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 present data indicating that aminoacylase I (EC 3.5.1.14) from porcine kidney and 'renal dipeptidase' (EC 3.4.13.11) are closely related. We show that, in situ, a considerable fraction of aminoacylase activity ist attached to membranes. Incubation of washed microsomal membranes with phospholipase C from B. cereus results in the rapid solubilization of aminoacylase I, suggesting that aminoacylase--as shown for renal dipeptidase before--bears a glycolipid 'membrane anchor'. In agreement with this assumption, purified aminoacylase was found to contain myo-inositol, a characteristic component of phosphatidylinositol-anchored membrane proteins. A reexamination of the molecular mass of purified aminoacylase yielded values (46,000 +/- 2,000 Da by SDS polyacrylamide electrophoresis, 98,000 +/- 5,000 Da by sedimentation equilibrium centrifugation) similar to those reported for renal dipeptidase. The enzymes coelute during most of the procedures applied in the purification of aminoacylase or renal dipeptidase, but can be separated by hydrophobic interaction chromatography. A survey of the literature revealed a series of additional features of aminoacylase I and renal dipeptidase (amino-acid composition, isoelectric points, metal dependence, and more) that are strikingly similar.
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PMID:Further characterization of porcine kidney aminoacylase I reveals close similarity to 'renal dipeptidase'. 322 87

The HlyA protein (Mr 110 kDa) which is the gene product of the hlyA gene encoded by the hemolysin determinant of Escherichia coli (Goebel, W. & Hedgpeth, J. (1982) J. Bacteriol. 151, 1290-1298) was observed to accumulate in the culture supernatant (in the presence of the three other Hly proteins HlyC, B and D) throughout the active growth cycle. However, the amount of extracellular HlyA protein did not correlate with the external hemolytic activity, which declined when the cells entered the stationary phase. External hemolytic activity was highly sensitive to phospholipase C and to ultrasonication. The size of the HlyA protein on SDS-PAGE was not changed by these treatments although the hemolytic activity was entirely abolished. On a polyacrylamide gel containing 2M urea but only 0.1% SDS hemolytically active HlyA migrated slightly ahead of the inactive HlyA suggesting that HlyA is more negatively charged than HlyA. Active hemolysin from unconcentrated hemolytic supernatants migrated on Sephacryl S-400 and on glycerol gradients as large complexes. Analysis of the hemolytically active fractions on SDS-PAGE yielded in both cases only HlyA (110 kDA) as major protein. An internal hemolytic activity appeared in most Escherichia coli K-12 strains in the stationary phase which was independent of the presence of HlyA or any other Hly gene product. This hemolytic activity which reached in some strains about 10% of the level determined by the hly genes was sensitive to proteinase K and disappeared upon shift of the cells to the logarithmic phase.
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PMID:Active and inactive forms of hemolysin (HlyA) from Escherichia coli. 327 76

The mechanism by which the rat T cell alloantigen, RT-6.2, is attached to the membrane was investigated. Treatment of rat lymph node and T-hybridoma cells with phosphatidylinositol-specific phospholipase C (PI-PLC) caused a substantial reduction in the amount of RT-6.2 on the cell surface. No significant release of a rat T helper marker (visualized by the mAb W3/25) was observed in response to PI-PLC treatment. This is in sharp contrast to the effects of trypsin, which removes most of the T helper marker but had little effect on RT-6.2. SDS-PAGE analysis of the RT-6.2 released by PI-PLC indicated that the Mr was not significantly changed by this treatment. Phase separation of the released RT-6.2 in Triton X-114 showed that the PI-PLC had converted it from an amphiphilic membrane form to a water-soluble form, apparently by removing its hydrophobic membrane anchoring domain. These results strongly suggest that RT-6.2, in common with Thy-1 and several other cell surface proteins, is anchored in the membrane by the 1,2-diacylglycerol moiety of a covalently attached phosphatidylinositol molecule.
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PMID:Release of the rat T cell alloantigen RT-6.2 from cell membranes by phosphatidylinositol-specific phospholipase C. 348 8

