Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.1.4.3 (phospholipase C)
 18,461 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Phosphatidylinositol-specific phospholipase C (PI-PLC) from Bacillus thuringiensis catalyzes the hydrolysis of phosphatidylinositol (PI) in discrete steps: (i) an intramolecular phosphotransferase reaction to form inositol 1,2-(cyclic)-phosphate (cIP), followed by (ii) a cyclic phosphodiesterase activity that converts cIP to inositol 1-phosphate. Water-soluble cIP was used as the substrate to study the cyclic phosphodiesterase activity and interfacial behavior of PI-PLC. Different detergent micelles and phospholipid vesicles were used to examine if "interfacial activation" of the enzyme could occur toward a soluble substrate. Almost all detergents examined activated the enzyme at least 2-fold, with PC species yielding the largest increases in PI-PLC specific activity. Kinetic parameters were measured in the absence and presence of several representative detergents (e.g., Triton X-100 and diheptanoylphosphatidylcholine (diC7PC)). Gel filtration experiments showed that, under these conditions, the cIP did not partition to any measurable extent with these detergent micelles. The concentration at which half the maximum activation was observed occurred near the detergent CMC. Both Km and Vmax were altered by the presence of a surface: Km decreased to different degrees depending on the detergent, while Vmax increased substantially. The Km for cIP was 90 mM without detergent and decreased to 29 mM with diC7PC micelles added; Vmax increased almost 7-fold in the presence of diC7PC micelles. The enzyme efficiency (Vmax/Km) in the presence of diC7PC increased more than 21-fold, but it was still 20-fold lower than initial phosphotransferase activity for monomeric dihexanoylphosphatidylinositol. The poor efficiency of the cyclic phosphodiesterase activity is largely due to substrate binding affinity. The dependence of rate on substrate concentration exhibits cooperative behavior, especially without detergent. This cooperativity is discussed in terms of protein aggregation and ligand binding sites on the enzyme.
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PMID:Activation of phosphatidylinositol-specific phospholipase C toward inositol 1,2-(cyclic)-phosphate. 900 87

The 5'-upstream region of the rat phospholipase C-beta 3 gene (PLC-beta 3) has been cloned and characterized. Sequence analysis of the 5'-upstream region showed that it contains a GC-rich region (-166 to +1: 79%) and multiple binding sites for the transcription factors Sp1, AP-1 and AP-2, but does not contain a canonical TATA box. Primer extension analysis of total RNA isolated from rat glial cell C6Bul revealed that single transcription start point (tsp) is located at an initiator (Inr) element similar to that found in the HIV promoter. Gel mobility shift and competitive mobility shift assays indicated that this Inr element forms a DNA-protein complex with the HIV Inr-binding protein, LBP-1/CP2 or a homologue. In order to localize functional elements of the 5'-upstream region of the rat PLC-beta 3 gene, 5'-deletion fragments were cloned into a chloramphenicol acetyltransferase (CAT) reporter vector. Transient transfection analyses of the 5'-deletion mutants identified a crucial promoter element located at -128 to -14. Supershift mobility assays, site-directed mutagenesis and DNase I footprints indicated that Sp1 binds to three GC boxes within the sequence between -128 and -14 of the PLC-beta 3 promoter. Transient transfection analyses of promoter constructs containing site-specific mutation(s) of these three GC boxes demonstrated that two GC boxes, located proximal to the tsp, are important elements for normal promoter activity.
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PMID:The 5'-upstream region of the rat phospholipase C-beta 3 gene contains two critical Sp1 sites and an HIV Inr-like element. 933 46

