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1.
In a previous paper (Rath, H. M., Doyle, G. A. R., and Silbert, D. F. (1989) J. Biol. Chem. 264, 13387-13390), we reported a selection for the isolation of Chinese hamster lung fibroblasts (CCL39) defective in thrombin-induced mitogenesis. One mutant, D1-6b, had decreased production of inositol phosphates when challenged with activators of phosphatidylinositol turnover and extracts of this mutant showed a marked decrease in phospholipase C (PLC) activity toward phosphatidylinositol. In the current studies, the PLC activities of wild type CCL39 and D1-6b cytosolic extracts are further characterized. Wild type cytosol had at least two phosphatidylinositol-specific PLC isoenzymes, which could be separated by anion exchange chromatography and behaved differently in thermal inactivation studies. Since gel filtration of PLC activity in wild type extracts gave Mr values similar to that of previously characterized PLCs (140,000-200,000), immunoblots with antibodies to bovine brain isoenzymes were used to show that the PLC activities obtained by anion exchange chromatography were PLC-delta and PLC-gamma. Immunoblots with mutant D1-6b cytosol confirmed the presence of the PLC-gamma but showed no detectable PLC-delta. This activity in the mutant extracts eluted at the same conductivity on anion exchange columns and had the same kinetics of thermal inactivation as the PLC-gamma found in the wild type extracts. PLC-gamma from mutant extracts was active in assays containing phospholipid detergent mixed micelles but not in assays utilizing phospholipid vesicles, in sharp contrast to PLC-gamma from CCL39 extracts, which was active under either condition. Thus, the phosphatidylinositol-specific phospholipase C activity of mutant D1-6b is diminished both by the loss of PLC-delta and by the compromised behavior of PLC-gamma.  相似文献   

2.
We demonstrated previously that Na+/H+ exchange activity was enhanced in rat parotid acinar cells following muscarinic (carbachol) or alpha-adrenergic (epinephrine) stimulation and that this activation of the exchanger was preserved in plasma membrane vesicles prepared from these cells (Manganel, M., and Turner, R. J. (1989) J. Membr. Biol. 111, 191-198). The carbachol dose dependence of this effect as determined here in intact acini is in good agreement with the carbachol dose dependence of the fluid secretory response in this tissue (K0.5 approximately 3 microM). In addition, we show here that the stimulation of the exchanger by secretagogues cannot be mimicked by active diacylglycerol analogues, nor can it be prevented by the protein kinase inhibitors H7 and HA1004, arguing strongly against the involvement of protein kinase C in this effect. However, stimulation of the exchanger is observed in both intact acini treated with the calcium ionophore A23187 and in vesicles prepared from these acini. Moreover, carbachol, epinephrine, and A23187 are without effect when extracellular calcium is not present during acinar pretreatment. These results indicate that the stimulation of the Na+/H+ exchanger studied here is a consequence of agonist (or A23187)-induced increases in intracellular calcium levels due to calcium influx from the extracellular solution. The calmodulin inhibitors trifluoperazine and W7 (10(-4) M) prevented the stimulation of the exchanger induced by carbachol or epinephrine, but W7 could not block the stimulation produced by A23187 arguing against the involvement of calmodulin in this effect. Taken together with previous data from this and other laboratories, these results strongly indicate that the Na+/H+ exchanger and its regulation are intimately involved in the fluid secretory response of the rat parotid.  相似文献   

3.
Brush-border membrane vesicles prepared from rabbit kidney cortex were incubated at 37 degrees C for 30 min with phosphatidylinositol-specific phospholipase C. This maneuver resulted in a release of approx. 85% of the brush-border membrane-linked enzyme alkaline phosphatase as determined by its enzymatic activity. Transport of inorganic [32P]phosphate (100 microM) by the PI-specific phospholipase C-treated brush-border membrane vesicles was measured at 20-22 degrees C in the presence of an inwardly directed 100 mM Na+ gradient. Neither initial uptake rates, as estimated from 10-s uptake values (103.5 +/- 6.8%, n = 7 experiments), nor equilibrium uptake values, measured after 2 h (102 +/- 3.4%) were different from controls (100%). Control and PI-specific phospholipase C-treated brush-border membrane vesicles were extracted with chloroform/methanol to obtain a proteolipid fraction which has been shown to bind Pi with high affinity and specificity (Kessler, R.J., Vaughn, D.A. and Fanestil, D.D. (1982) J. Biol. Chem. 257, 14311-14317). Phosphate binding (at 10 microM Pi) by the extracted proteolipid was measured. No significant difference in binding was observed between the two types of preparations: 31.0 +/- 9.37 in controls and 29.8 +/- 8.3 nmol/mg protein in the proteolipid extracted from PI-specific phospholipase C-treated brush-border membrane vesicles. It appears therefore that alkaline phosphatase activity is essential neither for Pi transport by brush-border membrane vesicles nor for Pi binding by proteolipid extracted from brush-border membrane. These results dissociate alkaline phosphatase activity, but not brush-border membrane vesicle transport of phosphate, from phosphate binding by proteolipid.  相似文献   

