共查询到20条相似文献,搜索用时 0 毫秒
1.
K K Griendling M B Taubman M Akers M Mendlowitz R W Alexander 《The Journal of biological chemistry》1991,266(23):15498-15504
Phosphoinositide-specific phospholipase C (PLC) activities have been partially purified from cultured vascular smooth muscle cells and analyzed for substrate specificity, calcium and pH requirements, and molecular weight. The purification procedure involved DEAE-cellulose and heparin-Sepharose chromatographies followed by Mono Q and size exclusion high performance liquid chromatography. This technique resolves multiple peaks of activity using phosphatidylinositol (PI) and PI 4,5-bisphosphate (PIP2) as substrates. The major peak was purified to near homogeneity as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. PLC activity in vascular smooth muscle cells can be divided into two types based on their calcium and pH requirements, substrate preferences, and molecular weights. The low molecular weight PLC hydrolyzes both PI and PIP2, has a molecular mass of 58 kDa, requires the most calcium for full activation, and has a PI-pH profile that shifts slightly with calcium concentration. Screening a cDNA library with oligonucleotides directed against several of the known PLCs identified a highly expressed PLC cDNA that is 99% homologous to PLC-alpha, suggesting that this low molecular weight peak in fact corresponds to PLC-alpha. The high molecular mass peak (157 kDa) shows much greater activity against PI than PIP2, is active at lower calcium concentrations, and has a PI-pH optimum of 5.0 regardless of calcium concentration. Each of the PIP2 PLC activities is strongly dependent on the relative levels of calcium and pH in the assay buffer. These observations suggest that vascular smooth muscle contains both a high and low molecular weight PLC whose activities are affected markedly by the changes in calcium and pH accompanying hormonal stimulation of the cell. 相似文献
2.
Characterization of phosphatidylinositol-specific phospholipase C defects associated with thrombin-induced mitogenesis 总被引:1,自引:0,他引: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
3.
Cocco L Faenza I Fiume R Maria Billi A Gilmour RS Manzoli FA 《Biochimica et biophysica acta》2006,1761(5-6):509-521
Over the last years, evidence has suggested that phosphoinositides, which are involved in the regulation of a large variety of cellular processes both in the cytoplasm and in the plasma membrane, are present also within the nucleus. A number of advances has resulted in the discovery that phosphoinositide-specific phospholipase C signalling in the nucleus is involved in cell growth and differentiation. Remarkably, the nuclear inositide metabolism is regulated independently from that present elsewhere in the cell. Even though nuclear inositol lipids hydrolysis generates second messengers such as diacylglycerol and inositol 1,4,5-trisphosphate, it is becoming increasingly clear that in the nucleus polyphosphoinositides may act by themselves to influence pre-mRNA splicing and chromatin structure. Among phosphoinositide-specific phospholipase C, the beta(1) isoform appears to be one of the key players of the nuclear lipid signaling. This review aims at highlighting the most significant and up-dated findings about phosphoinositide-specific phospholipase C beta(1) in the nucleus. 相似文献
4.
Crystallization of phosphatidylinositol-specific phospholipase C from Bacillus cereus. 总被引:1,自引:0,他引:1
Phosphatidylinositol-specific phospholipase C (PI-PLC) cleaves phosphoinositides into two parts, lipid-soluble diacylglycerol and the water-soluble phosphorylated inositol. Two crystal forms of Bacillus cereus PI-PLC have been obtained by the vapor diffusion technique. Hexagonal crystals were grown from solutions containing polyethylene glycol (PEG; 4,000 to 8,000 D). The space group of these hexagonal crystals is P6(1)22 (or the enantiomorphic space group P6(5)22), with cell constants a = b = 133 A, and c = 231 A. The crystals diffract to 2.8 A. The second crystalline form was grown from a two-phase PEG (600 D)-sodium citrate solution. The phase diagram and PI-PLC distribution between phases has been determined. The enzyme crystallizes from the PEG-rich phase. The crystals are orthorhombic with space group P2(1)2(1)2(1) (a = 45 A, b = 46 A, c = 160 A), and contain one PI-PLC monomer per asymmetric unit. The orthorhombic crystals diffract to 2.5 A. Both the hexagonal and orthorhombic forms are suitable for crystallographic studies. 相似文献
5.
Purification and characterization of phosphatidylinositol-specific phospholipase C from bovine platelets 总被引:8,自引:0,他引:8
Phosphatidylinositol-specific phospholipase C was purified to homogeneity from soluble fraction of bovine platelets by ammonium sulfate fractionation, hydrophobic chromatography, DEAE ion exchange chromatography and gel filtration. The purified enzyme has a narrow pH optimum ranging from 6.5 to 7.5 and the molecular weight of the enzyme was estimated to be 143,000 by sodium dodecyl sulfate slab gel electrophoresis. The purified enzyme requires Ca2+ strictly for activity, which was markedly enhanced in the presence of arachidonate. No enhancement of the activity was observed in the presence of purified calmodulin. The activity was markedly inhibited in the presence of quinacrine but no inhibition by indomethacin was observed. 相似文献
6.
