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)

A major glycoprotein of rat hepatoma plasma membranes was selectively released as a soluble form by incubating the membrane with phosphatidylinositol-specific phospholipase C. The soluble form corresponding to the glycoprotein was also prepared by butan-1-ol extraction of microsomal membranes at pH 5.5, whereas extraction at pH 8.5 yielded an electrophoretically different form with a hydrophobic nature. The soluble glycoprotein extracted at pH 5.5 was purified by sequential chromatography on concanavalin A-Sepharose, Sephacryl S-300 and anti-(alkaline phosphatase) IgG-Sepharose, the last step being used to remove a contaminating alkaline phosphatase. The glycoprotein thus purified was a single protein with Mr 130,000 in SDS/polyacrylamide-gel electrophoresis, although it behaved as a dimer in gel filtration on Sephacryl S-300. The glycoprotein was analysed for amino acid and carbohydrate composition. The composition of the carbohydrate moiety, which amounted to 64% by weight, suggested that the glycoprotein contained much larger numbers of N-linked oligosaccharide chains than those with O-linkage. It was confirmed that the purified glycoprotein was immunologically identical not only with that released by the phospholipase C but also with the hydrophobic form extracted with butan-1-ol at pH 8.5. The results indicate that the glycoprotein of rat hepatoma plasma membranes, which has an unusually high content of carbohydrate, is another membrane protein released by phosphatidylinositol-specific phospholipase C, as documented for alkaline phosphatase, acetylcholinesterase and Thy-1 antigen.
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PMID:Purification and characterization of a major glycoprotein in rat hepatoma plasma membranes. One of the membrane proteins released by phosphatidylinositol-specific phospholipase C. 303 62

Membrane anchoring of proteins by a covalently attached glycosyl-phosphatidylinositol moiety has been reported in many different eukaryotic cells including parasite protozoa. The diversity of proteins in which this phospholipid attachment is found suggests that it is functionally important and perhaps also functionally pleiotropic. Studies on the Thy-1 antigen of murine lymphocytes indicate that it can facilitate the lateral mobility of membrane proteins. It can also permit the rapid and specific release of the anchored proteins from the membrane following cleavage by a phosphatidylinositol-specific phospholipase C (PI-PLC). Here we show that this type of anchoring may be involved in the regulation of an enzymatic activity. PI-PLC releases a Plasmodium falciparum membrane protein of relative molecular mass (Mr) 76K (p76) from intact merozoites or isolated schizont membranes and induces a proteolytic activity associated with its soluble form. Endogenous activation of the proteolytic activity of p76 appears to occur at the end of the schizogony and could initiate a cascade of biochemical events associated with merozoite maturation.
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PMID:Induction of the proteolytic activity of a membrane protein in Plasmodium falciparum by phosphatidyl inositol-specific phospholipase C. 328 Oct 25

PC12 pheochromocytoma cells and cultures of early postnatal rat cerebellum were labeled with [3H]glucosamine, [3H]fucose, [3H]leucine, [3H]ethanolamine, or sodium [35S]sulfate and treated with a phosphatidylinositol-specific phospholipase C. Enzyme treatment of [3H]glucosamine- or [3H]fucose-labeled PC12 cells led to a 15-fold increase in released glycoproteins. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, most of the released material migrated as a broad band with an apparent molecular size of 32,000 daltons (Da), which was specifically immunoprecipitated by a monoclonal antibody to the Thy-1 glycoprotein. A second glycoprotein, with an apparent molecular size of 158,000 Da, was also released. After treatment with endo-beta-galactosidase, 40-45% of the [3H]glucosamine or [3H]fucose radioactivity in the phospholipase-released glycoproteins was converted to products of disaccharide size, and the molecular size of the 158-kDa glycoprotein decreased to 145 kDa, demonstrating that it contains fucosylated poly-(N-acetyllactosaminyl) oligosaccharides. The phospholipase also released labeled Thy-1 and the 158-kDa glycoprotein from PC12 cells cultured in the presence of [3H]ethanolamine, which specifically labels this component of the phosphatidylinositol membrane-anchoring sequence, while in the lipid-free protein residue of cells not treated with phospholipase, Thy-1 and a doublet at 46/48 kDa were the only labeled proteins. At least eight early postnatal rat brain glycoproteins also appear to be anchored to the membrane by phosphatidylinositol. Sulfated glycoproteins of 155, 132/134, 61, and 21 kDa are the predominant species released by phospholipase, which does not affect a major 44-kDa protein seen in [3H]ethanolamine-labeled brain cultures. The 44-48- and 155/158-kDa proteins may be common to both PC12 cells and brain.
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PMID:Phosphatidylinositol-anchored glycoproteins of PC12 pheochromocytoma cells and brain. 339 Apr 45

