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)

The alkaline phosphatase (AP) synthesized by human tumor cells closely resembles human placental AP (PLAP). Little is known about the molecular events that lead to the expression of a placenta-like AP in tumor cells. The complementary DNA encoding the AP expressed by a choriocarcinoma cell line, BeWo, was isolated and characterized. The complementary DNA is the product of the germ cell AP (Nagao isozyme) gene and not of the term PLAP gene. Like placental AP, the tumor AP can be released from the cell membrane by a phosphaditylinositol-specific phospholipase C and has a phosphaditylinositol-glycan (PI-glycan) moiety at the COOH terminus. Immunoprecipitation of phosphaditylinositol-specific phospholipase C-treated AP and analysis by polyacrylamide gel electrophoresis or isoelectric focusing demonstrates that at least 95% of the AP contains PI-glycan. Two-dimensional gel electrophoresis reveals two precursors of the mature AP. One of these does not bind an antibody against the Trypanosoma variable surface glycoprotein cross-reacting determinant and probably does not contain PI-glycan. This precursor had a shorter half-life than the more prominent PI-glycan-containing precursor in pulse-chase experiments, suggesting a precursor-product relationship between the two proteins. These data demonstrate that BeWo AP is the product of a gene normally expressed in testis, thymus, and germ cells, but not in placenta. Thus, the expression of BeWo AP results from the repression of the PLAP gene and derepression of the germ cell AP gene and, as such, the expression is ectopic. The BeWo AP (Nagao isozyme) is modified with PI-glycan that is added soon after translation, not cotranslationally.
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PMID:Expression of a Nagao-type, phosphatidylinositol-glycan anchored alkaline phosphatase in human choriocarcinomas. 216 49

Using the membrane form of variant surface glycoprotein from Trypanosoma equiperdum labelled with [3H]myristate as a substrate, we identified two glycosylphosphatidylinositol phospholipase C enzymic activities in mouse brain. These activities were associated with particulate membrane fractions. They were characterized by their pH activity maxima and sensitivity to activators and ion chelators. One of the activities was maximal at acidic pH, stimulated by butanol, sensitive to cation chelator and insensitive to manganese. The activity of the other was maximal at neutral pH, stimulated by the detergent deoxycholate and independent of the presence of cation chelator or calcium. On membrane subfractionation, the acidic butanol-stimulated activity was found mainly associated with the lysosomal compartment, whereas the neutral deoxycholate-stimulated activity sediments with the myelin and plasma membrane compartment. These activities could be differentiated from particulate phosphatidylinositol phospholipases C, whose acidic lysosomal form is sensitive to manganese and insensitive to cation chelator or butanol, whereas the deoxycholate-activated enzymes are Ca2(+)-dependent.
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PMID:Identification of glycosylphosphatidylinositol-specific phospholipases C in mouse brain membranes. 216 64

Temporal retinal axons growing in vitro on carpets of tectal membranes are deflected by cell membranes of posterior tectum. The activity responsible for this deflection can be abolished by antibodies raised against tectal membranes and the corresponding Fab fragments. Analysis of tectal membranes by two-dimensional gel electrophoresis and immunoblotting reveals a 33 kd glycoprotein that has a higher concentration in posterior than in anterior tectum. Its expression is developmentally regulated, and it is sensitive to phosphatidylinositol-specific phospholipase C. These are properties expected for a molecule responsible for the phenomena observed in experiments on in vitro guidance of retinal axons.
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PMID:Biochemical characterization of a putative axonal guidance molecule of the chick visual system. 217 92

