Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.1.4.3 (phospholipase C)
 18,461 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Biotin derivatives of methotrexate (biotin-SS-MTX) and folate (biotin-SS-folate), in which the functional components are joined by a dissociable disulfide-containing spacer, have been synthesized, purified by DEAE-Trisacryl chromatography, and characterized by HPLC, elemental analysis and mass spectrometry. These compounds provide a convenient means for the single-step purification of the folate transporters from L1210 cells. Parental L1210 murine leukemia cells, which contain only the microM transporter (the reduced folate/MTX transport protein) were treated with the N-hydroxysulfosuccinimide ester of biotin-SS-MTX, and a detergent extract of the plasma membranes was exposed to streptavidin-agarose beads to adsorb the labeled protein. Dithiothreitol cleavage of the disulfide linkage released the transporter, which migrated as a well-defined component (43 kDa) on SDS-PAGE gels; no other proteins were present. An L1210 subline (JF), obtained by adapting cells to grow on nanomolar concentrations of folate, contains both the microM transporter and the nM transporter (high-affinity folate binding protein). When these cells were treated with the N-hydroxysulfosuccimide ester of biotin-SS-folate and processed as described above, analysis on SDS-PAGE gels revealed the presence of two proteins, the microM transporter (43 kDa) and the nM transporter (39 kDa). Both transporters were characterized with respect to amino acid content; blocked N-termini precluded Edman sequencing. Treatment of the nM transporter with peptide:N-glycosidase F produced a smaller component (32 kDa); the microM transporter, conversely, was unchanged by this procedure. When the microM transporter in parental L1210 cells was labeled with fluorescein-MTX and then treated with phosphoinositol-specific phospholipase C (PI-PLC), no change in fluorescence was detected. Alternatively, when the nM transporter in the JF subline was labeled with fluorescein-folate and then treated with PI-PLC, complete loss of fluorescence was observed. These results indicate that the L1210 microM transporter is a non-glycosylated, integral membrane protein, while its nM counterpart is heavily glycosylated and anchored exofacially to the membrane by a glycosylphosphatidylinositol component.
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PMID:Multiple folate transport systems in L1210 cells. 132 5

Monoclonal antibody BM88 recognizes a neurospecific surface antigen in the CNS and the PNS. In the present study, the antigen recognized by BM88 was immunopurified from pig brain and shown to be a 22-kDa polypeptide by reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Under nonreducing conditions a protein of 40 kDa was obtained, a result indicating that the antigen is composed of two polypeptide chains of equal molecular weight linked by disulfide bridges. Gel filtration of the purified antigen in the presence of Emulphogene suggested that it may be either a monomeric or a dimeric protein. However, in the presence of Triton X-100 a monomeric structure was implied. N-Glycanase digestion indicated that the protein is probably not glycosylated. The purified antigen was characterized as an integral membrane protein by hydrophobic chromatography and phase-separation experiments with Triton X-114. The antigen, or at least the antibody binding region of the molecule, is very susceptible to protease attack, as judged by protease digestion experiments on brain membranes. By using very low concentrations of papain combined with short incubation times, the antigen was converted to a 16.3-kDa membrane-associated polypeptide as assessed by immunoblotting. This polypeptide contained the BM88 binding epitope. Soluble BM88 immunoreactive polypeptides were not obtained. Bacillus cereus phospholipase C was also unable to solubilize the antigen from the membrane. Our results suggest that the molecule, possessing at least one small extramembranous domain, is attached to the membrane via a polypeptide chain.
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PMID:Purification and characterization of neuron-specific surface antigen defined by monoclonal antibody BM88. 170 20

Neurothelin has recently been identified as a cell surface protein specific for chick endothelial cells forming the blood-brain barrier. Neurons of the adult brain are essentially devoid of neurothelin. In contrast, neurons of the chick retina, which lack blood vessels and accessory astrocytes, express neurothelin. Here we demonstrate that during chick brain development initially neurothelin is expressed probably in all neuroblasts. With proceeding cytodifferentiation, such as vascularization and gliogenesis, brain neurons become neurothelin negative. Coincidentally the endothelial cells forming the blood-brain barrier start to synthesize neurothelin. In contrast to brain neurons, in retina neurons, neurothelin expression increases by one order of magnitude during the course of histogenesis. Coculturing of chick retinal cells with purified rat astrocytes in vitro results in reduction of neural neurothelin expression as quantified by ELISA. Conversely, disruption of the glia-neuron interactions by culturing brain neurons as individualized cells in vitro leads to a reexpression of neurothelin. This is consistent with the hypothesis that astrocytes inhibit neurothelin expression in neurons. Biochemical characterization classifies neurothelin as an integral membrane protein. Temperature-induced-detergent phase separation, phospholipase C digestion and sodium carbonate treatment were employed to distinguish between integral membrane proteins, lipid-anchored proteins and peripheral membrane proteins. Two-dimensional gel electrophoresis reveals an isoelectric point of about 6.4 for neurothelin. Polysaccharide analysis by glycosidase digestion and lectin binding indicates that neurothelin is highly glycosylated. The relative molecular mass of glycosylated neurothelin is 41 x 10(3), whereas the peptide backbone is only 25 x 10(3). The very strict spatiotemporal regulation of neurothelin expression in the central nervous system suggests that neurothelin fulfils possibly a crucial function such as transport of low relative molecular mass components that are essential for neuronal metabolism. The proposed biological activity of neurothelin might be specifically affected by some of its distinct biochemical features.
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PMID:Neurothelin: molecular characteristics and developmental regulation in the chick CNS. 176 90

