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)

A cytoskeletal fraction of porcine tracheal smooth muscle (PTSM) was found to contain > 90% of total cellular aldolase (fructose 1,6-bisphosphate aldolase, EC 4.1.2.13) activity. PTSM aldolase was purified by DEAE and inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) affinity chromatography and found to react with an antibody directed against human aldolase C, but not anti-aldolase A and B. The molecular mass of native aldolase was about 138 kDa (on Sephacryl S-300); SDS-denatured enzyme was 35 kDa (comigrated with rabbit skeletal muscle aldolase). Total cellular aldolase tetramer (aldolase4) content was 34.5 pmol/100 nmol lipid P(i). Ins(1,4,5)P3) binding activity coeluted with aldolase during Sephacryl 300, DEAE, and Ins(1,4,5)P3 affinity chromatography. Ins(1,4,5)P3 bound to purified aldolase (at 0 degree C) in a dose-dependent manner over the range [Ins(1,4,5)P3] 20 nM to 20 microM, with maximal binding of 1 mol of Ins(1,4,5)P3/mol aldolase4 and a Kd of 12-14 microM. Fru(1,6)P2 and Fru(2,6)P2 displaced bound Ins(1,4,5)P3) with a 50% inhibition at 30 and 170 microM, respectively. Ins(1,3,4)P3 (20 microM) and glyceraldehyde 3-phosphate (2 mM) were also potent inhibitors of Ins(1,4,5)P3 binding, but not inositol 4-phosphate or inositol 1,4-bisphosphate (20 microM each). Aldolase-bound Ins(1,4,5)P3 may play a role in phospholipase C-independent increases in free [Ins(1,4,5)P3].
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PMID:Inositol 1,4,5-trisphosphate binding to porcine tracheal smooth muscle aldolase. 765 22

Urokinase plasminogen activator (u-PA) receptor (u-PAR) is a glycosyl-phosphatidylinositol-anchored membrane protein that promotes pericellular proteolysis and cellular migration. This investigation demonstrates that u-PAR is a substrate for the proteolytically active form of streptococcal pyrogenic exotoxin B (SPE B), a potent virulence factor secreted by Streptococcus pyogenes. Treatment of U937 monocyte-like cells with SPE B decreased specific 125I-labeled single-chain u-PA binding by up to 85%. Cysteine proteinase inhibitors neutralized SPE B without affecting the activity of phosphatidylinositol-specific phospholipase C. Due to decreased u-PA binding, SPE B-treated U937 cells expressed decreased activity against a u-PA-specific fluorogenic substrate and plasminogen. SPE B released single-chain u-PA that was noncovalently bound to U937 cells or cross-linked to cellular receptors with bis(sulfosuccinimidyl) suberate. The mass of the released u-PA-receptor complex was 100 kDa. Western blot analysis confirmed that the u-PA receptor that was cleaved by SPE B is u-PAR. After deglycosylation, the mass of SPE B-released u-PAR was 35 kDa, slightly smaller than the phosphatidylinositol-specific phospholipase C-derived form of this receptor. SPE B-released u-PAR retained the ability to bind u-PA, as determined by u-PA affinity chromatography. We conclude that SPE B may inhibit u-PA binding to monocytic cells by at least two mechanisms: (i) by decreasing the level of functional cell surface u-PAR and (ii) by releasing a soluble form of u-PAR that competes with the cellular receptor for ligand.
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PMID:Proteolytically active streptococcal pyrogenic exotoxin B cleaves monocytic cell urokinase receptor and releases an active fragment of the receptor from the cell surface. 798 88

Taurine, a beta amino acid, is a primary osmolyte in nucleated skate erythrocytes and is involved in the regulation of cell volume. Growth factors may be involved in the regulation of cell volume which occurs during cell division. Erythropoietin (EPO) is the primary growth factor controlling erythropoiesis. To investigate its mechanism of action, we used nucleated skate erythrocytes. EPO stimulates Na(+)-independent uptake of taurine in a concentration-dependent manner. The uptake was inhibited by the tyrosine kinase inhibitor genistein. Concomitantly, EPO stimulates tyrosine phosphorylation of a number of proteins, particularly ones of molecular masses 145, 120, 100, 80, 65, and 35 kDa. Using specific antibodies, the 145 kDa protein is identified as phospholipase C gamma-1 (PLC gamma-1) and the 100 kDa protein as the skate homolog of the anion exchanger band 3. Since PLC gamma-1 is activated, turnover of membrane lipids was determined. EPO increased 1,2-diacylglycerol formation from phosphatidylinositols (phosphatidylinositol-4-monophosphate and 4,5-biphosphate) during an early phase and later preferentially from phosphatidylcholine. The early hydrolysis of phosphoinositides was confirmed measuring generation of inositol-1,4,5-trisphosphate, demonstrating an activation of PLC gamma-1 activity. To determine if phospholipase D (PLD) stimulation also occurred, ethanol was included in the reactions. Phosphatidylethanol, synthesized by PLD-mediated transphosphatidylation, appeared at times longer than 5 min, suggesting delayed activation of PLD. These results demonstrate that EPO, via simulation of tyrosine phosphorylation, stimulates taurine transport in skate erythrocytes.
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PMID:Erythropoietin stimulates tyrosine phosphorylation and taurine transport in skate erythrocytes. 874 88

