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)

Inositol phospholipids (IPL) from epimastigote forms of Trypanosoma cruzi have been investigated by metabolic labelling with [3H]palmitic acid and by GLC-MS analysis of the lipids obtained from non-labelled parasites. The IPL fraction was separated into phosphatidylinositol (PI) and inositol-phosphoceramide subfractions, the latter accounting for 80-85% of the total IPL. The neutral lipids released from the IPLs by PI-specific phospholipase C (PI-PLC) from Bacillus thuringiensis were analysed by silica-gel and reverse-phase TLC for the radioactive lipids and by GLC-MS for the non-radioactive samples. Ceramides containing dihydrosphingosine and sphingosine with C16:0 and C18:0 fatty acids were identified. The main component in the [3H]palmitic acid-labelled ceramides was palmitoyldihydrospingosine, while in the non-labelled sample the ceramides contained mainly sphingosine. This could reflect partial uptake of phospholipid from the medium. The PI contain both alkylacyl- and diacyl-glycerol lipids, with the ether lipid being more abundant. The latter was identified as 1-O-hexadecylglycerol esterified by C18:2 and C18:1 fatty acids. Interestingly, the same lipid had been identified in the anchor of the 1G7 glycoprotein of T. cruzi metacyclic forms.
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PMID:Structural analysis of inositol phospholipids from Trypanosoma cruzi epimastigote forms. 764 54

Fluorescence dipolar resonance energy transfer between a receptor-bound fluorescent agonist, dansyl-C6-choline, and two membrane-partitioned fluorescent probes, C18-rhodamine and C12-eosin, was used to measure the transverse distance between the acetylcholine (ACh) binding sites on the intact Torpedo nicotinic acetylcholine receptor (nAChR) and the surface of the lipid membrane. Control experiments demonstrated that: (1) dansyl-C6-choline binds to cobra-alpha-toxin sensitive sites on the nAChR with a KD approximately 20 nM, (2) the quantum yield of dansyl-C6-choline increases 3.1-fold upon binding, and (3) the receptor-bound dansyl-C6-choline fluorescence is stable for at least 2 h. The calculated transverse distances between receptor-bound dansyl-C6-choline and the membrane-partitioned acceptors, C12-eosin and C18-rhodamine, were 31 and 39 A, respectively. Therefore, given the dimensions of the extracellular domain of the receptor, the ACh binding sites are located significantly below (approximately 25 A) the extracellular apex of the nAChR. These results are in agreement with the recent proposed location for the ACh binding sites in a pocket within each of the two alpha-subunits, approximately 30 A above the membrane surface (Unwin, N. (1993) J. Mol. Biol. 229: 1101-1124).
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PMID:Transverse distance between the membrane and the agonist binding sites on the Torpedo acetylcholine receptor: a fluorescence study. 801 98

The major macromolecules on the surface of the parasitic protozoan Leishmania major appear to be down-regulated during transformation of the parasite from an insect-dwelling promastigote stage to an intracellular amastigote stage that invades mammalian macrophages. In contrast, the major parasite glycolipids, the glycoinositol phospholipids (GIPLs), are shown here to be expressed at near-constant levels in both developmental stages. The structures of the GIPLs from tissue-derived amastigotes have been determined by h.p.l.c. analysis of the deaminated and reduced glycan head groups, and by chemical and enzymic sequencing. The deduced structures appear to form a complete biosynthetic series, ranging from Man alpha 1-4GlcN-phosphatidylinositol (PI) to Gal alpha 1-3Galf beta 1-3Man alpha 1-3Man alpha 1-4GlcN-PI (GIPL-2). A small proportion of GIPL-2 was further extended by addition of a Gal residue in either alpha 1-6 or beta 1-3 linkage. From g.c.-m.s. analysis and mild base treatment, all the GIPLs were shown to contain either alkylacylglycerol or lyso-alkylglycerol lipid moieties, where the alkyl chains were predominantly C18:0, with lower levels of C20:0, C22:0 and C24:0. L. major amastigotes also contained at least two PI-specific phospholipase C-resistant glycolipids which are absent from promastigotes. These neutral glycolipids were resistant to both mild acid and mild base hydrolysis, contained terminal beta-Gal residues and were not lost during extensive purification of amastigotes from host cell membranes. It is likely that these glycolipids are glycosphingolipids acquired from the mammalian host. The GIPL profile of L. major amastigotes is compared with the profiles found in L. major promastigotes and L. donovani amastigotes.
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PMID:Characterization of glycoinositol phospholipids in the amastigote stage of the protozoan parasite Leishmania major. 824 Feb 57

