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1.
The stimulatory and inhibitory activities in the crude preparation of protein kinase modulator from dog heart were separated by Sephadex G-100 gel filtration, and the stimulatory modulator was further purified by DEAE-cellulose chromatography. The isolated stimulatory modulator, as the crude modulator preparation, stimulated the activity of the purified guanosine 3':5'-monophosphate (cGMP)-dependent protein kinases of both mammalian and arthropod origins in the presence of cGMP. The cGMP-dependent protein kinases were not activated by cGMP in the absence of either the isolated stimulatory modulator or the crude modulator. The stimulatory modulator, unlike the crude modulator had no effect on the activity of adenosine 3':5'-monophosphate (cAMP)-dependent protein kinase. The stimulatory modulator was a protein since its activity was destroyed by trypsin but was resistant to hydrolysis by DNase, RNase, phospholipase C, and lysozyme. The isolated inhibitory modulator, presumably the same as the protein inhibitor of cAMP-dependent protein kinase reported by Walsh et al. (Wash. D.A., Ashby, C.D., Gonzalez, C., Calkins, D., Fischer. E.H., and Krebs, E.G. (1971) J. Biol. Chem. 246, 1977-1985), depressed the cAMP-stimulated activity of cAMP-dependent protein kinase as did the crude preparation of protein kinase modulator. The isolated inhibitory modulator, unlike the crude preparation, was without effect on cGMP-dependent protein kinase. The present findings provide evidence to support that in mammals there are separate proteins for the stimulatory and the inhibitory activities of protein kinase modulator, in contrast to the modulator from an arthropod tissue (lobster tail muscle, Donnelly et al. (Donnelly, T.E., Jr., Kuo, J.F., Reyes, P.L., Liu, Y.P., and Greengard, P. (1973) J. Biol. Chem. 248, 190-198) which has been shown to possess both activities.  相似文献   

2.
日本蝮蛇蛇毒碱性磷脂酶A2同源物的分离及鉴定   总被引:3,自引:0,他引:3  
We purified and characterizated a phospholipase A2 homologue from Agkistrodon blomhoffii ussurensis snake venom. We used Hitrap SP cation exchange and Superdex 75 columns chromatography to obtain a basic protein, used SDS-PAGE to analyse molecular mass, and IEF (Isoelectric focusing electrophoresis) IEF to identify isoelectric point. The molecular mass was 16 kDa, and the isoelectric point was 8.56. We detected its phospholipase A2 activity on egg yolk phospholipids, hemolytic activity on washed erythrocytes, and anticoagulant effect on pig platelet-rich plasma, as well as the N-terminal sequence with protein sequencer. The results showed that it had no phospholipase A2 activity and hemolytic activity, but had obvious anticoagulant effect on in witro. The N terminal sequence (21 amino acid residues) compared with other phospholipases A2 demonstrated that the protein was homogenous with BPLA2s from Agkistrodon halys Palls.  相似文献   

3.
The lysophosphoinositide-specific phospholipase C (lysoPI-PLase C) in porcine platelet membranes had an optimal pH of 9.2 and the activity at a physiological pH of 7.3 was 20% of the maximum in the absence of added divalent metals (Murase, S. et al. (1985) J. Biol. Chem. 260, 262). The activity was completely inhibited by 1 mM EGTA in the assay mixture but was restored by addition of excess Ca2+ or Mn2+, indicating that this is a metalloenzyme. However, membranes pretreated with 1 mM EGTA and washed with buffer retained full activity at a free Ca2+ concentration of 5 nM and no stimulation was observed by added Ca2+ at pH 9.2. In contrast to the results obtained at pH 9.2, addition of Ca2+ stimulated lysoPI-PLase C activity severalfold at pH 7.3, apparently by shifting down the optimal pH and broadening the pH profile. The effect of Ca2+ at pH 7.3 was to enhance Vmax with no significant change in Km value. The stimulatory effect of Ca2+ at pH 7.3 alone did not appear to be of physiological significance since millimolar concentrations of Ca2+ were necessary to reach the maximum activity. However, a shift in pH had a profound effect on the Ca2+-dependency of the activity. A rise in 2 pH units increased the apparent affinity for Ca2+ 10,000-fold. These results indicate that the alkalinization and the rise in free Ca2+ concentration known to occur in stimulated platelets could synergistically provide conditions under which the lysoPI-PLase C exerts its activity when the substrate lysoPI is generated by phospholipase A.  相似文献   

