -ITP; secretion is always observed but not inositol trisphosphate formation at this concentration. GTP, dGTP, UTP and CTP are inactive for both secretion and PPI-pde activation. Both GDP and dGDP are competitive inhibitors of both GTP gamma S-induced secretion and PPI-pde activation. Phorbol 12-myristate 13-acetate could not fully substitute for GTP gamma S in stimulating secretion, suggesting that the effect of GTP gamma S cannot result simply from the generation of diacylglycerol. In the absence of MgATP, secretion and PPI-pde activation is still evident, albeit at a reduced level. This also supports the hypothesis that protein kinase C-dependent phosphorylation is not essential for secretion. The effect of MgATP is to enhance secretion, and to reduce both the Ca2+ and GTP gamma S requirement for secretion. In conclusion, two roles for guanine nucleotides can be identified; one for activating PPI-pde (GP) and the other for activating exocytosis (GE), acting in series.  相似文献   

9.
Several G-proteins (GTP-binding proteins) were identified by SDS/PAGE in the cytosol (105,000 g supernatant) and membrane fractions of the oestrogen-dependent human mammary-tumour cell line ZR-75-1. These proteins, with molecular masses in the range 18-29 kDa, specifically bind [alpha-32P]GTP, which can be displaced by unlabelled GTP, GDP and their non-hydrolysable analogues guanosine 5'-[delta-thio]triphosphate (GTP[S]) and guanosine 5'-[beta-thio]diphosphate (GDP[S]), but not by GMP, ATP, ADP, AMP and other unrelated nucleotides. The apparent dissociation constant for GTP was approx. 2 x 10(-8)M. Homogenization of ZR-75-1 cells in high-salt buffer (1 M-KCl), and successive washing of the membrane fraction, suggested that, among the major G-proteins found, the 18 kDa protein is predominantly soluble, whereas the 27-29 kDa complex is primarily bound to the membrane fraction under the experimental conditions employed. Possible translocation of these G-proteins between membrane and cytosol was analysed. No redistribution of the 27-29 kDa complex was observed, whereas GTP[S] in the presence of Mg2+ caused apparent translocation of the 18 kDa protein to the membrane fraction. This effect was specific for GTP and stable GTP analogues, whereas GDP, GMP, ATP, ADP, AMP and other unrelated nucleotides were ineffective. GTP[S] and guanosine 5'-[beta gamma-imido]-triphosphate (p[NH]ppG) were equally potent (apparent Kd approximately 5 x 10(-6)M), whereas GTP was rather weak. The nucleotide effect is temperature-, time- and concentration-dependent. The translocation process was reversible, slow, and reached its maximum between 30 and 60 min at 37 degrees C. The apparent translocation of this small G-protein from the cytosol to the membrane fraction, and the specific effect of GTP analogues, suggest that this process may have functional significance in mammary-tumour cells.  相似文献   

10.
The guanine nucleotide binding properties of rap1 protein purified from human neutrophils were examined using both the protein kinase A-phosphorylated and the non-phosphorylated forms of the protein. Binding of GTP[S] (guanosine 5'-[gamma-thio]triphosphate) or GDP was found to be slow in the presence of free Mg2+, but very rapid in the absence of Mg2+. The binding of guanine nucleotides was found to correlate with the loss of endogenous nucleotide from the rap1 protein, which was rapid in the absence of Mg2+. The relative affinities of GTP and GDP for the binding site on rap1 were modulated by the presence of Mg2+, with a preferential affinity (approx. 15-fold) for GTP observed only in the absence of this bivalent cation. The dissociation of GDP from rap1 was not affected by the G-protein beta/gamma-subunit complex. Phosphorylation of rap1 in vitro by protein kinase A did not modify any of the observed nucleotide-binding parameters. Furthermore, the ability of a cytosolic rap1 GTPase-activating protein to stimulate neutrophil rap1 GTP hydrolysis was not modified by phosphorylation. These data suggest that the activation of rap in vivo may be regulated by the release of endogenous GDP, but that phosphorylation by protein kinase A does not affect guanine nucleotide binding or hydrolysis.  相似文献   