The heterotrimeric guanosine 5'-triphosphate (GTP)-binding protein Gq was suggested to couple the light receptor rhodopsin with the effector phospholipase C in visual cells of invertebrates. We indirectly linked Gq from Sepia officinalis to a concanavalin A-sepharose column via rhodopsin. Rhodopsin had been solubilized previously with 10 mM n-dodecyl-beta-maltoside from the purified photosensory membrane under illumination. All three subunits of the Gq were released almost pure by elution with 100 microM GTP. The alpha and beta subunits were identified by specific antipeptide antisera. The alpha subunit has a relative molecular mass of 46 kDa, and the beta subunit of 35 kDa. The gamma subunit corresponds to a 9 kDa polypeptide owing to the molecular mass, which is similar to the G gamma subunit of squid. The use of specific antibodies shows that neither actin nor G-protein related to transducin were in the fractions eluted with GTP or alpha-methyl mannoside. We demonstrate that all three subunits of Gq were associated with rhodopsin of invertebrates. Such use of a lectin column might be useful for further investigations of the interaction of rhodopsin and Gq.
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PMID:Interaction of guanosine 5'-triphosphate binding protein Gq from Sepia officinalis with illuminated rhodopsin bound to concanavalin A. 882 32

Neospora caninum is an apicomplexan parasite that is closely related to Toxoplasma gondii and has been found to be associated with neurological disorders in dogs and congenital infections and abortions in cattle. We have identified two surface proteins of 29 and 35 kDa (designated Ncp29 and Ncp35, respectively) from N. caninum tachyzoites that are the predominant antigens recognized by antisera from Neospora-infected animals. Monoclonal antibodies against Ncp29 and Ncp35 were used to analyze several independent and diverse N. caninum isolates; both antigens were recognized in all isolates, suggesting that they are well conserved. Localization studies and surface labeling with biotin demonstrated that Ncp29 and Ncp35 are membrane associated and displayed on the surface of the parasite. After treatment with phosphatidylinositol-specific phospholipase C, parasite lysates were analyzed with antibodies against the cross-reacting determinant of glycosylphosphatidylinositol anchors. Approximately six glycolipid-anchored surface proteins were identified, with the two most prominent corresponding to Ncp29 and Ncp35. Sequence comparisons of Ncp29 and Ncp35 with GenBank indicated that they are most similar to the T. gondii surface antigen 1 (SAG1) and surface antigen 1-related sequence 2 (SRS2), respectively. Consequently, Ncp29 has been designated NcSAG1 and Ncp35 has been designated NcSRS2. Both NcSAG1 and NcSRS2 contain a tandemly duplicated motif and 12 absolutely conserved cysteines which are also found in all of the SAG and SRS proteins of T. gondii. Maintenance of these motifs and the 12 cysteine residues suggests that these surface antigens share a similar secondary and tertiary structure that is presumably important for a conserved function that these antigens serve during infection.
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PMID:The p29 and p35 immunodominant antigens of Neospora caninum tachyzoites are homologous to the family of surface antigens of Toxoplasma gondii. 978 39