4.
The influence of nucleotides and pyrophosphate on phospholipase C from rabbit thymocytes was investigated by using two different methods for the determination of phospholipase C activity. In a first approach the release of radiolabeled inositol phosphates from [3H]inositol-labeled membranes was examined. By a second type of experiment the cleavage of exogenously added radiolabeled phosphatidylinositol 4,5-bisphosphate (PtdIns-4,5-P2) was measured. Using internally labeled membranes only guanosine 5'-O-(thiotriphosphate) exhibited a stimulatory effect on the phospholipase C suggesting the involvement of a G-protein. When exogenous [3H]PtdIns-4,5-P2 was used as substrate, cleavage of PtdIns-4,5-P2 was stimulated by all nucleotides investigated; in addition pyrophosphate showed a stimulatory effect. From these data we conclude that the increased cleavage of exogenous PtdIns-4,5-P2 induced by GTP analogues is not conclusive in terms of the involvement of a G-protein. Rather than induced by a G-protein this activation may be caused by an increased substrate accessibility. Our experiments with endogenous substrate clearly established the regulatory role of G-proteins for membrane-bound phospholipase C.  相似文献   

5.
Two novel phospholipase activities have been identified in the cytosolic fraction of canine myocardium. Neutral active phospholipase C activity was partially purified by anion exchange, hydroxylapatite, chromatofocusing, and gel filtration chromatographies. The partially purified enzyme had similar maximum velocities (237 versus 241 nmol/mg X h) and apparent Michaelis constants (20 versus 14 microM) utilizing either plasmenylcholine or phosphatidylcholine as substrate. Myocardial phospholipase C had a pH optimum between 7 and 8, required divalent cations for maximal activity, and did not hydrolyze phosphatidylinositol or sphingomyelin. Myocardial cytosol contained a potent inhibitor of phospholipase C which masked enzymic activity until it was removed during the purification procedure. A plasmalogen selective phospholipase A2 activity was also identified in the cytosolic fraction of canine myocardium. The protein catalyzing this activity was partially purified by DEAE-Sephacel-hydroxylapatite tandem chromatography and exhibited a maximum velocity of 5 nmol/mg X h for plasmenylcholine but only 1 nmol/mg X h for phosphatidylcholine, had a pH optimum between 6 and 7 for both substrates, and did not require calcium ion for activity. These results constitute the first demonstration of a neutral active phospholipase C specific for choline and ethanolamine glycerophospholipids and a plasmalogen selective phospholipase A2 in mammalian tissue.  相似文献   

6.
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   

7.
Mechanisms involved in regulating the activity of intracellular phospholipase A2 enzymes that function in eicosanoid and platelet-activating factor production are poorly understood. The properties of the substrate in the membrane may play a role in modulating phospholipase A2 activity. In this study, the effect of anionic phospholipids, diacylglycerol (DAG) and phosphatidylethanolamine (PE) on the activity of a partially purified, intracellular, arachidonoyl-hydrolyzing phospholipase A2 from the macrophage cell line, RAW 264.7 was studied. For these experiments phospholipase A2 activity was assayed in the presence of 1 microM calcium by measuring the hydrolysis of [3H]arachidonic acid from sonicated dispersions of the ether-linked substrate, 1-O-hexadecyl-2[3H]arachidonoylglycerophosphocholine. All the anionic phospholipids tested, including phosphatidylserine (PS), phosphatidic acid (PA), phosphatidylinositol (PI) and phosphatidylinositol-4,5-bisphosphate (PIP2), stimulated phospholipase A2 activity. At the lowest concentration of anionic phospholipids tested. PIP2 was the most stimulatory, resulting in a 7-fold increase in phospholipase A2 activity at 1 mol%. Co-dispersion of either DAG or PE with the substrate also induced a dose-dependent increase in phospholipase A2 activity, whereas sphingomyelin was inhibitory suggesting that the phospholipase A2 more readily hydrolyzed the ether-linked substrate when there was a decrease in the packing density of the bilayer. PIP2, together with either DAG or PE, synergistically stimulated phospholipase A2 activity by about 20-fold, and dramatically decreased the calcium concentration (from mM to nM) required for full activity of the enzyme. The results of this study demonstrate that the presence of anionic phospholipids and the packing characteristics of the bilayer can have pronounced effects on the activity and calcium requirement of an intracellular, arachidonoyl-hydrolyzing phospholipase A2 from macrophages.  相似文献   

