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)

1. Pretreatment of frozon cryostat sections with formaldehyde or calcium ions inhibits diffusion of the plasma membrane enzymes 5' nucleotidase, ATP-ase and alkaline phosphatase during incubation. 2. Treatment of fixed sections with different kinds of buffer at 37 degrees C induces diffusion of enzyme activity from the plasma membrane to other sites of the section and into the incubation medium. This buffer influence depends on temperature: at 4 degrees C only a slight diffusion occurs. Addition of phospholipase C, digitonin or taurocholate to the buffer opposes the buffer effect. 3. Pretreatment of frozen cryostat sections with a mixture of equal parts of chloroform and acetone give a good fixation of the plasma membrane enzymes 5'-nucleotidase, ATP-ase, alkaline phosphate and leucyl-beta-naphthylamidase. During this treatment the different kinds of lipids present in the membrane are ex-racted equally. After this fixation buffer treatment does not cause a visible diffusion of enzyme activity in the section. Only a slight diffusion (1 till 7 percent) into the buffer solution takes place. 4. The mentioned treatments open up possibilities to get insight into the membrane anchorage of plasma membrane enzymes.
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PMID:Influence of fixation and buffer treatment on the release of enzymes from the plasma membrane. 14 99

Low concentrations of protease and trypsin reduced the electrophoretic mobility (EPM) of thymocytes; with higher concentrations it was normal or above. Differences in membrane structure of thymocytes, T and B cells was found as B cells showed no reduction while T cells gave intermediate values. Further the reduction was greater with protease than with trypsin. Formalin fixation increased the EPM of all normal cell types to a similar degree. The EPM of proteolytically treated thymocytes and B cells was increased to a similar level and to a greater degree than neuraminidase-treated thymocytes. Small amounts of sialic acid were detected in the supernatant after proteolytic treatment of thymocytes. Protease reduced the binding of anti-lymphocyte serum, while no definite effect was obtained with trypsin. Neither sublytic doses of phospholipase C nor ribonuclease appeared to change the EPM.
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PMID:Electrophoresis of lymphoid cells. Differences in the cell membrane structure of murine thymocytes, T and B cells revealed by enzyme and formalin treatment. 76 20

Previous studies demonstrated that hen erythrocytes have an inoperative, latent sphingomyelinase which is activated when the cells are hemolyzed in a hypotonic medium. Within minutes after hemolysis about 60-80% of the sphingomyelin (SPM) of the RBC "ghost" membrane was hydrolyzed. In this paper, expression of sphingomyelinase activity was further investigated. The percentage of total SPM hydrolyzed depended on the volume of the hypotonic hemolyzing buffer. Thus, suspending the erythrocytes in 4 vol of the buffer resulted in clumping of the hemolyzed "ghosts" and no hydrolysis of SPM. In comparison, suspension in 19 vol of the hypotonic buffer showed no clumping and sphingomyelinase activity was fully expressed. But centrifugation of the latter or, alternatively, addition of concanavalin A induced clumping and elimination of sphingomyelinase activity. Hen RBC could also be hemolyzed in an isotonic medium in the presence of Triton X-100, mellitin, halothane, and phospholipase C. Activation of the latent sphingomyelinase occurred at concentrations of these reagents which caused cell lysis. Hen RBC were dispersed in an isotonic medium containing glutaraldehyde (0.1%) or formaldehyde (10%). This rendered the cells resistant to hemolysis, even when subsequently dispersed in a hypotonic medium or water. But incubation of the "fixed" cells in a hypotonic or isotonic medium activated the enzyme, resulting in hydrolysis of 60% of the cellular SPM. In contrast, when glutaraldehyde was included in the hypotonic buffer, hemolysis occurred but sphingomyelinase activity was eliminated.
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PMID:Utilization of membranous lipid substrates by membranous enzymes: activation of the latent sphingomyelinase of hen erythrocyte membrane. 300 52

The Fc receptor activity in placental extracts prepared using EDTA and 2-mercaptoethanol was assayed using an indirect hemagglutination technique with sheep erythrocytes sensitized with rabbit IgG. The agglutinating activity of the extract was not affected by storage at -70 degrees C, by rapid freezing and thawing, by treatment with periodic acid, formaldehyde, neuraminidase, trypsin, pronase, or phospholipase C. Papain abolished the activity, indicating that the receptor is a protein. Reduction and alkylation had no effect on the agglutinating activity, indicating that -S-S-bonds are not important for binding. In the presence of 0.6 M NaCl the agglutinating activity was abolished, indicating that electrostatic interactions are of significance. The solubilized Fc receptor shows so many similarities to the previously studied in situ Fc receptor that they are probably identical.
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PMID:Properties of the solubilized placental receptor for IgG. 621 64