4.
Fluorescence energy transfer [cf. F?rster, T. (1948) Ann. Phys. 6, 55-75] was tested for its suitability to study quantitative interactions of subunits of G0 with each other and these subunits or trimeric G0 with the beta 1-adrenoceptor in detergent micelles or after reconstitution into lipid vesicles [according to Feder, D., Im, M.-J., Klein, H. W., Hekman, M., Holzh?fer, A, Dees, C., Levitzki, A., Helmreich, E. J. M. & Pfeuffer, T. (1986) EMBO J. 5, 1509-1514]. For this purpose, alpha 0- and beta gamma-subunits and trimeric G0 purified from bovine brain, the beta gamma-subunits from bovine rod outer segment membranes and the beta 1-adrenoceptor from the turkey erythrocyte were all labelled with either tetramethylrhodamine maleimide or fluorescein isothiocyanate under conditions which leave the labelled proteins functionally intact. In the case of alpha 0- and beta gamma-interactions, specific high-affinity binding interactions (Kd approximately 10 nM) and nonspecific low-affinity binding interactions (Kd approximately 1 microM) could be readily distinguished by comparing fluorescence energy transfer before and after dissociation with 10 microM guanosine 5'-O-[gamma-thio]triphosphate and 10 mM MgCl2 where only low-affinity binding interactions remained. Interactions between alpha 0- and beta gamma-subunits from bovine brain or from bovine retinal transducin did not differ much. The beta gamma-subunits from bovine brain were found to bind with high transfer efficiency and comparable affinities to the hormone-activated and the nonactivated beta 1-receptor reconstituted in lipid vesicles: Kd = 100 +/- 20 and 120 +/- 20 nM, respectively; however, beta gamma-subunits from transducin appeared to bind more weakly to the beta 1-adrenoceptor than beta gamma-subunits from bovine brain. Separated purified homologous alpha 0- and beta gamma-subunits from bovine brain interfered mutually with each other in binding to the beta 1-adrenoceptor presumably because they had a greater affinity for each other than for the receptor. These findings attest to the suitability of fluorescence energy transfer for studying protein-protein interactions of G-proteins and G-protein-linked receptors. Moreover, they supported the previous finding [Kurstjens, N. P., Fr?hlich, M., Dees, C., Cantrill, R. C., Hekman, M. & Helmreich, E. J. M. (1991) Eur. J. Biochem. 197, 167-176] that beta gamma-subunits can bind to the nonactivated beta 1-adrenoceptor.  相似文献   

5.
The diacylglycerol (DAG) signal generated from membrane phospholipids by hormone-activated phospholipases is attenuated by mechanisms that include lipolysis or phospholipid resynthesis. To determine whether the DAG signal might also be terminated by incorporation of DAG into triacylglycerol (TAG), we studied the direct formation of TAG from endogenous DAG generated by bacterial phospholipase C (PLC). When Chinese hamster ovary (CHO) cells prelabeled with [(14)C]oleate were treated with PLC from Clostridium perfringens for 6 h, [(14)C]phospholipid decreased 15% and labeled TAG increased 60%. This transfer of (14)C label was even greater when the cells were simultaneously exposed to PLC and 100 microM oleic acid. PLC as well as oleate treatment concomitantly increased the TAG mass within the cell. Moreover, when phospholipids were prelabeled with [(3)H]glycerol, a subsequent increase in [(3)H]TAG indicated that an intact DAG moiety was channeled into the TAG structure. Incubating CHO cells with the diacylglycerol kinase inhibitor R59022 enhanced the formation of TAG from phospholipids hydrolyzed by PLC or by PLC in the presence of 100 microM oleate, but not by incubation with oleate alone, indicating that the DAG released from plasma membrane phospholipids does not require the formation of a phosphatidic acid precursor for TAG synthesis. Similarly, the diacylglycerol lipase inhibitor RHC 80267 did not alter TAG synthesis from plasma membrane DAG, further supporting direct incorporation of DAG into TAG.These studies indicate that DAG derived from plasma membrane phospholipid is largely used for TAG formation, and support the view that this mechanism can terminate DAG signals. The studies also suggest that a transport mechanism exists to move plasma membrane-derived DAG to the endoplasmic reticulum.-Igal, R. A., J. M. Caviglia, I. N. T. de Gómez Dumm, and R. A. Coleman. Diacylglycerol generated in CHO cell plasma membrane by phospholipase C is used for triacylglycerol synthesis. J. Lipid Res. 2001. 42: 88;-95.  相似文献   

