共查询到20条相似文献,搜索用时 15 毫秒
1.
We have examined the characteristics of the adenylate cyclase system from control and butyrate-treated cells. Butyrate treatment results in both an increased number of catecholamine receptors and an induction of a response to the hormone, as reported previously (Tallman, J.F., Smith, C.C., and Henneberry, R.C. (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 873-877); in addition, we found that the same treatment reduces the degree of activation of adenylate cyclase by GTP. We have demonstrated in two cell types that this decrease in GTP activation is inversely related to the degree of induction of the hormone response. Furthermore, in plasma membranes isolated from butyrate-treated cells, the hormone receptor is sensitive to GTP; i.e. GTP reduces the affinity of isoproterenol for the receptor. We propose that these changes reflect an interaction between the beta-adrenergic receptor and the nucleotide regulatory component and that this interaction represents, at least in part, the process of coupling. Several possible mechanisms which can account for the change in GTP activation are discussed in terms of our current understanding of the regulation of the adenylate cyclase system. 相似文献
2.
Incubation of blowfly salivary gland homogenates with 30 microM [gamma-32P]ATP resulted in a rapid, Mg2+-dependent, synthesis of [32P]polyphosphoinositides and [32P]phosphatidic acid. 5-Methyltryptamine, in the presence of 10 microM guanosine 5'-(3-O-thio)trisphosphate, reduced the net accumulation of 32P label into phosphatidylinositol-4,5-P2 and phosphatidylinositol-4-P by 35 and 20%, respectively. 5-Methyltryptamine did not affect synthesis of [32P]phosphatidic acid. Phosphorylation of polyphosphoinositides was not affected by 5-methyltryptamine. In membranes labeled in vitro with [gamma-32P]ATP, 5-methyltryptamine stimulated a rapid breakdown of the [32P]polyphosphoinositides. These results indicate that in blowfly salivary gland homogenates, hormone stimulates breakdown of the newly synthesized polyphosphoinositides. In the presence of hormone, the rate of polyphosphoinositide synthesis does not compensate for the rate of polyphosphoinositide degradation. 相似文献
3.
Phosphoinositide synthesis and Ca2+ gating in blowfly salivary glands exposed to 5-hydroxytryptamine. 总被引:1,自引:1,他引:0
下载免费PDF全文
![点击此处可从《The Biochemical journal》网站下载免费的PDF全文](/ch/ext_images/free.gif)
Blowfly salivary glands, previously exposed to 10 microM-5-hydroxytryptamine for 30 min, demonstrated a rapid compensatory resynthesis of [3H]inositol-labelled phosphatidylinositol 4,5-bisphosphate when allowed to recover in medium containing 3-5 microM-inositol. Phosphatidylinositol 4,5-bisphosphate comprised 70% of the total [3H]-phosphoinositide, and there was a corresponding decrease in the formation of [3H]-phosphatidylinositol. Subsequent addition of 5-hydroxytryptamine produced an equivalent breakdown of the newly synthesized phosphoinositides but little 45Ca2+ gating. Increasing the inositol concentration in the medium to 300 microM produced a 14-fold stimulation of phosphatidylinositol synthesis but only a 5-fold increase in phosphatidylinositol 4,5-bisphosphate synthesis. Increasing the inositol concentration in the medium from 3 microM to 300 microM resulted in a progressively greater recovery of the 45Ca2+-gating response. At 300 microM-inositol there was an 85% recovery of 45Ca2+-gating response. These results indicate that conversion of phosphatidylinositol into phosphatidylinositol 4,5-bisphosphate occurs in blowfly salivary glands and is secondary to an initial breakdown of the phosphoinositides. Recovery of Ca2+ gating is dependent on the restoration of both phosphatidylinositol and phosphatidylinositol 4,5-bisphosphate to appropriate concentrations. 相似文献
4.
5.