The pellet recovered after centrifugation (5000 X g) of human corneal endothelial homogenates was used as the source of membranes in these studies. A 66-kilodalton (kD) protein was identified as the most abundant protein in the particulate pellet by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. The de novo synthesis of the 66-kD protein by endothelial cells was observed during culturing of human corneas in the presence of 35S-methionine. The 66-kD protein was found to be a plasma membrane protein based on several of its properties, ie, its solubility in CHCl3:CH3OH, its labeling as surface glycoprotein, and during exposure to a photoaffinity hydrophobic probe: 1-azido-4-125I-iodobenzene. Furthermore this protein could be released from the particulate pellet after treatment with phosphatidylinositol-specific phospholipase C, suggesting its anchorage via a phosphatidylinositol glycan linkage in the plasma membrane. Such anchorage of this protein was further confirmed by its labeling during culture of corneas in the presence of 3H-myoinositol. The glycoprotein nature of the 66-kD protein was evident from its labeling during surface glycoprotein labeling of endothelial cells, staining with periodic acid-Schiff stain, and binding to peanut agglutinin (PNA), and lotus agglutinin (LTA) on SDS-acrylamide gels. The 66-kD protein of endothelial particulate pellets recovered from corneas of donors of different ages showed an age-related increase in binding to PNA and LTA. This suggested an increased glycosylation of the 66-kD protein with aging. A polyclonal anti-66-kD protein antibody was used as a probe to determine the presence of this protein in the rabbit and bovine corneal endothelia by the Western-blot analysis. The 66-kD protein was detected in both rabbit and bovine endothelia, but an additional immunoreactive species of 17 kD was also observed which may be a processed product of the 66-kD protein.
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PMID:Characterization of a 66-kilodalton surface glycoprotein of the human corneal endothelium. 221 Sep 94

Rat intestinal alkaline phosphatase (IAP) is unique among the brush-border membrane enzymes in that it is released bidirectionally (lumen and blood) and exists in either soluble (serum) or particulate (cellular) form. To elucidate the mechanism of membrane release, we examined the effects of phosphatidylinositol-specific phospholipase C (PtdIns-PLC) and serum anchor-specific phospholipase D (PLD) on the solubility of the various tissue forms of IAP. The "solubility" of cytosol IAP could be explained in part by intracellular PtdIns-PLC activity, detected by production of acidic IAP isomers, and by ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA)-sensitive PtdIns hydrolysis. Contamination with serum (abundant with anchor-specific PLD) was responsible for the complete or partial solubilization of IAP that was found during processing of light mucosal scrapings. Anchor-specific PLD activity was increased after fat feeding, and the IAP released did not react with antiserum that recognizes the PtdIns-PLC-released phospholipid portion of trypanosomal variable surface glycoprotein. These data are consistent with the hypothesis that, after secretion from the enterocyte bound to a phospholipid-rich membranous particle, IAP release into serum is mediated by serum anchor-specific PLD. The soluble forms of IAP in the lumen and the cytosol fraction appear to be due to a combination of endogenous PtdIns-PLC activity and anchor-specific PLD contamination that occurs during cell fractionation.
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PMID:Both tissue and serum phospholipases release rat intestinal alkaline phosphatase. 222 Oct 72

Uromodulin, originally identified as an immunosuppressive glycoprotein in the urine of pregnant women, has been previously shown to be identical to human Tamm-Horsfall glycoprotein (THP). THP is synthesized by the kidney and localizes to the renal thick ascending limb and early distal tubule. It is released into the urine in large quantities and thus represents a potential candidate for a protein secreted in a polarized fashion from the apical plasma membrane of epithelial cells in vivo. After introduction of the full-length cDNA encoding uromodulin/THP into HeLa, Caco-2, and Madin-Darby canine kidney cells by transfection, however, the expressed glycoprotein was almost exclusively cell-associated, as determined by immunoprecipitation after radioactive labeling of the cells. By immunofluorescence, THP was localized to the plasma membranes of transfected cells. In transfected cell extracts, THP also remained primarily in the detergent phase in a Triton X-114 partitioning assay, indicating that it has a hydrophobic character, in contrast to its behavior after isolation from human urine. Triton X-114 detergent-associated THP was redistributed to the aqueous phase after treatment of cell extracts with phosphatidylinositol-specific phospholipase C. Treatment of intact transfected HeLa cells with phosphatidylinositol-specific phospholipase C also resulted in the release of THP into the medium, suggesting that it is a glycosylphosphatidylinositol (GPI)-linked membrane protein. Similar to other known GPI-linked proteins, uromodulin/THP contains a stretch of 16 hydrophobic amino acids at its extreme carboxyl terminus which could function as a GPI addition signal and was shown to label with [3H]ethanolamine. The results indicate that THP is a member of this class of lipid-linked membrane proteins and is released into the urine after the loss of its hydrophobic anchor, probably by the action of a phospholipase or protease.
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PMID:Uromodulin (Tamm-Horsfall glycoprotein/uromucoid) is a phosphatidylinositol-linked membrane protein. 224 87