6.
Using [U-14C]phosphatidylinositol as substrate, Ca2+-dependent phospholipase C activity was detected in a group of bovine adrenal medullary proteins that bind to chromaffin granule membranes in the presence of Ca2+ ("chromobindins," Creutz, C. E., Dowling, L. G., Sando, J. J., Villar-Palasi, C., Whipple, J. H., and Zaks, W. J. (1983) J. Biol. Chem. 258, 14664-14674). The activity was maximal at neutral pH and represented an 80- to 240-fold enrichment of adrenal medullary cytosol phospholipase C activity measured at pH 7.3. The stimulation of activity by Ca2+ was complex; no activity was present in the absence of Ca2+, 25% activation occurred at 1 microM Ca2+, and full activation at 5 mM Ca2+. The enzyme bound to chromaffin granule membranes in the presence of 2 mM Ca2+ but was released at 40 microM Ca2+, suggesting that intrinsic enzyme activity may be regulated by [Ca2+] at 1 microM, but additional activation at higher concentrations of Ca2+ is seen in vitro as a result of Ca2+-dependent binding of the active enzyme to substrate-containing membranes. This enzyme may generate diacylglycerol and phosphorylated inositol to act as intracellular messengers in the vicinity of the chromaffin granule membrane during the process of exocytosis.  相似文献   

7.
Human platelets labelled with either [14C]arachidonic acid or [32P]orthophosphate were loaded or not with the Ca2+ fluorescent indicator quin 2. They were then incubated in the presence or in the absence of human thrombin (1 U/ml) in a medium where Ca2+ concentration was adjusted near zero or to 1 mM. Under these conditions, phospholipase A2 activity, as detected by the release of [14C]arachidonate and of its metabolites, or by the hydrolysis of [14C]phosphatidylcholine, was severely impaired in quin 2-loaded platelets upon removal of external Ca2+. However, Ca2+ was not required in non-loaded platelets, where a maximal phospholipase A2 activity was detected in the absence of external Ca2+. In contrast, phospholipase C action, as determined from the amounts of [14C]diacylglycerol, [14C]- or [32P]phosphatidic acid formed, appeared to be much less sensitive to the effects of quin 2 loading and of Ca2+ omission. By using various concentrations of quin 2, it was found that the inhibitory effect exerted against phospholipase A2 could be overcome by external Ca2+ only when the intracellular concentration of the calcium chelator did not exceed 2 mM. At higher concentrations averaging 3.5 mM of quin 2, phospholipase A2 activity was fully suppressed even in the presence of external Ca2+, whereas phospholipase C was still active, although partly inhibited. It is concluded that platelet phospholipase A2 requires higher Ca2+ concentrations than phospholipase C to display a maximal activity. By comparing platelet phospholipase A2 activity under various conditions with the values of cytoplasmic free Ca2+ as detected by quin 2 fluorescence, it is proposed that cytoplasmic free Ca2+ in control platelets stimulated with thrombin can attain concentrations above 1 microM, probably close to 5-10 microM, as recently determined with the photoprotein aequorin (Johnson, P.C., Ware, J.A., Cliveden, P.B., Smith, M., Dvorak, A.M. and Salzman, E.W. (1985) J. Biol. Chem. 260, 2069-2076).  相似文献   

8.
In the previous paper, we reported the identification of a 74-kDa G-protein that co-purifies with the alpha 1-adrenergic receptor following ternary complex formation. We report here on the purification and characterization of this 74-kDa G-protein (termed Gh) isolated de novo from rat liver membranes. After solubilization of rat liver membranes with the detergent sucrose monolaurate, Gh was isolated by sequential chromatography using heparin-agarose, Ultrogel AcA 34, hydroxylapatite, and heptylamine-Sepharose columns. The protein, thus isolated, is not a substrate for cholera or pertussis toxin but displays GTPase activity (turnover number, 3-5 min-1) and high-affinity guanosine 5'-O-3-thiotriphosphate (GTP gamma S) binding (half-maximal binding = 0.25-0.3 microM), which is Mg2(+)-dependent and saturable. The relative order of nucleotide binding by Gh is GTP gamma S greater than GTP greater than GDP greater than ITP much much greater than ATP greater than or equal to adenyl-5'-yl imidodiphosphate, which is similar to that observed for other heterotrimeric G-proteins involved in receptor signaling. Moreover, specific alpha 1-agonist-stimulated GTPase (turnover number, 10-15 min-1) and GTP gamma S binding activity could be demonstrated after reconstitution of purified Gh with partially purified alpha 1-adrenergic receptor into phospholipid vesicles. The alpha 1-agonist stimulation of GTP gamma S binding and GTPase activity was inhibited by the alpha-antagonist phentolamine. A 50-kDa protein co-purifies with the 74-kDa G-protein. This protein does not bind guanine nucleotides and may be a subunit (beta-subunit) of Gh. These findings indicate that Gh is a G-protein that functionally couples to the alpha 1-adrenergic receptor.  相似文献   