In our study, 5'-nucleotidase was released from bovine liver by the treatment with Bacillus thuringiensis phosphatidylinositol-specific phospholipase C and purified to a homogeneous state by concanavalin A-Sepharose and (diethylaminoethyl)-Toyopearl column chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Purified 5'-nucleotidase were then cleaved by cyanogen bromide (CNBr), and then inositol phosphoglycan-containing C-terminal peptides (IPG peptides) were separated by C18 reverse-phase liquid chromatography and analyzed by peptide sequencer, amino acid analyzer, gas chromatography (GC), and GC-mass spectrometry (MS). Ser523 of the amino acid sequence deduced from 5'-nucleotidase cDNA [Suzuki et al. (1993) J. Biochem. (Tokyo) 113, 607-613] is revealed to be the C-terminal amino acid to which a glycosylphosphatidylinositol is anchored. Separated peaks of CNBr-cleaved IPG peptides were then analyzed by electron spray ionization (ESI)-MS. Eight different molecular weight (MW) species of CNBr-cleaved IPG peptides were detected. Three fractions of CNBr-cleaved IPG peptides were separately treated by trypsin, and trypsinized IPG peptides were purified by C18 reverse-phase liquid chromatography. Finally, five different MW species of trypsinized IPG peptides (1629.4, 1752.7, 1791.8, 1832.8, and 1994.5) were detected by ESI-MS. Together with sequential exoglycosidase treatment and quantitative analysis of sugar moieties by GC and GC-MS, microheterogeneity in the structures of these five glycosylphosphatidylinositol (GPI) anchor species was determined. The common core structure was ethanolamine phosphate-mannose-mannose-mannose(-ethanolamine phosphate)-glucosamine-myoinositol phosphate. Variations observed in additional mannose, N-acetylhexosamine, and ethanolamine phosphate moieties form this heterogeneity.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Microheterogeneity in glycosylphosphatidylinositol anchor structures of bovine liver 5'-nucleotidase. 830 28

We have previously demonstrated the potential usefulness of capillary columns coated with a high temperature polarizable phenylmethylsilicone liquid phase in plasma lipid profiling (Kuksis, A., J.J. Myher, and P. Sandra. 1990. J. Chromatogr. 500: 427-441). The present study reports improved operating conditions along with a practical application to the analysis of a series of human plasma samples in comparison to capillary gas-liquid chromatography on nonpolar columns. For this purpose the plasma lipids were dephosphorylated with phospholipase C and converted to the trimethylsilyl ethers. The molecular species of the plasma lipids were identified by comparing the relative retention times to reference standards. The species were quantitated using tridecanoylglycerol as internal standard. The recoveries of the lipid classes were determined by summing the molecular species within each carbon number and comparing the proportion of the carbon numbers obtained on polar and nonpolar columns. The relative recoveries varied with the lipid class and sample load and averaged as follows: FA C16, 78%; FA C18, 78%; FC, 99%; tridecanoylglycerol (TD), 100% (by definition); CER 34:1, 92%; DG C34, 103%; DG C36, 98%; CE C16, 73%; CE C18, 61%; TG C50, 93%; TG C52, 60%; TGC54, 32%. We conclude that high temperature polarizable capillary columns are suitable for qualitative and quantitative assessment of plasma lipids and provide more information per man-hour, instrument time, and unit cost than any other analytical method known.
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PMID:Quantitation of plasma lipids by gas-liquid chromatography on high temperature polarizable capillary columns. 835 50

