Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.4.3 (phospholipase C)
18,461 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

SH3 (Src homology 3) domains are found in many signaling proteins and appear to function as binding modules for cytoplasmic target proteins. The solution structure of the SH3 domain of human phospholipase C-gamma (PLC-gamma) was determined by two-dimensional 1H NMR analysis. This SH3 domain is composed of eight antiparallel beta strands consisting of two successive "Greek key" motifs, which form a barrel-like structure. The conserved aliphatic and aromatic residues form a hydrophobic pocket on the molecular surface, and the conserved carboxylic residues are localized to the periphery. The hydrophobic pocket may serve as a binding site for target proteins. Analysis of the slowly exchanging amide protons by NMR measurements indicates that despite containing a high content of beta structure, the SH3 domain of PLC-gamma is flexible.
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PMID:Solution structure of the SH3 domain of phospholipase C-gamma. 768 65

We have recently demonstrated that phospholipase C (PLC) activity on membranes decreases in the presence of membrane-active peptides such as alamethicin, gramicidin S, and melittin [Rao, N. M. (1992) Biochem. Biophys. Res. Commun. 182, 682-688]. Since these peptides affect lipid packing in the membrane and induce nonbilayer phases depending on the lipid composition, we tested for the sensitivity of PLC activity to lipid packing. We monitored PLC activities on four lipid systems which demonstrate a transition from the bilayer to the nonbilayer phase as a function of one of the components. The four model systems are (1) dioleoylphosphatidylcholine (DOPC) and dioleoylphosphatidylethanolamine (DOPE); (2) DOPE, DOPC, and cholesterol; (3) DOPE and lysophosphatidylcholine; and (4) DOPC and gramicidin D. On all four lipid systems, the PLC activity was high for lipid in the bilayer phase and decreased as the phase changed to the nonbilayer phase. The phase changes were also monitored in PLC assay conditions on the four model systems by 31P NMR to confirm the observations made with PLC. These results suggest that the lipid in bilayer and nonbilayer phases was differentially susceptible to PLC; hence, PLC activity may be used to monitor isothermal phase transitions at physiological conditions.
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PMID:Sensitivity of phospholipase C (Bacillus cereus) activity to lipid packing in sonicated lipid mixtures. 768 34

We have determined the solution structure of an alpha-toxin, CsE-V, isolated from the venom of the New World scorpion Centruroides sculpturatus Ewing (CsE). This toxin causes spontaneous rhythmic contractions in muscle. Unlike other New World toxins from CsE, this protein exhibits amino acid insertions and deletions at locations similar to Old World toxins and may thus represent a transition protein between the New World and Old World scorpion alpha-toxins. Sequence-specific assignments were made using 600 MHz 1H two-dimensional NMR data. NOESY, PH-COSY and amide-exchange data were used to deduce constraints for molecular modeling calculations. Distance geometry and dynamical simulated annealing calculations were performed to generate a family of 70 structures free of constraint violations. With respect to this family of structures, the energy-minimized average structure had root-mean-square deviations of 0.74 and 1.32 A for backbone and all atoms, respectively (excluding the C-terminal dipeptide, which is disordered). As with other scorpion toxins, the secondary structure of CsE-V consists of an alpha-helix, a three-strand anti-parallel beta-sheet, four beta-turns, and a hydrophobic patch that includes tyrosine residues in herringbone configuration. Unlike the CsE-v3 and -v1 proteins from C. sculpturatus, all of the proline residues were found to be in the trans configuration. The alpha-helix is slightly longer in CsE-V. The overall structure is more similar to the Old World alpha-toxin AaH-II from Androctonus australis Hector (r.m.s.d 1.59 A for backbone atoms of matching residues) than to the New World alpha-toxin CsE-v3 (r.m.s.d. 1.91 A). These structural data on CsE-V add further to our knowledge of the conformational repertoire exhibited by these sodium channel-binding neurotoxins.
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PMID:Solution structure of an Old World-like neurotoxin from the venom of the New World scorpion Centruroides sculpturatus Ewing. 773 52

13C nuclear magnetic resonance spectroscopy of lipoproteins, isolated from the insect Manduca sexta, has been employed to probe the microenvironment of diacylglycerol (DG), their major neutral lipid component. Natural abundance 13C NMR spectra of high density lipophorin exhibited several well-separated resonances derived from its lipid moiety, including those for the carbonyl carbon atoms of phospholipid and DG fatty acyl chains in the region of 175-180 ppm. To verify the assignment of the DG acyl chain carbonyl carbon resonances, di[1-13C]oleoylglycerol high density lipophorin was isolated after instilling a bolus of tri[1-13C]oleoylglycerol into the midgut of larvae fed a fat-free diet. 13C NMR spectra of the isolated lipoprotein revealed a specific and dramatic enrichment of resonances at 175.5 ppm. Expansion of this region revealed two resonances separated by 0.08 ppm. These were assigned as 1,2- and 1,3- isomers of DG, the latter presumably arising from spontaneous acyl chain migration of 1,2-DG following lipoprotein isolation. On the basis of compositional and structural analysis of this lipoprotein, it is postulated that these DG species are localized predominantly in the hydrophobic core of the particle. By contrast, natural abundance 13C NMR spectra of the DG-rich, low density lipophorin (LDLp) subspecies revealed two additional resonances, separated by 0.2 ppm, that were tentatively assigned as 1,2- and 1,3-DG present at the surface of the particle. The verify this assignment, experiments employing phospholipase C, to convert lipophorin surface associated phospholipid into DG, were performed.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Identification and localization of two distinct microenvironments for the diacylglycerol component of lipophorin particles by 13C NMR. 775 6