9.
Transglutaminase type 2 (TG2; also known as G(h)) is a multifunctional protein involved in diverse cellular processes. It has two well characterized enzyme activities: receptor-stimulated signaling that requires GTP binding and calcium-activated transamidation or cross-linking that is inhibited by GTP. In addition to the GDP binding residues identified from the human TG2 crystal structure (Liu, S., Cerione, R. A., and Clardy, J. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 2743-2747), we have previously implicated Ser171 in GTP binding, as binding is lost with glutamate substitution (Iismaa, S. E., Wu, M.-J., Nanda, N., Church, W. B., and Graham, R. M. (2000) J. Biol. Chem. 275, 18259-18265). Here, we have shown that alanine substitution of homologous residues in rat TG2 (Phe174 in the core domain or Arg476, Arg478, or Arg579 in barrel 1) does not affect TG activity but reduces or abolishes GTP binding and GTPgammaS inhibition of TG activity in vitro, indicating that these residues are important in GTP binding. Alanine substitution of Ser171 does not impair GTP binding, indicating this residue does not interact directly with GTP. Arg579 is particularly important for GTP binding, as isothermal titration calorimetry demonstrated a 100-fold reduction in GTP binding affinity by the R579A mutant. Unlike wild-type TG2 or its S171E or F174A mutants, which are sensitive to both trypsin and mu-calpain digestion, R579A is inherently more resistant to mu-calpain, but not trypsin, digestion, indicating reduced accessibility and/or flexibility of this mutant in the region of the calpain cleavage site(s). Basal TG activity of intact R579A stable SH-SY5Y neuroblastoma cell transfectants was slightly increased relative to wild-type transfectants and, in contrast to the TG activity of the latter, was further stimulated by muscarinic receptor-activated calcium mobilization. Thus, loss of GTP binding sensitizes TG2 to intracellular calcium concentrations. These findings are consistent with the notion that intracellularly, under physiological conditions, TG2 is maintained largely as a latent enzyme, its calcium-activated cross-linking activity being suppressed allosterically by guanine nucleotide binding.  相似文献   

10.
Treatment of canine cardiac sarcolemmal vesicles with phospholipase D resulted in a large stimulation (up to 400%) of Na+-Ca2+ exchange activity. The phospholipase D treatment decreased the apparent Km (Ca2+) for the initial rate of Nai+-dependent Ca2+ uptake from 18.2 +/- 2.6 to 6.3 +/- 0.3 microM. The Vmax increased from 18.0 +/- 3.6 to 31.5 +/- 3.6 nmol of Ca2+/mg of protein/s. The effect was specific for Na+-Ca2+ exchange; other sarcolemmal transport enzymes ((Na+, K+)-ATPase; ATP-dependent Ca2+ transport) are inhibited by incubation with phospholipase D. Phospholipase D had little effect on the passive Ca2+ permeability of the sarcolemmal vesicles. After treatment with 0.4 unit/ml of phospholipase D (20 min, 37 degrees C), the sarcolemmal content of phosphatidic acid rose from 0.9 +/- 0.2 to 8.9 +/- 0.4%; simultaneously, Na+-Ca2+ exchange activity increased 327 +/- 87%. It is probable that the elevated phosphatidic acid level is responsible for the enhanced Na+-Ca2+ exchange activity. In a previous study (Philipson, K. D., Frank, J. S., and Nishimoto, A. Y. (1983) J. Biol. Chem. 258, 5905-5910), we hypothesized that negatively charged phospholipids were important in Na+-Ca2+ exchange, and the present results are consistent with this hypothesis. Stimulation of Na+-Ca2+ exchange by phosphatidic acid may be important in explaining the Ca2+ influx which accompanies the phosphatidylinositol turnover response which occurs in a wide variety of tissues.  相似文献   

