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)

A cysteine-to-phenylalanine mutation in the third transmembrane domain of the alpha 1B-adrenergic receptor constitutively activates the receptor, resulting in G protein coupling in the absence of agonist and activation of only a single effector pathway (phospholipase C but not phospholipase A2). This mutant receptor displays a higher affinity for the catecholamines, norepinephrine, and epinephrine, as well as for other phenethylamines, but not for imidazolines, a class of structurally distinct alpha agonists. Dose-response studies demonstrate a higher potency and intrinsic activity of phenethylamines for polyphosphoinositide turnover but not for arachidonic acid release. Imidazolines have wild-type potencies and intrinsic activities for both pathways. These data indicate that a single receptor subtype forms multiple conformations (i.e., exhibits induced conformational pleiotropy) for G protein interactions (high affinity states) that are specific for a particular G protein/effector pathway and that multiple binding sites exist for agonists, which promote or induce these specific interactions. Pharmacological diversity may, thus, be achieved through a single receptor by the development of compounds that induce a single activated conformer. This has major ramifications for the eventual development of signaling-specific therapeutics.
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PMID:Constitutive activation of a single effector pathway: evidence for multiple activation states of a G protein-coupled receptor. 856 96

Purified bovine erythrocyte acetylcholinesterase (AChE) was radiomethylated on its amine groups and incubated with bacterial phosphatidylinositol-specific phospholipase C to remove the lipid portion of the AChE glycoinositol phospholipid (GPI) anchor, and a C-terminal tryptic fragment that contained the residual GPI glycan was isolated by HPLC. Analysis by electrospray-ionization mass spectrometry revealed a parent ion of m/z 3798. The fragmentation patterns produced by collision-induced dissociation mass spectrometry of the +4 and +5 states of the parent ion indicated a 23-amino acid peptide in amide linkage to ethanolamine-P04-Hex-Hex-Hex(PO4-ethanolamine)(HexNAc)-Hex N(Me)2-inositol phosphate. The glycan structure is completely consistent with that obtained previously for the GPI anchor of human erythrocyte AChE except for the addition of the HexNAc substituent. A nearly complete peptide sequence was deduced from the fragmentation patterns, although four assignments were based only on single fragments of very low abundance. To resolve this uncertainty, a segment of bovine genomic DNA corresponding to the C-terminal AChE sequence was amplified by PCR. DNA sequencing established the 23-amino acid peptide sequence to be FLPKLLSATASEAPCTCSGPAHG, in agreement with the MS data and consistent with results from Edman protein sequencing. Dimerization of AChE polypeptides is mediated by intersubunit disulphide bonding in this C-terminal segment, but the bovine AChE contained two cysteine residues in a ...CTC... motif, in contrast with human AChE which contains only a single cysteine in this segment. Although bovine AChE contained no free thiol groups reactive with iodo[14C]acetamide, partial reduction and alkylation with iodo[14C]acetamide revealed that conversion into monomers occurred with an overall incorporation of only one alkyl group per monomer. An identical level of alkylation was observed when dimeric human AChE was converted into monomers by partial reduction. The question of whether the bovine AChE contains one or two intersubunit disulphide linkages is considered.
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PMID:Glycoinositol phospholipid anchor and protein C-terminus of bovine erythrocyte acetylcholinesterase: analysis by mass spectrometry and by protein and DNA sequencing. 861 75

Staphylococcus aureus alpha-toxin is a hydrophilic polypeptide of 293 amino acids that produces heptameric transmembrane pores. During assembly, the formation of a pre-pore precedes membrane permeabilization; the latter is linked to a conformational change in the oligomer. Here, 41 single-cysteine replacement toxin mutants were thiol-specifically labelled with the polarity-sensitive fluorescent probe acrylodan. After oligomerization on membranes, only the mutants with acrylodan attached to residues in the sequence 118-140 exhibited a marked blue shift in the fluorescence emission maximum, indicative of movement of the fluorophore to a hydrophobic environment. Within this region, two functionally distinct parts could be identified. For mutants at positions 126-140, the shifts were partially reversed after membrane solubilization by detergents, indicating a direct interaction of the label with the membrane lipids. Membrane insertion of this sequence occurred together with the final pre-pore to pore transition of the heptamer. Thus residues 126-140 constitute a transmembrane sequence in the pore. With labelled residues 118-124, pre-pore assembly was the critical event to induce the spectral shifts, which persisted after the removal of membrane lipids and hence probably reflects protomer-protomer contacts within the heptamer. Finally, a derivative of the mutant N121C yielded occluded pores which could be opened by reductive reversal of the modification. Therefore this residue probably lines the lumen of the pore.
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PMID:Molecular architecture of a toxin pore: a 15-residue sequence lines the transmembrane channel of staphylococcal alpha-toxin. 861 32