It is generally thought that formation of inositol 1,2-cyclic phosphate (IcP) on agonist-stimulated "breakdown" of endogenous phosphatidylinositol in intact cells would provide strong evidence for the direct phosphodiesteratic cleavage of phosphatidylinositol. We report here that on ionophoresis of extracts of pancreatic minilobules incubated with the cholecystokinin/pancreozymin congener, caerulein, the usual inositol phosphates, i.e. inositol 1-phosphate (IP), inositol 4,5-bisphosphate (IP2), and inositol 1,4,5-trisphosphate (IP3) were seen. In addition, an [3H]inositol-labeled unknown was present with the correct electrophoretic mobility of IcP. There was only a trace of "IcP" in the unstimulated pancreatic minilobules. Several lines of evidence indicate that the unknown peak was IcP. 1) It ran on ionophoresis with standard [14C]IcP, and the ratio of 3H to 14C for each point on the peak was a constant within experimental error. 2) The putative IcP peak which had been eluted from the electropherogram also coincided with standard [14C]IcP on paper chromatography. 3) On mild acid hydrolysis in the presence of standard 14C-labeled IP, the putative [3H] IcP peak disappeared and appeared in the exact position of the standard [14C]IP peak, as to be predicted of IcP. The formation of IcP on agonist stimulation supports direct phosphodiesteratic cleavage of phosphatidylinositol on stimulation of phosphoinositide breakdown in pancreatic minilobules. 相似文献
6.
Development of muscarinic-cholinergic stimulation of inositol phosphate production in cultured embryonic chick atrial cells. Evidence for a switch in guanine-nucleotide-binding protein coupling.
下载免费PDF全文
![点击此处可从《The Biochemical journal》网站下载免费的PDF全文](/ch/ext_images/free.gif)
J V Barnett S M Shamah B Lassegue K K Griendling J B Galper 《The Biochemical journal》1990,271(2):443-448
We have demonstrated that muscarinic stimulation of inositol phosphate production in cultured atrial cells from chicks at 14 days in ovo is partially sensitive to inhibition by pertussis toxin. In these cells, muscarinic agonist binding is coupled to phospholipase C activity via at least two guanine-nucleotide-binding proteins (G-proteins), one sensitive to pertussis toxin and the other (Gp) insensitive to pertussis toxin [Barnett, Shamah, Lassegue, Griendling & Galper (1990) Biochem. J. 271, 437-442]. In the current study we demonstrate that during embryonic development of the chick heart, muscarinic stimulation of inositol phosphate production decreases by 50% between days 5 and 14 in ovo in cells cultured from both atrium and ventricle. In atrial cells, however, pertussis toxin-sensitive muscarinic stimulation of inositol phosphate production increased from undetectable levels at day 5 in ovo to 40% of total stimulation at day 12 in ovo. Muscarinic stimulation of inositol phosphate production in the ventricle did not become sensitive to pertussis toxin at any age studied. In permeabilized atrial cells from embryonic chicks at 5 days in ovo, guanosine 5'-[gamma-thio]triphosphate (GTP[S]) stimulated InsP1 levels by 40 +/- 10% (mean +/- S.E.M., n = 3), InsP2 levels by 117 +/- 18% and InsP3 levels by 51 +/- 8%, suggesting that at day 5 in ovo all of the muscarinic-stimulated inositol phosphate production was coupled to phospholipase C via Gp. H.p.l.c. analysis demonstrated that, in spite of these changes in coupling of phospholipase C to different G-proteins, no changes could be demonstrated in the isomers of InsP3 produced in response to carbamylcholine at both days 5 and 14 in ovo. These data demonstrate that embryonic development of the chick atrium is associated with a switch in coupling of muscarinic receptors to phospholipase C from Gp to a pertussis toxin substrate. This developmental switch in coupling of G-proteins may be related to possible developmental switches in levels of muscarinic receptor isoforms or switches in the subtype of phospholipase C. 相似文献
7.
Muscarinic cholinergic stimulation of inositol phosphate production in cultured embryonic chick atrial cells. Evidence for a role of two guanine-nucleotide-binding proteins.