Female macaques produced isoantibodies to a limited number of sperm surface proteins following immunization with sperm components released by phosphatidylinositol-specific phospholipase C (PI-PLC). Washed, acrosome-intact, fixed sperm injected into rabbits elicited a major immune response to one of the same PI-PLC-released proteins, which was shown to be a sperm surface-coating protein. After purification and digestion of the glycoprotein, four peptides were analyzed for amino acid sequence, and all had 100% homology with an epididymal secretory protein, ESP13.2, reported previously to be a small, cationic-rich peptide and a member of the beta-defensin family. Antibodies to purified ESP13.2 recognized a number of protein bands on Western blots of nonreduced PI-PLC-released sperm components and nonreduced whole-sperm extracts. After chemical disulfide reduction, only a single, broad band from 31 to 35 kDa was recognized by anti-ESP13.2 antibodies. Indirect immunofluorescence showed ESP13.2 over the entire surface of ejaculated macaque sperm. Fluorescence was only slightly reduced after sperm were washed through 80% Percoll. A 24-h incubation in capacitating medium significantly reduced the amount of ESP13.2 over the head and midpiece, whereas exposure of the incubated sperm to dbcAMP and caffeine (capacitation activators) resulted in almost complete loss of ESP13.2 from the sperm surface. After activation, ESP13.2 was the primary component released into the medium as judged electrophoretically. Lignosulfonic acid, a potent inhibitor of macaque fertilization in vitro, completely blocked release of ESP13.2 from the sperm surface, even following treatment with activators. These findings suggest that the beta-defensin, ESP13.2, has a function in the capacitation of macaque spermatozoa and may modulate sperm surface-receptor presentation at the time of fertilization.
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PMID:ESP13.2, a member of the beta-defensin family, is a macaque sperm surface-coating protein involved in the capacitation process. 1277 4

Sphingomyelin synthase is the enzyme that synthesizes sphingomyelin (SM) in mammalian cells by transferring a phosphorylcholine moiety from phosphatidylcholine to ceramide. Despite its importance, the gene and/or the protein responsible for this activity has not yet been identified. Here we report the purification, identification, and biochemical characterization of an enzymatic activity that synthesizes SM in Pseudomonas aeruginosa. SM synthase-like activity was found secreted in the culture medium of P. aeruginosa, strains PA01 and PAK, whereas it could not be detected in cultures of Escherichia coli. From the medium of PAK cultures, SM synthase was purified through sequential chromatographic columns. After separation on polyacrylamide-SDS gels and visualization by silver staining, the purified enzyme showed two bands, one of approximately 75 kDa and one of 30-35 kDa. Interestingly, the highly purified SM synthase preparation also showed neutral sphingomyelinase activity. We therefore investigated whether the protein we purified as SM synthase could actually be the previously identified PlcH, a 78-kDa phospholipase C known to hydrolyze phosphatidylcholine and SM in P. aeruginosa. First, the purified SM synthase preparation contained a 78-kDa protein that reacted with monoclonal antibodies raised against purified PlcH. Second, purified PlcH showed SM synthase activity. Third, using different knockout mutant strains for the PlcH operon, PlcH was found to be necessary for SM synthase activity in P. aeruginosa. Interestingly, SM synthase activity was specific to the Pseudomonas PlcH as other bacterial phospholipases did not display SM synthase activity. Biochemical studies on the Pseudomonas SM synthase confirmed that it is a transferase, similar to the mammalian enzyme, that specifically recognizes the choline head-group and the primary hydroxyl on ceramide. This SM synthase did not have reverse transferase activity. In conclusion, the Pseudomonas PlcH also exerts SM synthase activity; therefore, for the first time, we have identified a structural gene for a SM synthase.
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PMID:Purification, characterization, and identification of a sphingomyelin synthase from Pseudomonas aeruginosa. PlcH is a multifunctional enzyme. 1279 77

The cell-associated and extracellular peptidases of Herpetomonas megaseliae grown in brain-heart infusion and in modified Roitman's complex media were analyzed by measuring peptidase activity on gelatin, casein and hemoglobin in zymograms. Casein was the best proteinaceous substrate for the peptidase detection on both growth conditions. However, no proteolytic activity was detected when hemoglobin was used. Our results showed that cellular cysteine peptidase (115-100, 40 and 35 kDa) and metallopeptidase (70 and 60 kDa) activities were detected on both media in casein and gelatin zymograms. Additionally, the use of casein in the gel revealed a distinct acidic metallopeptidase of 50 kDa when the parasite was cultured in the modified Roitman's complex medium. Irrespective of the culture medium composition, H. megaseliae released metallopeptidases exclusively in the extracellular environment. The presence of gp63-like molecules on the H. megaseliae surface was shown by flow cytometry using anti-gp63 antibody raised against recombinant gp63 from Leishmania mexicana. The pre-treatment of parasites with phospholipase C reduced the number of gp63-positive cells, suggesting that these molecules were glycosylphosphatidylinositol-anchored to the surface. Additionally, the supernatant obtained from phospholipase C-treated cells and probed with anti-cross-reacting determinant confirmed that at least a 52 kDa gp63-like molecule is glycosylphosphatidylinositol-anchored. Furthermore, we assessed a possible function for the gp63-like molecules in H. megaseliae on the interaction with explanted guts of its original host, Megaselia scalaris, and with an experimental model employing Aedes aegypti. Parasites pre-treated with either anti-gp63 antibody or phospholipase C showed a significant reduction in the adhesion to M. scalaris and A. aegypti guts. Similarly, the pre-treatment of the explanted guts with purified gp63 diminished the interaction process. Collectively, these results corroborate the ubiquitous existence of gp63 homologues in insect trypanosomatids and the potential adhesion of these molecules to invertebrate host tissues.
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PMID:Peptidases and gp63-like proteins in Herpetomonas megaseliae: possible involvement in the adhesion to the invertebrate host. 1650 Jun 61