8.
Ethanol decreases protein synthesis in cells, although the underlying regulatory mechanisms of this process are not fully established. In the present study incubation of C2C12 myocytes with 100 mm EtOH decreased protein synthesis while markedly increasing the phosphorylation of eukaryotic elongation factor 2 (eEF2), a key component of the translation machinery. Both mTOR and MEK pathways were found to play a role in regulating the effect of EtOH on eEF2 phosphorylation. Rapamycin, an inhibitor of mammalian target of rapamycin, and the MEK inhibitor PD98059 blocked the EtOH-induced phosphorylation of eEF2, whereas the p38 MAPK inhibitor SB202190 had no effect. Unexpectedly, EtOH decreased the phosphorylation and activity of the eEF2 upstream regulator eEF2 kinase. Likewise, treatment of cells with the inhibitor rottlerin did not block the stimulatory effect of EtOH on eEF2, suggesting that eEF2 kinase (eEF2K) does not play a role in regulating eEF2. In contrast, increased eEF2 phosphorylation was correlated with an increase in AMP-activated protein kinase (AMPK) phosphorylation and activity. Compound C, an inhibitor of AMPK, suppressed the effects of EtOH on eEF2 phosphorylation but had no effect on eEF2K, indicating that AMPK regulates eEF2 independent of eEF2K. Finally, EtOH decreased protein phosphatase 2A activity when either eEF2 or AMPK was used as the substrate. Thus, this later action may partially account for the increased phosphorylation of eEF2 in response to EtOH and the observed sensitivity of AMPK to rapamycin and PD98059 treatments. Collectively, the induction of eEF2 phosphorylation by EtOH is controlled by an increase in AMPK and a decrease in protein phosphatase 2A activity.  相似文献   

9.
We have previously characterized a hormonally regulated soluble form of phospholipase A2 (PLA2) in the cultured renal mesangial cell which is similar and possibly identical to the major form in rat kidney. In an attempt to further characterize the mechanisms of regulation of this enzyme we have used epidermal growth factor (EGF), which does not activate polyphosphoinositide-specific phospholipase C in these cells. EGF-enhanced PLA2 activity as assayed by the ability of the soluble extracts of cells to cleave arachidonic acid from the sn-2 position of phosphatidylcholine and phosphatidylethanolamine. This represents a direct demonstration of EGF-induced PLA2 activation which is preserved in a cell-free extract. Phorbol myristate acetate (PMA), as well as 1-oleoyl-2-acetylglycerol, also enhanced PLA2 activity. By contrast, the calcium ionophore A23187 had no effect on extract PLA2 activity. The EGF- and PMA-induced enhanced activity was recovered following fractionation by Mono-Q anion exchange chromatography. The peak of activity comigrated for both agonists, suggesting that both EGF and PMA stimulated the same form of the enzyme. Down-regulation of protein kinase C by pretreatment with PMA resulted in loss of the PMA-induced, but not the EGF-induced, enhancement in PLA2 activity. 8-Bromo-cAMP had no effect upon the PLA2 activity, and did not modulate the EGF effect. Pertussis toxin induced G protein ADP-ribosylation but had no effect upon PLA2 activity, and did not alter the EGF effect. In summary, EGF results in a stable modification of PLA2 activity in glomerular mesangial cells. This enhanced activity is independent of polyphosphoinositide hydrolysis, insensitive to protein kinase C down-regulation, and is not affected by cAMP or pertussis toxin pretreatment of the cells.  相似文献   