下载免费PDF全文
![点击此处可从《The Biochemical journal》网站下载免费的PDF全文](/ch/ext_images/free.gif)
J V Barnett S M Shamah B Lassegue K K Griendling J B Galper 《The Biochemical journal》1990,271(2):437-442
These studies demonstrate a novel mechanism for the coupling of the muscarinic receptor to phospholipase C activity in embryonic chick atrial cells. In monolayer cultures of atrial cells from hearts of embryonic chicks at 14 days in ovo, carbamylcholine stimulated the sequential appearance of InsP3, InsP2 and InsP1 with an EC50 (concn. causing 50% of maximal stimulation) of 30 microM. In the presence of 15 mM-Li, a 5 min exposure to carbamylcholine (0.1 mM) increased InsP3 levels to a maximum of 47 +/- 12% over basal, InsP2 to 108 +/- 13% over basal and InsP1 to 42 +/- 5% over basal. This effect was blocked by 5 microM-atropine. Incubation of these cells with pertussis toxin (15 h; 0.5 ng/ml) inhibited carbamylcholine-stimulated InsP3, InsP2 and InsP1 formation by 42 +/- 7%, 30 +/- 3% and 48 +/- 7% respectively. The IC50 (concn. causing 50% inhibition) for pertussis toxin inhibition of all three inositol phosphates was 0.01 ng/ml, with a half-time of 6 h at 0.5 ng/ml. This partial sensitivity to pertussis toxin was not due to incomplete ADP-ribosylation of the guanine-nucleotide-binding protein (G-protein), since autoradiography of polyacrylamide gels of cell homogenates incubated with [32P]NAD+ in the presence of pertussis toxin demonstrated that incubation of cells with 0.5 ng of pertussis toxin/ml for 15 h resulted in complete ADP-ribosylation of pertussis toxin substrates by endogenous NAD+. In cells permeabilized with saponin (10 micrograms/ml), 0.1 mM-GTP[S] (guanosine 5'-[gamma-thio]triphosphate) stimulated InsP1 by 102 +/- 15% (mean +/- S.E.M., n = 4), InsP2 by 421 +/- 67% and InsP3 by 124 +/- 33% above basal. Incubation of cells for 15 h with 0.5 ng of pertussis toxin/ml decreased GTP[S]-stimulated InsP1 production in saponin-treated cells by 30 +/- 10% (n = 3), InsP2 production by 45 +/- 7% (n = 4) and InsP3 production by 49 +/- 6% (n = 4). These data demonstrate that in embryonic chick atrial cells at least two independent G-proteins, a pertussis toxin-sensitive G-protein and a pertussis toxin-insensitive G-protein, play a role in coupling muscarinic agonist binding to phospholipase C activation and to inositol phosphate production. 相似文献
8.
Endothelial inositol phosphate generation and prostacyclin production in response to G-protein activation by AlF4-.
下载免费PDF全文
![点击此处可从《The Biochemical journal》网站下载免费的PDF全文](/ch/ext_images/free.gif)
In order to elucidate the role of guanine-nucleotide-binding proteins (G-proteins) in endothelial prostacyclin (PGI2) production, human umbilical vein endothelial cells, prelabelled with either [3H]inositol or [3H]arachidonic acid, were stimulated with the non-specific G-protein activator aluminium fluoride (AlF4-). AlF4- caused a dose- and time-dependent generation of inositol phosphates, release of arachidonic acid and production of PGI2. The curves for the three events were similar. When the cells were stimulated in low extracellular calcium (60 nM), they released [3H]arachidonic acid and produced PGI2, but depleting the intracellular Ca2+ stores by pretreatment with the Ca2+ ionophore A23187 totally inhibited both events, although the cells still responded when extracellular Ca2+ was added. The Ca2+ ionophore did not inhibit the generation of inositol phosphates in cells maintained at low extracellular Ca2+. Pertussis toxin pretreatment (14 h) altered neither inositol phosphate nor PGI2 production in response to AlF4-. To investigate the functional role of the diacylglycerol/protein kinase C arm of the phosphoinositide system, the cells were pretreated with the protein kinase C activator 12-O-tetradecanoylphorbol 13-acetate (TPA) or the protein kinase C inhibitor 1-(5-isoquinolinylsulphonyl)-2-methylpiperazine (H7). TPA inhibited the AlF4(-)-induced inositol phosphate generation but stimulated both the release of arachidonic acid and the production of PGI2. H7 had opposite effects both on inositol phosphate generation and on PGI2 production. These results suggest that AlF4(-)-induced PGI2 production is mediated by a pertussis-toxin-insensitive G-protein which activates the phosphoinositide second messenger system. This production of PGI2 can be modulated by protein kinase C activation, both at the level of inositol phosphate generation and at the level of arachidonic acid release. 相似文献
9.
