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)

Rat ovarian granulosa cells synthesize two distinct species of plasma membrane-intercalated heparan sulfate (HS) proteoglycans; glycosylphosphatidylinositol (GPI)-anchored and core protein-intercalated HS proteoglycans. Both species of HS proteoglycans are primarily localized on the plasma membrane. Cell surface localization of GPI-anchored and protein-intercalated HS proteoglycans can be determined by their accessibility to exogenously added phosphatidylinositol-specific phospholipase C (PI-PLC) and trypsin, respectively. Kinetic parameters for the processes involving their transfer from the Golgi to the cell surface, endocytosis and secretion, and the modes of intracellular degradation were determined by metabolic labeling experiments using [35S]sulfate and various chase protocols in combination with the use of PI-PLC and trypsin in rat ovarian granulosa cells. The experiments demonstrated that (i) both HS proteoglycan species are transferred from the Golgi to the cell surface with an average transit time of approximately 12 min. (ii) GPI-anchored HS proteoglycans are endocytosed with a t1/2 approximately 3 h, without being shed into the medium, and they are rapidly degraded, t1/2 approximately 25 min, without generating recognizable degradation intermediates. (iii) Protein-intercalated HS proteoglycans are partly (approximately 30%) shed from the cell surface into the medium and the remaining approximately 70% are endocytosed with a t1/2 approximately 4 h. After endocytosis, they undergo a slow (t1/2 approximately 4 h) stepwise degradation generating distinct HS oligosaccharides as degradation intermediates. These results indicate that the GPI-anchored and the protein-intercalated HS proteoglycans have distinct secretory, endocytotic, and intracellular degradation pathways probably due to the differences in their anchor structures.
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PMID:Glycosylphosphatidylinositol-anchored and core protein-intercalated heparan sulfate proteoglycans in rat ovarian granulosa cells have distinct secretory, endocytotic, and intracellular degradative pathways. 153 36

The antigen recognized by the MOv18 MAb (Ca-MOv18) was recently shown to be a glycosylphosphatidylinositol (GPI)-linked protein. In this report we show that GPI-anchorage is not limited to IGROVI cells nor to other ovary carcinoma cell lines, but Ca-MOv18 was also found to be sensitive to phosphatidylinositol-specific phospholipase C (PI-PLC) treatment on fresh ovarian cancer cells. Furthermore, we found a heterogeneous sensitivity of Ca-MOv18 to PI-PLC cleavage, not only among the different cells studied but also in different experiments performed on the same cell line, during extended periods of time in culture. Sensitivity to PI-PLC cleavage was determined by immunofluorescence on live cells and by double-determinant radioimmunoassay of the antigen released in the supernatant. The specificity of the PI-PLC cleavage was demonstrated as follows: (a) TX114 solubilized Ca-MOv18 shifts from the detergent to the aqueous phase after treatment with PI-PLC; (b) on membrane preparations, PI-PLC specifically released a fraction of the antigen, which is distinct from the weakly associated form released by high-salt treatment; (c) Ca-MOv18 from IGROVI expressed the cross-reacting determinant (CRD), which is characteristic of GPI-linked molecules. The absence of CRD expression on the spontaneously released protein and the possibility of artificially inducing antigen shedding during the resynthesis of Ca-MOv18 which follows bacterial PI-PLC treatment are interesting points which need to be further investigated in order to understand the physiology of the Ca-MOv18 tumor antigen.
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PMID:Membrane association and shedding of the GPI-anchored Ca-MOv18 antigen in human ovary carcinoma cells. 153 20