Two immunologically distinct forms of phosphoinositide-specific phospholipase C were purified to near homogeneity from bovine brain. Their molecular weights determined by SDS-PAGE are 150,000 (enzyme I) and 145,000 (enzyme II), respectively. Under a nondenaturing condition, purified enzyme I exists mainly in dimeric form and as tetramer to a small extent, while enzyme II is predominantly in monomer, to a small extent as dimer and to a very small extent as trimer. Multiple forms of phosphoinositide-specific phospholipase C in brain tissue described in the literature might be, therefore attributed to the oligomerization of the two independent forms.
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PMID:Two forms of phosphatidylinositol-specific phospholipase C from bovine brain. 354 24

Staphylococcus aureus (Wood 46) was grown aerobically and anaerobically in supplemented 3% (w/v) Tryptone Soya Broth medium for 24 h at 37 degrees C. Although the bacterial density achieved was 9 times higher in the aerobic culture, the exoprotein produced per unit of bacterial dry weight was only 1.4 times higher than in the anaerobic culture. However, the SDS-PAGE patterns of extracellular proteins were quite different: the aerobic products occurred almost exclusively in the mol. wt range 15-30000 compared with 30-60000 for those produced anaerobically. The only major component common to both preparations was alpha-toxin which accounted for 2.4 times more of the total exoprotein under aerobic than under anaerobic conditions.
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PMID:A comparison of the patterns of extracellular proteins produced by the high alpha-toxin-secreting organism Staphylococcus aureus (Wood 46) during aerobic and anaerobic growth. 398 Nov 33

An effective concentration of alpha-toxin from Staphylococcus aureus Wood 46, directly from the culture supernatant, could be achieved by adsorption on digitonin-sepharose and elution with 3 mol/l sodium thiocyanate (NaSCN). The toxin was further purified by gelchromatography. The purified product yielded 1 single protein band upon SDS-polyacrylamide electrophoresis. It was nonhemolytic, but reacted with anti-alpha-toxin under complement fixation. Dialysis against 0.14 mol/l NaCl with hydrophobic amino acids partially reactivated the alpha-hemolytic activity of the toxin. Ultracentrifugal analysis yielded sedimentation coefficients for the purified toxin of approximately 3,7 S when dissolved in 3 mol/l NaSCN and of about 12 S after dialysis against 0.14 mol/l NaCl (Table 1). The spontaneous oligomerization of the alpha-toxin during dialysis against 0.14 mol/l NaCl possibly resulted from a change in configuration induced by its adsorption to digitonin-sepharose.
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PMID:Purification of oligomeric staphylococcal alpha-toxin by affinity chromatography on digitonin-sepharose. 400 34

In cell culture, a partially purified commercial preparation of phospholipase C (PLC) from Clostridium welchii inhibited fusion of myoblasts at concentrations of 12-50 microg per ml. At lower concentrations, PLC-treated cultures were indistinguishable from controls, and at concentrations above 100 microg per ml, PLC-treated cells detached from their substrates. The effect was reversible and fusion resumed approximately one cell cycle time after removal of the enzyme. Neither the percent of cells in the mitotic cycle nor the duration of the different phases of the cycle were altered by PLC at concentrations which inhibited fusion. Cell motility was not reduced by the enzyme. Unfused, PLC-treated myoblasts were virtually indistinguishable in ultrastructure from untreated cells just before fusion. In the presence of PLC, mononucleated myogenic cells did not synthesize thick (150 A) filaments. Treatment of culture medium with insolubilized commercial PLC did not abolish the capacity of the medium to support myogenesis. Chondrocytes treated with PLC divided repeatedly but failed to synthesize metachromatic matrix and failed to incorporate labeled sulfate into chondroitin sulfate. PLC was further purified by chromatography on Sephadex G-100. The resulting preparation was free of detectable protease, yielded one band on SDS-acrylamide gel electrophoresis, and displayed all of the biological activities of the less pure material.
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PMID:Inhibition of cellular differentiation by phospholipase C. I. Effects of the enzyme on myogenesis and chondrogenesis in vitro. 435 37