Phallolysin, a mixture of two to three cytolytic proteins (all of Mr 34 000), has been isolated from Amanita phalloides mushrooms and purified to homogeneity (specific activity 24 000 hemolytic units/mg of protein). After separation by isoelectric focusing, the amino acid composition of two of these proteins has been determined. They are rich in water-soluble amino acids and contain one tryptophan residue each, but no cysteine or methionine. Mr was determined to be 34 000 in the native form as well as under denaturing conditions, indicating that the native proteins exist as monomers. Many of the physical properties of phallolysin are strikingly similar to those of staphylococcal alpha-toxin, e.g., molecular weight, existence of multiple forms, pI values, amino acid composition, and thermolability (60 degrees C). Pure phallolysin allowed us to prepare a radioactively labeled toxin. Labeling was achieved by reaction with formaldehyde, followed by reduction with sodium [3H]borohydride. With the labeled toxin (specific activity 7-14 Ci/mmol, ca. 60% biological activity), we investigated its binding to human A2 erythrocytes. We determined the number of receptors on these cells (2 X 10(4) per cell) as well as their affinity to the toxin (KD = 4 X 10(-9) M). In studies on the mechanism of cytolytic activity, we were able to distinguish at least three sequential events: binding of the toxin to human erythrocytes, K+ release, and membrane rupture (hemoglobulin release). These steps could be characterized by different kinetics as well as by different temperature dependencies. Again, the kinetic data for phallolysin are very closely related to those obtained for staphylococcal alpha-toxin.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Physical properties and function of phallolysin. 662 15

The aims of the present study were to demonstrate FcR activity of dental periapical granulomas and to correlate the activity with the degree of lymphoreticular cell infiltration. Cryostat sections of 46 out of 51 granulomas adsorbed sheep erythrocytes(E) sensitized with rabbit IgG antibodies (A) (EA). No adsorption occurred using erythrocytes sensitized with F(ab')2 fragments of IgG. IgG and Fc fragments of human of rabbit IgG inhibited the binding of EA, whereas F(ab')2 fragments, human IgA, IgM or albumin did not, indicating the presence of receptors for the Fc region of IgG. Periodate, neutral formaldehyde and phospholipase C abolished the FcR activity whereas neuraminidase had no effect. Comparison of sections binding EA and adjacent sections stained with haematoxylin and eosin showed that EA adhered to areas infiltrated with mononuclear cells. The degree of binding of EA coincided with the density of mononuclear cell infiltration. Point attachments between the tissue sections and the adsorbed EA could be demonstrated by scanning electron microscopy. Sections with no infiltrates did not bind EA.
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PMID:In situ characterization of cell infiltrates in human dental periapical granulomas. 1. Demonstration of receptors for the Fc region of IgG. 680 Dec 41

Properties of guinea pig peritoneal macrophage Fc receptor for IgG are described. It was found that the receptor was binding both monomeric and aggregated rabbit IgG. The values of apparent affinity constants were 2.6 +/- 0.6 x 10(8) M-1 and 3.84 +/- x 10(8) m-1 for IgG monomers and aggregates, respectively. The number of monomeric IgG molecules bound was calculated to be 3.3 +/- 0.2 x 10(5) per cell and of aggregated IgG 3.95 +/- 0.12 x 10(5) per cell. When the homologous system: guinea pig IgG2 and guinea pig macrophages was investigated, the affinity constant found was 1.66 +/- 0.45 x 10(8) M-1 and 2.6 +/- 0.24 x 10(5) molecules of IgG were bound per cell. Both, rabbit IgG and, guinea pig IgG2, interacted wih the same receptor binding sites on macrophages. Treatment of macrophages with 2-mercaptoethanol, formaldehyde, and iodoacetamide was without any effect on the IgG binding properties of cells. Periodate, trypsin, pronase, phospholipase C considerably diminished the number of IgG molecules bound to macrophages. Treatment of macrophages with neuraminidase increased the number of IgG molecules bound per cell. The results obtained suggests that both sugar and protein components are important for the IgG binding activity of guinea pig peritoneal macrophages. Studies on the effect of pH, ionic strength, and temperature on the interaction of macrophages with IgG showed that electrostatic interactions are important for binding of IgG to the macrophage Fc receptor.
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PMID:Studies on properties of guinea pig peritoneal macrophage Fc receptor. 744 40

