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 previously shown that exposure of responding cells to vitamin A leads to profound modifications of chromatin structure as revealed by an increased susceptibility to DNase I digestion, modified patterns of histone acetylation, and impaired synthesis of a nonhistone chromosomal protein (Ferrari, N., and Vidali, G. (1985) Eur. J. Biochem. 151, 305-310). The present results show that these effects are most probably due to the direct interaction between retinol and chromatin, and analysis of mononucleosomes and higher oligomers obtained from retinol-treated cells shows that retinol is indeed tightly bound to chromatin. Enzymatic digestions of vitamin A containing nucleosomes with proteinase K, phospholipase C, and phospholipase A2 support a model where the final binding of retinol to chromatin is mediated by a lipoprotein: the recognition of the binding sites on DNA being dictated by the proteic component while the hydrophobic retinol is solubilized in the fatty acid moiety.
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PMID:In vivo binding of retinol to chromatin. The binding is mediated by a lipoprotein. 333 5

We have previously shown that membranes from the retinal pigment epithelium can transform added all-trans-retinol into a mixture of 11-cis-retinoids, demonstrating the "missing reaction" in the visual cycle for the first time (Bernstein, P. S., Law, W. C., and Rando, R. R. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 1849-1853). In this article, this isomerase activity is further characterized. Double-label experiments with [15-3H]- and [15-14C]all-trans-retinol as the substrate show that the tritium label is retained in the 11-cis-retinol and 11-cis-retinyl palmitate products. This requires that isomerization occur at the alcohol level of oxidation. All-trans-retinyl esters, such as the palmitate, acetate, butyrate, and hexanoate esters, are not directly transformed into their 11-cis counterparts by the membranes. The data are consistent with the presence of an all-trans-retinol isomerase enzyme system or enzyme complex, which produces 11-cis-retinol. Other isomeric retinols were tested for substrate activity. Neither 9-cis-retinol(al) nor 13-cis-retinol were processed by the isomerase. Since the membranes containing the isomerase possess other retinol metabolizing activities, such as retinyl ester synthetase and dehydrogenase activities, further purification was attempted. Appreciable quantities of all detergents tested led to the disappearance of isomerase activity, and high salt or EDTA did not dissociate isomerase activity from the membranes. However, extensive sonication of the membranes did produce a 100,000 x g supernatant fraction of light membranes depleted of other all-trans-retinol processing activities. The isomerase activity in these membranes was saturable with all-trans-retinol, as required for a biologically significant process, and showed a Vmax of 5 pmol/h/mg of protein, a KM of 0.8 microM, and a pH optimum of 8. The isomerase was destroyed by proteinase K, by phospholipase C, by heating, or by ethanol at concentrations greater than 1%. The addition of high energy compounds, such as MgATP, MgGTP, or palmitoyl-CoA, did not appear to stimulate isomerase activity in the 100,000 x g supernatant.
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PMID:Biochemical characterization of the retinoid isomerase system of the eye. 350 Jan 73

Model systems simulating the cementum portion of teeth were used to characterize the attachment process by which certain species of oral Cytophaga initiate the colonization of the tooth root surface in vitro. The adsorption of these bacteria to spheroidal hydroxyapatite beads and mechanically powdered root material followed Langmuir isotherm kinetics. From such data, the number of binding sites per 20 mg of substrate and the affinity constants were evaluated for two strains of Cytophaga sp. Resting cells of the two strains tested adhered relatively tenaciously to hydroxyapatite beads in numbers similar to those observed with cells of Streptococcus sanguis. Attachment of bacteria to the substrates was partially inhibited by (i) coating the substrates with human serum or saliva, (ii) pretreating cell suspensions with proteinase K or phospholipase C or D, or (iii) exposing the cells to temperatures greater than 60 degrees C for 15 min. Treating resting cell suspensions with pronase, neuraminidase, phospholipase A2, or 0.1 M ethylenediaminetetraacetic acid had no effect on the attachment process.
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PMID:Attachment of oral Cytophaga species to hydroxyapatite-containing surfaces. 721 36

Glycosylphosphatidylinositol (GPI) membrane protein anchors are synthesized from sugar nucleotides and phospholipids in the ER and transferred to newly synthesized proteins destined for the cell surface. The topology of GPI synthesis in the ER was investigated using sealed trypanosome microsomes and the membrane-impermeant probes phosphatidylinositol-specific phospholipase C, Con A, and proteinase K. All the GPI biosynthetic intermediates examined were found to be located on the external face of the microsomal vesicles suggesting that the principal steps of GPI assembly occur in the cytoplasmic leaflet of the ER. Protease protection experiments showed that newly GPI-modified trypanosome variant surface glycoprotein was primarily oriented towards the ER lumen, consistent with eventual expression at the cell surface. The unusual topographical arrangement of the GPI assembly pathway suggests that a biosynthetic intermediate, possibly the phosphoethanolamine-containing anchor precursor, must be translocated across the ER membrane bilayer in the process of constructing a GPI anchor.
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PMID:The GPI anchor of cell-surface proteins is synthesized on the cytoplasmic face of the endoplasmic reticulum. 792 79

