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 catalytic subunits of asymmetric and hydrophobic forms of acetylcholinesterase arise from a single gene by alternative mRNA splicing. Each protein is encoded in three exons, with exons 1 and 2 encoding sequence common to both forms and exons 3A and 3H specifying unique carboxyl-terminal domains. We examined the expression of cDNAs for the two forms by transient transfection in COS-1 cells. The catalytic subunit of the asymmetric form expressed by transfected cells exhibits low activity and is retained within the cell. The cDNA encoding hydrophobic acetylcholinesterase directs the synthesis of enzyme with much greater activity, which is expressed on the outer surface of the cell membrane and can be released by phosphatidylinositol-specific phospholipase C. A mutant truncated acetylcholinesterase which lacks either carboxyl-terminal sequence encoded by the alternative exons is secreted into the medium. An exon 1-3H fusion mutant, created by deletion of coding exon 2 from the hydrophobic form cDNA, is glycophospholipid-linked. The 30-amino acid carboxyl-terminal domain specified by exon 3H appears necessary and sufficient to direct glycophospholipid attachment. Thus, heterologous expression of wild-type and mutant acetylcholinesterase proteins indicates that the carboxyl-terminal domains specified by alternative coding exons determine the cellular dispositions of acetylcholinesterase.
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PMID:Biosynthesis of Torpedo acetylcholinesterase in mammalian cells. Functional expression and mutagenesis of the glycophospholipid-anchored form. 216 68

The chemical properties of human renal dipeptidase (hrDP) purified from the membrane fraction of kidney have been characterized. When treated with phosphatidylinositol-specific phospholipase C, hrDP was released from renal membrane fractions. After digestion with trypsin, carboxyl-terminal peptide was isolated employing anhydrotrypsin-agarose column chromatography and reversed-phase high performance liquid chromatography. The amino acid sequence of the peptide was identified at positions 363-369 in the primary structure deduced from the cDNA sequence (Adachi, H., Tawaragi, Y., Inuzuka, C., Kubota, I., Tsujimoto, M., Nishihara, T., And Nakazato, H. (1990) J. Biol. Chem. 265, 3992-3995). Further examination of the chemical composion of the peptide showed that it contained, respectively, 2, 1, 5, 1, and 1 mol of ethanolamine, glucosamine, mannose, inositol, and phosphate in addition to amino acids. These results suggest that the mature hrDP molecule lacks the carboxyl-terminal hydrophobic peptide extension predicted from the cDNA sequence and is anchored at Ser369 via glycosylphosphatidylinositol to the membrane. To characterize further the action of the enzyme, we have established expression systems for both secretory and membrane anchored forms of hrDP using COS-1 cells and found that both recombinant forms were as active as natural enzyme. Our expression system made it possible to prepare large amounts of soluble enzyme, and will contribute toward elucidation of the physiological roles of the enzyme.
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PMID:Identification of membrane anchoring site of human renal dipeptidase and construction and expression of a cDNA for its secretory form. 216 7

Endothelin-1 was initially identified as a 21-residue potent vasoconstrictor peptide produced by vascular endothelial cells, but was subsequently found to have many effects on both vascular and non-vascular tissues. The discovery of three isopeptides of the endothelin family, ET-1, ET-2 and ET-3, each possessing a diverse set of pharmacological activities of different potency, suggested the existence of several different endothelin receptor subtypes. Endothelins may elicit biological responses by various signal-transduction mechanisms, including the G protein-coupled activation of phospholipase C and the activation of voltage-dependent Ca2+ channels. Thus, different subtypes of the endothelin receptor may use different signal-transduction mechanisms. Here we report the cloning of a complementary DNA encoding one subtype belonging to the superfamily of G protein-coupled receptors. COS-7 cells transfected with the cDNA express specific and high-affinity binding sites for endothelins, responding to binding by the production of inositol phosphates and a transient increase in the concentration of intracellular free Ca2+. The three endothelin isopeptides are roughly equipotent in displacing 125I-labelled ET-1 binding and causing Ca2+ mobilization. A messenger RNA corresponding to the cDNA is detected in many rat tissues including the brain, kidney and lung but not in vascular smooth muscle cells. These results indicate that this cDNA encodes a 'nonselective' subtype of the receptor which is different from the vascular smooth muscle receptor.
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PMID:Cloning of a cDNA encoding a non-isopeptide-selective subtype of the endothelin receptor. 217 94