Lipids of Drosophila heads were extracted and separated by high-performance thin-layer chromatography. Fatty acid compositions of major phospholipids as well as of triglycerides were analyzed by gas-liquid chromatography. Proportions of the major fatty acids (14:0, 16:0, 16:1, 18:0, 18:1, 18:2, 18:3) varied depending on the lipid analyzed. Docosahexaenoic acid (22:6), common in vertebrate photoreceptors and brain, and arachidonic acid (20:4), a precursor of eicosanoids, were lacking. A comparison of the fatty acid composition of the diet vs. the head suggested that Drosophila can desaturate but may not be able to elongate fatty acid carbon chains. Fatty acid analyses were carried out after the following visual system alterations: i) the transduction mutant where no receptor potential results from a deficit in phospholipase C; ii) an allele of eyes absent; iii) the mutant outer rhabdomeres absent which lacks visual pigment and rhabdomeres in the predominant type of compound eye receptor, rhabdomeres 1 through 6; and iv) carotenoid deprivation which reduces opsin and rhabdomere size. We also evaluated aging by comparing newly-emerged vs. aged wild-type flies. Alterations in fatty acid composition based on some of these manipulations were found. Based on comparisons between flies reared on media differing in C16 and C18, there is an indication that diet readily affects tissue fatty acid composition.
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PMID:Fatty acids in the lipids of Drosophila heads: effects of visual mutants, carotenoid deprivation and dietary fatty acids. 848 27

Significant changes in platelet-activating factor (PAF; 1-O-alkyl-2-acetyl-sn-glycero-3-phosphorylcholine) concentration have been observed in rabbit endometrium during the preimplantation period, but, under in vitro conditions, constitutive PAF biosynthesis by isolated endometrial tissues was not easily demonstrable. Relative changes in enzymes involved in the synthesis and metabolism of PAF in the tissues may account for this disparity. In addition, during this period of preimplantation, marked changes in PAF receptor concentration have been noted. The present study examines the factors that may modulate the metabolism of exogenous [3H]PAF in the endometrium of rabbits on day 6 of pregnancy. Since preferential [3H]PAF binding in situ by the glandular epithelial, but not by the stromal, cells was demonstrated, their cell-specific metabolism of exogenous [3H]PAF was also examined. After entry into the endometrial cell, [3H]PAF was rapidly metabolized by the sequential action of cytosolic Ca(2+)-independent acetylhydrolase to [3H]lyso-PAF and this was in turn acylated by membrane-associated transacylase to [3H]alkylacyl-glycerylphosphorylcholine. PAF resynthesis was not observed and, in stromal cells, there was a significant build-up of [3H]lyso-PAF, suggesting that lyso-PAF:acetyl-CoA acetyl-transferase may be a limiting factor. In the glandular epithelial cells, however, there was a significant accumulation of a neutral lipid without a significant build-up of [3H]lyso-PAF or [3H]PAF. The neutral lipid co-migrated with the product of phospholipase C-catalysed metabolism of PAF and authentic 1-O-hexadecyl-2-acetyl-glycerol. In addition, the elution times of phospholipase C digestion of C18 PAF and the neutral lipid produced by cellular metabolism of [3H]PAF, determined by gas chromatography/flame ionization detection, were similar. It seems that it is the synthesis of the neutral lipid from reacetylated [3H]lyso-PAF that prevented [3H]PAF accumulation under in vitro conditions. This is the first documentation of the synthesis of this lipid in the mammalian uterus. The lipid may serve as the precursor for de novo PAF synthesis in the glandular epithelial cells during endometrial proliferation.
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PMID:Differential metabolism of exogenous platelet-activating factor by glandular epithelial and stromal cells of rabbit endometrium. 856 77