10.
Eighty-three percent of polyphosphoinositide-specific phospholipase C activity was recovered in a cytosolic fraction after nitrogen cavitation of turkey erythrocytes. This activity has been purified approximately 50,000-fold when compared to the starting cytosol with a yield of 1.7-5.0%. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the phospholipase C preparation revealed a major polypeptide of 150 kDa. The specific activity of the purified enzyme was 6.7-14.0 mumol/min/mg of protein with phosphatidylinositol 4,5-bisphosphate or phosphatidylinositol 4-phosphate as substrate. Phospholipase C activity was markedly dependent on the presence of Ca2+. The phospholipase C showed an acidic pH optimum (pH 4.0). At neutral pH, noncyclic inositol phosphates were the major products formed by the phospholipase C, while at pH 4.0, substantial formation of inositol 1:2-cyclic phosphate derivatives occurred. Properties of the purified 150-kDa turkey erythrocyte phospholipase C were compared with the approximately 150-kDa phospholipase C-beta and -gamma isoenzymes previously purified from bovine brain (Ryu, S. H., Cho, K. S., Lee, K. Y., Suh, P. G., and Rhee, S. G. (1987) J. Biol. Chem. 262, 12511-12518). The turkey erythrocyte phospholipase C differed from the two mammalian phospholipases with respect to the effect of sodium cholate on the rate of polyphosphoinositide hydrolysis observed. Moreover, when presented with dispersions of pure inositol lipids, phospholipases C-beta and -gamma displayed comparable maximal rates of polyphosphoinositide and phosphatidylinositol hydrolysis. By contrast, the turkey erythrocyte phospholipase C displays a marked preference for polyphosphoinositide substrates.  相似文献   

11.
The effect of various detergents on polyphosphoinositide-specific phospholipase C activity in highly purified wheat root plasma membrane vesicles was examined. The plasma membrane-bound enzyme was solubilized in octylglucoside and purified 25-fold by hydroxylapatite and ion-exchange chromatography. The purified enzyme catalyzed the hydrolysis of phosphatidylinositol 4-phosphate (PIP) and phosphatidylinositol 4,5-bisphosphate (PIP2) with specific activities of 5 and 10 mumol/min per mg protein, respectively. Phosphatidylinositol (PI) was not a substrate. Optimum activity was between pH 6-7 (PIP) and pH 6-6.5 (PIP2). The enzyme was dependent on micromolar concentrations of Ca2+ for activity, and millimolar Mg2+ further increased the activity. Other divalent cations (4 mM Ca2+, Mn2+ and Co2+) inhibited (PIP2 as substrate) or enhanced (PIP as substrate) phospholipase C activity.  相似文献   

12.
The hydrolytic activity of phosphatidylcholine phospholipase D in the synaptosomes from canine brain was examined using a radiochemical assay with 1,2-dipalmitoyl-sn-glycerol-3-phosphoryl[3H]choline as the exogenous substrate. The involvement of G protein(s) in regulation of this enzyme was demonstrated by a 2- to 3-fold stimulation of the basal activity (4.81 +/- 0.44 nmol choline released/mg protein/h) with guanosine 5'-(3-O-thiol)triphosphate (GTP gamma S), guanyl-5'-yl-(beta, gamma-methylene)diphosphonate, aluminum fluoride, or cholera toxin. The stimulation of phospholipase D hydrolytic activity by GTP gamma S was inhibited by 2 mM guanosine 5'-(2-O-thiol)diphosphate. GTP gamma S at the maximum stimulatory concentration (10 microM) had an additive effect on the maximum cholera toxin stimulation of phospholipase D activity. However, the reverse was not true, thus indicating the possibility that more than one G protein may be involved. Furthermore, cholinergic agonists, including acetylcholine, carbachol, and muscarine, were able to increase the phospholipase D hydrolytic activity at low but not maximally stimulatory concentrations of guanine nucleotide. These cholinergic stimulations were antagonized by atropine, a muscarinic blocker. In addition, O-tetradecanoylphorbol 13-acetate, a protein kinase C activator, was able to stimulate the hydrolytic activity of phospholipase D more than 300% in the presence of 0.2 microM GTP gamma S. However, in the absence of GTP gamma S, stimulation was less than 60%. Our results not only indicate that the receptor-G protein-regulated phospholipase D may be directly responsible for the rapid accumulation of choline and phosphatidic acid in the central nervous system but also reveal that muscarinic acetylcholine receptor-G protein-regulated phospholipase D is a novel signal transduction process coupling the neuronal muscarinic receptor to cellular responses.  相似文献   

