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)

We have isolated a set of complementary DNA (cDNA) clones that together encode the alkaline phosphatase of human colon cancer LS174T cells. These clones include two cDNAs isolated from a conventionally prepared oligodeoxythymidylate-primed lambda ZAP cDNA library and three cDNA clones prepared by using the polymerase chain reaction. The deduced amino acid sequence of the alkaline phosphatase primary transcript contains 532 amino acids. This enzyme is similar to, but not identical with, placental alkaline phosphatase (PLAP); it exhibits 12-19 amino acid substitutions when compared to the various alleles of PLAP. Also, it is similar to PLAP in that it is apparently attached to the cell membrane by a phosphatidylinositol-containing anchor as judged by the ability of phosphatidylinositol-specific phospholipase C to release it from membranes. It is different from PLAP however, in terms of its signal sequence which only contains 19 amino acids as compared to 22 for PLAP. Moreover, the 3'-untranslated region of the LS174T cell alkaline phosphatase message diverges considerably from the PLAP message. The LS174T cell alkaline phosphatase cDNAs are actually much more similar to the "germ cell" alkaline phosphatase gene than they are to PLAP. Only 7 amino acid substitutions exist between the LS174T cell enzyme and the alkaline phosphatase encoded by the germ cell alkaline phosphatase genomic DNA clone isolated by Millan and Manes (Proc. Natl. Acad. Sci. USA, 85: 3024-3028, 1988). Furthermore, the 3'-untranslated region of the LS174T cell alkaline phosphatase message is very similar to the sequence immediately downstream of the coding region of the germ cell alkaline phosphatase genomic DNA clone. Thus, these results indicate that this colon cancer cell alkaline phosphatase is likely to represent an allelic variant encoded at the germ cell alkaline phosphatase locus.
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PMID:Molecular cloning of complementary DNAs encoding alkaline phosphatase in human colon cancer cells. 229 57

Placental alkaline phosphatase [orthophosphoric-monoester phosphohydrolase (alkaline optimum), EC 3.1.3.1] is a member of a diverse group of membrane proteins whose attachment to the lipid bilayer is mediated by a phosphatidylinositol-glycan. To investigate structural aspects of the glycolipid anchor, cultured WISH cells were used because we found that they produce the enzyme in abundant quantities. When cell suspensions were incubated with purified phosphatidylinositol-specific phospholipase C, most of the placental alkaline phosphatase was released from membranes in a hydrophilic form. On incubation of the cells with [14C]ethanolamine, [14C]myristic acid, or myo-[3H]inositol, each was incorporated into the phosphatase near the carboxyl terminus, showing that these components, which are found in other phosphatidylinositol membrane-linked proteins, are also present in placental alkaline phosphatase.
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PMID:Characterization of the phosphatidylinositol-glycan membrane anchor of human placental alkaline phosphatase. 281 64

The biosynthesis and post-translational modification of placental alkaline phosphatase were studied in human choriocarcinoma cells, JEG-3. Pulse-chase experiments with [35S]methionine demonstrated that placental alkaline phosphatase was synthesized as a major precursor form with Mr 63,000, which was then converted to a mature form with Mr 66,000, by processing of its N-linked oligosaccharides from the high-mannose type to the complex type. In addition, the two forms of the protein were found to be modified by a glycophospholipid, components of which were characterized by metabolic incorporation into placental alkaline phosphatase of 3H-labeled compounds such as myo-inositol, palmitic acid, stearic acid, mannose, glucosamine, and ethanolamine. When placental alkaline phosphatase labeled with these compounds was treated with phosphatidylinositol-specific phospholipase C or papain, the phospholipase C removed only the 3H-labeled fatty acids, whereas papain, that is known to cleave the C-terminal region, released all the radioactive glycolipid components including [3H]ethanolamine. More detailed analysis with shorter pulse-chase experiments demonstrated that placental alkaline phosphatase was primarily synthesized as a form with Mr 64,500 which was not yet labeled with [3H]palmitic acid. This form was converted by papain digestion to the above-mentioned major precursor with Mr 63,000. Taken together, these results suggest that placental alkaline phosphatase is initially synthesized as the precursor with Mr 64,500, which is immediately converted to the intermediate form with Mr 63,000 by simultaneously occurring proteolysis of the C terminus and replacement by the glycophospholipid, and finally to the mature form with Mr 66,000 by terminal glycosylation of its N-linked oligosaccharides. The glycophospholipid thus attached is considered to function as the membrane-anchoring domain of placental alkaline phosphatase.
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PMID:Biosynthesis of placental alkaline phosphatase and its post-translational modification by glycophospholipid for membrane-anchoring. 334 38