Rat brain cytosol contains proteins that markedly inhibit the activity of partially purified brain membrane phospholipase D (PLD) stimulated by ADP-ribosylation factor (Arf) and phosphatidylinositol 4,5-bisphosphate (PIP2). Sequential chromatography of the brain cytosol yielded four inhibitor fractions, which exhibited different kinetics to heat treatment at 70 degrees C. Purification of the most heat-labile inhibitor to homogeneity yielded two preparations, which displayed apparent molecular masses of 150 kDa and 135 kDa, respectively, on SDS-polyacrylamide gels. Tryptic digests of the 150- and 135-kDa proteins yielded similar elution profiles on a C18 reverse-phase column, suggesting that the 135-kDa form is a truncated form of the 150-kDa form. Sequences of two tryptic peptides were determined. A data base search revealed no proteins with these sequences. The purified 150-kDa inhibitor negated the PLD activity stimulated by Arf, RhoA, or Cdc42. The concentration required for half-maximal inhibition was 0.4 nM. Concentration dependence on the 150-kDa inhibitor was not affected by changes in the concentrations of Arf, PIP2, or phosphatidylcholine used in the assays, suggesting that the inhibition is not due to competition with the activators or substrate for PLD. The purified inhibitor did not affect the PIP2-hydrolyzing activity of a phospholipase C isozyme that was measured with substrate vesicles of lipid composition identical with that used for the PLD assay. Thus, the mechanism of inhibition appears to be a specific allosteric modification of PLD rather than disruption of substrate vesicles.
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PMID:Multiple forms of phospholipase D inhibitor from rat brain cytosol. Purification and characterization of heat-labile form. 862 62

In vivo labeling experiments with [3H]palmitic acid, [3H]inositol, and [3H]glucose allowed the identification of two main classes of inositolphospholipids (IPLs) from the trypomastigote stage of Trypanosoma cruzi. Purification of these compounds was achieved by ion-exchange chromatography, high performance liquid chromatography and thin layer chromatography. Specific phosphatidyl-inositol phospholipase C digestion, dephosphorylation and acid methanolysis showed a ceramide structure for the lower migrating IPL1. Palmitoyldihydrosphingosine and palmitoylsphingosine were detected by reverse-phase thin-layer chromatography. On the other hand, IPL2 showed to be a mixture of diacylglycero- and alkylacylglycero-phospholipids in a 1:1 ratio. After PI-PLC digestion, the lipids were separated by preparative TLC and individually analysed. The diacylglycerol contained mainly C18:0 fatty acid together with a low amount of C16:0. Hexadecylglycerol esterified with the C18:0 fatty acid was the only alkylacylglycerol detected. The C18:2 and C18:1 fatty acids, preponderant in the PI molecules of epimastigote forms, were not detected in trypomastigote forms. This is the first report on inositol phospholipids, putative precursors of lipid anchors in the infective stage of T. cruzi.
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PMID:Characterization of inositolphospholipids in Trypanosoma cruzi trypomastigote forms. 867 89

A reversed-phase high-performance liquid chromatography technique was developed to separate, identify, and quantify individual phosphatidylglycerol (PG) molecular species in thylakoid membranes isolated from higher plant leaves. PG was first separated by thin-layer chromatography; then the dinitrobenzoyl derivatives of diacylglycerols produced after phospholipase C hydrolysis of PG were separated by a C18 reversed-phase column and detected at 254 nm. A linear response of the detector was observed in the range of 0.025 to 12 nmol of PG molecular species. It was established that there was an excellent correlation (r = 0.996) between the carbon and double-bond number in the aliphatic residues and the relative retention time of dinitrobenzoyl derivatives. A new equivalent carbon number value (ECN*) which takes into consideration the number of cis-(nc) and trans-(nt) double bonds per molecular species was defined as ECN* = CN - 2nc - nt, where CN is the number of carbon atoms in the aliphatic residues. The logarithm of the retention time increased linearily as a function of ECN* value. However, in this type of correlation, it may happen that two molecular species of PG having distinct relative retention times had the same ECN* value. In this case, the two molecular species can be identified by the linear correlation (r = 1) existing between the reciprocal of the relative retention time and the number of double bonds (0 < or = n < or = 3) in the separate 18:n/delta 3-trans-hexadecenoic acid -16:1(3t)- and 18:n/16:0 molecular species series. The advantages of this method are good separation, cohort elution time, quantitative precision, and predictable retention times of PG molecular species from chloroplast membranes. The method has been used routinely to identify the ten PG molecular species of thylakoid membranes in squash, potato, lettuce, and spinach leaf: 18:3/16:1(3t), 18:3/16:0, 18:2/16:1(3t), 18:2/16:0, 18:1/16:1(3t), 18:1/16:0, 18:0/16:1(3t), 18:0/16:0, 16:0/16:1(3t), and 16:0/16:0.
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PMID:Phosphatidylglycerol molecular species of photosynthetic membranes analyzed by high-performance liquid chromatography: theoretical considerations. 883 12


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