13.
Retinoic acid, a derivative of vitamin A, is shown to inhibit the levels of inositol phosphates and diacylglycerol by 25-30% when added to intact HL-60 cells at concentrations which induce differentiation. The onset of inhibition occurs after 10 min and reaches a maximum at 45 min. To study the mechanism and the site of action of retinoic acid, the activity of the phosphatidylinositol bisphosphate-specific phospholipase C was studied in cells permeabilized with streptolysin O and in membrane preparations. Phospholipase C activity was stimulated either via the guanine nucleotide regulatory protein (G-protein) or directly by Ca2+. Retinoic acid treatment, in a time- and concentration-dependent manner, led to a decrease in phospholipase C activity when stimulated with either GTP gamma S or NaF, both of which activate the enzyme via the G-protein. By contrast, it had no effect on the enzyme activity when stimulated with Ca2+ alone. This indicates that retinoic acid interferes with the coupling of the G-protein and phospholipase C. A relationship between the inhibition of phospholipase C activity and the induction of differentiation by retinoic acid was investigated. Only a small inhibition of GTP gamma S-stimulated phospholipase C activity was observed when an analogue of retinoic acid, etretine or Ro10-1670, with low differentiating activity, was used. Moreover, no inhibition of the GTP gamma S-stimulated phospholipase C activity was observed in an HL-60 sub-line resistant to retinoic acid. These results suggest that phospholipase C inhibition is an important step in the induction of differentiation.  相似文献   

14.
A phosphatidylinositol-specific phospholipase C (PI-PLC) has been isolated from bovine brain (purification factor of 5.6 x 10(4)). By sodium dodecyl sulfate-polyacrylamide gel electrophoresis, it had a Mr of 57,000. Neither amino nor neutral sugars were detected in the purified enzyme. The pH optimum was 7.0-7.5, and the activity decreased only slightly at pH 8.0. When phosphatidylinositol was used as a substrate, the optimum Ca2+ requirement was 4 mM, and Km was 260 microM. When phosphatidylinositol 4,5-bisphosphate was used, the optimum Ca2+ requirement was 10(-7) M, and the Km was reduced to 90 microM. Lipid specificity studies showed that equal amounts of inositol phosphate and diacylglycerol were released from phosphatidylinositol but 4 times as much inositol 1,4,5-trisphosphate was released from phosphatidylinositol 4,5-bisphosphate. Other lipids, phosphatidylcholine, phosphatidylethanolamine, and sphingomyelin, were not substrates. Failure to detect phosphatidic acid confirmed the absence of a phospholipase D activity in the purified enzyme. Myelin basic protein (MBP) stimulated the PI-PLC activity between 2- and 3-fold. Histone had a small effect only, whereas bovine serum albumin and cytochrome C had no effect. Phosphorylation of MBP reduced the stimulatory effect. Protein-protein interactions between MBP and PI-PLC have been demonstrated both immunologically and by sucrose density gradients. A stoichiometry of 1:1 has been suggested by the latter method. A number of peptides have been prepared by chemical, enzymatic, and synthetic methods. Peptides containing the MBP sequences consisting of residues 24-33 and 114-122 stimulated the PI-PLC but were less effective than the intact protein.  相似文献   