Insulin sensitive glycosylated phosphatidylinositol (GPI) from chick embryo fibroblasts was isolated and partially characterized. [(3)H]Ethanolamine was incorporated into lipids different from phosphatidylethanolamine, as shown by two sequential thin layer chromatographies (TLC) using an acidic solvent system followed by a basic solvent system. Other isotopes, myo-[(3)H]inositol, [(3)H]glucosamine, [(3)H]galactose, and [(3)H]palmitic acid were also incorporated into these lipids. These lipids were separated into two peaks on the second basic TLC, designated as peaks I and II from the origin. Insulin stimulation of cells caused a rapid breakdown of these two lipids. These two lipids were treated by nitrous acid and phosphatidylinositol-specific phospholipase C (PI-PLC). The radioactivity of peak I lipid was decreased by both treatments, and that of peak II lipid was also decreased by PI-PLC treatment but not significantly by nitrous acid treatment. Peak II lipid did not fulfill the criteria for GPI. Tritium released by the treatment of PI-PLC of peak I lipid was recovered in the aqueous phase. [(3)H]Ethanolamine-labeled peak I lipid was hydrolyzed by acid treatment and the hydrolysis products were analyzed by TLC and high performance liquid chromatography (HPLC). Tritium label was recovered as native label at the rate of 95%. [(3)H]Ethanolamine of peak I lipid was reductively methylated completely with formaldehyde and cyanoborohydride, as shown by HPLC analysis. The results indicate that peak I lipid contains primary ethanolamine as a glycan component and is insulin-sensitive free GPI.
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PMID:Incorporation of ethanolamine into insulin-sensitive glycosylated phosphatidylinositol of chick embryo fibroblasts. 1108 35

Granulocyte-macrophage colony-stimulating factor (GM-CSF) can induce the generation and activation of dendritic cells (DCs), the most potent of antigen presenting cells (APCs). Tumors secreting GM-CSF have been shown to induce strong anti-tumor immune responses. In this report, we have constructed a glycosylphosphatidyl-inositol (GPI) anchored form of GM-CSF (GPI-GM-CSF). This protein subsequently was found expressed on the cell membrane and sensitive to phosphatidyl-inositol-specific phospholipase C (PIPLC), confirming that it is GPI-anchored. However, GM-CSF was also found in the culture supernatant of cells expressing GPI-GM-CSF. Inhibition studies using brefeldin A and para-formaldehyde fixation revealed that GM-CSF found in the supernatant was not secreted, but due to shedding or proteolytic cleavage. Accumulation of GM-CSF in the media from isolated membranes was time and temperature-dependent. The released portion represented 10-15% of all membrane-bound GM-CSF after 72h under culture conditions. GPI-GM-CSF retained functional activity to induce bone marrow cell proliferation and administration of GPI-GM-CSF expressing membranes induced the generation of DCs in vivo. These results demonstrate that GPI-anchored GM-CSF retains all functional activity of native GM-CSF while gaining the ability to attach to cell membranes. The ability of GPI-GM-CSF to be expressed on membranes and be partially released, can possibly lead to formation of a cytokine gradient, while retaining ability to target associated membrane antigens to DCs. This novel form of GM-CSF may have wide range of clinical applicability.
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PMID:GPI-anchoring of GM-CSF results in active membrane-bound and partially shed cytokine. 1192 38

We previously found that Merkel cells (MCs) of the rat and monkey show a strong immunoreaction of the alpha-subunit of Gq protein. The Galphaq-subunit isoform activates isozymes of phospholipase C-beta (PLC-beta), which produces inositol-(1,4,5)-triphosphate (IP3) which mobilizes intracellular Ca(++) from calcium stores via IP3 receptors. Glutamate and adenosine triphosphate (ATP), which are candidates for neurotransmitters in Merkel endings, are known to couple to Galphaq. Although MCs showed positive immunoreactions of metabotropic glutamate receptor 5 (mGluR5) in our preliminary study, these cells were not reactive to all antibodies to PLC-beta isozymes. We, therefore, reinvestigated immunohistochemical affinities to MCs of antibodies to PLC-beta isozymes and mGluRs using frozen sections of rat sinus hair follicles that were briefly postfixed in formaldehyde. We also studied the immunohistochemical expressions of P2Y receptors for ATP and IP3 receptor subtypes using similar sections. Merkel cells showed positive immunoreactions of PLC-beta1 and mGluR5. It was also found that MCs show positive immunoreactions of P2Y2, IP3R-I, and IP3R-II receptors. These results suggest that the Galphaq isoform in MCs couples to both the P2Y2 receptor and mGluR5 and regulates the intracellular Ca(++) concentration via the PLC-beta-IP3 cascade.
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PMID:Immunohistochemical expressions of mGluR5, P2Y2 receptor, PLC-beta1, and IP3R-I and -II in Merkel cells in rat sinus hair follicles. 1280 96


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