Human placental alkaline phosphatase (PALPase) has been transiently expressed in simian (COS) cells by transfection with a eukaryotic expression vector containing the corresponding cDNA. The level of expression of PALPase was high, and it was produced in an enzymatically active form. The bulk of PALPase was associated with the cell membrane as shown by immunocytochemistry and subcellular fractionation studies. The PALPase produced by transfected COS cells, like PALPase in human tissue, was specifically released from the intact cells in a hydrophilic form by phosphatidylinositol-specific phospholipase C and is, therefore, apparently attached to the outer membrane by means of a phosphatidylinositol-glycan. Transfected COS cells appear to be an excellent model for elucidating the mechanism of attachment of this phosphatidylinositol-glycan to a protein moiety.
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PMID:Expression of active, membrane-bound human placental alkaline phosphatase by transfected simian cells. 347 33

Previous studies have shown that some cells (e.g. SKG3a) express human placental alkaline phosphatase (AP) in a form which can be released from the membrane by bacterial PtdIns-specific phospholipase C (PI-PLC) while others (e.g. HeLa) are relatively resistant to this enzyme. Chemical and enzymic degradation studies have suggested that the PI-PLC resistance of AP is due to inositol acylation of its glycosylphosphatidylinositol (GPI) anchor. In order to identify the biosynthetic origin of PI-PLC resistance we determined the PI-PLC sensitivity of AP in 35S-labelled cells (10 min pulse; 0-60 min chase) by Triton X-114 phase separation. At the beginning of the chase period, the majority of the AP synthesized was hydrophilic, indicating that it had not acquired a GPI anchor. The concentration of hydrophilic AP species decreased with a t1/2 of 30-60 min but was not processed to an endoglycosidase H-resistant species or secreted into the medium. In both SKG3a and HeLa cells all of the hydrophobic, GPI-anchored AP detectable at the beginning of the chase was PI-PLC sensitive. PI-PLC-resistant species of AP were only observed in HeLa cells and these only appeared after about 30 min. The delayed appearance of PI-PLC resistance was unexpected as previous studies have suggested that candidate GPI-anchor precursors are PI-PLC-resistant as a result of inositol acylation. This work reveals unanticipated complexities in the biosynthesis of AP and its GPI anchor.
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PMID:Biosynthesis of glycosylphosphatidylinositol-anchored human placental alkaline phosphatase: evidence for a phospholipase C-sensitive precursor and its post-attachment conversion into a phospholipase C-resistant form. 803 72

The ability of epidermal growth factor, insulin or guanosine thiotriphosphate to induce the release of two glycosyl-phosphatidylinositol-linked proteins from isolated human placental syncytiotrophoblast plasma membrane vesicles was investigated. Epidermal growth factor induced the ATP-dependent release of a fraction of syncytiotrophoblast plasma membrane placental alkaline phosphatase, whereas no release was detected following insulin treatment. This effect of epidermal growth factor was apparent at 30 min but not at 5 min. Guanosine thiotriphosphate stimulated the release of a small amount of syncytiotrophoblast plasma membrane placental alkaline phosphatase and appeared to have an additive effect when applied together with epidermal growth factor. Guanosine thiodiphosphate did not induce phosphatase release, but partially inhibited the epidermal growth factor response. 28.7% of syncytiotrophoblast plasma membrane 5'-nucleotidase was solubilized using glycosyl-phosphatidylinositol-specific phospholipase C. However, unlike placental alkaline phosphatase, no detectable release of 5'-nucleotidase was observed following treatment of syncytiotrophoblast plasma membrane vesicles with epidermal growth factor or guanosine thiotriphosphate. These results indicate (i) the presence of at least two placental alkaline phosphatase-releasing pathways in syncytiotrophoblast plasma membrane vesicles, and (ii) the presence of subpopulations of glycosyl-phosphatidylinositol-linked proteins sensitive to growth factor-induced release.
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PMID:Growth factor-induced release of placental alkaline phosphatase from human syncytiotrophoblast membranes. 818 14