15.
GTP-binding proteins have been implicated to function as key transducing elements in the mechanism underlying receptor activation of a membrane-associated phospholipase C activity. In the present study, the regulation of phospholipase C activity by GTP-binding proteins has been characterized in a detergent-solubilized system derived from bovine brain membranes. Guanosine-5'-(3-O-thio)triphosphate (GTP-gamma-S) and guanyl-5'-yl imidodiphosphate (Gpp(NH)p) stimulated a dose-dependent increase in phospholipase C activity with half-maximal activation at 0.6 microM and 10 microM, respectively. The maximal degree of stimulation due to Gpp(NH)p or GTP-gamma-S was comparable. 100 microM GTP had only a slight stimulatory effect on phospholipase C activity. Adenine nucleotides, 100 microM adenylyl-imidodiphosphate and ATP, did not stimulate phospholipase C activity, indicating that specific guanine nucleotide-dependent regulation of phospholipase C activity was preserved in the solubilized state. Gpp(NH)p or GTP-gamma-S stimulation of phospholipase C activity was time-dependent and required Mg2+.Mg2+ regulated the time course for activation of phospholipase C by guanine nucleotides and the ability of guanine nucleotides to promote an increase in the Ca2+ sensitivity of phospholipase C. 200 microM GDP-beta-S or 5 mM EDTA rapidly reversed the activation due to GTP-gamma-S or Gpp(NH)p. These findings demonstrate that G protein regulation of phospholipase C activity in a bovine brain membrane- solubilized system occurs through a Mg2+ and time-dependent mechanism. Activation is readily reversible upon addition of excess GDP-beta-S or removal of Mg2+.  相似文献   

16.
The activation of protein kinase C was investigated in digitonin-permeabilized human neuroblastoma SH-SY5Y cells by measuring the phosphorylation of the specific protein kinase C substrate myelin basic protein4-14. The phosphorylation was inhibited by the protein kinase C inhibitory peptide PKC19-36 and was associated to a translocation of the enzyme to the membrane fractions of the SH-SY5Y cells. 1,2-Dioctanoyl-sn-glycerol had no effect on protein kinase C activity unless the calcium concentration was raised to concentrations found in stimulated cells (above 100 nM). Calcium in the absence of other activators did not stimulate protein kinase C. Phorbol 12-myristate 13-acetate was not dependent on calcium for the activation or the translocation of protein kinase C. The induced activation was sustained for 10 min, and thereafter only a small net phosphorylation of the substrate could be detected. Calcium or dioctanoylglycerol, when applied alone, only caused a minor translocation, whereas in combination a marked translocation was observed. Arachidonic acid (10 microM) enhanced protein kinase C activity in the presence of submaximal concentrations of calcium and dioctanoylglycerol. Quinacrine and p-bromophenacyl bromide did not inhibit calcium- and dioctanoylglycerol-induced protein kinase C activity at concentrations which are considered to be sufficient for phospholipase A2 inhibition.  相似文献   

17.
Membranes from human placenta contain proteins which inhibit the activity of phospholipases A2 by binding to phospholipid thus impeding substrate availability. We used unilamellar mixed liposomes and a partially purified cytosolic phospholipase A2 from placenta for characterizing this substrate-depleting activity. A major portion of these inhibitory proteins was released by extracting washed membranes with a Ca+(+)-chelator. Biochemical fractionation and systematic analysis resulted in the unequivocal identification of a series of annexin proteins. We describe a straightforward procedure which allows to obtain 8 annexins from placenta either in pure form or as a mixture of two annexins. One of them was obtained in two forms which had the same molecular mass of 68 kDa but differed in charge. We also present suggestive evidence for a novel annexin I-related polypeptide of Mr 45,000 which is an excellent in vitro substrate for protein kinase C. We estimate that about 2% of the total placental membrane proteins are annexins. For achieving half inhibition of phospholipase A2 activity with pure annexins, up to a 6.5-fold difference in the amounts of protein was observed when calculated on a molar basis. This suggests specificity of individual annexin species.  相似文献   