Aggregation of the receptor with high affinity for immunoglobulin E (IgE) in rat basophilic leukemia cells leads to a calcium-dependent and a calcium-independent hydrolysis of phosphoinositides. The increase in the levels of inositol phosphates induced in the absence of calcium is only 25% of that observed with 1 mM Ca2+. The inositol phosphates reach a new steady state level 2 min after stimulation in EGTA, whereas with calcium they continue to increase up to 15 min. A similar response is observed when the receptors are aggregated due to the interaction of bound IgE with antigen or with anit-IgE, or by the binding of IgE cross-linked chemically. The antigen-mediated response is inhibited by hapten and disruption of such antigen-antibody aggregates late after stimulation leads to a rapid decline in the levels of the inositol phosphates to basal values. Separation of the inositol phosphates by Dowex columns shows that there is a fast rise in inositol trisphosphate which peaks at 15 s and slowly declines to a lower plateau within 2 min. Analysis by high pressure liquid chromatography reveals a 5-fold increase in the levels of inositol 1,4,5-trisphosphate in less than 10 s after stimulation, which precedes any major change in the other inositol phosphates. Aggregation of the receptor in the absence of external calcium induces a transient increase in cytoplasmic calcium which reaches a maximum of approximately 25 nM over basal levels after activation. The onset of the rise in Ca2+ lags after the initial rise in the inositol 1,4,5-trisphosphate. 相似文献
10.
The murine receptor for luteinizing hormone (LHR) was cloned and expressed in L cells. This LHR (mature protein of 674 amino acids) is very similar to that of the rat (same length, 36 amino acid differences) but differs significantly more from that of man (673 amino acids, 109 differences). Expression of the murine LHR in L cells led to the appearance of binding sites for human chorionic gonadotropin (hCG) with a Kd of 150 pM and an LH- and hCG-stimulable adenylyl cyclase activity (EC50 = 50-100 pM hCG). Upon labeling pools of phosphoinositides with [3H]myo-inositol, L cells expressing the murine LHR responded to hCG with an increase in their rate of phosphoinositide hydrolysis (EC50 = 2,400 pM hCG). This was accompanied by an increase in intracellular Ca2+ [( Ca2+]i), as determined by the Fura2 method. This increase in [Ca2+]i in response to hCG was dependent on the LHR, for HCG did not affect [Ca2+]i in L cells not expressing the LHR. The effect was not due to the cAMP-forming activity of the LH receptor, for neither forskolin nor prostaglandin E1, which both increase cAMP levels in L cells, had a similar effect in either control or LHR-expressing cells and isoproterenol had no effect in L cells expressing a functionally active hamster beta-adrenergic receptor. The effect was also not due to overexpression of a Gs-coupled receptor, for L cells expressing 8-fold higher levels of the human V2 vasopressin receptor did not mimic the Ca(2+)-mobilizing response of the LH receptor. We conclude that the LH receptor has the capability of activating two intracellular signaling pathways: one leading to stimulation of adenylyl cyclase and resulting in increases in cAMP and a second leading to stimulation of phospholipase C and resulting in formation of inositol phosphates and elevations in [Ca2+]i. These data correlate positively with and provide a mechanistic explanation for previous reports on the ability of hCG to mobilize phosphoinositides and increasing [Ca2+]i in luteal and granulosa cells (e.g. Davis, J. S., West, L. A., and Farese, R. V. (1984) J. Biol. Chem. 259, 15028-15034). 相似文献
11.