The protein-coding capacities of rat and human catechol O-methyltransferase (COMT) DNA clones were analysed by in vitro transcription and translation using bacteriophage RNA polymerase and rabbit reticulocyte lysate. Two types of clones corresponding to the structures of human placental cDNA clones were used. The shorter clones, containing the 663-residue open reading frame for the soluble COMT (S-COMT), produced 24-kDa (rat) and 26-kDa (human) polypeptides. Translation of the longer clones, containing 43 (rat) or 50 (human) amino acid amino-terminal extensions to the S-COMT polypeptides, yielded 28-kDa (rat) and 30-kDa (human) putative membrane-bound COMT (MB-COMT) polypeptides as the main products. These clones also yielded low amounts of the S-COMT polypeptides. Labelling time or ionic conditions during translation did not eliminate the shorter products, suggesting translation initiation from the second S-COMT AUG codon. In accordance with this postulation, the relative amount of S-COMT could be affected by changing the translation initiation contexts preceding the first AUG codon. The 28-kDa and 30-kDa products, but not the 24-kDa and 26-kDa products, associated with microsomal membranes cotranslationally, indicating that the amino-terminal extensions were functional signal sequences. However, the presence of membranes did not affect the mobilities of the proteins in SDS/polyacrylamide gels. The MB-COMT polypeptides could not be released from the microsomes by treatments with phospholipase C or alkali and were not protected by the microsomes against proteinase K digestion. These results indicate that MB-COMT synthesized in vitro is an integral membrane protein having an amino-terminal signal-anchor sequence.
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PMID:Cell-free synthesis of rat and human catechol O-methyltransferase. Insertion of the membrane-bound form into microsomal membranes in vitro. 176 63

Subcellular fractionation of pig kidney cortex revealed that aminoacylase I (EC 3.5.1.14, N-acyl-L-amino-acid aminohydrolase) is predominantly a soluble enzyme with only 0.5% of the total activity being recovered in the membrane fraction. The aminoacylase I activity associated with the membrane preparations displayed neither rapid release following incubation with phosphatidylinositol-specific phospholipase C from Bacillus thuringiensis nor the distinctive differential pattern of detergent solubilization which was seen with glycosyl-phosphatidylinositol-anchored proteins (renal dipeptidase, alkaline phosphatase). When fractionated by phase separation in Triton X-114, integral membrane proteins of kidney microvillar membranes partitioned predominantly (greater than 90%) into the detergent-rich phase. In contrast, only 3.7% of aminoacylase I activity associated with microvillar membranes partitioned into the detergent-rich phase. Aminoacylase I activity of pig kidney would therefore appear to be a hydrophilic protein in nature and is not, as suggested previously, a G-PI-anchored integral membrane protein.
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PMID:Aminoacylase I is not a glycolipid-anchored ectoenzyme in pig kidney. 182 88

Sm25, a major antigen in the surface tegument of the parasitic helminth Schistosoma mansoni, is a 25 kDa N-glycosylated glycoprotein which co-purifies with isolated surface membranes and behaves as an integral membrane protein in Triton X-114 (TX-114). The deduced amino acid sequence of Sm25 shows a short C-terminal hydrophobic domain between residues 163 and 180, containing six uncharged polar amino acids and followed by a Lys181-Ser192 dipeptide. We were interested in whether or not this marginal C-terminal amphiphilic domain is responsible for the association of Sm25 with the membrane or whether a post-translational modification such as the addition of glycosyl phosphatidyl inositol (GPI) represents the membrane anchor for this molecule. We find that treatment with phosphatidyl inositol-specific phospholipase C, which cleaves many GPI anchors, does not reveal Cross Reacting Determinant (CRD) on Sm25, nor affect the association of this protein with membranes, providing no support for the addition of GPI. However, Sm25 is palmitoylated via a thioester bond to the single Cys residue, at position 168, which lies within the C-terminal hydrophobic domain. Removal of palmitate by reduction results in a marked decrease in the hydrophobicity of Sm25, as demonstrated by its partitioning into the aqueous rather than detergent phase of TX-114 and its quantitative release from membrane preparations. The hydrophobicity of several membrane proteins in addition to Sm25 is also decreased by reduction, raising the possibility that fatty acylation by thioester linkage is an important mechanism used by schistosomes to stabilize protein-membrane interactions.
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PMID:Sm25, a major schistosome tegumental glycoprotein, is dependent on palmitic acid for membrane attachment. 183 82

