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)

Cyclic AMP-dependent protein kinases from several mammalian sources inhibit Na+-dependent alpha-aminoisobutyric acid transport by membrane vesicles isolated from 3T3 cells. Evidence is provided that phosphorylation of membrane proteins by the enzyme is responsible for the inhibition. Lysis of the vesicles, or a reduction in the intravesicular volume is not the cause of reduced transport. The cyclic AMP-dependent protein kinase and its catalytic subunit phosphorylate a number of membrane proteins. Most of these proteins are phosphorylated, but to a lesser extent in the absence of protein kinase or cyclic AMP. The phosphorylated proteins remain associated with the membranes during hypotonic lysis treatments, which would be expected to release intravesicular contents and loosely associated membrane proteins. 32P-labeled bands detected on sodium dodecyl sulfate polyacrylamide gels after phosphorylation of membranes by the catalytic subunit of the cyclic AMP-dependent kinase are eliminated by treatment with either pronase or 1 N NaOH, but not by ribonuclease nor by phospholipase C. The stability of the incorporated radioactivity to hot acid and hydroxylamine relative to hot base suggests that most of the 32P from [gamma-32P]ATP is incorporated into protein phosphomonoester linkages.
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PMID:Inhibition of alpha-aminoisobutyric acid transport in membrane vesicles from mouse fibroblasts after phosphorylation by cyclic AMP-dependent protein kinase. 22 60

Sodium azide, hydroxylamine, and phenylhydrazine at concentrations of 1 mM increased the activity of soluble guanylate cyclase from rat liver 2- to 20-fold. The increased accumulation of guanosine 3':5'-monophosphate in reaction mixtures with sodium azide was not due to altered levels of substrate, GTP, or altered hydrolysis of guanosine 3':5'-monophosphate by cyclic nucleotide phosphodiesterase. The activation of guanylate cyclase was dependent upon NaN3 concentration and temperature; preincubation prevented the time lag of activation observed during incubation. The concentration of NaN3 that resulted in half-maximal activation was 0.04 mM. Sodium azide increased the apparent Km for GTP from 35 to 113 muM. With NaN3 activation the enzyme was less dependent upon the concentration of free Mn2+. Activation of enzyme by NaN3 was irreversible with dilution or dialysis of reaction mixtures. The slopes of Arrhenius plots were altered with sodium azide-activated enzyme, while gel filtration of the enzyme on Sepharose 4B was unaltered by NaN3 treatment. Triton X-100 increased the activity of the enzyme, and in the presence of Triton X-100 the activation by NaN3 was not observed. Trypsin treatment decreased both basal guanylate cyclase activity and the responsiveness to NaN3. Phospholipase A, phospholipase C, and neuraminidase increased basal activity but had little effect on the responsiveness to NaN3. Both soluble and particulate guanylate cyclase from liver and kidney were stimulated with NaN3. The particulate enzyme from cerebral cortex and cerebellum was also activated with NaN3, whereas the soluble enzyme from these tissues was not. Little or no effect of NaN3 was observed with preparations from lung, heart, and several other tissues. The lack of an effect with NaN3 on soluble GUANYLATE Cyclase from heart was probably due to the presence of an inhibitor of NaN3 activation in heart preparations. The effect of NaN3 was decreased or absent when soluble guanylate cyclase from liver was purified or stored at -20degrees. The activation of guanylate cyclase by NaN3 is complex and may be the result of the nucleophilic agent acting on the enzyme directly or what may be more likely on some other factor in liver preparations.
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PMID:Activation of guanylate cyclase from rat liver and other tissues by sodium azide. 24 Aug 48

The type of membrane association of acetylcholinesterase (AChE, EC 3.1.1.7) was studied in rabbit lymphocytes and erythrocytes. In both cases, the unique AChE molecular form was an amphiphilic dimer (referred to as G2a) anchored in the membrane by a glycosylphosphatidylinositol. In lymphocytes, G2a AChE was directly converted into its hydrophilic G2h counterpart by a treatment with Bacillus thuringiensis phosphatidylinositol-phospholipase C (PI-PLC, EC 3.1.4.10). In erythrocytes, AChE was resistant to PI-PLC but was rendered sensitive by a prior deacylation with alkaline hydroxylamine. This observation suggests that, as previously reported for human erythrocyte AChE, an acylation of the inositol ring in the glycolipid anchor of rabbit erythrocyte AChE (that does not occur in lymphocytes) prevents the cleavage.
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PMID:Glycolipid-anchored acetylcholinesterases from rabbit lymphocytes and erythrocytes differ in their sensitivity to phosphatidylinositol-specific phospholipase C. 132 66