Oestradiol stimulates tyrosine phosphorylation and hormone binding activity of its own receptor in a cell-free system. 总被引:1,自引:1,他引:0
下载免费PDF全文
![点击此处可从《The EMBO journal》网站下载免费的PDF全文](/ch/ext_images/free.gif)
Recent experiments have shown that calf uterus oestrogen receptor exists in a tyrosine-phosphorylated hormone binding form and in non-phosphorylated, non-hormone binding form. We report here that physiological concentrations of oestradiol in complex with the receptor stimulate the calf uterus receptor kinase that converts the non-hormone binding receptor into hormone binding receptor through phosphorylation of the receptor on tyrosine. The activity of this enzyme has been followed by reactivation of hormone binding sites and phosphorylation on tyrosine of calf uterus phosphatase-inactivated receptor. Phosphorylation of the receptor has been demonstrated by interaction of kinase 32P-phosphorylated proteins with anti-receptor antibody followed either by sucrose gradient centrifugation or SDS-PAGE of the immunoprecipitated proteins. Hormone stimulation of the kinase is inhibited by receptor occupancy of the anti-oestrogen tamoxifen. Oestradiol-receptor complex increases the affinity of the kinase for the dephosphorylated receptor. Findings of this report are consistent with the observation that several protein tyrosine kinases that are associated with peptide hormone receptors are stimulated by the binding of the hormone to the receptor. This is the first report on the activation of a tyrosine kinase by a steroid hormone. The finding that hormones can regulate their own receptor binding activity through a tyrosine kinase is also new. 相似文献
12.
Cd2+ and other divalent metals mobilized cell Ca2+ in human skin fibroblasts. The divalent metals produced a large spike in cytosolic free Ca2+ and strikingly increased net Ca2+ efflux similarly to bradykinin. One-tenth microM Cd2+ half-maximally increased 45Ca2+ efflux. The potency order of the Ca2+ mobilizing metals was: Cd2+ greater than Co2+ greater than Ni2+ greater than Fe2+ greater than Mn2+. Cd2+ probably acts at an extracellular site because loading the cells with a heavy metal chelator only slightly inhibited Cd2+-evoked 45Ca2+ efflux. Cd2+ increased [3H]inositol polyphosphates; [3H]inositol trisphosphate increased 4-fold in 15 s. Zn2+ reversibly blocked 45Ca2+ efflux evoked by Cd2+ but not that produced by bradykinin. Zn2+ competitively (Ki = approximately 0.4 microM) inhibited net Ca2+ efflux produced by Cd2+. Cd2+ also evoked Ca2+ mobilization in umbilical artery muscle, endothelial, and neuroblastoma cells, and the divalent cation agonist and antagonist specificities were similar to those in the fibroblasts. The divalent metals appear to trigger Ca2+ mobilization via a reversible interaction with an external site on the cell surface, which may be considered a "Cd2+ receptor." 相似文献
13.