Rabbit or human erythrocytes lysed with Staphylococcus aureus alpha-toxin were solubilized with Triton X-100, and the toxin was subsequently isolated by gel chromatography, sucrose density gradient centrifugation, and reincorporation into liposomes. In the presence of Triton X-100, the toxin exhibited a sedimentation coefficient of 11S and eluted at a position between those of IgG and alpha 2-macroglobulin in gel chromatography. A single polypeptide subunit of 34,000 mol wt was found in SDS PAGE. In the electron microscope, ring-shaped or cylindrical structures were observed, 8.5-10 nm in diameter, harboring central pits or channels 2-3 nm in diameter. An amphiphilic nature of these structures was evident from their capacity to bind lipid and detergent, aggregation in the absence of detergents, and low elutability from biological and artificial membranes through ionic manipulations. In contrast to the membrane-derived form of alpha-toxin, native toxin was a water-soluble, 34,000 mol wt, 3S molecule, devoid of an annular structure. Because studies on the release of radioactive markers from resealed erythrocyte ghosts indicated the presence of circumscribed lesions of approximately 3-nm effective diameter in toxin-treated membranes, the possibility is raised that native alpha-toxin oligomerizes on and in the membrane to form an amphiphilic annular complex that, through its partial embedment within the lipid bilayer, generates a discrete transmembrane channel.
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PMID:On the mechanism of membrane damage by Staphylococcus aureus alpha-toxin. 627 94

Rabbit TNF has been purified 2000-fold by a series of salt precipitations, gel filtrations, ion exchange chromatography, and lectin affinity chromatography to a single species on SDS-polyacrylamide gel electrophoresis (SDS-PAGE). TNF activity could be recovered from nondenaturing gel systems and has been shown to be an alpha-globulin with an isoelectric point of 5.1. The m.w. was estimated to be 68,000 d by SDS-PAGE, 55,000 by gel filtration, and 52,000 by glycerol gradient centrifugation. TNF activity was stable over the pH range of 6 to 10 and was relatively heat stable, not being inactivated at 70 degrees C for 1 hr. TNF activity was pronase sensitive, but relatively trypsin resistant. Neuraminidase and phospholipase C treatment did not destroy TNF activity. Partially purified TNF was still capable of eliciting hemorrhagic necrosis in susceptible tumors. Crude TNF serum had an interferon titer of 3000 U, whereas the partially purified sample had a titer of <30 U.
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PMID:Purification and physico-chemical characterization of rabbit tumor necrosis factor. 699 83

Human E express two surface forms of decay-accelerating factor (DAF; CD55). On SDS-PAGE under reducing conditions the major form, DAF-1, migrates as a 70-kDa protein and the minor form, DAF-2, present at < 10% the amount of DAF-1, migrates as a 140-kDa protein (Kinoshita, T., S. I. Rosenfeld, and V. Nussenzweig. 1987. J. Immunol. 138:2994). Both forms possess decay-accelerating activity and, after purification from solubilized E, reinsert into sheep E, indicating a glycosylphosphatidylinositol anchor. In contrast to human cells, these two forms of DAF from orangutan E are expressed in approximately equal amounts (Nickells, M. W., and J. P. Atkinson. 1990. J. Immunol. 144:4262). An orangutan B lymphocyte cell line, CP81, also expresses similar quantities of both forms. These sources of orangutan DAF were utilized for further characterization of DAF-2. Orangutan and human DAF-1 were 98% and 95% homologous at the nucleotide and amino acid levels, respectively. Northern and Southern analyses of orangutan DAF were also similar to those for human DAF. Tryptic peptide maps of DAF-1 and DAF-2 were identical. After treatment with phosphatidylinositol-specific phospholipase C and glycosidases, the change in M(r) of DAF-2 was consistent with it possessing two glycosylphosphatidylinositol anchors and twice as much oligosaccharide as DAF-1. Biosynthetic analysis demonstrated a single 46-kDa precursor for both forms. Taken together, these data indicate that DAF-2 is a covalently cross-linked dimer of DAF-1. Analysis of a series of human DAF deletion mutants localized the cross-link(s) within the short consensus repeat domains.
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PMID:Characterization of DAF-2, a high molecular weight form of decay-accelerating factor (DAF; CD55), as a covalently cross-linked dimer of DAF-1. 750 31


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