In this study, we investigated whether vitamin E at concentrations achievable in blood after supplementation inhibits platelet function in humans. Gel-filtered platelets were incubated 30 minutes with scalar concentrations (50 to 250 mmol/L) of vitamin E and then stimulated with collagen. Compared with controls, vitamin E inhibited collagen-induced platelet aggregation and thromboxane A2 formation in a dose-dependent manner. Furthermore, vitamin E inhibited, in a dose-dependent manner, Ca(2+) mobilization and formation of inositol 1,4,5-triphosphate. Because it was previously shown that hydrogen peroxide formation mediates arachidonic acid metabolism and phospholipase C activation in collagen-induced platelet activation, we investigated whether vitamin E was able to blunt hydrogen peroxide. In experiments performed in unstimulated platelets supplemented with hydrogen peroxide and in collagen-stimulated platelets, vitamin E was able to blunt hydrogen peroxide. In 6 healthy subjects given vitamin E for 2 weeks (600 mg/d), we found a significant decrease of collagen-induced H(2)O(2) formation, platelet aggregation, and calcium mobilization. This study demonstrated in vitro and ex vivo that vitamin E inhibits collagen-induced platelet activation by blunting hydrogen peroxide formation.
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PMID:Vitamin E inhibits collagen-induced platelet activation by blunting hydrogen peroxide. 1052 85

Regulation of toxin production in the gram-positive anaerobe Clostridium perfringens occurs at the level of transcription and involves a two-component signal transduction system. The sensor histidine kinase is encoded by the virS gene, while its cognate response regulator is encoded by the virR gene. We have constructed a VirR expression plasmid in Escherichia coli and purified the resultant His-tagged VirR protein. Gel mobility shift assays demonstrated that VirR binds to the region upstream of the pfoA gene, which encodes perfringolysin O, but not to regions located upstream of the VirR-regulated plc, colA, and pfoR genes, which encode alpha-toxin, collagenase, and a putative pfoA regulator, respectively. The VirR binding site was shown by DNase I footprinting to be a 52-bp core sequence situated immediately upstream of the pfoA promoter. When this region was deleted, VirR was no longer able to bind to the pfoA promoter. The binding site was further localized to two imperfect direct repeats (CCCAGTTNTNCAC) by site-directed mutagenesis. Binding and protection analysis of these mutants indicated that VirR had the ability to bind independently to the two repeated sequences. Based on these observations it is postulated that the VirR positively regulates the synthesis of perfringolysin O by binding directly to a region located immediately upstream of the pfoA promoter and activating transcription.
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PMID:The VirR response regulator from Clostridium perfringens binds independently to two imperfect direct repeats located upstream of the pfoA promoter. 1061 63

To date, little is known about the molecular mechanisms controlling the regulation of phospholipase C-delta1 (PLC-delta1) gene expression. To understand the mechanisms responsible for the regulation of PLC-delta1 gene expression, the 5'-flanking region of the mouse PLC-delta1 gene was isolated from a mouse genomic DNA library. Primer extension analysis revealed that there is a single transcriptional start site located at 127 bases upstream from the translation start codon in the mouse PLC-delta1 gene. DNA sequence analysis showed that the sequence around the transcriptional start site is very GC-rich and has no TATA or CAAT boxes. Transient expression of a luciferase reporter gene under the control of serially deleted 5'-flanking sequences revealed that the 160-base-pair region from -622 to -462 upstream of the transcriptional start site includes a positive cis-acting element(s) for the efficient expression of the PLC-delta1 gene. Gel retardation analysis suggests that multiple transcription factors bind to separate sites on the promoter region. Based on these results, our study suggests that the minimal essential region located at -622 to +70 is fully sufficient to confer high-level transcriptional activity and contains high-affinity binding elements for multiple transcription factors.
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PMID:Cloning and characterization of the 5'-flanking region for the mouse phospholipase C-delta1 gene. 1087 10