Qa-2, a cell-surface glycoprotein anchored by phosphatidylinositol (PI), is structurally related to the class I transplantation antigens H-2 K, D, and L, which are integral membrane glycoproteins. The predicted transmembrane segment of Qa-2 differs from those of H-2 K, D, and L by the presence of an aspartate in place of a valine at position 295. A single base change that replaced this aspartate with valine resulted in cell-surface Qa-2 molecules that were insensitive to hydrolysis by a PI-specific phospholipase C and more resistant to papain cleavage, properties shared by H-2D. Cells expressing Asp----Val mutant Qa-2 proteins were still able to attach a PI anchor to endogenous proteins such as Thy-1 and J11D. It therefore appears that this single amino acid change converts Qa-2 from a PI-linked form into an integral membrane protein.
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PMID:Conversion of a PI-anchored protein to an integral membrane protein by a single amino acid mutation. 339 1

The mechanism by which the rat T cell alloantigen, RT-6.2, is attached to the membrane was investigated. Treatment of rat lymph node and T-hybridoma cells with phosphatidylinositol-specific phospholipase C (PI-PLC) caused a substantial reduction in the amount of RT-6.2 on the cell surface. No significant release of a rat T helper marker (visualized by the mAb W3/25) was observed in response to PI-PLC treatment. This is in sharp contrast to the effects of trypsin, which removes most of the T helper marker but had little effect on RT-6.2. SDS-PAGE analysis of the RT-6.2 released by PI-PLC indicated that the Mr was not significantly changed by this treatment. Phase separation of the released RT-6.2 in Triton X-114 showed that the PI-PLC had converted it from an amphiphilic membrane form to a water-soluble form, apparently by removing its hydrophobic membrane anchoring domain. These results strongly suggest that RT-6.2, in common with Thy-1 and several other cell surface proteins, is anchored in the membrane by the 1,2-diacylglycerol moiety of a covalently attached phosphatidylinositol molecule.
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PMID:Release of the rat T cell alloantigen RT-6.2 from cell membranes by phosphatidylinositol-specific phospholipase C. 348 8

We searched for mouse thymocyte surface proteins attached to the cell membrane through a phosphatidylinositol (PI)-containing glycolipid similar to that identified in the T cell-activating Thy-1 glycoprotein. Our approach was to biochemically analyse the supernatants of 125I surface-labeled thymocytes treated with 60 U/ml of Staphylococcus aureus PI-specific phospholipase C (PI-PLC). In addition to Thy-1, two molecules of Mr 13,000 and 52,000 were found to be specifically solubilized by the enzymatic treatment. The 52,000 structure is a single basic polypeptide of Mr 50,000 under non-reducing conditions. Two-dimensional gel electrophoresis analyses resolved the 13,000 mol. wt molecules in three relatively basic components including (i) a monomeric molecule(s), a fraction of which exhibited slower migration in reducing gels, and (ii) disulfide-linked multimeric structures comprising a major component of Mr 30,000 and a minor one of Mr 45,000. These 52,000 and 13,000 mol. wt molecules could be released from thymocytes and Escherichia coli lipopolysaccharide (LPS)-stimulated B cell blasts, but not from a variety of mature T cell populations. These data add new members to the list of PI-linked rodent lymphoid cell differentiation markers, which already includes three activation signal-transducing T cell molecules (i.e. Thy-1, Ly-6-linked T cell-activating proteins, and RT-6).
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PMID:Two novel phospholipid-linked mouse thymocyte surface molecules released by phosphatidylinositol-specific phospholipase C. 350 37