11.
Upon fertilization, ascidian eggs release a cell surface glycosidase used in the block to polyspermy and undergo cortical contractions resulting from increased intracellular calcium levels. The glycosidase is released by fertilization, calcium ionophores or added phospholipase C (PLC) activity. The PLC inhibitor D609 blocks glycosidase release. Intact Ascidia ceratodes eggs cleave 4-methylumbelliferyl-phospho-choline when it is added to seawater. This yields highly fluorescent 4-methylumbelliferone. Authentic phospholipase C but not phospholipase D can cleave this substrate. Thus, the authors believe that cleavage of the substrate is specific for PLC activity. Eggs incubated in the fluorogenic substrate after having been washed and detergent extracted were not fluorescent. Therefore the substrate failed to enter intact cells. Glycosidase release and PLC activity were stimulated by ionomycin. Octylglucoside or Triton X-100 extracts of ascidian eggs had two forms of phospholipase activity as shown by ion affinity chromatography: PL1 eluting at 0.25 mol/L NaCl and PL2 eluting at 0.6mol/L NaCl. The PL1 appeared to be isolated as a single protein. When surface proteins were labeled with non-penetrating biotin and were subsequently reacted with streptavidin, half of the PLC activity bound. This demonstrates that half the ascidian egg PLC activity is located on the surface of either the egg or follicle cell, and half is located within the egg.  相似文献   

12.
Long chain bases (sphinganine and sphingosine) are potent inhibitors of protein kinase C in an in vitro mixed micelle-reconstituted system (Hannun, Y. A., Loomis, C. R., Merrill, A. H. J., and Bell, R. M. (1986) J. Biol. Chem. 261, 12604-12609) and block activation of the superoxide-generating respiratory burst in human neutrophils (Wilson, E., Olcott, M. C., Bell, R. M., Merrill, A. H., Jr., and Lambeth, J. D. (1986) J. Biol. Chem. 261, 12616-12623). In the present studies, we have investigated the effects of sphinganine on cellular levels of the second messengers related to phosphoinositide turnover: diacylglycerol and calcium. We find that sphinganine added from a stock solution containing equimolar or greater bovine serum albumin had no effect on either formyl-methionyl-leucyl-phenylalanine-stimulated calcium fluxes or diacylglycerol generation, at levels which completely blocked activation of superoxide generation. In addition, there was no effect of sphinganine on cell viability in this concentration range. These data indicate an inhibitory effect subsequent to the generation of second messengers and are consistent with protein kinase C as the locus of action. When sphinganine was added from a stock in dimethyl sulfoxide, significant cytotoxic effects (assayed by trypan blue exclusion, release of cellular lactate dehydrogenase, and leakage of Quin2) were seen at concentrations nearer those which inhibited the respiratory burst. Cytotoxicity was inversely proportional to cell concentration and was probably due to detergent micelle formation which occurs in the absence of albumin. These studies emphasize the importance of the method of delivery and the consideration of cytotoxic effects, but indicate that long-chain bases possess potent inhibitory properties which make them useful probes of signal transduction mechanisms.  相似文献   

13.
Platelet activation by the prostaglandin endoperoxide (PGH2)/thromboxane (Tx) A2 analog, U46619, involves stimulation of phospholipase (PL) C and an increase in intracellular calcium via distinct receptor subtypes. Agents which stimulate adenylate cyclase inhibit platelet function. We demonstrate that PGH2/TxA2 receptor desensitization is associated with enhanced stimulation of platelet cyclic AMP by the prostacyclin analog, iloprost and by forskolin. Sensitization of adenylate cyclase is mediated via the PGH2/TxA2 receptor subtype which activates PLC, as it is blocked by the specific antagonist, GR32191 (Takahara, K., Murray, R., FitzGerald, G. A., and Fitzgerald, D. J. (1990) J. Biol. Chem. 265, 6838-6844). This effect is not observed in platelets desensitized with thrombin or platelet activating factor and is not mediated by protein kinase C. Prior exposure of platelets to platelet activating factor results in much greater desensitization of PGH2/TxA2-induced aggregation (mean 64%) compared with calcium stimulation (mean 18%), consistent with selective heterologous desensitization of the PLC-linked PGH2/TxA2 receptor subtype. Platelet activation by PGH2/TxA2 is a tightly regulated process, involving both homologous desensitization of at least two receptor subtypes and sensitization of the platelet adenylase cyclase system.  相似文献   

