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)

A 247 000 x g particulate fraction from a moderately halophilic halotolerant bacterium incorporated [14C] glucose added as UDP[14C]glucose and 32P-labeled phosphatidylglycerol into glucosylphosphatidylglycerol. Exogenously added phosphatidylglycerol was available to the enzyme only when dispersed in a detergent, preferably Triton X-405, by sonication. The 14C- or 32P-labeled glucosylphosphatidylglycerol was degraded with phospholipase C. The water soluble product formed was isolated and identified by paper chromatography as glucosylglycerolphosphate. The system required Mg2+ or Ca2+ for activity. KCl and NaCl were inhibitory even when added at low concentrations.
 ...
PMID:Glycolipids of a halotolerant, moderately halophilic bacterium. 69 36

The effect of modification of photoreceptor membranes of the bovine retina on the termodynamical parameters that characterize heat denaturation of rodopsin was studied. The highest increase of the rate constant and the corresponding maximal drop of the free energy change of heat denaturation of the pigment were obtained by using 7 M urea or 25% Triton X-100 in the presence of 5.10(-4) M EDTA. After chipping off one third of the protein from the rodopsin molecule by papain treatment a significant decrease of the slope of the Arrenius curve and a maximal decrease of entropy change compared to the parameters known for heat denaturation of the pigment in native photoreceptor membranes were found. Modification of the lipid components of the photoreceptor membranes (treatment with Triton X-100 and phospholipase C) reduced the thermostability of rodopsin. Maximal changes were obtained at Triton X-100 concentrations 0.1--1%, further concentration increas (1--25%) did not lead to significant changes. Phospholipase C treatment resulted in a decrease of free energy change and an increase of entropy change without affecting entalpy changes, accompaning the heat denaturation of rodopsin. Bivalent cations (Ca2+, Mg2+) increased the termostability of rodopsin both in photoreceptor membranes and in solutions to 25% Triton X-100.
 ...
PMID:[Modification of the retina photoreceptor membranes and temperature stability of rhodopsin]. 73 88

Fixation with glutaraldehyde at a concentration above 0.05 per cent for 1 h at neutrality rendered erythrocytes resistant towards surfactants as Triton X-100, Tween-80 and sodium dodecyl sulphate. Fixation of erythrocytes with low concentrations of glutaraldehyde abolished the lytic effect of membrane active proteins as complement, phospholipase C and staphylococcal alpha-toxin. The fixation procedure did not alter significantly the receptor groups on erythocytes for viruses of the ortho- and para-myxo group and rubella virus. The fixation reduced the agglutinability of human O-erythrocytes by reovirus.
 ...
PMID:The effect of glutaraldehyde on the stability of erythrocytes and on virus receptor substances. 82 91

Nicotinic acetylcholine receptor protein (nAChR) has been solubilized from rat cerebral cortices by extracting a crude membrane fraction with the nonionic detergent Triton X-100 (polyoxyethylene-p-t-octylphenol). The solubilized nAChR was partially purified by affinity chromatography (Naja naja siamensis alpha-toxin affinity arm, linked to Sepharose 4B) and characterized by binding of 125I-labeled alpha-bungarotoxin. The reaction of labeled toxin and nAChR appears to be second order with a rate constant (k1) equal to 0.38 X 10(5) M-1 S-1 at 20 degrees. The toxin-nAChR complex dissociates with a dissociation rate constant (k-1) of 1.23 X 10(-5) S-1 at 20 degrees (t 1/2 = 15.6 h). The kinetically determined dissociation constant (Kd) for the complex is 3.24 X 10(-10) M. A variety of cholinergic ligands were studied for their ability to inhibit binding of labeled toxin. The results indicate that the brain receptor is indeed nicotinic. The s20, w and v of the toxin-nAChR complex in 0.1% Triton were determined by velocity sedimentation in D2O and H2O sucrose gradients. The values are 12.9 S and 0.80 cm3 g-1. The Stokes radius of the complex determined by gel filtration equals 7.5 nm. The Mr of the complex calculated from the hydrodynamic parameters, and corrected for bound detergent, equals 357,000.
 ...
PMID:Nicotinic acetylcholine receptor from rat brain. Solubilization, partial purification, and characterization. 97 72