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

Protein sequence analysis of a bovine brain phosphoinositide-specific phospholipase C (PI-PLC; PLC-154) has permitted the isolation of a cDNA that appears to code for this protein. Transient expression of this cDNA in COS-1 cells demonstrates that the cDNA encodes a functional phospholipase C that migrates at approximately 150,000 daltons. A transcript of approximately 7 kb is observed in RNA derived from bovine brain and a related transcript of the same size is present in certain human cell lines. Southern blot analysis indicates that one or possibly two genes hybridize with a PLC-154 probe. Regions of homology between PLC-154 and the previously described PLC-148 allow the assignment of a putative catalytic domain to the central region of PLC-154.
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PMID:Determination of the primary structure of PLC-154 demonstrates diversity of phosphoinositide-specific phospholipase C activities. 245 1

A cDNA clone encoding the human monocyte antigen CD14 was isolated by transient expression in COS cells of a cDNA library prepared from phorbol diester-treated HL60 cells. RNA blot analysis showed abundant expression of a single mRNA species in mature monocytes and an increased expression of the mRNA following induction of differentiation in leukemic cell lines. The DNA blot hybridization pattern was consistent with a single-copy gene. The predicted amino acid sequence lacks the characteristic transmembrane domain and stop transfer motif of conventionally anchored membrane proteins. COS cells transfected with the CD14 cDNA released virtually all CD14 protein in soluble form following treatment with glycosyl phosphatidylinositol-specific phospholipase C, and CD14 immunoreactivity was absent from the affected monocytes of a patient with paroxysmal nocturnal hemoglobinuria (PNH). The data show that, to the limit of experimental sensitivity, all monocyte CD14 is joined to the plasma membrane by a phosphatidylinositol phospholipid.
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PMID:Monocyte antigen CD14 is a phospholipid anchored membrane protein. 246 37

A novel cell surface antigen has been identified on a wide range of lymphoid cells and erythrocytes. A mAb YTH 53.1 (CD59) against this antigen enhanced the lysis of human red cells and lymphocytes by homologous complement. Studies of reactive lysis using different species of C56, and of whole serum used as a source of C7-9, indicated that the inhibitory activity of the CD59 antigen is directed towards the homologous membrane attack complex. CD59 antigen was purified from human urine and erythrocyte stroma by affinity chromatography using the mAb YTH 53.1 immobilized on Sepharose, and, following transient expression of a human T cell cDNA library in COS cells, the corresponding cDNA also identified using the antibody. It was found that the CD59 antigen is a small protein (approximately 20 kD as judged by SDS-PAGE, 11.5 kD predicted from the isolated cDNA) sometimes associated with larger components (45 and 80 kD) in urine. The sequence of CD59 antigen is unlike that of other complement components or regulatory proteins, but shows 26% identity with that of the murine LY-6 antigen. CD59 antigen was released from the surface of transfected COS cells by phosphatidylinositol-specific phospholipase C, demonstrating that it is attached to the cell membrane by means of a glycolipid anchor; it is therefore likely to be absent from the surface of affected erythrocytes in the disease paroxysmal nocturnal hemoglobinuria.
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PMID:CD59, an LY-6-like protein expressed in human lymphoid cells, regulates the action of the complement membrane attack complex on homologous cells. 247 70