Four major glycolipids were extracted from Toxoplasma gondii tachyzoites which were metabolically labeled with tritiated glucosamine, mannose, palmitic and myristic acid, ethanolamine, and inositol. Judging from their sensitivity to a set of enzymatic and chemical tests, these glycolipids share the following properties with the glycolipid moiety of the glycosylphosphatidylinositol anchor (GPI anchor) of the major surface protein, P30, of T. gondii: 1) a nonacetylated glucosamine-inositol phosphate linkage; 2) sensitivity toward phosphatidylinositol-specific phospholipase C and nitrous acid; 3) identity of HF-dephosphorylated GPI glycan backbone between three glycolipids and the HF-dephosphorylated core glycan of the GPI anchor of the major surface protein P30; 4) the presence of a linear core glycan structure blocked by an ethanolamine phosphate residue(s). Taken together with the nature of radiolabeled precursors incorporated into these glycolipids, the data indicate that these GPIs are involved in the biosynthesis of the GPI-membrane anchors of T. gondii.
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PMID:A family of glycolipids from Toxoplasma gondii. Identification of candidate glycolipid precursor(s) for Toxoplasma gondii glycosylphosphatidylinositol membrane anchors. 153 1

The structure of a major ether polar lipid of the methanogenic archaeon Methanosarcina barkeri was identified as glucosaminyl archaetidylinositol. This lipid had archaeol (2,3-di-O-phytanyl-sn-glycerol) as a core lipid portion, and the polar head group consisted of 1 mol each of phosphate, myo-inositol and D-GlcN. The polar head group was identified by means of chemical degradations, phosphatidylinositol-specific phospholipase C treatment, permethylation analysis, and fast atom bombardment-mass spectrometry as glucosaminylinositol phosphate, which was linked to the glycerol backbone via a phosphodiester bond. The stereochemical configuration of the phospho-myo-inositol residue of glucosaminyl archaetidylinositol was determined to be 1-D-myo-inositol 1-phosphate by measuring optical rotation of phospho-myo-inositol prepared by nitrous acid deamination and alkaline hydrolysis from the lipid. 1H NMR of the intact lipid showed that GlcN was linked to C-6 position of myo-inositol as an alpha-anomer. It is, finally, concluded that the complete structure of this lipid is 2,3-di-O-phytanyl-sn-glycero-1-phospho- 1'[6'-O-(2"-amino-2"-deoxy-alpha-D-glucopyranosyl)]-1'-D-myo-inositol. This lipid has a hybrid nature of an archaeal feature in alkyl glycerol diether core portion and an eucaryal feature in the polar head group identical to the conserved core structure (GlcNp(alpha 1-6)-myo-inositol 1-phosphate) of glycosylated phosphatidylinositol which serves as a membrane protein anchor in eucaryal cells.
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PMID:Archaea contain a novel diether phosphoglycolipid with a polar head group identical to the conserved core of eucaryal glycosyl phosphatidylinositol. 153 21

The membrane-bound form of aminopeptidase P (aminoacylprolyl-peptide hydrolase) (EC 3.4.11.9) was purified to apparent homogeneity from bovine lung microsomes. The enzyme was solubilized using phosphatidylinositol-specific phospholipase C (Bacillus thuringiensis), indicating that bovine lung amino-peptidase P is attached to membranes via a glycosylphosphatidylinositol anchor. The enzyme was purified 1900-fold with a yield of 25% by chromatography on decyl-agarose, omega-aminodecyl-agarose, a second decylagarose column, DEAE-Sephacel, and an ultrafiltration step. Native gradient polyacrylamide gel electrophoresis revealed a single stained protein band whose position in the gel corresponded to cleavage of the Arg1-Pro2 bond of bradykinin. The Mr was 360,000 by gel permeation chromatography and 95,000 by reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The substrate specificity of aminopeptidase P was determined using approximately 50 peptides with proline in the second position. The enzyme could hydrolyze lower NH2-terminal homologs of bradykinin, including Arg-Pro-Pro, which was used as the routine substrate in a rapid fluorescence assay performed in the absence of added Mn2+. Some peptides having NH2-terminal amino acids other than arginine were also cleaved. Aminopeptidase P appeared to favor peptides that had 2 proline residues or proline analogs in positions 2 and 3 of the substrate. In general, tripeptides having a single proline residue in position 2 were poor substrates. Aminopeptidase P was inhibited by a series of peptides, 3-8 residues long, having an NH2-terminal Pro-Pro sequence. The enzyme was also inhibited by metal-chelating agents, 2-mercaptoethanol (4 mM), p-chloromercuribenzenesulfonic acid, and NaCl at concentrations greater than or equal to 0.25 M. The purified enzyme had a pH optimum of 6.5-7.0 and was most stable in the basic pH range. A role for membrane-bound aminopeptidase P in the pulmonary inactivation of circulating bradykinin is proposed.
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PMID:Membrane-bound aminopeptidase P from bovine lung. Its purification, properties, and degradation of bradykinin. 153 67