11.
A method of membrane permeabilization of T lymphocytes with the bacterial cytotoxin streptolysin O has allowed the effect of guanine nucleotide analogues on phosphatidylinositol metabolism and protein kinase C (PKC) activation to be investigated. The data demonstrate that, in permeabilized cells, phosphorylation of the gamma subunit of the CD3 antigen can be induced in response to the PKC activator phorbol 12,13-dibutyrate, the polyclonal mitogen phytohaemagglutinin (PHA) and the stimulatory guanine nucleotide analogue guanosine 5'-[gamma-thio]triphosphate (GTP[S]). Application of a pseudo-substrate inhibitor of PKC indicated that CD3gamma-chain phosphorylation induced in response to all three agonists was mediated by PKC. PHA and GTP[S] also stimulated inositol phospholipid turnover and inositol phosphate accumulation. The kinetics and concentration-dependence of PHA-induced inositol phospholipid hydrolysis correlated with PHA-induced CD3gamma phosphorylation, suggesting that PHA may regulate CD3gamma phosphorylation via diacylglycerol produced as a consequence of inositol phospholipid hydrolysis. However, there was an inconsistency in that PHA induced greater (greater than 200%) levels of inositol phospholipid turnover than did GTP[S], but much weaker (less than 50%) levels of CD3-antigen phosphorylation. There was also a discrepancy between GTP[S] effects on phosphatidylinositol turnover and PKC activation, in that the half-maximal GTP[S] concentration for inositol phosphate production and CD3gamma-chain phosphorylation was 0.75 microM and 75 microM respectively. Moreover, 10 microM-GTP[S] induced maximal inositol phosphate production, but only 10% of maximal CD3gamma-chain phosphorylation. The data are consistent with the idea that other signal-transduction pathways, in addition to those involving inositol phosphate production, exist for the regulation of PKC in T lymphocytes.  相似文献   

12.
Known nucleoside diphosphate kinases (NDPKs) are oligomers of 17-23-kDa subunits and catalyze the reaction N1TP + N2DP --> N1DP + N2TP via formation of a histidine-phosphorylated enzyme intermediate. NDPKs are involved in the activation of heterotrimeric GTP-binding proteins (G-proteins) by catalyzing the formation of GTP from GDP, but the properties of G-protein-associated NDPKs are still incompletely known. The aim of our present study was to characterize NDPK in soluble preparations of the retinal G-protein transducin. The NDPK is operationally referred to as transducin-NDPK. Like known NDPKs, transducin-NDPK utilizes NTPs and phosphorothioate analogs of NTPs as substrates. GDP was a more effective phosphoryl group acceptor at transducin-NDPK than ADP and CDP, and guanosine 5'-[gamma-thio]triphosphate (GTP[S]) was a more effective thiophosphoryl group donor than adenosine 5'-[gamma-thio]triphosphate (ATP[S]). In contrast with their action on known NDPKs, mastoparan and mastoparan 7 had no stimulatory effect on transducin-NDPK. Guanosine 5'-[beta, gamma-imido]triphosphate (p[NH]ppG) potentiated [3H]GTP[S] formation from [3H]GDP and ATP[S] but not [3H]GTP[S] formation from [3H]GDP and GTP[S]. Depending on the thiophosphoryl group acceptor and donor, [3H]NTP[S] formation was differentially regulated by Mg2+, Mn2+, Co2+, Ca2+ and Zn2+. [gamma-32P]ATP and [gamma-32P]GTP [32P]phosphorylated, and [35S]ATP[S] [35S]thiophosphorylated, a 36-kDa protein comigrating with transducin-beta. p[NH]ppG potentiated [35S]thiophosphorylation of the 36-kDa protein. 32P-labeling of the 36-kDa protein showed characteristics of histidine phosphorylation. There was no evidence for (thio)phosphorylation of 17-23-kDa proteins. Our data show the following: (a) soluble transducin preparations contain a GDP-prefering and guanine nucleotide-regulated NDPK; (b) transducin-beta may serve as a (thio)phosphorylated NDPK intermediate; (c) transducin-NDPK is distinct from known NDPKs and may consist of multiple kinases or a single kinase with multiple regulatory domains.  相似文献   