Molecular characterization of a novel metabotropic glutamate receptor mGluR5 coupled to inositol phosphate/Ca2+ signal transduction. 总被引:22,自引:0,他引:22
T Abe H Sugihara H Nawa R Shigemoto N Mizuno S Nakanishi 《The Journal of biological chemistry》1992,267(19):13361-13368
A cDNA clone for a new metabotropic glutamate receptor, mGluR5, was isolated through polymerase chain reaction-mediated DNA amplification by using primer sequences conserved among the metabotropic glutamate receptor (mGluR) family and by the subsequent screening of a rat brain cDNA library. The cloned receptor consists of 1171 amino acid residues and exhibits a structural architecture common to the mGluR family, possessing a large extracellular domain preceding the seven putative membrane-spanning segments. mGluR5 shows the highest sequence similarity to mGluR1 among the mGluR members and is coupled to the stimulation of phosphatidylinositol hydrolysis/Ca2+ signal transduction in Chinese hamster ovary cells transfected with the cloned cDNA. This receptor also resembles mGluR1 in its agonist selectivity and antagonist responses; the potency rank order of agonists for mGluR5 was determined to be quisqualate greater than L-glutamate greater than or equal to ibotenate greater than trans-1-aminocyclopentane-1,3-dicarboxylate. Blot and in situ hybridization analyses indicated that mGluR5 mRNA is widely distributed in neuronal cells of the central nervous system and is expressed differently from mGluR1 mRNA in many brain regions. This investigation thus demonstrates that there is an additional mGluR subtype which closely resembles mGluR1 in its signal transduction and pharmacological properties and is expressed in specialized neuronal cells in the central nervous system. 相似文献
14.
Inhibition of inositol phosphate production is a late, Ca2+-dependent effect of D2 dopaminergic receptor activation in rat lactotroph cells 总被引:1,自引:0,他引:1
The effect of dopamine, working through the activation of D2 receptors, on inositol phosphate production induced by thyrotropin-releasing hormone (TRH) was investigated in rat pituitary lactotroph cells. Dopamine (10 microM) did not modify the initial rapid stimulation of inositol 1,4,5-triphosphate and inositol bisphosphate observed within the first 15 s after TRH addition, but progressively inhibited the later inositol phosphate production induced by the neurohormone. This kinetics of inhibition was independent of dopamine preincubation time (from 2 to 10 min). The effect was still visible when dopamine was added after TRH. It was sensitive to pertussis toxin, was unchanged by increasing cellular cAMP levels with 8-Br-cAMP, but was greatly affected by treatments that modify the cytosolic free Ca2+ concentration. Specifically, the dopamine-induced inhibition was prevented by treatment of the cells with the Ca2+ ionophore ionomycin (100-200 nM) and was mimicked either by withdrawal of Ca2+ from the incubation medium or by blockade of voltage-gated Ca2+ channels with verapamil. The dopamine treatment did not decrease the cellular levels of the various phosphoinositides, strongly suggesting that the inhibition of inositol phosphate production is not due to precursor depletion. In isolated membranes, however, dopamine was unable to counteract the inositol phosphate accumulation triggered by TRH. Taken together, the data indicate that inhibition of inositol phosphate production is not a primary event triggered by D2 receptor activation, but is a late consequence, due to the previously demonstrated (Malgaroli, A., Vallar, L., Reza Elahi, F., Pozzan, T., Spada, A., and Meldolesi, J. (1987) J. Biol. Chem. 262, 13920-13927) inhibition by dopamine of the prolonged cytosolic free Ca2+ concentration increase induced by TRH via the activation of voltage-gated Ca2+ channels. These results are inconsistent with the possibility of a direct inhibitory coupling of D2 receptors to phospholipase C in rat pituitary lactotroph cells. 相似文献
15.
Klco JM Wiegand CB Narzinski K Baranski TJ 《Nature structural & molecular biology》2005,12(4):320-326
More than 90% of G protein-coupled receptors (GPCRs) contain a disulfide bridge that tethers the second extracellular loop (EC2) to the third transmembrane helix. To determine the importance of EC2 and its disulfide bridge in receptor activation, we subjected this region of the complement factor 5a receptor (C5aR) to random saturation mutagenesis and screened for functional receptors in yeast. The cysteine forming the disulfide bridge was the only conserved residue in the EC2-mutated receptors. Notably, approximately 80% of the functional receptors exhibited potent constitutive activity. These results demonstrate an unexpected role for EC2 as a negative regulator of C5a receptor activation. We propose that in other GPCRs, EC2 might serve a similar role by stabilizing the inactive state of the receptor. 相似文献
16.