14.
It was reported previously that rat platelets release phospholipase A2 upon in vitro stimulation by thrombin, ADP, or A23187 (Horigome, K., Hayakawa, M., Inoue, K., & Nojima, S. (1987) J. Biochem. 101, 53-61). Secretion of phospholipase A2 was also observed with rabbit platelets. Rabbit platelets seem to release phospholipase A2 upon stimulation in vivo, because the rabbit plasma taken immediately after intravenous injection of PAF contained an appreciable level of phospholipase A2 activity and fewer platelets. Rabbit platelet phospholipase A2 released in vitro was purified by column chromatography using Sepharose CL-4B conjugated with anti-rat platelet derived phospholipase A2 monoclonal antibody, followed by reversed-phase HPLC. The purified enzyme was subjected to structural analysis by HPLC peptide mapping and primary sequence determination of the separated peptides. Based on the homology with rat platelet secretory phospholipase A2 (Hayakawa, M., Kudo, I., Tomita, M., Nojima, S., & Inoue, K. (1988) J. Biochem. 104, 767-772), a partial primary structure (62 amino acid residues) of the rabbit enzyme was tentatively determined; the two sequences were highly homologous (72%). The rabbit sequence was also nearly identical to that of rabbit ascitic fluid phospholipase A2, which was determined by Forst et al. (Forst, S., Weiss, J., Elsbach, P., Maraganore, J.M., Reardon, I., & Heinrikson, R.L. (1986) Biochemistry 25, 8381-8385). Phospholipase A2 from the membrane fraction of rabbit platelets was also purified; it had the same characteristics and th same amino-terminal sequence as the purified secretory enzyme. Secretory and membrane-bound phospholipase A2 of rabbit platelets may in fact be identical.(ABSTRACT TRUNCATED AT 250 WORDS)  相似文献   

15.
A 150-kDa phospholipase C has previously been purified from turkey erythrocytes and has been shown by reconstitution with turkey erythrocyte membranes to be a receptor- and G-protein-regulated enzyme (Morris, A. J., Waldo, G. L., Downes, C.P., and Harden, T. K. (1990) J. Biol. Chem. 265, 13501-13507; Morris, A.J., Waldo, G.L., Downes, C.P., and Harden, T.K. (1990) J. Biol. Chem. 265, 13508-13514). Combination of this 150-kDa protein with phosphoinositide substrate-containing phospholipid vesicles prepared with a cholate extract from purified turkey erythrocyte plasma membranes resulted in conferrence of AlF4- sensitivity to the purified phospholipase C. Guanosine 5'-3-O-(thio)triphosphate also activated the reconstituted phospholipase C in a manner that was inhibited by guanosine 5'-2-O-(thio)-diphosphate. The magnitude of the AlF4- stimulation was increased with increasing amounts of plasma membrane extract, and was also dependent on the concentration of purified phospholipase C. Using reconstitution of AlF4- sensitivity as an assay, the putative G-protein conferring regulation to the 150-kDa phospholipase C was purified to near homogeneity by sequential chromatography over Q-Sepharose, Sephacryl S-300, octyl-Sepharose, hydroxylapatite, and Mono-Q. Reconstituting activity co-purified with an approximately 43-kDa protein identified by silver staining; lesser amounts of a 35-kDa protein was present in the final purified fractions, as was a minor 40-kDa protein. The 43-kDa protein strongly reacted with antiserum against a 12-amino acid sequence found at the carboxyl terminus of Gq and G11, the 35-kDa protein strongly reacted with G-protein beta-subunit antiserum, and the 40-kDa protein reacted with antiserum that recognizes Gi3. Immunoprecipitation of the 43-kDa protein resulted in loss of phospholipase C-stimulating activity of the purified fraction. The idea that this is a phospholipase C-regulating G-protein is further supported by the observation that co-reconstitution of G-protein beta gamma-subunit with the purified phospholipase C-activating fraction resulted in a beta gamma-subunit-dependent inhibition of AlF(4-)-stimulated phospholipase C activity in the reconstituted preparation.  相似文献   

16.
Familial Alzheimer's disease (FAD) presenilin 1 (PS1) mutations give enhanced calcium responses upon different stimuli, attenuated capacitative calcium entry, an increased sensitivity of cells to undergo apoptosis, and increased gamma-secretase activity. We previously showed that the FAD mutation causing an exon 9 deletion in PS1 results in enhanced basal phospholipase C (PLC) activity (Cedazo-Minguez, A., Popescu, B. O., Ankarcrona, M., Nishimura, T., and Cowburn, R. F. (2002) J. Biol. Chem. 277, 36646-36655). To further elucidate the mechanisms by which PS1 interferes with PLC-calcium signaling, we studied the effect of two other FAD PS1 mutants (M146V and L250S) and two dominant negative PS1 mutants (D257A and D385N) on basal and carbachol-stimulated phosphoinositide (PI) hydrolysis and intracellular calcium concentrations ([Ca2+]i) in SH-SY5Y neuroblastoma cells. We found a significant increase in basal PI hydrolysis in PS1 M146V cells but not in PS1 L250S cells. Both PS1 M146V and PS1 L250S cells showed a significant increase in carbachol-induced [Ca2+]i as compared with nontransfected or wild type PS1 transfected cells. The elevated carbachol-induced [Ca2+]i signals were reversed by the PLC inhibitor neomycin, the ryanodine receptor antagonist dantrolene, the general aspartyl protease inhibitor pepstatin A, and the specific gamma-secretase inhibitor N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester. The cells expressing either PS1 D257A or PS1 D385N had attenuated carbachol-stimulated PI hydrolysis and [Ca2+]i responses. In nontransfected or PS1 wild type transfected cells, N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester and pepstatin A also attenuated both carbachol-stimulated PI hydrolysis and [Ca2+]i responses to levels found in PS1 D257A or PS1 D385N dominant negative cells. Our findings suggest that PS1 can regulate PLC activity and that this function is gamma-secretase activity-dependent.  相似文献   