Vascular endothelial growth factor (VEGF) induces adhesion molecules on endothelial cells during inflammation. Here we examined the mechanisms underlying VEGF-stimulated expression of intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1), and E-selectin in human umbilical vein endothelial cells. VEGF (20 ng/ml) increased expression of ICAM-1, VCAM-1, and E-selectin mRNAs in a time-dependent manner. These effects were significantly suppressed by Flk-1/kinase-insert domain containing receptor (KDR) antagonist and by inhibitors of phospholipase C, nuclear factor (NF)-kappaB, sphingosine kinase, and protein kinase C, but they were not affected by inhibitors of mitogen-activated protein/extracellular signal-regulated kinase kinase (MEK) 1/2 or nitric-oxide synthase. Unexpectedly, the phosphatidylinositol (PI) 3'-kinase inhibitor wortmannin enhanced both basal and VEGF-stimulated adhesion molecule expression, whereas insulin, a PI 3'-kinase activator, suppressed both basal and VEGF-stimulated expression. Gel shift analysis revealed that VEGF stimulated NF-kappaB activity. This effect was inhibited by phospholipase C, NF-kappaB, or protein kinase C inhibitor. VEGF increased VCAM-1 and ICAM-1 protein levels and increased leukocyte adhesiveness in a NF-kappaB-dependent manner. These results suggest that VEGF-stimulated expression of ICAM-1, VCAM-1, and E-selectin mRNAs was mainly through NF-kappaB activation with PI 3'-kinase-mediated suppression, but was independent of nitric oxide and MEK. Thus, VEGF simultaneously activates two signal transduction pathways that have opposite functions in the induction of adhesion molecule expression. The existence of parallel inverse signaling implies that the induction of adhesion molecule expression by VEGF is very finely regulated.
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PMID:Vascular endothelial growth factor expression of intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1), and E-selectin through nuclear factor-kappa B activation in endothelial cells. 1110 18

A few years ago, it was shown that intrahemocoelic injection of the insect apolipoprotein apolipophorin III (apoLp-III) stimulates an immune response in larvae of the greater wax moth, Galleria mellonella. Since the mode of action of this activation process is unknown, we followed apoLp-III's pathway in the early phase of the immune-stimulating process, using biotin as a probe. Biotinylated apoLp-III was injected and localized using avidin-coupled horseradish peroxidase. The labeled protein was fully functional; the added amount of biotin per apoLp-III molecule used in this study only slightly decreased its ability to associate with phospholipase C-treated human low-density lipoprotein, as well as the immune-stimulating capability of apoLp-III.Gel electrophoresis with subsequent staining of biotin moieties and lipids revealed that apoLp-III undergoes lipid association in vivo within the first few minutes after injection. After two hours, no biotinylated apoLp-III was detectable in cell-free hemolymph. At this time, a subpopulation of hemocytes showed a distinct peroxidase staining. Control injections of biotinylated bovine serum albumin did not lead to similar results, giving evidence for the specificity of the phenomena observed. The results indicate that lipid association of apoLp-III occurs prior to endocytosis by immune-competent hemocytes, which is followed by the induction of a humoral immune response.
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PMID:Localization of injected apolipophorin III in vivo - new insights into the immune activation process directed by this protein. 1135 26

The PRIP [phospholipase C related, but catalytically inactive protein] family has been isolated as a novel inositol 1,4,5-trisphosphate binding protein with a domain organization similar to phospholipase C-delta but lacking the enzyme activity, comprising PRIP-1 and PRIP-2. The PRIP-1 gene is expressed predominantly in the brain, while PRIP-2 exhibits a relatively ubiquitous expression in rats and mice. We also found that PRIP-1 plays an important role in type A receptor signaling for gamma-aminobutyric acid in the brain. In this study, we investigated PRIP-1 gene structure and the possible mechanisms involved in the expression. The tissue distribution pattern of PRIP gene expression in humans was similar to that in rodents. 5'RACE (rapid amplification of cDNA ends) analysis using PRIP-1 gene specific primers with human brain mRNA revealed the presence of three new exons, indicating that the PRIP-1 gene is organized into 8 exons intervened by 7 introns. Although three transcripts resulting from the alternative splicing of exon 2 and/or 3 were detected, a transcript lacking exons 2 and 3 was predominantly expressed in humans, suggesting that the translation start codon of human PRIP-1 exists in exon 1. To characterize the human PRIP-1 promoter, transient luciferase assay was carried out with luciferase constructs including various lengths of the 5' flanking region of the PRIP-1 gene. The results indicated that the positive regulatory region is located -237 to -108 bp upstream from the transcription start site. Gel shift assay revealed the specific binding of some nuclear proteins to this region, suggesting that the existence of transcription factors contributes to the positive regulation of PRIP-1 gene expression. Mutation analyses revealed that the binding of a transcription factor, MAZ to the regulatory site leads to the promoter activity, indicating that MAZ is involved in the expression regulation of the human PRIP-1 gene.
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PMID:Characterization of the human PRIP-1 gene structure and transcriptional regulation. 1695 28


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