The gamma subunit of heterotrimeric G proteins is isoprenylated and methylated on its carboxyl terminal cysteine residue. While retinal transducin is farnesylated, all other gamma subunits are modified by geranylgeranylation. An immobilized form of pig liver esterase (iPLE) is able to hydrolyze the methyl ester of a geranylgeranylated beta gamma isoform (beta 1 gamma 2). Since methylation is the only reversible reaction in the isoprenylation pathway, it could be a site of regulation of G protein activity. With both the methylated and demethylated beta 1 gamma 2 now available, the role of methylation for a geranylgeranylated heterotrimeric G protein may be addressed. Here, it is reported that methylation has no effect on the ability of beta gamma to interact with an alpha subunit, as probed by ADP-ribosylation studies with pertussis toxin, and has a small effect (less than 2-fold) on the ability of geranylgeranylated beta gamma to activate phosphatidylinositol-specific phospholipase C (PIPLC) and phosphoinositide 3 kinase (PI3K). In binding studies, demethylation only slightly decreased the ability of beta 1 gamma 2 to adhere to azolectin vesicles. Therefore, methylation of heterotrimeric G proteins appears to have only a minor effect in signal transduction processes which can be correlated to a decrease in hydrophobicity of the beta gamma subunit.
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PMID:The role of G protein methylation in the function of a geranylgeranylated beta gamma isoform. 865 28

Post-translational modifications such as phosphorylation and palmitoylation play important roles for the function and regulation of receptors coupled to heterotrimeric guanyl nucleotide-binding proteins. Here we demonstrate that the human endothelin receptor A (ETA) incorporates [3H]palmitate. Mutation of a cluster of five cysteine residues present in the cytoplasmic tail of ETA into serine or alanine residues completely prevented palmitoylation of the receptor. The ligand binding affinity of the non-palmitoylated ETA mutants was essentially unchanged as compared to the palmitoylated wild type ETA suggesting that the replacement of the cysteine residues did not alter the overall structure of the receptor. Furthermore, the ligand-induced stimulation of adenylyl cyclase by the mutant ETA was unaffected by the mutation. In contrast, the mutated non-palmitoylated receptors but not the wild type receptor failed to stimulate phosphatidylinositol hydrolysis by phospholipase C activation upon challenge by endothelin-1. Furthermore, the mutant receptors failed to stimulate the ligand-induced transient increase in the cytoplasmic calcium seen with the wild type ETA. Endothelin-1 induced mitogenic stimuli via the wild type receptors but not through the mutated receptors suggesting an important role for phospholipase C in this signal transduction pathway. The differential regulation of distinct signal transduction pathways by post-translational modification suggests that palmitoylation of the ETA provides a novel mechanism of modulating ETA receptor activity.
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PMID:Palmitoylation of endothelin receptor A. Differential modulation of signal transduction activity by post-translational modification. 870 36