Avirulent mutant strains of Listeria monocytogenes which fail to produce phosphatidylinositol-specific phospholipase C, or which produce reduced amounts of hemolytic listeriolysin O, are incapable of causing progressive infection in normal mice. However, both strains can grow progressively in mice that have been rendered incapable of focusing neutrophils at sites of infection as a result of being treated with monoclonal antibody 5C6, specific for the type 3 complement receptor of myelomonocytic cells. In 5C6-treated mice, phospholipase C-negative and listeriolysin-defective mutant strains of L. monocytogenes, like the wild-type strain, give rise in the liver to large numbers of discrete foci of infected hepatocytes that retain their morphological integrity during the first 24 h, despite their large bacterial burden. In normal mice, in contrast, sites of infection in the liver are indicated by discrete focal accumulations of neutrophils that occupy the space originally occupied by infected hepatocytes. It is apparent that in normal mice neutrophils function to lyse infected hepatocytes and thereby to release L. monocytogenes for ingestion and killing by neutrophils themselves and by macrophages. However, whereas a proportion of wild-type organisms survive this early mechanism of defense to give rise to progressive infection, the phospholipase C-negative organisms are totally eliminated. On the basis of these and other results, it is suggested that virulence factors other than listeriolysin are needed by L. monocytogenes to counteract the early neutrophil-mediated mechanism of defense. Listeriolysin, itself, is an intrinsic virulence factor that allows L. monocytogenes to survive and multiply in a proportion of the fixed phagocytes of the liver (permissive phagocytes) and which enables the organism to go on to infect and replicate in adjacent hepatocytes. It was found that a mutant strain of L. monocytogenes incapable of producing any listeriolysin was incapable of establishing progressive infection, even in 5C6-treated mice.
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PMID:Roles of Listeria monocytogenes virulence factors in survival: virulence factors distinct from listeriolysin are needed for the organism to survive an early neutrophil-mediated host defense mechanism. 154 69

To study surface molecules of Entamoeba histolytica we produced monoclonal antibodies from mice immunized with lysates from the pathogenic amebic strain HM1:IMSS, and screened them for the ability to inhibit E. histolytica adhesion. One monoclonal antibody, CC 8.6, was a potent inhibitor of amebic adhesion to a Chinese hamster ovary cell line, and was capable of inhibiting HM1:IMSS mediated cytotoxicity by 50%. We found that monoclonal antibody CC 8.6 bound to an amebic glycoconjugate. The glycoconjugate is present only in E. histolytica and not in other Entamoeba sp. It migrates as a polydisperse band on SDS-PAGE, and can be metabolically radiolabeled with [14C]glucose, [32P]phosphate, and [3H]palmitate. The glycoconjugate can be purified by hydrophobic interaction chromatography on octyl-Sepharose; enzymatic hydrolysis with phosphatidylinositol-specific phospholipase C alters the hydrophobic properties of the molecule. HPLC analysis of [14C]glucose-labeled glycoconjugate saccharides revealed that approximately 82% of the incorporated label was in glucose and 12% in galactose. Our studies demonstrate that one of the immunogenic surface molecules of E. histolytica is a phosphorylated, lipid-containing, glycoconjugate, and that antibodies to this antigen may have the potential to protect against E. histolytica adhesion and cytotoxicity.
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PMID:Isolation and partial characterization of a surface glycoconjugate of Entamoeba histolytica. 154 7