Binding of canine parvovirus (CPV) to the susceptible feline T cell line 3201 was quantitated by fluorescence-activated cell sorter (FACS) analysis. CPV bound to the cells in a dose-dependent manner, while no binding to the non-permissive MSB-1 avian lymphoma cell line was detected. Binding could be competitively inhibited by addition of excess unlabeled empty capsids, or by pre-incubation of virus with a CPV-specific monoclonal antibody. To characterize the biochemical nature of this binding, live cells were treated with a variety of enzymes prior to use in the binding assay. Treatment with neuraminidase removed a significant proportion of the wild-type virus binding activity, while both proteinase K and phosphatidylinositol-specific phospholipase C (PI-PLC) prevented binding of a non-hemagglutinating (non-HA), non-sialic acid binding mutant to 3201 cells. This suggests that CPV binds to sialic acid expressed on host cells as well as erythrocyte membranes, and that it also binds a protein moiety which is glycosylphosphatidylinositol (GPI)-anchored. The role of these components in CPV infection was also examined by pretreating cells with neuraminidase or PI-PLC prior to inoculating them with either wild-type CPV or the non-hemagglutinating mutant. Neuraminidase treatment had no effect on the ability of CPV to infect the cells, while infectivity was severely compromised by pretreating the cells with either proteinase K or PI-PLC. GPI-anchored proteins on 3201 cells were further characterized by Triton X-114 extraction and reactivity to anti-CRD after PI-PLC treatment.
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PMID:Characterization of canine parvovirus (CPV) interactions with 3201 T cells: involvement of GPI-anchored protein(s) in binding and infection. 808 Dec 56

Sm23, a surface protein of the human parasite Schistosoma mansoni, belongs to the family of "cysteine-rich, hydrophobic proteins," which are expressed on mammalian hematopoietic cells or tumor cells. Sm23 shares the highly conserved hydrophobicity profile of these proteins, which predicts four transmembrane segments, but is in addition linked to the membrane by a glycosylphosphatidylinositol (GPI) anchor. Our results suggest that Sm23 uses both the potential transmembrane domains and the GPI anchor for membrane insertion: (a) Sm23 was not released from the surface after cleavage with phosphatidylinositol-specific phospholipase C (PIPLC). (b) In a Triton X-114 phase-separation system, native [3H]ethanolamine- or [35S]methionine-labeled Sm23 partitioned into the detergent phase. Upon removal of the GPI anchor by PIPLC, the majority of the molecules stayed in the detergent-phase as expected of a transmembrane protein. (c) When full-length recombinant Sm23 was transcribed and translated in vitro, the polypeptide chain was inserted into microsomal membranes: Sm23 stayed associated with the membranes when they were incubated with carbonate buffer at pH 11.5, and membrane bound Sm23 was protected from digestion with proteinase K. (d) Recombinant Sm23, when expressed in the baculovirus expression system, was transported to the surface of infected insect cells, and similarly to the native protein it was not released from these cells after cleavage with PIPLC.
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PMID:Schistosoma mansoni: Sm23 is a transmembrane protein that also contains a glycosylphosphatidylinositol anchor. 816 Nov 93

Staphylococcal alpha-toxin is a single-chain polypeptide with a molecular weight of 34,000 that hexamerizes in lipid bilayers to form pores of 1-1.5 nm effective diameter in membranes. We demonstrate that limited proteolysis of purified alpha-toxin with proteinase K generates a hemolytically active product that yields one major protein band of 17-18 kDa in SDS-polyacrylamide gel electrophoresis. The 17-18-kDa protein band harbors two major fragments of similar size representing the N- and C-terminal halves, which remain associated with each other in non-denaturing buffers but dissociate in 6 M urea. Dissociation in urea leads to loss of hemolytic activity. In contrast, unnicked alpha-toxin is not inactivated by urea. Nicked, hemolytically active alpha-toxin forms hexamers on erythrocyte membranes and on lymphocytes and monocytes. However, the nicked toxin can only lyse erythrocytes and fails to permeabilize nucleated cells. Osmotic protection experiments indicate that the size of pores generated by the nicked toxin is considerably smaller (0.6-0.9 nm effective diameter) than that generated by native toxin. The collective results do not support a previous proposal that different functions of alpha-toxin are contained in separate domains of the molecule.
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PMID:Altered pore-forming properties of proteolytically nicked staphylococcal alpha-toxin. 850 21