Biotin derivatives of methotrexate and folate (2-(biotinamido)ethyl-1,3'-dithiopropionyldiaminopentyl methotrexate and/or folate), in which carboxyl groups of the functional components are joined by a disulfide-containing spacer, have been synthesized, purified by DEAE-Trisacryl chromatography, and characterized by high pressure liquid chromatography and mass spectrometry. These bifunctional, dissociable probes were utilized for the single-step purification to homogeneity of two folate transport proteins (43 and 39 kDa) from L1210 cells. Treatment of the 39-kDa protein with peptide N-glycosidase F produced a smaller component (32 kDa); the 43-kDa protein, conversely, was unchanged by this procedure. When the 39-kDa transporter in intact cells was labeled with a fluorescein derivative of folate and then treated with phosphoinositol-specific phospholipase C, complete loss of fluorescence was observed. Alternatively, there was no change in fluorescence when the 43-kDa transporter was labeled with a fluorescein derivative of methotrexate and treated with the enzyme. These results indicate that the 43-kDa transporter is a nonglycosylated, integral membrane protein, whereas the 39-kDa counterpart is heavily glycosylated and anchored exofacially to the membrane by a glycosylphosphatidylinositol component.
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PMID:Biotin derivatives of methotrexate and folate. Synthesis and utilization for affinity purification of two membrane-associated folate transporters from L1210 cells. 186 24

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

Previous studies have determined that various Qa2 serologic determinants can be removed from the surface of spleen cells by treatment with a phospholipase C. Our studies have determined that the class I molecule Qa2, expressed on the surface of spleen cells and activated T cells, behaves as an integral membrane protein based on its ability to associate with detergent micelles. Studies utilizing two purified phospholipase C have revealed that although most (90 to 95%) of the Qa2 molecules expressed on the surface of resting spleen cells are released as intact 40-kDa polypeptides associated with beta 2-microglobulin, activated T cells contain a major cell subpopulation expressing lipase-resistant Qa2 molecules. Flow cytometric analysis revealed that L3T4+-activated T cells expressed lipase-sensitive Qa2 molecules, whereas Lyt-2+ cells express lipase-resistant forms of the Qa2 molecule. The relationship between the secreted form of the Qa2 molecule and the lipase-generated soluble Qa2 molecule was investigated. Based on SDS-PAGE analysis, the secreted Qa2 molecules has a Mr of 39 kDa whereas the cell surface form released from either resting spleen or activated T cells by phosphatidylinositol-specific phospholipase C has a Mr of approximately equal to 40 kDa. Furthermore, the secreted Qa2 molecule lacks an epitope, cross-reacting determinant, often present on lipase-solubilized cell surface molecules. Thus, based on serologic and biochemical criteria, the soluble Qa2 molecules generated by an exogenous phospholipase C and the secreted Qa2 molecule are structurally distinct.
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PMID:Further characterization of the membrane anchor found on the tissue-specific class I molecule Qa2. 245 59

Recently we isolated a protein growth factor of 34 kDa from trophoblastic membranes of human placenta. A fraction (approximately equal to 50%) of the membrane-associated 34-kDa protein is peripherally associated--i.e., it can be released by high salt treatment. The remainder shows the characteristics of an integral membrane protein--i.e., its release requires detergent treatment. Here we report studies on the structural basis for membrane anchorage of the protein. Phospholipase C was found to release an immunoreactive 34-kDa polypeptide from intact isolated cytotrophoblasts. Studies with isolated trophoblastic membranes showed that phospholipase C specifically released the salt-resistant fraction of the 34-kDa polypeptide. The polypeptide released by phospholipase C showed the same electrophoretic mobility in NaDodSO4/PAGE as the polypeptide prior to phospholipase C treatment. The identity of the released protein with the 34-kDa growth factor has been established by both immunologic and receptor-binding assays. Other studies show that there is biosynthetic incorporation of [3H]myristate into the 34-kDa protein. The myristate label is labile to phospholipase C treatment. These results suggest that some of the 34-kDa protein is anchored to the plasma membrane via a posttranslationally added phospholipid. This mode of anchorage has been observed for some other membrane proteins and raises interesting questions regarding the role of this novel linkage in the mitogenic function of the 34-kDa polypeptide.
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PMID:A phospholipid is the membrane-anchoring domain of a protein growth factor of molecular mass 34 kDa in placental trophoblasts. 316 24


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