17.
Thiol:protein-disulfide oxidoreductase catalyzes the GSH reduction of protein disulfides to sulfhydryls. Chromatography of solubilized hepatic microsomes on Mono Q yielded two peaks, Q-2 and Q-5, which contained all the thiol:protein-disulfide oxidoreductase activity. These were further purified by chromatofocusing giving specific activities of 14.4 and 45.9 nmol/mg of protein/min, respectively with purifications of 45.0- and 143.6-fold. Amino acids 1-18 of Q-5 were the same as previously reported for Thiol:protein-disulfide oxidoreductase (Edman, J. C., Ellis, L., Blacher, R. W., Roth, R. A., and Rutter, W. J. (1985) Nature 317, 267-270), except amino acid 1 was leucine instead of aspartate and amino acid 6 was asparagine instead of glutamate. The N-terminal amino acid sequence of Q-2 differed markedly from Q-5 but Q-2 showed 100% identity at amino acids 25-54, 258-269, 285-310, 347-350, 412-419, and 434-463 for the reported sequence of rat, hepatic, cytosolic phosphatidylinositol-specific phospholipase C form 1a (PLC) (Bennett, C. F., Balcarek, J. M., Varrichio, A., and Crooke, S. T. (1988) Nature 334, 268-270). PLC activity was found in the elution from the Mono Q column, but none was found in purified Q-2 or Q-5. Antibodies to Q-5 reacted with Q-2, but anti-Q-2 did not react with Q-5. Anti-Q-2 antibody showed immunoreactivity with 55- and 60-kDa microsomal proteins, whereas Q-5 antibody reacted with a number of microsomal proteins. Although Q-2 was immunoreactive with a polyclonal antibody to guinea pig, uterine cytosolic PLC, partially purified PLCs from rat liver cytosol did not react to this antibody. Our data would suggest that the published sequence for PLC form 1a may actually be the sequence for Q-2.  相似文献   

18.
The alpha 2-C10 adrenergic receptor from human platelets was expressed permanently in Rat-1 fibroblasts. A series of clones that varied in expression of the receptor from 0 to 3.5 pmol/mg of membrane protein were isolated. We have demonstrated recently in cells of one of these clones (1C) that the alpha 2-C10 receptor interacts directly with two distinct pertussis toxin-sensitive G-proteins, Gi2 and Gi3 (Milligan, G., Carr, C., Gould, G. W., Mullaney, I., and Lavan, B.E. (1991) J. Biol. Chem. 266, 6447-6455). High affinity GTPase activity in membranes of cells from the various clones was stimulated by the addition of the alpha 2-adrenergic agonist UK14304, defining that the receptor coupled productively to the G-protein signaling system. Maximal stimulation of high affinity GTPase activity correlated with the levels of receptor expressed. Clones expressing the receptor also demonstrated agonist-mediated inhibition of adenylylcyclase. Futhermore, the alpha 2-C10 receptor in one clone (1C), but not other clones, promoted a marked stimulation in the generation of water-soluble products derived from phosphatidylcholine. The concentration of UK14304 required to produce half-maximal regulation of GTPase activity (20-30 nM), of forskolin-amplified adenylylcyclase activity (30-40 nM), and of choline generation (30-40 nM) were similar. Transphosphatidylation experiments with cells of clone 1C indicated that the receptor-mediated hydrolysis of phosphatidylcholine was via the action of a phospholipase D. All of these effects were attenuated by pretreatment of the cells with pertussis toxin. Dose-effect curves of pertussis toxin-treatment demonstrated similar effective concentrations of the toxin in causing endogenous ADP-ribosylation of both Gi2 and Gi3, inhibition of receptor-stimulated GTPase activity, and phospholipase D activity. Receptor activation of phospholipase D activity was not dependent upon prior phospholipase C-dependent activation of protein kinase C, as alpha 2-adrenergic stimulation of inositol phosphate production was negligible and the presence of the selective protein kinase C inhibitor RO-31-8220, at concentrations up to 10 microM, had no effect on UK14304-mediated production of phosphatidylbutanol. These results demonstrate that expression of the alpha 2-C10 receptor in a heterologous system can result in receptor regulation of signaling elements that appear not to be primary targets for the receptor in vivo. Such results are important in respect to recent observations that transfection of a single defined receptor into separate cell lines can lead to the regulation of distinct effector systems (Vallar, L., Muca, C., Magni, M., Albert, P., Bunzow, J., Meldolesi, J. and Civelli, O. (1990) J. Biol. Chem. 265, 10320-10326).(ABSTRACT TRUNCATED AT 400 WORDS)  相似文献   