Transformation of cercariae of Schistosoma mansoni into schistosomula is accompanied by release of a soluble 28-kDa serine protease (s28) from the acetabular glands. The postulated activities of s28 include cleavage of skin connective tissue proteins (elastin, etc.), release of the cercarial glycocalyx, and cleavage of complement proteins. Our previous results demonstrated the presence of an antigenically cross-reactive protein on the surface of mechanically transformed schistosomula. As shown here, schistosomula express on their surface a 28-kDa serine protease (m28) which can be immunoprecipitated with anti-s28 antibodies. m28 eluted from the schistosomular tegumental membrane with NP-40 was purified to homogeneity in one step by adsorption on a chymotrypsin inhibitor column: 6-aminocaproyl-D-tryptophan methyl ester-Sepharose. Proteolytic activity of m28 was completely inhibited by the chymotrypsin inhibitor N-succinyl-Ala-Ala-Pro-Phe-chloromethyl ketone. Efficient removal of m28 from schistosomula was achieved with NP-40, deoxycholate, cholate, Tween 20, and phospholipases A2 and C, but not with papain, trypsin, pronase, or proteinase K. Furthermore, treatment with phosphatidyl inositol-specific phospholipase C (PI-PLC) followed by hydroxylamine also released m28. Anti-cross-reactive determinant antibodies which recognize a neo epitope exposed in glycosyl phosphatidyl inositol-containing molecules cleaved by PI-PLC bind to purified m28. The latter results suggest that m28 is anchored to the tegumental membrane of schistosomula by a lipid anchor and that perhaps some of the m28 molecules are bound via glycosylphosphatidyl inositol. Based on inhibitor sensitivity and antigenic cross-reactivity, it is conceivable that s28 and m28 are related, if not identical, proteins. Finally, m28 was detected antigenically also on lung-stage and adult worms of S. mansoni.
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PMID:Schistosoma mansoni: evidence for a 28-kDa membrane-anchored protease on schistosomula. 865 54

Staphylococcal alpha-toxin is a single-chain protein with a molecular mass of 33.2 kDa, which can form large water-filled pores both in lipid bilayers and in erythrocyte membranes. Limited proteolysis of the purified toxin with proteinase K led to time-dependent changes of all the functional features of the channels formed by the toxin. Single-channel conductance in planar bilayers was decreased about threefold. The anion selectivity of the channel was replaced with cation selectivity and the asymmetry in the current-voltage relationship of the channel became more pronounced. At the same time the nicked toxin kept its full ability to form ion channels in lipid bilayers, although it lost a considerable part of its hemolytic activity. In planar bilayers and in erythrocyte membranes, the proteolytically nicked toxin actually formed channels with a slightly smaller diameter (approximately 1.2 times) than that formed by the native toxin. This decrease was not marked enough to explain changes in the biological effects of the nicked toxin. The change in channel selectivity induced by the cleavage is considered to be the major determinant of the changes in the biological effects of the nicked toxin.
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PMID:Pore-forming properties of proteolytically nicked staphylococcal alpha-toxin: the ion channel in planar lipid bilayer membranes. 925 67

Syrian hamster prion protein (PrPC) and a truncated Syrian hamster prion protein lacking the glycosylphosphatidylinositol (GPI) anchor C-terminal signal sequence (GPI-) were expressed in Chinese hamster ovary cells using a glutamine synthetase selection and amplification system. The CHO cell clones expressing the GPI- PrP secreted the majority of the protein into the media, whereas most of the PrP produced by clones expressing the full-length protein with the GPI anchor was located on the cell surface, as demonstrated by its release upon treatment with phosphatidylinositol-specific phospholipase C (PIPLC). A cell clone that expressed the highest levels of full length PrP was subcloned to obtain clone 30C3-1. PrP from clone 30C3-1 was shown to be sensitive to proteolysis by proteinase K and to react with monoclonal and polyclonal antibodies that recognize native PrPC. The recombinant PrP migrated as a diffuse band of 19-40 kDa but removal of the N-linked oligosaccharides with peptide N-glycosidase F (PNGase F) revealed three protein species of 19, 17 and 15 kDa. The 19 kDa band corresponding to deglycosylated full-length PrP was quantified and found to be expressed at a level approximately 14-fold higher than that of PrPC found in Syrian hamster brain.
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PMID:Prion protein expression in Chinese hamster ovary cells using a glutamine synthetase selection and amplification system. 954 9


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