13.
A mechanism by which protein kinase C potentiates arachidonic acid (AA) liberation in rabbit platelets was examined using [3H]AA-labeled, saponin (7 micrograms/ml)-permeabilized rabbit platelets. Pretreatment of the [3H]AA-labeled platelets with 4 beta-phorbol 12-myristate 13-acetate (PMA, 10-40 nM) or 1,2-dioctanoylglycerol (DOG, 20 microM) enhanced [3H]AA liberation induced by an addition of Ca2+ (1 mM) after cell permeabilization, whereas 4 alpha-phorbol 12,13-didecanoate (80 nM) did not exert such an effect. The potentiating effects of PMA and DOG were inhibited by staurosporine (200 nM). PMA (40 nM) also potentiated [3H]AA liberation induced by guanosine 5'-[gamma-thio]triphosphate (GTP gamma S, 100 microM), 5'-guanylyl imidodiphosphate (200 microM) or NaF (20 mM) plus AlCl3 (10 microM) in the presence of Ca2+ (100 microM). The enhancement by PMA of the GTP gamma S-induced AA liberation was also inhibited by staurosporine (200 nM). Furthermore, guanosine 5'-[beta-thio]diphosphate (GDP beta S, 0.5-2 mM) suppressed the PMA (40 nM)- and DOG (20 microM)-enhanced, Ca2+ (1 mM)-dependent [3H]AA liberation. This inhibitory effect of GDP beta S was reversed by a further addition of GTP gamma S (200 microM). However, pertussis toxin (0.2-1 micrograms/ml) had no effect on the PMA-enhanced [3H]AA liberation. These results indicate a possibility that protein kinase C may potentiate AA liberation through a guanine-nucleotide-binding protein-mediated mechanism in saponin-permeabilized rabbit platelets.  相似文献   

14.
Guanosine 5'-[gamma-thio]triphosphate and guanosine 5'-[beta,gamma-imido]triphosphate enhance Ca2+-dependent 5-hydroxytryptamine secretion from electropermeabilised human platelets. GTP has little such effect except when the platelets are permeabilised, and incubated with this nucleotide, at 2 degrees C and pH 7.4. The lag phase observed in the time course of 5-hydroxytryptamine secretion induced by addition of guanosine 5'-[gamma-thio]triphosphate is markedly longer than that characterising secretion induced by Ca2+ alone, by thrombin +/- GTP or by guanosine 5'-[gamma-thio]triphosphate in the presence of thrombin. GTP causes competitive inhibition of the enhancement of the Ca2+-dependent secretory response induced by guanosine 5'-[gamma-thio]triphosphate when both nucleotides are added simultaneously. The extent of inhibition is decreased if guanosine 5'-[gamma-thio]triphosphate is added prior to GTP. GTP markedly enhances the effect of thrombin on Ca2+-dependent 5-hydroxytryptamine secretion by increasing the maximal extent of the response and decreasing the thrombin concentration required to give half-maximal response. A similar effect is observed on addition of guanosine 5'-[gamma-thio]triphosphate in the presence of thrombin at short incubation times. On more prolonged incubation the effects of thrombin and guanosine 5'-[gamma-thio]triphosphate are additive. Guanosine 5'-[beta-thio]diphosphate completely inhibits the response induced by guanosine 5'-[gamma-thio]triphosphate or guanosine 5'-[beta,gamma-imido]triphosphate but has little effect on the response induced by Ca2+ when added alone or in the presence of thrombin. Partial inhibition is observed for the response induced by thrombin + GTP. Cyclic-AMP effectively inhibits the response induced by thrombin + GTP but has little effect on that induced by guanosine 5'-[gamma-thio]triphosphate or guanosine 5'-[beta,gamma]imidotriphosphate. The results provide further support for the proposal [Haslam, R.J. & Davidson, M.M.L. (1984) FEBS Lett. 170, 90-95], that receptor--phospholipase-C coupling in platelets is mediated in part by a guanine-nucleotide-binding (Np) protein but that a coupling mechanism may also exist which is independent of such a protein. The properties of guanine-nucleotide-dependent coupling resemble those previously described for receptor--adenylate-cyclase coupling.  相似文献   

15.
The requirement for Ca2+ for the activation of polyphosphoinositide phosphodiesterase was studied with the guanine nucleotide analogue guanosine 5'-[gamma-thio]triphosphate (GTP gamma S). Levels of Ca2+ that pertain in unstimulated neutrophils (100 nM) are obligatory for the full expression of enzyme activity stimulated with GTP gamma S. Reduction of Ca2+ to 1 nM leads to inhibition. Increasing the level of Ca2+ from 100 nM to 1000 nM does not alter enzyme activity. Guanosine 5'-[beta-thio]diphosphate (GDP beta S) does not stimulate the phosphodiesterase but is an effective inhibitor of activation by GTP gamma S. Ca2+ in the millimolar range can also activate the phosphodiesterase alone and this is not inhibited by GDP beta S. It is also shown that Sr2+ in the millimolar range can stimulate enzyme activity similarly to Ca2+.  相似文献   