We showed that some of Thy-1 molecules on murine thymocytes are resistant to phosphatidylinositol-specific phospholipase C (PI-PLC) derived from Bacillus thuringiensis. Both immature thymocytes with low CD3 expression and mature thymic T lymphocytes with high CD3 expression carried the PI-PLC-resistant Thy-1, and the PI-PLC-sensitivity of Thy-1 extensively varied among thymocyte subpopulations. In contrast, the same PI-PLC fully hydrolysed the anchor of Thy-1 on peripheral T lymphocytes. When the latter cells were activated with mitogen in vitro, however, some Thy-1 on them became resistant to PI-PLC. We then found that virtually all Thy-1 molecules on thymocytes became sensitive to PI-PLC when they were treated with hydroxylamine that should cleave ester-linked lipids. The result ruled out the possibility that the PI-PLC-resistant Thy-1 had a transmembranous peptide sequence, and suggested the presence of an additional fatty acyl group on the inositol ring of the Thy-1 anchor. In addition, the molecular size of the PI-PLC-resistant membrane-bound Thy-1 was only marginally larger than that of the PI-PLC-sensitive solubilized Thy-1 in detergent-partitioning SDS-PAGE analysis.
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PMID:T cell maturation stage-linked heterogeneity of the glycosylphosphatidylinositol membrane anchor of Thy-1. 136 Apr 44

CD59, the membrane regulator of autologous C5b-9 channel formation, exhibits variable sensitivity to cleavage by phosphatidylinositol-specific phospholipase C (PI-PLC), an enzyme that releases glyco-inositolphospholipid (GPI)-anchored proteins from cell surfaces. To determine whether the GPI-anchor phospholipid of CD59 is similar to that of decay-accelerating factor (DAF) and whether variation in its structure underlies its variable enzyme susceptibility, the GPI anchors of the two proteins expressed on erythrocytes, polymorphonuclear and mononuclear leucocytes were compared in situ and after purification. Flow cytometric analyses of PI-PLC-treated cells showed parallel cell type specific release of both proteins as a function of enzyme concentration. Non-denaturing PAGE analyses of alkaline/hydroxylamine-treated proteins (affinity-purified from [125I]-surface-labelled cells) provided evidence for (i) comparable proportions of GPI-anchor acylation, and (ii) alkali-resistant rather than alkali-sensitive lipid substituents in erythrocytes. These findings argue that the differential C5b-9 sensitivity that distinguishes paroxysmal nocturnal haemoglobinuria II and III erythrocytes does not derive from expression of CD59 molecules with alternative GPI-anchor phospholipid structures.
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PMID:Structural properties of the glycoplasmanylinositol anchor phospholipid of the complement membrane attack complex inhibitor CD59. 137 55

Human decay-accelerating factor (DAF) proteins expressed on E and nucleated cells differ in their susceptibility to phosphatidylinositol (PI)-specific phospholipase C (PLC) cleavage/release. To investigate the mechanism of this difference, the glycoinositol-phospholipid anchoring moieties of E DAF, and of HeLa cell, polymorphonuclear cell, and lymphocyte DAF were structurally compared. Labeling of PI-PLC-resistant E DAF with 3-(trifluoromethyl)-3-(m-[125I]-iodophenyl)-diazirine ([125I]TID) and TLC analysis of nitrous acid deamination anchor fragments showed a predominant phospholipid species with less polar migration than the 125I-TID-labeled PI. Gas chromatographic analyses of methanolyzed E protein revealed 2.20 +/- 0.16 mol of fatty acids [16:0, 18:0, 18:1, 20:4, 22:4, and 22:5 (0.76, 0.36, 0.25, 0.15, 0.40, 0.28 mol, respectively)] and 0.86 +/- 0.05 mol of inositol per mol of N-terminal Asp. Gas chromatography-mass spectroscopy demonstrated principally myo-inositol but also variable amounts of the chiro-isomer. Nondenaturing polyacrylamide gel electrophoresis of 14C-radiomethylated E protein revealed that pretreatment with hydroxylamine, a reagent which removes ester-linked lipids, rendered it PI-PLC susceptible. In contrast, parallel analyses of 35S-cys-labeled PI-PLC-sensitive HeLa DAF protein revealed only minor amounts of the hydroxylamine-sensitive phospholipid species. Similar results were obtained with 125I-surface-labeled DAF from polymorphonuclear cells, as well as from unstimulated peripheral blood and anti-CD3-activated lymphocytes. These findings demonstrate that, rather than PI, the E DAF anchor contains an inositol alkylacylglycerol-phospholipid which is heterogeneous with respect to acyl groups and inositol isomers, that an ester-linked substitution in this inositolphospholipid underlies the resistance of E DAF protein to PI-PLC cleavage/release, and that this structural modification is cell-specific.
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PMID:Structural basis for variations in the sensitivity of human decay accelerating factor to phosphatidylinositol-specific phospholipase C cleavage. 168 88