gp65 and gp55 are glycoprotein components of CNS synapses that are recognised by a single monoclonal antibody, SMgp65. This antibody has now been used to investigate the molecular properties of these two glycoproteins and the structural relationship between them. Both gp65 and gp55 occur in most brain regions as doublets of apparent molecular masses of 63 and 67 kDa, and 52 and 57 kDa, respectively. Striatal samples, however, are enriched in a novel gp65 isoform of 69 kDa. Removal of oligosaccharide residues from gp65 and gp55 with trifluoromethanesulphonic acid shows that gp65 and gp55 are composed of single polypeptide chains of 40 and 28 kDa, respectively. Removal of sialic acid residues with neuraminidase lowers the apparent molecular mass of both glycoproteins by 5-6 kDa. Triton X-114 phase partitioning and alkaline extraction of synaptic membranes indicate that both gp65 and gp55 are integral membrane glycoproteins. Treatment of synaptic membranes with phosphatidylinositol-specific phospholipase C does not solubilise either glycoprotein. One-dimensional peptide and epitope maps obtained by digestion of gp65 and gp55 with endoproteinase lys C or subtilisin are consistent with a close structural relationship between the two molecules. Tryptic digestion of samples enriched in gp65 and/or gp55 results in the formation of a novel immunoreactive 53-kDa species that is resistant to further trypsin degradation except in the presence of 0.1% (wt/vol) sodium dodecyl sulphate. Trypsin treatment of cultures of forebrain neurones in situ lowers the apparent molecular mass of gp65 to 53 kDa.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Molecular characterisation and structural relationship of the synapse-enriched glycoproteins gp65 and gp55. 157 91

Applying a new four-step isolation procedure, we have purified butyrylcholinesterase (BChE) from chicken serum to homogeneity with more than 250 U/mg specific activity. The serum enzyme was used for producing monoclonal antibodies. These BChE-specific also recognize BChE from brain, and thus enabled us to isolate the enzymes from embryonic and adult brain that occur only in minute amounts. More than 50% of the brain BChE is membrane-bound. The catalytic and inhibition properties of brain BChE are similar to those of serum BChE. However on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the serum enzyme is represented by a double-band of 79/82 kDa, whereas the brain enzyme has a size of 74 kDa. Limited digestion of the serum and brain preparations by V8-protease leads to similar peptide patterns. Enzymatic deglycosylation shows that their core proteins consist of 59-kDa subunits and that the different molecular weights are due to different glycosylation patterns. The differently sized glycosylation parts of brain and serum BChE may indicate that they subserve different functions. Furthermore, the membrane-bound brain BChE can be solubilized by Pronase or protease K, but not by phosphatidylinositol-specific phospholipase C.
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PMID:Butyrylcholinesterase from chicken brain is smaller than that from serum: its purification, glycosylation, and membrane association. 157 4

Interleukin-2 (IL-2) plays a central role in the immune system by regulating the proliferation and differentiation of T lymphocytes. However, the molecular mechanism of the signal transduction through the IL-2 receptor is poorly understood. We have studied the role of phosphatidic acid (PA) on IL-2 signal transduction using cloned T lymphocytes. IL-2 stimulated a transient increase in the PA concentration in resting CTLL-2 cells prelabeled with [3H]palmitic acid. This effect was detected as early as 1 min after IL-2 addition and peaked at 5 min. IL-2 similarly increased phospholipase D activity in intact CTLL-2 cells, as inferred by phosphatidylethanol production. By contrast, IL-2 did not affect [3H]palmitic acid-labeled diacylglycerol levels. Furthermore, exogenous addition of several natural or synthetic PA to T cells mimicked IL-2 activity. Thus, PA were able to induce DNA synthesis on CTLL-2 cells, although this effect was only 10%-20% of that observed with IL-2. PA showed a synergistic effect with low doses of IL-2. In addition, PA was able to induce c-myc RNA transcription in CTLL-2 cells as well as IL-2 receptor (CD25) expression on the cell membrane with equal potency as saturating doses of IL-2. It is likely that IL-2-induced PA accumulation is a consequence of phospholipase D activation. This hypothesis is further supported by the fact that the addition of exogenous phospholipase D but not phosphatidylinositol-specific phospholipase C also reproduced the IL-2 or PA effects mentioned above. In summary, our results suggest a role of phospholipase D activation and PA formation as second messengers of IL-2 activity.
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PMID:Regulation of interleukin-2 responses by phosphatidic acid. 162 28


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