S P Kennedy R I Sha'afi E L Becker 《Biochemical and biophysical research communications》1988,155(1):189-196
Rabbit peritoneal neutrophils, permeabilized with Triton X-100, contain inositol phosphate 5-phosphomonoesterase activity capable of converting [3H]inositol 1,4,5-trisphosphate (Ins-1,4,5-P3) to [3H]inositol 1,4-bisphosphate. This activity is found predominantly associated with the soluble component of fractionated neutrophils. It is comprised of specific and nonspecific activities toward Ins-1,4,5-P3 which can be separated by cation exchange chromatography. Treatment of neutrophils with phorbol 12-myristate 13-acetate (PMA) prior to permeabilization does not affect the rate of Ins-1,4,5-P3 breakdown by these cells. In addition, activation of endogenous protein kinase C in a soluble fraction prepared from neutrophils does not affect the specific inositol phosphate 5-phosphomonoesterase activity of this fraction. Taken together, these results provide evidence that activation of protein kinase C in the neutrophil does not affect its 5-phosphomonoesterase activity. Unlike platelets, the phosphorylation of a 5-phosphomonoesterase, if it occurs, may not play a role in the inhibitory effects of PMA on neutrophil responsiveness. 相似文献
17.
Cytochrome c activation of CPP32-like proteolysis plays a critical role in a Xenopus cell-free apoptosis system. 总被引:10,自引:2,他引:8
下载免费PDF全文
![点击此处可从《The EMBO journal》网站下载免费的PDF全文](/ch/ext_images/free.gif)
R M Kluck S J Martin B M Hoffman J S Zhou D R Green D D Newmeyer 《The EMBO journal》1997,16(15):4639-4649
In a cell-free system based on Xenopus egg extracts, Bcl-2 blocks apoptotic activity by preventing cytochrome c release from mitochondria. We now describe in detail the crucial role of cytochrome c in this system. The mitochondrial fraction, when incubated with cytosol, releases cytochrome c. Cytochrome c in turn induces the activation of protease(s) resembling caspase-3 (CPP32), leading to downstream apoptotic events, including the cleavage of fodrin and lamin B1. CPP32-like protease activity plays an essential role in this system, as the caspase inhibitor, Ac-DEVD-CHO, strongly inhibited fodrin and lamin B1 cleavage, as well as nuclear morphology changes. Cytochrome c preparations from various vertebrate species, but not from Saccharomyces cerevisiae, were able to initiate all signs of apoptosis. Cytochrome c by itself was unable to process the precursor form of CPP32; the presence of cytosol was required. The electron transport activity of cytochrome c is not required for its pro-apoptotic function, as Cu- and Zn-substituted cytochrome c had strong pro-apoptotic activity, despite being redox-inactive. However, certain structural features of the molecule were required for this activity. Thus, in the Xenopus cell-free system, cytosol-dependent mitochondrial release of cytochrome c induces apoptosis by activating CPP32-like caspases, via unknown cytosolic factors. 相似文献
18.
Activation of the O2.- generating oxidase in neutrophils can be achieved with a cell-free oxidase-activating system, which consists of a high speed supernatant (cytosol), a particulate fraction enriched in plasma membrane, GTP-gamma-S, arachidonic acid and Mg ions. Cytosolic proteins from bovine neutrophils were fractionated by chromatography on Mono Q and Mono S columns into two fractions, neither of which was able to activate efficiently the membrane-bound oxidase. However, when combined and added to the cell-free system under optimized conditions, they acted synergistically, activating the oxidase to virtually the same extent as crude cytosol. This synergism demonstrates that more than one cytosolic factor is required for oxidase activation, and can be used to trace the cytosolic factors during the course of their purification. 相似文献
19.