A synthetic tripeptide (pGLU-LEU-TRP-OCH3) Pol 509, derived from snake venom, was studied directly by analyzing the interactions with synthetic lipid bilayers using NMR spectroscopy. Functional studies were also performed by measuring the effects: i), on early biochemical events (adenyl cyclase and phospholipase C activation products), intermediate (surface Ag expression) and late (DNA synthesis) parameters following B-cell activation elicited by PPD-linkage to specific membrane Ig; and ii), on the presentation of PPD to Ag-specific T-cell lines. Comparative experiments using PMA and IFN-gamma were also performed. We found that all parameters studied were affected by Pol 509 treatment. In fact, while PPD linkage to mlg reversed the balance between cAMP and IP3 existing in unstimulated EBV-B cells, Pol 509 reduced the PPD-induced accumulation of cAMP to control values and induced a further decrease of IP3 level. Pol 509-mediated decrease of these second messenger levels was accompanied by a slight increase of HLA-DR molecule expression and DNA synthesis inhibition. Furthermore, Pol 509 enhanced the efficiency of PPD presentation to T-cell lines. Taken together, these observations suggest that Pol 509, which enhances Ag presentation by modifying second messenger levels, may be considered as a new immunomodulatory drug with immunopotentiating activity.
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PMID:A new tripeptide, Pol 509, influences biochemical events associated with antigen presentation efficiency of PPD-specific EBV-B cells. 810 May 58

A series of mixed-chain diacyl-PCs which contain an omega-COOH on the sn-2 chain [1-Cx-2-Cy-(COOH)-PC] and bolaform (1-Cx-2,2'-Cy-1'-Cx-PC) phosphatidylcholines were synthesized and examined as substrates for phospholipase A2 (Naja naja naja) and C (Bacillus cereus). There is very little detectable phospholipase A2 activity toward pure micellar 1-acyl-2-acyl-(omega-COOH) species. In addition, when these same omega-COOH species are present at concentrations above their CMCs, they are potent inhibitors of phospholipase A2 hydrolysis of other micellar lipids. In contrast, phospholipase C hydrolysis of the same 1-acyl-2-acyl-omega-COOH)-PC species proceeds with rates comparable to that of diheptanoyl-PC. The bolaform lipids, which are tethered through a common sn-2 acyl chain, (e.g., 1-C8-2,2'-C12-1'-C8-PC) display quite different kinetic results. Under limiting Ca2+ conditions (100 microM) all the available sn-2 acyl bonds of the dimer are hydrolyzed. However, at high Ca2+ concentrations (1-10 mM) the reaction curves have a biphasic nature, characterized by an initial burst of activity followed by much slower rate. This is consistent with only the micellar 1-acyl-2-acyl-(omega-COOH)-PC produced in situ from phospholipase A2 hydrolysis of the dimer acting as an inhibitor of subsequent phospholipase A2 activity. Phospholipase C hydrolysis of the PC dimer and the sn-2 omega-COOH PC is rapid, with both available glycerophosphate groups cleaved at presumably the same rate. These results are discussed in terms of the unique physical properties (as measured by NMR and fluorescence experiments) of these phospholipids.
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PMID:Micellar bolaform and omega-carboxylate phosphatidylcholines as substrates for phospholipases. 817 75

SH2 and SH3 domains are small protein modules that mediate protein-protein interactions in signal transduction pathways that are activated by protein tyrosine kinases. SH2 domains bind to short phosphotyrosine-containing sequences in growth factor receptors and other phosphoproteins. SH3 domains bind to target proteins through sequences containing proline and hydrophobic amino acids. SH2 and SH3 domain containing proteins, such as Grb2 and phospholipase C gamma, utilize these modules in order to link receptor and cytoplasmic protein tyrosine kinases to the Ras signaling pathway and to phosphatidylinositol hydrolysis, respectively. The three-dimensional structures of several SH2 and SH3 domains have been determined by NMR and X-ray crystallography, and the molecular basis of their specificity is beginning to be unveiled.
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PMID:SH2/SH3 signaling proteins. 819 36