Membrane bound and soluble forms of a high-affinity folate binding protein have been found in kidney, placenta, serum, milk, and in several cell lines. The two forms have similar binding characteristics for folates, are immunologically cross-reactive and based upon limited amino acid sequence data, are nearly identical. Based upon pulse-chase experiments, a precursor-product relationship has been suggested. The membrane form has been shown to mediate the transport of folate in cells grown in physiological concentrations of folate. A function for the soluble form has not yet been identified. We constructed a cDNA library from a human carcinoma cell line, Caco-2, which expresses the membrane form abundantly. The library was screened and a near full-length cDNA for the folate binder was isolated. Transfection of COS cells with the cDNA inserted in an expression vector resulted in marked overexpression of a membrane-associated folate binder as assessed by direct binding of radiolabeled folate and by indirect immunofluorescence. The deduced amino acid sequence is not consistent with a typical membrane spanning domain but rather with a signal for anchoring via a glycosyl-phosphatidylinositol linkage. Release of the binder with a phosphatidylinositol-specific phospholipase C strongly supports this hypothesis.
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PMID:Complementary DNA for the folate binding protein correctly predicts anchoring to the membrane by glycosyl-phosphatidylinositol. 252 52

The signal transduction pathways of the cloned human 5-HT1A receptor have been examined in two mammalian cell lines transiently (COS-7) or permanently (HeLa) expressing this receptor gene. In both systems, 5-hydroxytryptamine (5-HT, serotonin) mediated a marked inhibition of beta 2-adrenergic agonist-stimulated (80% inhibition in COS-7 cells) or forskolin-stimulated cAMP formation (up to 90% inhibition in HeLa cells). This serotonin effect (EC50 = 20 nM) could be competitively antagonized by metitepine and spiperone (Ki = 81 and 31 nM, respectively) and could also be blocked by pretreatment of cells with pertussis toxin. In both cell types, 5-HT failed to stimulate adenylyl cyclase through the expressed receptors. In HeLa cells, 5-HT also stimulated phospholipase C (approximately 40-75% stimulation of formation of inositol phosphates). Again, this effect was inhibited by metitepine. However, the EC50 of 5-HT was considerably higher (approximately 3.2 microM) than that found for inhibition of adenylyl cyclase. Both pathways were demonstrated to be similarly affected by pertussis toxin. These findings indicate that like the M2 and M3 muscarinic cholinergic receptors, the 5-HT1A receptor can couple to multiple transduction pathways with varying efficiencies via pertussis toxin-sensitive G-proteins. The lack of stimulation of cAMP formation by this 5-HT1A receptor may suggest the existence of another pharmacologically closely related receptor.
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PMID:Effector coupling mechanisms of the cloned 5-HT1A receptor. 254 39

Neural cell adhesion molecules (N-CAMs) are a family of cell surface sialoglycoproteins encoded by a single copy gene. A full-length cDNA clone that encodes a nontransmembrane phosphatidylinositol (PI) linked N-CAM of Mr 125 x 10(3) has been isolated from a human skeletal muscle cDNA library. The deduced protein sequence encodes a polypeptide of 761 amino acids and is highly homologous to the N-CAM isoform in brain of Mr 120 x 10(3). The size difference between the 125 x 10(3). The size difference between the 125 x 10(3) Mr skeletal muscle form and the 120 x 10(3) Mr N-CAM form from brain is accounted for by the insertion of a block of 37 amino acids called MSD1, in the extracellular domain of the muscle form. Transient expression of the human cDNA in COS cells results in cell surface N-CAM expression via a putative covalent attachment to PI-containing phospholipid. Linked in vitro transcription and translation experiments followed by immunoprecipitation with anti-N-CAM antibodies demonstrate that the full-length clone of 761 amino acid coding potential produces a core polypeptide of Mr 110 x 10(3) which is processed by microsomal membranes to yield a 122 x 10(3) Mr species. Taken together, these results demonstrate that the cloned cDNA sequence encodes a lipid-linked, PI-specific phospholipase C releasable surface isoform of N-CAM with core glycopeptide molecular weight corresponding to the authentic muscle 125 x 10(3) Mr N-CAM isoform. This is the first direct correlation of cDNA and deduced protein sequence with a known PI-linked N-CAM isoform from skeletal muscle.
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PMID:Complete sequence and in vitro expression of a tissue-specific phosphatidylinositol-linked N-CAM isoform from skeletal muscle. 325 57


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