Amphiphilic,glycophosphatidylinositol-specific phospholipase C (PI-PLC)-insensitive monomers and dimers of acetylcholinesterase 总被引:5,自引:0,他引:5
Suzanne Bon Terrone L. Rosenberry Jean Massoulié 《Cellular and molecular neurobiology》1991,11(1):157-172
1. In a recent study, we distinguished two classes of amphiphilic AChE3 dimers in Torpedo tissues: class I corresponds to glycolipid-anchored dimers and class II molecules are characterized by their lack of sensitivity to PI-PLC and PI-PLD, relatively small shift in sedimentation with detergent, and absence of aggregation without detergent. 2. In the present report, we analyze the amphiphlic or nonamphiphilic properties of globular AChE forms in T28 murine neural cells, rabbit muscle, and chicken muscle. The molecular forms were identified by sucrose gradient sedimentation in the presence and absence of detergent and analyzed by nondenaturing charge-shift electrophoresis. Some amphiphilic forms showed an abnormal electrophoretic migration in the absence of detergent, because of the retention of detergent micelles. 3. We show that the amphiphilic monomers (G1a) from these tissues, as well as the amphiphilic dimers (G2a) from chicken muscle, resemble the class II dimers of Torpedo AChE. We cannot exclude that these molecules possess a glycolipidic anchor but suggest that their hydrophobic domain may be of a different nature. We discuss their relationship with other cholinesterase molecular forms. 相似文献
7.
1. The distribution of phosphatidylinositol3, phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate hydrolysis or phosphatidylinositol-specific phospholipase C (PI-PLC), activity in the bull reproductive system showed the highest specific activity in the isolated spermatozoa (SZ) followed by testis and different epididymal segments. Both the head and tail fractions of SZ were active. 2. The optimal solubilization of the enzyme from SZ was obtained with 0.2% Triton X-100 or at 0.05% detergent concentration when combined with a 60 sec sonication. The sucrose gradient centrifugation showed that PI-PLC was enriched in membrane fraction distinct from mitochondria and acrosomes. 3. The enzyme was purified by ammonium sulphate precipitation and fractionations by hydrophobic interaction chromatography, gel filtration, Con A-Sepharose affinity and chromatofocusing columns. The purified enzyme was able to hydrolyse all phosphatidylinositol substrates with optimum at pH 7.0 and activation by Ca2+, Cd2+ and Mn2+ but not phospholipids lacking the inositol residue. 4. In PAGE (8-25% gradient) the purified (aggregated) enzyme did not enter the gel. In SDS-PAGE two closely located bands were found with Mr-values of 15,000 and 18,000. Isoelectric focusing showed a wide band at pl 4.5-5.1. 5. Gel filtration resulted in a broad elution peak indicating multiple molecular forms (aggregates); the basic form had an apparent molecular weight of 100,000. The binding of the enzyme to Con A-Sepharose indicated that the enzyme is a glycoprotein. 相似文献
8.
Pollen tubes of Lilium longiflorum Thunb. cv. White Europe contain three distinguishable phosphatidylinositol phospholipase C activities (EC 3.1.4.10). Two of these are particulate and have optima at pH 5.2 and 7.0, respectively. The third one, a cytosolic activity, has an optimum at pH 6.0. The distribution of radioactivity in reaction products from phosphatidylinositol, labeled in either the inositol, glycerol or phosphate moiety, indicates that the three phospholipase activities cleave only the bond between glycerol and phosphate. The dependence on divalent cations slightly differs, though Ca2+ is the most stimulatory ion species for all the three enzyme activities. Activity is not observed in the presence of EDTA. When anionic phospholipids are mixed with phosphatidylinositol substrate an increase in phosphatidylinositol phospholipase C activities is observed, except for the particulate activity with an optimum at pH 5.2. Phosphatidylcholine and phosphatidylethanolamine are inhibitory. 相似文献
9.
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
10.
Adibhatla RM 《Biochemical and biophysical research communications》2012,419(2):447; author reply 448-447; author reply 449
11.