19.
The effects of thrombin and GTP gamma S on the hydrolysis of phosphoinositides by membrane-associated phospholipase C (PLC) from human platelets were examined with endogenous [3H]inositol-labeled membranes or with lipid vesicles containing either [3H]phosphatidylinositol or [3H]phosphatidylinositol 4,5-bisphosphate. GTP gamma S (1 microM) or thrombin (1 unit/mL) did not stimulate release of inositol trisphosphate (IP3), inositol bisphosphate (IP2), or inositol phosphate (IP) from [3H]inositol-labeled membranes. IP2 and IP3, but not IP, from [3H]inositol-labeled membranes were, however, stimulated 3-fold by GTP gamma S (1 microM) plus thrombin (1 unit/mL). A higher concentration of GTP gamma S (100 microM) alone also stimulated IP2 and IP3, but not IP, release. In the presence of 1 mM calcium, release of IP2 and IP3 was increased 6-fold over basal levels; however, formation of IP was not observed. At submicromolar calcium concentration, hydrolysis of exogenous phosphatidylinositol 4,5-bisphosphate (PIP2) by platelet membrane associated PLC was also markedly enhanced by GTP gamma S (100 microM) or GTP gamma S (1 microM) plus thrombin (1 unit/mL). Under identical conditions, exogenous phosphatidylinositol (PI) was not hydrolyzed. The same substrate specificity was observed when the membrane-associated PLC was activated with 1 mM calcium. Thrombin-induced hydrolysis of PIP2 was inhibited by treatment of the membranes with pertussis toxin or pretreatment of intact platelets with 12-O-tetradecanoyl-13-acetate (TPA) prior to preparation of membranes. Pertussis toxin did not inhibit GTP gamma S (100 microM) or calcium (1 mM) dependent PIP2 breakdown, while TPA inhibited GTP gamma S-dependent but not calcium-dependent phospholipase C activity.(ABSTRACT TRUNCATED AT 250 WORDS)  相似文献   

20.
Ligand stimulation of the platelet-derived growth factor receptor (PDGF-R) results in rapid activation of the receptor tyrosine kinase, stimulation of phosphoinositide hydrolysis, an increase in intracellular free Ca2+ concentration ([Ca2+]i), and, ultimately, cellular proliferation. In a previous study, we demonstrated that staurosporine, a known inhibitor of protein kinase C, blocked PDGF-induced [Ca2+]i increases in Swiss mouse 3T3 fibroblasts by a mechanism that appeared unrelated to inhibition of protein kinase activity (Olsen, R., Melder, D., Seewald, M., Abraham, R., and Powis, G. (1990) Biochem. Pharmacol. 39, 968-972). In the present study, we report that staurosporine inhibits ligand-dependent PDGF-R tyrosine kinase activation in cell-free receptor preparations and in intact Swiss 3T3 cells. At the same concentrations (10(-8)-10(-6) M), staurosporine suppressed both the tyrosine phosphorylation of phospholipase C activity and the hydrolysis of phosphoinositides induced by PDGF stimulation of intact cells. In contrast, guanine nucleotide-binding protein-dependent phospholipase C activation induced by bradykinin or fluoroaluminate anion was relatively insensitive to staurosporine. A preferential inhibitory effect of staurosporine on signal generation by the PDGF-R was indicated by findings that epidermal growth factor receptor (EGF-R) tyrosine kinase activity and EGF-dependent phospholipase C in A-431 carcinoma cells were approximately 100-fold less sensitive to this drug. These data indicate that submicromolar concentrations of staurosporine inhibit PDGF-dependent phosphoinositide hydrolysis and Ca2+ mobilization through a proximal inhibitory effect on ligand-induced activation of the PDGF-R tyrosine kinase.  相似文献   

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