Gel filtration studies in the presence of Triton X-100 showed that treatment with phosphatidylinositol-specific phospholipase C reduced the apparent molecular size of the 100 kDa folate binding protein from human milk, choroid plexus and semen to 25 kDa. Cleavage of a hydrophobic glycosyl phosphatidylinositol domain (a membrane anchor) inserting the protein into Triton X-100 micelles could account for this phenomenon.
 ...
PMID:Conversion of an apparent 100 kDa folate binding protein from human milk, choroid plexus and semen to a 25 kDa molecular species by phosphatidylinositol-specific phospholipase C. 133 54

Phosphatidylinositol (PtdIns) kinase and phosphatidylinositol 4-phosphate (PtdIns4P) kinase have been studied in a purified sarcolemmal fraction isolated from rat heart. Both enzymes were Mg(2+)-dependent and their activities were maximal at 2.5 mM Mg2+ and pH 7.5. Kinetic analysis of endogenous substrate phosphorylation by ATP showed that the apparent Km and Vmax values for PtdIns kinase were 292 +/- 17 microM and 1390 +/- 80 pmol.mg-1.min-1, respectively, while the apparent Km and Vmax values for PtdIns4P kinase were 398 +/- 25 microM and 382 +/- 24 pmol.mg-1.min-1. Under normal conditions, the activity of PtdIns4P kinase was lower than that of PtdIns kinase; however, the former activity increased several fold in the presence of PtdIns4P as an exogenous substrate. The enzymatic synthesis of intramembranal PtdIns4P and phosphatidylinositol 4,5-bisphosphate (PtdIns (4,5)P2) was maximally enhanced by 0.1% Triton X-100 and inhibited by micromolar concentrations of Ca2+. Inhibition of PtdIns and PtdIns4P kinase showed IC50 values for Ca2+ of 20 and 6 microM, respectively, and was independent of either Ca(2+)-induced activation of phospholipase C and polyphosphoinositide monophosphoesterases or low ATP concentrations. The results indicate that purified rat heart sarcolemmal membranes contain a very active phosphoinositide phosphorylation system which is regulated by micromolar levels of Ca2+. The Ca2+ effect may contribute to the feedback inhibition of the receptor-activated formation of inositol 1,4,5-trisphosphate.
 ...
PMID:Phosphoinositide kinases in rat heart sarcolemma: biochemical properties and regulation by calcium. 133 11

Glycosyl phosphatidylinositol-specific phospholipase C (GPI-PLC) from Trypanosoma brucei cleaves the glycosyl phosphatidylinositol (GPI) anchor of the trypanosome variant surface glycoprotein (VSG) and other GPI structures. We have expressed this enzyme in Escherichia coli, using a protocol designed to produce the native enzyme rather than a fusion protein. We have purified large amounts of GPI-PLC from E. coli membranes, using a single step immunoaffinity technique. The expressed enzyme is identical to its trypanosome counterpart in enzymatic specificity, mobility on SDS-PAGE, and isoelectric point. Recombinant GPI-PLC is a membrane enzyme; it associates with E. coli membranes and, like the T. brucei GPI-PLC, partitions into the detergent phase in Triton X-114 phase separation experiments. The Michaelis constants for the two enzymes are similar (400 nM, with VSG as substrate). The turnover number (kcat, 72 min-1) of the recombinant enzyme (expressed from a. T. brucei rhodesiense WRATat 1.1 cDNA) is about one-tenth that of GPI-PLC from T. brucei brucei (ILTat 1.3).
 ...
PMID:Glycosyl phosphatidylinositol-specific phospholipase C of Trypanosoma brucei: expression in Escherichia coli. 136 51

A number of cell surface proteins have been shown to be anchored to the plasma membrane by a covalently attached glycoinositol phospholipid (GPL) in amide linkage to the C-terminus of the mature protein. We applied several criteria to establish that folate binding protein (FBP) in brush border membranes of rat kidney contains a GPL anchor. Brush border membranes were isolated and labeled with [3H]folate, and the complex of FBP and [3H]folate was shown to be released to the supernatant by incubation with purified bacterial phosphatidylinositol-specific phospholipase C (PIPLC) but not by incubation with a purified bacterial phosphatidylcholine-specific phospholipase C. The FBP-[3H]folate complex both in crude extracts and after FBP purification by ligand-directed affinity chromatography interacted with Triton X-114 micelles, and prior incubation with PIPLC prevented this detergent interaction. Individual residues characteristic of GPL anchors were found to be covalently associated with FBP following polyacrylamide gel electrophoresis in sodium dodecyl sulfate. These included glucosamine and ethanolamine, which were radiolabeled by reductive methylation and identified by chromatography on an amino acid analyzer, and inositol phosphate, which was inferred by Western blotting with an anti-CRD antisera. This antisera gave positive immunostaining only after FBP had been cleaved by PIPLC, a reliable diagnostic of a GPL anchor. The relationship between GPL-anchored FBP in biological membranes and soluble FBP in biological fluids also is discussed.
 ...
PMID:Folate binding protein from kidney brush border membranes contains components characteristic of a glycoinositol phospholipid anchor. 137 26