Ectoenzyme release from rat liver and kidney by phosphatidylinositol-specific phospholipase C 总被引:3,自引:0,他引:3
Ectoenzyme release from rat liver and kidney by phosphatidylinositol (PI)-specific phospholipase C of Bacillus thuringiensis was studied. Alkaline phosphatase and 5'-nucleotidase were released from rat kidney slices to extents of up to 60% and 30%, respectively. Release of alkaline phosphatase was observed at lower amounts of PI-specific phospholipase C than that of 5'-nucleotidase. Both enzymes were more easily released from microsomal fractions or free cells. From kidney cells, alkaline phosphatase was released without cell lysis, and more than 80% release of alkaline phosphatase was observed at 3.8% hydrolysis of PI. Isoelectric focusing profiles of alkaline phosphatase released by PI-specific phospholipase C were significantly different from the control in the cases of both rat liver and kidney. Lubrol-solubilized alkaline phosphatase was eluted at the void volume of a Toyopearl HW-55 column, while the enzyme obtained by further treatment with PI-specific phospholipase C was eluted in the lower-molecular-weight region corresponding to 100,000-110,000 daltons. Furthermore, Lubrol-solubilized phosphatase became more thermostable on treatment with PI-specific phospholipase C. 相似文献
12.
Release of alkaline phosphatase from membranes by a phosphatidylinositol-specific phospholipase C. 总被引:16,自引:6,他引:10
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Purified phosphatidylinositol-specific phospholipase C from Staphylococcus aureus released a substantial proportion of the total alkaline phosphatase activity from a wide range of tissues from several mammalian species. Co-purification of the phospholipase C and alkaline phosphatase-releasing activities and the inhibition of both these activities by iso-osmotic salt solutions suggested that the releasing effect was unlikely to be due to a contaminant. 相似文献
13.
Improved purification and biochemical properties of phosphatidylinositol-specific phospholipase C from Bacillus thuringiensis 总被引:4,自引:0,他引:4
Monophosphatidylinositol inositol phosphohydrolase (phosphatidylinositol-specific phospholipase C. PtdIns-PLC. EC 3.1.4.10) has been purified from a Bacillus thuringiensis culture supernatant and from the cellular fraction of a recombinant Escherichia coli clone containing the PtdIns-PLC gene from B. thuringiensis. The two-step purification procedure involved ion-exchange chromatography on DEAE-Sepharose followed by separation on a Mono-Q/FPLC-column with yields of 32% and 50%, respectively. The molecular mass was determined to be 34 kDa by SDS/PAGE. The isoelectric point of the enzyme was 5.15. The amino-terminal sequences were shown to be identical for the enzymes purified from both organisms. PtdIns-PLC was inhibited by divalent cations using mixed micelles of Triton X-100 and pure phosphatidylinositol. PtdIns-PLC activity was detectable on polyacrylamide gels by activity staining on phosphatidylinostiol-containing agarose. 相似文献
14.
Inhibition of phosphatidylinositol-specific phospholipase C by phosphonate substrate analogues 总被引:1,自引:0,他引:1
M S Shashidhar J J Volwerk J F Keana O H Griffith 《Biochimica et biophysica acta》1990,1042(3):410-412
Non-hydrolysable analogues of phosphatidylinositol were synthesized and tested as inhibitors of phosphatidylinositol-specific phospholipase C from Bacillus cereus. In these molecules, the phosphodiester bond of phosphatidylinositol hydrolyzed by the phospholipase was replaced by a phosphonate linkage and a simpler hydrophobic group replaced the diacylglycerol moiety. One of the phosphonates also contained a carboxylate functional group suitable for matrix attachment. All three synthetic phosphonates inhibited the phospholipase C activity in a concentration-dependent manner, with the analogue most closely resembling the structure of the natural substrate, phosphatidylinositol, being the most potent inhibitor. The data indicate that phosphonate analogues of phosphatidylinositol may be useful for study of phospholipase C and other proteins interacting with myo-inositol phospholipids. 相似文献
15.
The interactions of PI-PLC with nonsubstrate zwitterionic [phosphatidylcholine (PC)] and anionic [phosphatidylmethanol (PMe), phosphatidylserine, phosphatidylglycerol, and phosphatidic acid] interfaces that affect the catalytic activity of PI-PLC have been examined. PI-PLC binding is strongly coupled to vesicle curvature and is tighter at acidic pH for all of the phospholipids examined. PI-PLC binds to small unilamellar vesicles (SUVs) of anionic lipids with much higher affinity (K(d) is 0.01-0.07 microM for a site consisting of n = 100 +/- 25 lipids when analyzed with a Langmuir adsorption isotherm) than to zwitterionic PC SUVs (K(d) is 5-20 microM and n = 8 +/- 3). The binding to PC surfaces is dominated by hydrophobic interactions, while binding to anionic surfaces is dominated by electrostatic interactions. The contributions of specific cationic side chains and hydrophobic groups at the rim of the alpha beta-barrel to zwitterionic and anionic vesicle binding have been assessed with mutagenesis. The results are used to explain how PC activates the enzyme for both phosphotransferase and cyclic phosphodiesterase activities. 相似文献
16.