With respect to normal tissues, 31P NMR spectra of tumors usually exhibit elevated phosphomonoester (PME) and phosphodiester (PDE) signals, arising from phospholipid metabolites such as phosphocholine (PCho) and glycerophosphocholine (GroPCho) (and/or ethanolamine analogues). PME and PDE resonances may undergo significant alterations during tumor growth, at early stages of tumor response to treatment or following cell differentiation and maturation. The enzymatic mechanisms which regulate these alterations are scarcely understood. Recent studies on agonist-induced phosphatidylcholine (PC) hydrolysis by PC-specific phospholipase C (PC-plc) in cells stimulated by hormones or growth factors suggest the hypothesis that repeated transient activations of this enzyme may also contribute to the elevation of PCho levels in tumor NMR spectra. This paper reports the first direct evidence on neutral active PC-plc activity in a tumour cell system, Friend leukemia cells, either in the undifferentiated (FLC) or differentiated state (dFLC). Cell homogenates were incubated in the presence of mixed diheptanoylphosphatidylcholine/sphingomyelin unilamellar vesicles (SLUV), which were previously shown to represent a good substrate for bacterial plc. 31P NMR analyses allowed the simultaneous detection and quantification of phosphorylated metabolites produced in tumor cell homogenates by PC-plc activity, as well by enzymes active in the PC deacylation pathway. With respect to FLC, dFLC homogenates exhibited higher PC-plc activity and lower accumulation of a deacylation product, GroPCho, in agreement with the elevation in the [PCho]/[GroPCho] ratio, already reported in 31P NMR spectra of intact differentiated cells. The direct detection of PC-plc in this cell system opens novel biochemical interpretations on a series of oncological observations, such as a) transient increases in the levels of PCho and PC-derived diacylglycerols reported in immature or in transformed cells in response to agonist-receptor interactions and b) accumulation of mobile lipids in tumor cell membranes and tissues.
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PMID:Detection of neutral active phosphatidylcholine-specific phospholipase C in Friend leukemia cells before and after erythroid differentiation. 829 51

The action of phospholipase C (PLC) from Bacillus cereus on phosphatidylglycerol (PG), derived from egg yolk phosphatidylcholine (PC), was examined in an ether-water mixture. The PLC cleavage of PG and PC followed a Michaelis-Menten kinetics with apparent Vmax values per 1 microgram enzyme of 0.26 and 0.91 mumol.min-1 and Km values of 10 and 12 mM, respectively. When the same enzymic reaction was carried out in minimally buffered aqueous solution of 1% Triton X-100, the decrease in pH with respect to phospholipid cleavage was as expected with PC but much less pronounced with PG. This could be accounted for by the formation of a cyclic glycerophosphate, rather than alpha-glycerophosphate, in the PLC hydrolysis of PG. Examination of the chemical nature of the water-soluble product of PG by phosphorus nuclear magnetic resonance (31P NMR) revealed a single band at 2.31 ppm, while the bands of alpha-glycerophosphate and beta-glycerophosphate appeared at 5.12 and 4.57 ppm, respectively. Basic hydrolysis of the phospholipase cleavage product of PG (0.1 M NaOH for 1 min at 80 degrees C) followed by neutralization shifted its 31P NMR band to 5.18 ppm, which practically coincided with that of alpha-glycerophosphate. Analogous experiments were carried out with PG labeled with 3H at the carbon 2 of the glycerol headgroup ([3H]PG). Autoradiography of thin layer chromatography (TLC) of the [3H]PG enzymic hydrolyzate displayed a single 3H-labeled compound, which could be converted to alpha-glycerophosphate by basic hydrolysis. These results strongly suggest that the phosphate headgroup of PG is cleaved off by PLC as 1,3-cyclic glycerophosphate. A series of PLC experiments with phosphatidyl dihydroxyacetone and phosphatidyl 1,3-propanediol as model substrates supported this assignment. Two-dimensional homonuclear 1H NMR correlated spectra as well as infrared spectra carried out on the isolated sodium salt of this product could further confirm such a structure. The unique structure and chemical nature of 1,3-cyclic glycerophosphate may bear a distinct physiological function.
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PMID:Formation of 1,3-cyclic glycerophosphate by the action of phospholipase C on phosphatidylglycerol. 831 78

Hydrolysis of phospholipids in biological membranes by phospholipase C (PLC) produces an important second messenger molecule, 1,2-diacylglycerol (DAG), that is essential for the activation of protein kinase C (PKC). While the effects of DAG on model membranes have been investigated earlier, studies on physical properties of DAG introduced into phospholipid bilayers by PLC have been lacking. We present an NMR approach for studying structural and kinetic aspects of PLC-mediated hydrolysis of 13 carbonyl-enriched phospholipids in model membranes (small unilamellar vesicles). The product DAG is readily detected by 13C NMR, and its structural properties as well as those of the model membrane can be monitored continuously. PLC hydrolysis was limited to a low proportion of the model membrane by incorporating a small amount of ester phospholipid into a nonhydrolyzable ether-linked phospholipid matrix. Under these conditions, PLC (Bacillus cereus) hydrolyzed only the monolayer of phosphatidylcholine to which it was exposed (the outer monolayer). The 1,2-DAG product remained associated with the membrane bilayer and did not alter bilayer structure in any detectable way. From the chemical shift data, it is inferred that the DAG has an interfacial conformation similar to that of phosphatidylcholine. These results show that DAG could activate PKC by direct interaction with the enzyme rather than by perturbation of the membrane bilayer.
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PMID:NMR studies of phospholipase C hydrolysis of phosphatidylcholine in model membranes. 842 17


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