Thy-1 has the structure of a single variable-type immunoglobulin domain anchored to the external face of the plasma membrane via a glycophosphatidylinositol moiety. When the lipid is removed from this anchor by either phospholipase C or D, the reactivity of the delipidated Thy-1 for a range of antibodies, including those known to be determined by amino acid residues, is impaired. We have investigated in detail the effect of delipidation on the reaction with the OX7 monoclonal antibody, determined by the allelic variant residue Arg 89. Analysis of the kinetics of OX7 binding shows that delipidation affects primarily the dissociation of antibody, increasing the dissociation rate constant kdiss from 0.27 x 10(-3) s-1 to 2.39 x 10(-3) s-1. Addition of phospholipase to preformed antibody-antigen complex causes an immediate change from the slow to the faster dissociation rate, implying that delipidation induces a conformational change in the Thy-1 protein that is sufficiently strong to dissociate bound antibody. This conformational change can be demonstrated directly by the circular dichroism spectrum of human Thy-1 that detects changes in the environment of Tyr residues located near the antigenic epitopes. Molecular dynamics studies suggest that, on delipidation, a conformational change occurs in the glycan chain that affects the protein in the region of the antigenic epitopes. This study thus demonstrates that the glycophosphatidylinositol anchor strongly influences the conformation of Thy-1 protein by a mechanism that could occur generally with membrane proteins of this class.
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PMID:The glycophosphatidylinositol anchor affects the conformation of Thy-1 protein. 753 35

The cation-independent mannose-6-phosphate/insulin-like growth factor II receptor has been observed to bind to soluble forms of glycosyl-phosphatidylinositol-linked molecules, one of mammalian origin (rat Thy-1) and two of protozoan origins. Of the two phosphate groups found on the soluble forms of the protozoan glycosyl-phosphatidylinositol-linked molecules: (i) the internal mannose-6-phosphate diester (which forms a part of the ethanolamine bridge) and (ii) the inositol-1,2 cyclic phosphate group (which arises after cleavage of the membrane associated form with phosphatidylinositol-specific phospholipase C), only the former appears to be recognized by the mannose-6-phosphate/insulin-like growth factor II receptor, as mild acid hydrolysis which destroys the latter has been observed not to affect the receptor binding site.
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PMID:The cation-independent mannose-6-phosphate receptor binds to soluble GPI-linked proteins via mannose-6-phosphate. 787 96

The biological significance of the action of glycosylphosphatidylinositol (GPI)-anchored proteins in cell physiology and pathology when stimulated with their natural agonists is not known. Here we provide evidence that GPI-anchored proteins play a crucial role in the recently defined heavy metal (HgCl2)-triggered signal delivery to T lymphocytes. Thiol-reactive HgCl2, a multi-potent crosslinker of cell membrane proteins, induced heavy aggregation of Thy-1, a representative GPI-anchored protein, on murine thymocytes, and delivered a signal to induce heavy tyrosine phosphorylation of cellular proteins. This rather unusual signal delivery by HgCl2 is diminished by the pre-treatment of cells with phosphatidylinositol-specific phospholipase C, which partially cleaved GPI-anchored proteins from the cell surface. Direct evidence for the involvement of GPI or GPI-anchored proteins in the HgCl2-mediated signaling is provided by the loss of signaling in a mutant thymoma cell line defective in the phosphatidylinositol glycan-class A gene (PIG-A), and its restoration in a transfectant with PIG-A.
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PMID:Direct evidence of involvement of glycosylphosphatidylinositol-anchored proteins in the heavy metal-mediated signal delivery into T lymphocytes. 769 41

To evaluate whether a glycosylphosphatidylinositol (GPI) anchor can function as a protein sorting signal in polarized intestinal epithelial cells, the GPI-attachment sequence from Thy-1 was fused to bacterial endoglucanase E' (EGE') from Clostridium thermocellum and polarity of secretion of the chimeric EGE'-GPI protein was evaluated. The chimeric EGE'-GPI protein was shown to be associated with a GPI anchor by TX-114 phase-partitioning and susceptibility to phosphoinositol-specific phospholipase C. In polarized MDCK cells, EGE' was localized almost exclusively to the apical cell surface, while in polarized intestinal Caco-2 cells, although 80% of the extracellular form of the enzyme was routed through the apical membrane over a 24 hour period, EGE' was also detected at the basolateral membrane. Rates of delivery of EGE'-GPI to the two membrane domains in Caco-2 cells, as determined with a biotinylation protocol, revealed apical delivery was approximately 2.5 times that of basolateral. EGE' delivered to the basolateral cell surface was transcytosed to the apical surface. These data indicate that a GPI anchor does represent a dominant apical sorting signal in intestinal epithelial cells. However, the mis-sorting of a proportion of EGE'GPI to the basolateral surface of Caco-2 cells provides an explanation for additional sorting signals in the ectodomain of some endogenous GPI-anchored proteins.
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PMID:Epithelial sorting of a glycosylphosphatidylinositol-anchored bacterial protein expressed in polarized renal MDCK and intestinal Caco-2 cells. 773 11


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