We found that 35S-labeled recombinant human interleukin-1beta (rhIL-1beta) binds phosphatidylinositol-specific phospholipase C-treated human placental alkaline phosphatase, phosphatidylinositol-specific phospholipase C-treated trypanosome surface variant glycoproteins, and urinary uromodulin immobilized on plates or immobilized on CNBr-activated Sepharose 4B. The interaction between rhIL-1beta and these glycoproteins was lectin-like, since it was inhibited in the presence of specific saccharides, i.e. mannose 6-phosphate or synthetic Ac-NH.CH2.CH2. PO4--->6Manalpha1-->(+/-2Manalpha1-->+/-6Manalpha1-->) propyl at about 1 microM. On the other hand, a wide variety of compounds including biantennary sugar chains derived from these glycoproteins as well as ethanolamine phosphate, inositol phosphate, mannose 6-sulfate, mannose 1-phosphate, glucose 6-phosphate, and mannitol 6-phosphate did not show any inhibitory effect at concentrations up to 1 mM. These results indicate that rhIL-1beta interacts with these glycoproteins via the mannose 6-phosphate diester of glycans on the glycosylphosphatidylinositol (GPI) anchor. Furthermore, when monolayers of polarized Madin-Darby canine kidney cells on polycarbonate filter membranes were incubated with 35S-rhIL-1beta in either the apical or basolateral chamber, 35S-interleukin-1beta was found to bind specifically to the apical membranes with a Ka value of 4.6 x 10(7) M-1, and the specific interaction was inhibited by 1 microM mannose 6-phosphate. Since the mannose 6-phosphate diester moiety exists only in the GPI glycans on plasma membranes, it was evident that interleukin-1beta can directly interact with the mannose 6-phosphate diester component of the intact glycan of GPI anchors on plasma membranes.
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PMID:Lectin-like characteristics of recombinant human interleukin-1beta recognizing glycans of the glycosylphosphatidylinositol anchor. 909 4

A novel cell surface phosphoinositide-cleaving phospholipase C (ecto-PLC) activity was isolated from cultured cells by exploiting its presumed external exposure. Biotinylation of intact cells followed by solubilization of the biotinylated proteins from a membrane fraction and recovery onto immobilized-avidin beads, allowed assay of this cell surface enzyme activity apart from the background of the substantial family of intracellular PLCs. Several cell lines of differing ecto-PLC expression were examined as well as cells stably transfected to overexpress the glycosylphosphatidylinositol (GP) anchored protein human placental alkaline phosphatase (PLAP) as a cell surface enzyme marker. The resulting bead preparations from ecto-PLC positive cells possessed calcium-dependent PLC activity with preference for lysophosphatidylinositol (lysaPI) rather than phosphatidylinositol (PI). The function of ecto-PLC of intact cells evidently is not to release GPI-anchored proteins at the cell surface, as no detectable Ca(2+)-dependent release of overexpressed PLAP from ecto-PLC-positive cells was observed. To investigate the cell surface linkage of the ecto-PLC itself, intact cells were treated with bacterial PI-PLC to cleave simple GPI anchors, but no decrease in ecto-PLC activity was observed. High ionic strength washes of biotinylated membranes prior to the generation of bead preparations did not substantially reduce the lysoPI-PLC activity. The results verify that the ecto-PLC is truly cell surface-exposed, and unlike other members of the PLC family that are thought to be peripheral membrane proteins, this novel lysoPI-PLC is most likely a true membrane protein.
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PMID:Partial isolation from intact cells of a cell surface-exposed lysophosphatidylinositol-phospholipase C. 917 4