Staphylococcus aureus alpha-toxin opens an ion channel in planar phospholipid bilayers, which is selective for anions over cations, supposedly because of the presence of positively charged groups along the ion pathway. To remove some positive charges of this protein toxin, we chemically modified part of its lysine residues either with diethylpyrocarbonate, followed by histidine regeneration with hydroxylamine, or with trinitrobenzenesulfonic acid. The extent of chemical modification can be followed accurately by native polyacrylamide gel electrophoresis and isoelectric focusing. Ethoxyformilation of two to three lysine residues per toxin monomer does not impair hemolysis of rabbit red blood cells nor formation of pores in model membranes. It reduces the conductance and the anion selectivity of the channel and changes the shape of its current-voltage characteristic. This indicates that positively charged lysine residues are actually important in determining the electrical properties of the pore. Ethoxyformilation of channels preassembled in planar bilayers produces the same changes as modification of toxin monomers before channel formation. Furthermore, it can be performed by adding diethylpyrocarbonate on either side of the bilayer. This suggests that the lysine residues relevant for the electrical properties of the pore are located inside its lumen where they can be reached by diethylpyrocarbonate diffusing from either entrance of the channel.
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PMID:Modification of lysine residues of Staphylococcus aureus alpha-toxin: effects on its channel-forming properties. 170 80

1. We describe two simple procedures for the rapid identification of certain structural features of glycolipid anchors in acetylcholinesterases (AChEs). 2. Treatment with alkaline hydroxylamine (that cleaves ester-linked acyl chains but not ether-linked alkyl chains) converts molecules possessing a diacylglycerol, but not those with an alkylacylglycerol, into hydrophilic derivatives. AChEs in human and bovine erythrocytes possess an alkylacylglycerol (Roberts et al., J. Biol. Chem. 263:18766-18775, 1988; Biochem. Biophys. Res. Commun. 150:271-277, 1988) and are not converted to hydrophilic dimers by alkaline hydroxylamine. Amphiphilic dimers of AChE from Drosophila, from mouse erythrocytes, and from the human erythroleukaemia cell line K562 also resist the treatment with hydroxylamine and likely possess a terminal alkylacylglycerol. This indicates that the cellular pool of free glycolipids used as precursors of protein anchors is distinct from the pool of membrane phosphatidylinositols (which contain diacylglycerols). 3. Pretreatment with alkaline hydroxylamine is required to render the amphiphilic AChE from human erythrocytes susceptible to digestion by Bacillus thuringiensis phosphatidylinositol-specific phospholipase C (PI-PLC) (Toutant et al., Eur. J. Biochem. 180:503-508, 1989). We show here that this is also the case for the AChE from mouse erythrocytes, which therefore likely possesses an additional acyl chain in the anchor that prevents the action of PI-PLC. 4. In two sublines of K562 cells (48 and 243), we observed that AChE either was directly susceptible to PI-PLC (243) or required a prior deacylation by alkaline hydroxylamine (48). This suggests that glycolipid anchors in AChE of K562-48 cells, but not those in AChE of K562-243 cells, contain the additional acylation demonstrated in AChE from human erythrocytes. These observations illustrate the cell specificity (and the lack of species-specificity) of the structure of glycolipid anchors.
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PMID:Rapid analysis of glycolipid anchors in amphiphilic dimers of acetylcholinesterases. 184 55