18.
Peroxiredoxin 6 (Prdx6) is a bifunctional protein with glutathione peroxidase and phospholipase A(2) (PLA(2)) activities, and it alone among mammalian peroxiredoxins can hydrolyze phospholipids. After identifying a potential catalytic triad (S32, H26, D140) from the crystal structure, site-specific mutations were used to evaluate the role of these residues in protein structure and function. The S32A mutation increased Prdx6 alpha-helical content, whereas secondary structure was unchanged by mutation to H26A and D140A. Lipid binding by wild-type Prdx6 to negatively charged unilamellar liposomes showed an apparent rate constant of 11.2 x 10(6) M(-1) s(-1) and a dissociation constant of 0.36 microM. Both binding and PLA(2) activity were abolished in S32A and H26A; in D140A, activity was abolished but binding was unaffected. Overoxidation of the peroxidatic C47 had no effect on lipid binding or PLA(2) activity. Fluorescence resonance energy transfer from endogenous tryptophanyls to lipid probes showed binding of the phospholipid polar head in close proximity to S32. Thus, H26 is a site for interfacial binding to the liposomal surface, S32 has a key role in maintaining Prdx6 structure and for phospholipid substrate binding, and D140 is involved in catalysis. This putative catalytic triad plays an essential role for interactions of Prdx6 with phospholipid substrate to optimize the protein-substrate complex for hydrolysis.  相似文献   

19.
In has been found that sphingosine, propranolol, imipramine and phorbol ester (12-O-tetradecanoylphorbol-13-acetate, TPA) have a stimulatory effect on phospholipase D activity in glioma C6 cells. The cells were prelabelled with [1-(14)C]palmitic acid and phospholipase D-mediated synthesis of [(14)C]phosphatidylethanol was measured. The enhancing effect of TPA was almost completely blocked by a specific protein kinase C inhibitor, GF 109203X. In contrast, GF 109203X failed to inhibit the sphingosine, imipramine and propranolol stimulatory effects, indicating that their stimulation was independent of protein kinase C. The effect of TPA on phospholipase D was also blocked by imipramine and propranolol, whereas sphingosine additively potentiated TPA-mediated phospholipase D activity, both at shorter and longer (2-60 min) times of incubation. These results suggest that in glioma C6 cells, sphingosine is not only involved in a different phospholipase D activation than the TPA regulatory system, but also that it operates in a different compartment of the cell.  相似文献   

20.
Turkey erythrocyte membranes possess a phospholipase C that is markedly activated by P2Y-purinergic receptor agonists and guanine nucleotides. Reconstitution of [3H]inositol-labeled turkey erythrocyte membranes with guanine nucleotide regulatory protein (G-protein) beta gamma subunits resulted in inhibition of both AlF-4-stimulated adenylate cyclase and AlF-4-stimulated phospholipase C activities. The apparent potency (K0.5 approximately 1 microgram or 20 pmol of beta gamma/mg of membrane protein) of beta gamma subunits for inhibition of each enzyme activity was similar and occurred with beta gamma purified by different methodologies from turkey erythrocyte, bovine brain, or human placenta membranes. In contrast to the effect on AlF-4-stimulated activity, the stimulatory effect on phospholipase C of the P2Y-purinergic receptor agonist 2-methylthioadenosine 5'-triphosphate in the presence of guanine nucleotides was potentiated by 50-100% in a concentration-dependent manner by reconstitution of beta gamma subunits. beta gamma subunits did not affect the K0.5 value of 2-methylthioadenosine 5'-triphosphate for the stimulation of phospholipase C activity. These results indicate that beta gamma subunits influence phospholipase C activity in a concentration range similar to that necessary for regulation of adenylate cyclase activity and suggest the involvement of a G-protein possessing an alpha beta gamma heterotrimeric structure in coupling hormone receptors to phospholipase C.  相似文献   

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