To clarify the molecular structures of the nonspecific cross-reacting antigens (NCAs) produced by human granulocytes, we cloned cDNAs from libraries of normal white blood cells. A clone, NCA-W272, was found to code a protein similar to NCA of tumor cells. The protein consisted of a signal peptide (34 aa), domain-N (108 aa), -A1 (92 aa), -B1 (86 aa) and -M (29 aa). Similarity of the amino acid sequence of each domain to that of the tumor NCA was 72, 92, 76 and 79%, respectively. COS-1 cells transfected with an expression vector carrying the cDNA synthesized a 70 kDa glycoprotein, which was reactive with anti-NCA antibody and released from cell surface by phosphatidylinositol-specific phospholipase C. Thus the clone NCA-W272 was indicated to encode a new species of NCA distinct from the tumor NCA.
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PMID:Characterization of a cDNA clone encoding a new species of the nonspecific cross-reacting antigen (NCA), a member of the CEA gene family. 230 28

Carboxypeptidase M, a plasma membrane-bound enzyme, is present in many human organs and differs from other carboxypeptidase that cleave basic COOH-terminal amino acids. Cultured Madin-Darby canine kidney (MDCK) distal tubular cells contain a kininase I-type enzyme that inactivates bradykinin by releasing Arg9. We found the properties of this kininase to be identical with carboxypeptidase M. In fractionated cells, carboxypeptidase activity sediments with membranes; and detergents, trypsin, and phosphatidylinositol-specific phospholipase C solubilize it, similar to results with human placental carboxypeptidase M. Ten microM 2-mercaptomethyl-3-guanidinoethylthiopropanoic acid and 1 mM o-phenanthroline inhibit, whereas 1.0 mM CoCl2 activates the enzyme. It has a neutral pH optimum and cleaves COOH-terminal Arg or Lys in bradykinin and in shorter peptides. The relative hydrolysis rates of peptides in the presence or absence of 1 mM CoCl2 were similar to those obtained with human carboxypeptidase M. The carboxypeptidase in MDCK cells (54 kDa) cross-reacts with antibodies to human carboxypeptidase M in Western blotting, but not with antibodies to plasma carboxypeptidase N. The enzyme is a glycoprotein; chemical deglycosylation reduced the size to 48 kDa. The presence of the enzyme on the cell membrane of MDCK cells was also shown with transmission electron microscopy using immunogold, which indicated that the enzyme is on the apical side. In addition, MDCK cells contain neutral endopeptidase 24.11 (enkephalinase) and prolylcarboxypeptidase (angiotensinase C) activities. Partitioning of solubilized carboxypeptidase M into Triton X-114 and water indicates that trypsin and phospholipase C remove a hydrophobic tail, while detergent solubilization leaves the hydrophobic moiety intact. Labeling of MDCK cells with [3H]ethanolamine resulted in the synthesis of radiolabeled carboxypeptidase M as determined by immunoprecipitation and fluorography. Thus, MDCK cells contain membrane-bound carboxypeptidase M, which is anchored to the plasma membrane via phosphatidylinositol-glycan. As a major kininase of the distal tubules, it may regulate salt and water excretion.
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PMID:Carboxypeptidase M in Madin-Darby canine kidney cells. Evidence that carboxypeptidase M has a phosphatidylinositol glycan anchor. 239 13