Acetylcholinesterase (AChE) in K562 cells exists in two molecular forms. The major form, an amphiphilic dimer (G2a) which sediments at 5.3 S, and the minor form, an amphiphilic monomer (G1a) which sediments at 3.5 S. Extraction in the presence of the sulfhydryl alkylating agent N-ethylmaleimide was required to preserve the G2a form. In Triton X-100 extracts of the subline K562-243, phosphatidylinositol-specific phospholipase C (PtdIns-PLC) from Bacillus thuringiensis converted most of the G2a AChE into a hydrophilic dimer (G2h), indicating that the G2a form possessed a hydrophobic glycoinositol phospholipid that mediated its attachment to the membrane. Treatment of intact K562-243 cells with PtdIns-PLC released approximately 60% of the total AChE activity and provided an estimate of the externally exposed AChE. The direct conversion from an amphiphilic to a hydrophilic dimeric form by PtdIns-PLC was not obtained in extracts or intact cells of the subline K562-48. Instead, pretreatment with alkaline hydroxylamine was necessary to render the amphiphilic G2 form of this subline susceptible to digestion by the phospholipase. In this respect, the amphiphilic dimer of K562-48 AChE resembles the G2a form of human erythrocyte AChE, which is resistant to PtdIns-PLC because of the direct palmitoylation of an inositol hydroxyl group in the anchor [Roberts et al. (1988) J. Biol. Chem. 263, 18766-18775]. Release of this acyl chain by hydroxylamine renders the enzyme susceptible to PtdIns-PLC [Toutant et al. (1989) Eur. J. Biochem. 180, 503-508]. In both K562 sublines, sialidase decreased the migration of the G2a form but not of the G1a form of AChE. G1a forms thus appear to represent an intracellular pool of newly synthesized molecules residing in a compartment proximal to the trans-Golgi apparatus. The sialidase-resistant G1a molecules were also resistant to PtdIns-PLC digestion; possible explanations for this resistance are presented.
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PMID:Molecular forms of acetylcholinesterase in two sublines of human erythroleukemia K562 cells. Sensitivity or resistance to phosphatidylinositol-specific phospholipase C and biosynthesis. 229 8

Each catalytic subunit in the amphiphilic dimer of human erythrocyte acetylcholinesterase (AChE) is anchored in the plasma membrane exclusively by a glycoinositol phospholipid. In contrast to erythrocyte AChEs in other mammalian species, the human enzyme is resistant to direct cleavage by phosphatidylinositol-specific phospholipase C (PtdIns-specific PLC). The resistance is due to the existence of an additional fatty acyl chain on the inositol ring which blocks the action of PtdIns-specific PLC [Roberts et al. (1988) J. Biol. Chem. 263, 18766-18775]. In this report, nondenaturing polyacrylamide gel electrophoresis was applied to permit rapid and unambiguous distinction between amphiphilic AChE, in which each catalytic subunit binds one nonionic detergent micelle, and hydrophilic AChE, which does not interact with detergent. Deacylation of human erythrocyte AChE by an alkaline treatment with hydroxylamine rendered the amphiphilic AChE susceptible to PtdIns-specific PLC with the consequent release of hydrophilic AChE. Although serum anchor-specific phospholipase D (PLD) cleaves the intact human erythrocyte AChE anchor, this treatment, as judged by nondenaturing electrophoresis, did not release hydrophilic AChE. Hydroxylamine treatment before or after PLD digestion was necessary to achieve the conversion. These observations indicate that binding of a single detergent micelle was maintained when any of the three fatty acyl or alkyl groups in the human erythrocyte AChE anchor phospholipid were retained. For proteins that can be identified following nondenaturing gel electrophoresis, these procedures provide methods both for detecting glycoinositol phospholipid anchors resistant to PtdIns-specific PLC and for indicating fatty acyl and/or alkyl chains in these anchors.
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PMID:Conversion of human erythrocyte acetylcholinesterase from an amphiphilic to a hydrophilic form by phosphatidylinositol-specific phospholipase C and serum phospholipase D. 254 Sep 62


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