Study of fibroblast origins and lineages is complicated by the lack of unambiguous markers that could be used to identify discrete subpopulations on the basis of functional attributes. We have studied the role of the membrane-anchored hydrolytic enzyme tissue-nonspecific alkaline phosphatase (TN-AP) and the placental alkaline phosphatase (PL-AP) in collagen phagocytosis and in the deletion of cells by apoptosis. Rat-2 cells, which do not constitutively express AP, were transfected with full-length rat TN-AP or PL-AP cDNAs to determine the impact of the TN-AP collagen-binding domain on cell function. Various levels of expression were driven by early (strong) or late (weak) SV40 promoters in the plasmid construct. Controls were transfected with plasmids that did not contain AP cDNA. AP expression in transfected cells was confirmed by Northern blotting, histochemical analysis, and SDS-PAGE analysis of membrane-anchored enzyme released by phosphatidyl inositol phospholipase C. Low levels of TN-AP expression increased cell spreading slightly, nearly doubled the percentage of collagen phagocytic cells (up to 80%), and increased the number of internalized collagen-coated fluorescence beads per cell. In cells transfected with PL-AP (i.e., no collagen-binding domain), collagen phagocytosis was not affected. Internalization of BSA beads was also not affected by either AP isozyme, indicating that AP was selective for integrin-mediated phagocytosis. In single cells, histochemically demonstrable TN-AP activity on cell membranes was colocalized with the binding of collagen beads, but this colocalization was not detected in cells transfected with PL-AP. Phagocytosis was inhibited by antibodies to the alpha 2 integrin and to AP but not by levamisole, an inhibitor of AP phosphohydrolytic activity. High-level TN-AP expression caused a fivefold reduction of cell proliferation and was associated with the development of cells with sub-G1 DNA content, nuclear condensation, and nuclear budding. In AP-positive cultures, there was a greatly increased number of floating cells; nick-labeling of DNA by terminal transferase and biotinylated dUTP showed a 15-fold increase of stained cells. These data indicate that low-level TN-AP expression enhances collagen phagocytosis, presumably through the TN-AP collagen-binding domain. High-level AP expression promotes cell deletion by apoptosis. We suggest that the expression of AP by fibroblasts indicates a novel role for this enzyme in collagen degradation by phagocytosis.
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PMID:Collagen phagocytosis and apoptosis are induced by high level alkaline phosphatase expression in rat fibroblasts. 928 52

A retroviral vector DAP that encodes the human placental alkaline phosphatase (PLAP) and the neomycin-resistant gene was used to transduce the salivary gland-derived cell line A5 in vitro and acinar cells in rat submandibular gland in vivo. Expression of the transduced PLAP gene was established by histochemical staining for heat-resistant AP and by determination of enzyme activity. From the in vitro experiments, we concluded that the salivary gland-derived cell line A5 can be infected by the retroviral vector DAP. In the transduced cells the viral long terminal repeat (LTR) promoter was effective, and the cells expressed heat-stable PLAP which was localized mostly in the plasma membrane and could be released by treatment with bromelain or phosphatidyinositol-specific phospholipase C. A5-DAP cells secreted PLAP into the medium. Clones of A5-DAP cells expressed various levels of the enzyme. The level of enzyme activity in different clones was unrelated to growth rate. Retrograde ductal injection of the viral vector into the duct of the submandibular gland of rats resulted in integration and long-term expression of PLAP gene in acinar cells. Expression of PLAP was seen up to 25 days, the limit of the observation period. To facilitate integration of the viral DNA, cell division of acinar cells was induced by administration of the beta-adrenergic agonist isoproterenol before administration of the virus. PLAP was secreted into submandibular saliva. The data support the notion that salivary glands are suitable targets for gene transfer in vivo by a retroviral vector.
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PMID:Retrovirus-mediated gene transfer into rat salivary gland cells in vitro and in vivo. 935 55


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