We have purified to homogeneity the 33-kDa phosphatidylinositol-specific phospholipase C (PI-PLC) from the culture fluid of Listeria monocytogenes, a facultative intracellular pathogen. The protein was overexpressed, and secretion of PI-PLC was further enhanced by the addition of divalent cations to the culture medium. The basic protein (pI, approximately 9.4) was complexed with anionic proteins in the crude culture fluid. It bound to DEAE-Sepharose and was eluted from Sephacryl S-200 near the void volume in low-ionic-strength buffer, suggesting aggregates of greater than or equal to 150 kDa. Gel filtration chromatography on Sephacryl S-200 in the presence of 1 M ammonium sulfate resulted in disaggregation and complete separation of PI-PLC, which interacted with the column matrix. Amino-terminal sequencing of the pure protein gave results consistent with the previously deduced sequence and showed that the signal cleavage site was between alanine 29 and tyrosine 30. The enzyme was specific for PI and showed no activity with phosphatidylethanolamine, phosphatidylcholine, or phosphatidylserine. It did not cleave PI-4-phosphate or PI-4,5-bisphosphate, but it was active on the membrane form of the variable surface glycoprotein from Trypanosoma brucei, a PI-glycan-anchored protein. When assayed with deoxycholate-mixed micelles of PI, activity was highly dependent on added salt. Activation by salt was also observed with Triton X-100-mixed micelles. The optimal concentration of CaCl2 or MgCl2 was lower than that of KCl or (NH4)2SO4, but activity was not specifically dependent on divalent cations and was not inhibited by addition of EDTA. With deoxycholate, the optimum pH was 7.0. A broader pH optimum ranging from 5.5 to 6.5 was observed with Triton X-100-mixed micelles. These results are consistent with a postulated role for secreted PI-PLC in the acidified primary phagocytic vesicle of infected cells.
 ...
PMID:Purification and characterization of Listeria monocytogenes phosphatidylinositol-specific phospholipase C. 139 18

The properties of a cholinesterase from mucosal cells of rat intestine have been characterized. The enzyme was identified as butyrylcholinesterase because it was more sensitive to iso-OMPA (IC50 = 1.0 x 10(-6) M) than to BW284C51 (IC50 = 5.5 x 10(-5) M) and was not inhibited by substrate excess. It displayed a higher affinity for acetylthiocholine than for butyrylthiocholine. A major molecular form was observed sedimenting at 5.9 S. Two other minor molecular forms were identified as a hydrophilic tetramer (G4, sedimenting at 10.5 S) and a monomer (G1, sedimenting at 4.3 S). The 5.9 S component was referred to as "G" form (G for globular) and not "G2" as usual dimers for the following reasons: (i) the G form was unaffected by the reducing agents, beta-mercaptoethanol and dithiothreitol, which converted disulfide-linked dimers of acetylcholinesterase into monomers, (ii) the G form was shifted from 5.9 to 3.4 S when the sucrose gradient contained Triton X-100. This value of 3.4 S (in Triton X-100) appeared too low for a typical G2 form. The shift in the S value was partly reversible: the 3.4 S form resedimented at 5.2 S in the absence of detergent. The behavior of the G form in sucrose gradients indicated that it was amphiphilic. This was confirmed in nondenaturing electrophoreses and also by quantitative binding of the G form to octyl-Sepharose. The hydrophobic domain of the G form was not a glycolipid, as shown by its insensitivity to Bacillus thuringiensis phosphatidylinositol-specific phospholipase C and its nonaggregating properties in the absence of nondenaturing detergent.(ABSTRACT TRUNCATED AT 250 WORDS)
 ...
PMID:Amphiphilic forms of butyrylcholinesterase in mucosal cells of rat intestine. 142 Feb 1


<< Previous 1 2 3 4 5 6 7 8 9 10 Next >>