H Oberhuber K Maly F Uberall J Hoflacher A Kiani H H Grunicke 《The Journal of biological chemistry》1991,266(3):1437-1442
Expression of a transforming Ha-ras gene in NIH 3T3 cells transfected with an inducible Ha-ras construct leads to a rapid desensitization of the intracellular Ca2(+)-mobilizing system to bombesin and serum growth factors. Half-maximal depression of the Ca2+ response is observed 2 h after induction of p21ras. A maximum is obtained after 6 h. Bombesin-induced elevation of inositol 1,4,5-trisphosphate formation is also depressed in cells expressing Ha-ras. This, however, is a relatively late phenomenon and not yet detectable when maximal depression of the Ca2+ signal is observed. We conclude that the rapid densensitization of the Ca2(+)-releasing system to bombesin by Ha-ras is not caused by down-modulation or uncoupling of phospholipase C-coupled bombesin receptors. The inositol 1,4,5-trisphosphate-mediated release of intracellular Ca2+ is reduced in permeabilized cells expressing the Ha-ras oncogene. A depletion of intracellular Ca2+ stores by Ha-ras is unlikely since (i) the Ha-ras-induced growth factor-independent stimulation of inositol phosphate formation occurs several hours after reduction of the Ca2+ response and (ii) the Ca2+ load of intracellular nonmitochondrial Ca2+ stores was found to be unaffected by Ha-ras. We conclude that the desensitization of the Ca2(+)-mobilizing system is caused either by partial inhibition of inositol 1,4,5-trisphosphate-regulated Ca2+ channels or by interference of Ha-ras with Ca2+ translocation between intracellular Ca2+ compartments. 相似文献
20.
Masuda K Itoh H Sakihama T Akiyama C Takahashi K Fukuda R Yokomizo T Shimizu T Kodama T Hamakubo T 《The Journal of biological chemistry》2003,278(27):24552-24562
To investigate the coupling selectivity of G proteins and G protein-coupled receptors (GPCRs), we developed a reconstitution system made up of GPCR and heterotrimeric G proteins on extracellular baculovirus particles (budded virus (BV)). BV released from Sf9 cells infected with a recombinant baculovirus coding for human leukotriene B4 receptor (BLT1) cDNA exhibited a high level of BLT1 expression (27.3 pmol/mg of protein) and specific [3H]leukotriene B4 binding activity (Kd = 3.67 nm). The apparent low affinity of the expressed BLT1 is thought to be due to relative non-availability of the Galphai isoform, which couples to BLT1, in BV. Co-infection of heterotrimeric G protein recombinant viruses led to co-expression of BLT1 and G protein subunits on BV. A guanosine-5'-(beta,gamma-imido)triphosphate-sensitive, high affinity ligand binding was observed in the BLT1 BV co-expressing Galphai1beta1gamma2 (Kd = 0.17 nm). A relatively large amount of high affinity receptor protein was recovered in the co-expressing BV fraction (6.81 pmol/mg of protein). A combination of BLT1 and Galphai1 without Gbeta1gamma2 did not exhibit high affinity ligand binding on BV, indicating the low background environment for the GPCR-G protein coupling in this BV reconstitution system. To test other G proteins for coupling, various Galpha subunits were combinatorially expressed in BV with BLT1 and Gbeta1gamma2. The BLT1 BV co-expressing GalphaoAbeta1gamma2 exhibited a comparably high affinity ligand binding as well as ligand-stimulated guanosine 5'-3-O-(thio)triphosphate binding to Galphai1beta1gamma2. Co-expression of other Galpha isoforms such as Galphas, Galpha11, Galpha14, Galpha16, Galpha12, or Galpha13 did not exhibit any significant effects on ligand binding affinity in this system. These results reveal that BLT1 and coupled trimeric G proteins were functionally reconstituted on BV and that Galphao as well as Galphai couples to BLT1. This expression system should prove highly useful for pharmacological characterization, biosensor chip applications, and also drug discovery directed at highly important targets of the membrane receptor proteins. 相似文献