16.
Whereas the chemotactic peptide, N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMet-Leu-Phe), induced NADPH-oxidase-catalyzed superoxide (O2-) formation in human neutrophils, purine and pyrimidine nucleotides per se did not stimulate NADPH oxidase but enhanced O2- formation induced by submaximally and maximally stimulatory concentrations of fMet-Leu-Phe up to fivefold. On the other hand, FMet-Leu-Phe primed neutrophils to generate O2- upon exposure to nucleotides. At a concentration of 100 microM, purine nucleotides enhanced O2- formation in the effectiveness order adenosine 5'-O-[3-thio]triphosphate (ATP[gamma S]) greater than ITP greater than guanosine 5'-O-[3-thio]triphosphate (GTP[gamma S]) greater than ATP = adenosine 5'-O-[2-thio]triphosphate (Sp-diastereomer) = GTP = guanosine 5'-O-[2-thio]diphosphate (GDP[beta S] = ADP greater than adenosine 5'-[beta, gamma-imido]triphosphate = adenosine 5'-O-[2-thio]triphosphate] (Rp-diastereomer). Pyrimidine nucleotides stimulated fMet-Leu-Phe-induced O2- formation in the effectiveness order uridine 5'-O-[3-thio]triphosphate (UTP[gamma S]) = UTP greater than CTP. Uracil (UDP[beta S]) = uridine 5'-O[2-thio]triphosphate (Rp-diastereomer) (Rp)-UTP[beta S]) = UTP greater than CTP. Uracil nucleotides were similarly effective potentiators of O2- formation as the corresponding adenine nucleotides. GDP[beta S] and UDP[beta S] synergistically enhanced the stimulatory effects of ATP[gamma S], GTP[gamma S] and UTP[gamma S]. Purine and pyrimidine nucleotides did not induce degranulation in neutrophils but potentiated fMet-Leu-Phe-induced release of beta-glucuronidase with similar nucleotide specificities as for O2- formation. In contrast, nucleotides per se induced aggregation of neutrophils. Treatment with pertussis toxin prevented aggregation induced by both nucleotides and fMet-Leu-Phe. Our results suggest that purine and pyrimidine nucleotides act via nucleotide receptors, the nucleotide specificity of which is different from nucleotide receptors in other cell types. Neutrophil nucleotide receptors are coupled to guanine-nucleotide-binding proteins. As nucleotides are released from cells under physiological and pathological conditions, they may play roles as intercellular signal molecules in neutrophil activation.  相似文献   

17.
1. Slowly hydrolysable analogues of GTP were introduced into hepatocytes by incubating the cells in the absence of Mg2+ and in the presence of ATP4-. Experiments using guanosine 5'-[gamma-[35S]thio]triphosphate (GTP[35S])indicated that about 50% of the GTP[S] loaded into the cells was subsequently hydrolysed. 2. In cells loaded with GTP[S] and incubated in the absence of added extracellular Ca2+ (Ca2+o), the rate of activation of glycogen phosphorylase observed after addition of 1.3 mM-Ca2+o was 250% greater than the rate observed in unloaded cells. Smaller effects (130%) were observed in cells loaded with either guanyl-5'-yl imidodiphosphate or guanosine 5-[beta-thio]diphosphate (GDP[S]). Cells loaded with adenosine 5'-[gamma-thio]triphosphate showed no increase in glycogen phosphorylase activity on addition of Ca2+o. 3. The effect of a submaximal concentration of GTP[S] on the Ca2+-induced activation of glycogen phosphorylase was additive with that of a half-maximally effective concentration of vasopressin. GTP[S] did not increase the effect of a maximally effective concentration of the hormone. 4. Cells loaded with GTP[S] exhibited an increased initial rate of 45Ca2+ exchange measured at 1.3 mM-Ca2+o. 5. GTP[S] did not affect the amount of 45Ca2+ exchanged by cells incubated at 0.1 mM-Ca2+o or the ability of vasopressin to release 45Ca2+ from these cells. 6. It is concluded that the introduction of slowly hydrolysable analogues of GTP to the liver cell cytoplasmic space stimulates the inflow of Ca2+ across the plasma membrane through a channel similar to that activated by vasopressin.  相似文献   