Shao C Shi X Wehbi H Zambonelli C Head JF Seaton BA Roberts MF 《The Journal of biological chemistry》2007,282(12):9228-9235
The crystal structure of the W47A/W242A mutant of phosphatidylinositol-specific phospholipase C (PI-PLC) from Bacillus thuringiensis has been solved to 1.8A resolution. The W47A/W242A mutant is an interfacially challenged enzyme, and it has been proposed that one or both tryptophan side chains serve as membrane interfacial anchors (Feng, J., Wehbi, H., and Roberts, M. F. (2002) J. Biol. Chem. 277, 19867-19875). The crystal structure supports this hypothesis. Relative to the crystal structure of the closely related (97% identity) wild-type PI-PLC from Bacillus cereus, significant conformational differences occur at the membrane-binding interfacial region rather than the active site. The Trp --> Ala mutations not only remove the membrane-partitioning aromatic side chains but also perturb the conformations of the so-called helix B and rim loop regions, both of which are implicated in interfacial binding. The crystal structure also reveals a homodimer, the first such observation for a bacterial PI-PLC, with pseudo-2-fold symmetry. The symmetric dimer interface is stabilized by hydrophobic and hydrogen-bonding interactions, contributed primarily by a central swath of aromatic residues arranged in a quasiherringbone pattern. Evidence that interfacially active wild-type PI-PLC enzymes may dimerize in the presence of phosphatidylcholine vesicles is provided by fluorescence quenching of PI-PLC mutants with pyrene-labeled cysteine residues. The combined data suggest that wild-type PI-PLC can form similar homodimers, anchored to the interface by the tryptophan and neighboring membrane-partitioning residues. 相似文献
17.
In the culture supernatant of Cytophaga sp. we detected an enzyme that converted glycosylphosphatidyl-inositol-anchored acetylcholinesterase to the hydrophilic form. This enzyme had a cleavage specificity of a phospholipase C. It hydrolyzed phosphatidylinositol but did not act on phosphatidylcholine. On gel filtration the enzyme migrated with an apparent molecular mass of about 17 kDa. It displayed maximal activity between pH 6-6.5 and did not require cofactors for the expression of catalytic activity. Mercurials and zinc ions inhibited the enzyme and its activity also decreased with increasing ionic strength in the assay. With acetylcholinesterase as substrate optimal activity was obtained in pure micelles of Triton X-100, whereas in mixed micelles containing Triton X-100 and phosphatidylcholine the activity was reduced. The enzyme from Cytophaga sp. showed little activity towards acetylcholinesterase embedded in intact membranes where more than 1000-times higher concentrations of phosphatidylinositol-specific phospholipase C was necessary to solubilize acetylcholinesterase as compared to acetylcholinesterase in detergent micelles. 相似文献
18.
19.
In the present paper we studied the involvement of the phosphatidylinositol-specific PLC (PI-PLC)/protein kinase C (PKC) pathway in (Na+ + K+)ATPase stimulation by heme in Leishmania amazonensis promastigotes. Heme stimulated the PKC-like activity with a concentration of 50 nM. Interestingly, the maximal stimulation of the PKC-like activity promoted by phorbol ester was of the same magnitude promoted by heme. However, the stimulatory effect of heme is completely abolished by ET-18-OCH3 and U73122, specific inhibitors of PI-PLC. (Na+ + K+)ATPase activity is increased in the presence of increased concentrations of heme, being maximally affected at 50 nM. This effect was completely reversed by 10 nM calphostin C, an inhibitor of PKC. Thus, the effect of 50 nM heme on (Na+ + K+)ATPase activity is completely abolished by ET-18-OCH3 and U73122. Taken together, these results demonstrate that the heme receptor mediates the stimulatory effect of heme on the (Na+ + K+)ATPase activity through a PI-PLC/PKC signaling pathway. 相似文献
20.
We investigated the effect of thyroid hormone on phosphatidylinositol-specific phospholipase C activity in rat liver. Thyroidectomy increased the activity of the enzyme. Thyroid hormone (T4, 40 micrograms) administration to thyroidectomized-rats decreased phospholipase C activity. The inhibition induced by thyroid hormone was of a non-competitive type. The higher concentration of Ca2+ strongly inhibited the activity of the enzyme obtained from thyroidectomized-rats' liver in vitro. The diminished activity of the enzyme obtained from thyroxine-treated-thyroidectomized-rats was recovered by pretreatment of the enzyme with EGTA. The activity of the enzyme derived from thyroidectomized-rats was not affected by EGTA treatment. These results suggest that thyroid hormone decreases the activity of phosphatidylinositol-specific phospholipase C activity through the mobilization of Ca2+ in the intracellular space. 相似文献