Decay-accelerating factor (DAF) is an integral membrane protein that inhibits amplification of the complement cascade on the cell surface. We and other investigators have shown that DAF is part of a newly characterized family of proteins that are anchored to the cell membrane by phosphatidylinositol (PI). The group includes the variant surface glycoprotein (VSG) of African trypanosomes, the p63 protein of Leishmania, acetylcholinesterase (AChE), alkaline phosphatase, Thy-1, 5'-nucleotidase, and RT6.2--an alloantigen from rat T cells. The structure of the membrane anchor has been best characterized for VSG, but chemical studies of the membrane anchors of AChE and Thy-1 suggest that similar glycolipid moieties anchor these proteins to the cell surface. In the VSG, the membrane anchor consists of an ethanolamine linked covalently to an oligosaccharide and glucosamine; the entire complex is anchored to the cell membrane by PI. Immunologically, this glycolipid defines an epitope, the cross-reacting determinant (CRD), that is only revealed after removal of the diacyl glycerol anchor by a phospholipase C. By Western blotting, we show here that DAF-S (DAF released from the membrane by PI-specific phospholipase C [PIPLC]) also contains CRD. Using a newly developed immunoradiometric assay (IRMA) in which the solid-phase capturing antibody is a monoclonal antibody to DAF and the second antibody is anti-CRD, we have been able to quantitate DAF-S. By IRMA, we show that the reaction between anti-CRD and DAF-S is specific, since the binding is competitively inhibited only by the soluble form of the VSG. These observations further support the concept that the glycolipid anchors of this new family of proteins have similar structures. DAF is also found as a soluble protein in various tissue fluids as well as in Hela cell supernatants. No evidence for the presence of the CRD epitope was found on these proteins, suggesting that these forms of DAF are not released from the surface of cells by endogenous phospholipases.
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PMID:Decay-accelerating factor (DAF) shares a common carbohydrate determinant with the variant surface glycoprotein (VSG) of the African Trypanosoma brucei. 243 27

Decay-accelerating factor (DAF) of human erythrocytes is a glycoprotein with a Mr of 65,000 that is anchored in the membrane via a glycolipid tail. During the purification of DAF, two lower m.w. forms were noted. DAF-A had an Mr of 63,000, and DAF-B had an Mr of 55,000. In a fluid phase assay, both forms accelerated the decay of the classical and the alternative C3 convertases with a specific activity similar to that of DAF. However, the decay-accelerating activity for the cell-bound C3 convertases was abolished, suggesting that neither could insert into E membranes and therefore that the glycolipid tail is altered. Analysis by molecular sieve high-pressure liquid chromatography demonstrated that DAF-A eluted with a Mr of approximately 450,000, similar to native DAF, and was thus in an aggregated form. In contrast, DAF-B eluted as a monomer with a Mr of approximately 60,000. DAF-A, but not DAF-B, bound to a hydrophobic column. To further characterize these two forms, surface-labeled human erythrocytes were incubated with phosphatidyl inositol-specific phospholipase C or papain. The phospholipase inefficiently released a form of DAF that was slightly larger (Mr of 64,000) than DAF-A. Papain efficiently released a 55,000 fragment that had the same Mr as DAF-B. To determine if DAF was cleaved by endogenous enzymes, surface-labeled erythrocytes were incubated with leukocytes. The kinetics of the leukocyte-induced degradation was similar to those observed with papain, and the released fragment aligned on seizing gels with the papain-derived fragment. We hypothesize that endogenous phospholipases and proteases cleave DAF to produce fragments similar to DAF-A and DAF-B, respectively.
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PMID:Additional forms of human decay-accelerating factor (DAF). 244 Sep 50


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