18.
The effect of the GTP analogue guanosine 5'-[gamma-thio]triphosphate (GTP[S]) on the polyphosphoinositide phospholipase C (PLC) of rat liver was examined by using exogenous [3H]phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2]. GTP[S] stimulated the membrane-bound PLC up to 20-fold, with a half-maximal effect at approx. 100 nM. Stimulation was also observed with guanosine 5'-[beta gamma-imido]triphosphate, but not with adenosine 5'-[gamma-thio]triphosphate, and was inhibited by guanosine 5'-[beta-thio]diphosphate. Membrane-bound PLC was entirely Ca2+-dependent, and GTP[S] produced both a decrease in the Ca2+ requirement and an increase in activity at saturating [Ca2+]. The stimulatory action of GTP[S] required millimolar Mg2+. [8-arginine]Vasopressin (100 nM) stimulated the PLC activity approx. 2-fold in the presence of 10 nM-GTP[S], but had no effect in the absence of GTP[S] or at 1 microM-GTP[S]. The hydrolysis of PtdIns(4,5)P2 by membrane-bound PLC was increased when the substrate was mixed with phosphatidylethanolamine, phosphatidylcholine or various combinations of these with phosphatidylserine. With PtdIns(4,5)P2, alone or mixed with phosphatidylcholine, GTP[S] evoked little or no stimulation of the PLC activity. However, maximal stimulation by GTP[S] was observed in the presence of a 2-fold molar excess of phosphatidylserine or various combinations of phosphatidylethanolamine and phosphatidylserine. Hydrolysis of [3H]phosphatidylinositol 4-phosphate by membrane-bound PLC was also increased by GTP[S]. However, [3H]phosphatidylinositol was a poor substrate, and its hydrolysis was barely affected by GTP[S]. Cytosolic PtdIns(4,5)P2-PLC exhibited a Ca2+-dependence similar to that of the membrane-bound activity, but was unaffected by GTP[S]. It is concluded that rat liver plasma membranes possess a Ca2+-dependent polyphosphoinositide PLC that is activated by hormones and GTP analogues, depending on the Mg2+ concentration and phospholipid environment. It is proposed that GTP analogues and hormones, acting through a guanine nucleotide-binding protein, activate the enzyme mainly by lowering its Ca2+ requirement.  相似文献   

19.
The effects of guanosine 5'-[beta-thio]diphosphate (GDP[S]) on the kinetics of activation of rat liver membrane adenylate cyclase by guanosine 5'-[beta,gamma-imido]triphosphate (p[NH]ppG) were examined. GDP[S] caused immediate inhibition of the activation by p[NH]ppG at all time points tested. Substantial inhibition by GDP[S] was observed even after the time required for the enzyme to reach its steady-state activity, but the extent of inhibition became progressively smaller as the preincubation time with p[NH]ppG increased. The rate at which adenylate cyclase became quasi-irreversibly activated was a strictly first-order process. In the presence of glucagon, the formation of the irreversibly activated state was much slower. A combination of GDP[S] and glucagon could partially reverse the quasi-irreversible activation by p[NH]ppG. Glucagon decreased the lag time required for p[NH]ppG to activate adenylate cyclase and increased the extent of activation by p[NH]ppG. This stimulatory effect of the hormone on top of guanine nucleotide decreased on preincubation with p[NH]ppG, but not with GTP. Our results suggest that the activation of adenylate cyclase by non-hydrolysable GTP analogues is a two-stage process: the formation of a reversibly activated form (G rev) is a rapid process, followed by a much slower formation of the quasi-irreversibly activated form (G irr). Glucagon can stimulate G rev but not G irr, and can partially facilitate the formation of the G rev from the G irr state.  相似文献   

20.
Fluoride and guanosine 5'-[gamma-thio]triphosphate (GTP gamma S) both activate the hepatocyte membrane polyphosphoinositide phosphodiesterase (PPI-pde) in a concentration-dependent manner. AlCl3 enhances the fluoride effect, supporting the concept that [A1F4]- is the active species. Analysis of the products of inositol lipid hydrolysis demonstrate that phosphatidylinositol bisphosphate is the major lipid to be hydrolysed. Guanosine 5'-[beta-thio]diphosphate (GDP beta S) is an inhibitor of activation of PPI-pde by both fluoride and GTP gamma S. These observations suggest that the guanine nucleotide regulatory protein (termed Gp) bears a structural resemblance to the well-characterized G-proteins of the adenylate cyclase system and the cyclic GMP phosphodiesterase system in phototransduction.  相似文献   

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