Synthetic peptides corresponding to the C-terminus of auxin-binding protein 1 (ABP1) have been shown to function as auxin agonists. To define a C-terminal receptor, photoaffinity crosslinking experiments were performed using an azido derivative of a C-terminal peptide and plasma membranes from maize (Zea mays L.). The crosslinking reaction was monitored by immunoblotting using anti-ABP1 antibodies. The crosslinked proteins were isolated by 2D gel electrophoresis and identified by mass spectrometric analysis. Further, the noncrosslinked forms of these proteins were also identified. Two proteins with apparent molecular masses of 73 kDa (termed C-terminal peptide-binding protein 1, CBP1) and 35 kDa (CBP2) were specifically linked with the C-terminal peptide. CBP2 is a cytoplasmic protein that consists of two conserved domains that are characteristic of a ricin-type lectin domain. CBP2 remained in the detergent-insoluble particles and was released from the particles by the addition of monosaccharides such as methyl-beta-D-galactopyranoside. CBP1 was released from the membranes by treatment with phosphatidylinositol-specific phospholipase C, indicating that CBP1 is a glycosylphosphatidylinositol (GPI)-anchored plasma membrane protein. CBP1 was found to be a copper-binding protein, and is highly homologous to Arabidopsis thaliana SKU5 that contributes to directional root growth processes. Further, it is similar to A. thaliana SKS6 that contributes to cotyledon vascular patterning and to Nicotiana tabacum NTP303 that contributes to pollen tube growth. The present results indicate that ABP1 may contribute to directional cell growth processes via the GPI-anchored plasma membrane protein SKU5 and its family members.
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PMID:Identification of a glycosylphosphatidylinositol-anchored plasma membrane protein interacting with the C-terminus of auxin-binding protein 1: a photoaffinity crosslinking study. 1664 5

Heterotrimeric G-proteins transduce signals from activated G-protein-coupled receptors (GPCR) to appropriate downstream effectors and thereby play an important role in signaling. A role of G-proteins in salinity and heat stress tolerance has not heretofore been described. We report isolation of cDNAs of two isoforms of Galpha (Galpha1, 1152 bp; Galpha2, 1152 bp), one Gbeta (1134 bp), two isoforms of Ggamma (Ggamma1, 345 bp; Ggamma2, 303 bp) and a GPCR (1008 bp) from Pisum sativum, and purification of all the encoded recombinant proteins (Galpha, 44 kDa; Gbeta, 41 kDa; Ggamma, 14 kDa; GPCR, 35 kDa). The transcript levels of Galpha and Gbeta were upregulated following NaCl, heat and H(2)O(2) treatments. Protein-protein interaction studies using an in vitro yeast two-hybrid system and in planta co-immunoprecipitation showed that the Galpha subunit interacted with the pea Gbeta subunit and pea phospholipase C (PLCdelta) at the calcium-binding domain (fn1). The GTPase activity of the Galpha subunit increased after interaction with PLCdelta. The GPCR protein interacted with all the subunits of G-proteins and with itself. Transgenic tobacco plants (T(0) and T(1)) constitutively over-expressing Galpha showed tolerance to salinity and heat, while Gbeta-over-expressing plants showed only heat tolerance, as tested by leaf disk senescence assay and germination/growth of T(1) seeds/seedlings. These findings provide direct evidence for a novel role of Galpha and Gbeta subunits in abiotic stress tolerance and possible cross-talk between PLC- and G-protein-mediated signaling pathways.
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PMID:Heterotrimeric G-protein complex and G-protein-coupled receptor from a legume (Pisum sativum): role in salinity and heat stress and cross-talk with phospholipase C. 1758 33


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