It was previously shown that the Haemonchus contortus apical gut surface proteins p46, p52, and p100 induced protective immunity to challenge infections in goats. Here, it is shown that the three proteins are all encoded by a single gene (GA1) and initially expressed in adult parasites as a polyprotein (p100GA1). p46GA1 and p52GA1 are related proteins with 47% sequence identity, including a cysteine-containing region, which appears to confer secondary structure to these proteins, and a region with sequence similarity to bacterial Tolb proteins. GA1 protein expression is regulated during the life cycle at the level of transcript abundance. Only p52GA1 has characteristics of a glycosylinositolphospholipid membrane-anchored protein. However, both p46GA1 and p52GA1 were released from the gut membrane by phosphatidylinositol specific-phospholipase C, suggesting that p46GA1 membrane association depends on interactions with a glycosylinositolphospholipid gut membrane protein. Finally, GA1 proteins occur in abomasal mucus of infected lambs, demonstrating possible presentation to the host immune system during H. contortus infection. The results identify multiple characteristics of the GA1 proteins that should be considered for design of recombinant antigens for vaccine trials and that implicate a series of cellular processes leading to modification and expression of GA1 proteins at the nematode apical gut surface.
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PMID:Haemonchus contortus GA1 antigens: related, phospholipase C-sensitive, apical gut membrane proteins encoded as a polyprotein and released from the nematode during infection. 871 Sep 24

1. Phospholipase D (PLD) is the key enzyme in a signal transduction pathway leading to the formation of the second messengers phosphatidic acid and diacylglycerol. In order to define the pharmacological profile of PLD-coupled metabotropic glutamate receptors (mGluRs), PLD activity was measured in slices of adult rat brain in the presence of mGluR agonists or antagonists. Activation of the phospholipase C (PLC) pathway by the same agents was also examined. 2. The mGluR-selective agonist (1S,3R)-l-aminocyclopentane-1,3-dicarboxylic acid [(1S,3R)-ACPD] induced a concentration-dependent (10-300 microM) activation of PLD in the hippocampus, neocortex, and striatum, but not in the cerebellum. The effect was particularly evident in hippocampal slices, which were thus used for all subsequent experiments. 3. The rank order of potencies for agonists stimulating the PLD response was: quisqualate > ibotenate > (2S,3S,4S)-alpha-(carboxycyclopropyl)-glycine > (1S,3R)-ACPD > L-cysteine sulphinic acid > L-aspartate > L-glutamate. L-(+)-2-Amino-4-phosphonobutyric acid and the ionotropic glutamate receptor agonists N-methyl-D-aspartate, alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid, and kainate failed to activate PLD. (RS)-3,5-dihydroxyphenylglycine (100300 microM), an agonist of mGluRs of the first group, stimulated PLC but inhibited the PLD response elicited by 100 microM (1S,3R)-ACPD. 4. (+)-alpha-Methyl-4-carboxyphenylglycine (0.1-1 mM), a competitive antagonist of mGluRs of the first and second group, elicited a significant PLD response. L-(+)-2-Amino-3-phosphonopropionic acid (1 mM), an antagonist of mGluRs of the first group, inhibited the 100 microM (1S,3R)-ACPD-induced PLC response but produced a robust stimulation of PLD. 5. 12-O-Tetradecanoylphorbol 13-acetic acid and phorbol 12,13-dibutyrate (PDBu), activators of protein kinase C, at 1 microM had a stimulatory effect on mGluRs linked to PLD but depressed (1S,3R)-ACPD-induced phosphoinositide hydrolysis. The protein kinase C inhibitor, staurosporine (1 and 10 microM) reduced PLD activation induced by 1 microM PDBu but not by 100 microM (1S,3R)-ACPD. 6. Our results suggest that PLD-linked mGluRs in rat hippocampus may be distinct from any known mGluR subtype coupled to PLC or adenylyl cyclase. Moreover, they indicate that independent mGluRs coupled to the PLC and PLD pathways exist and that mGluR agonists can stimulate PLD through a PKC-independent mechanism.
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PMID:Pharmacological characterization of metabotropic glutamate receptors coupled to phospholipase D in the rat hippocampus. 879 79

Myonecrosis induced in vivo by cardiotoxin, melittin, and Asp49 and Lys49 phospholipase A2 (PLA2) myotoxins involves rapid lysis of the sarcolemma, myofibril clumping, and hypercontraction of sarcomeres. In contrast, skeletal muscle necrosis induced by crotamine and myotoxin a is much slower, consisting of mitochondrial and sarcoplasmic reticulum swelling, myofibril degeneration, and lack of sarcolemma or transverse tubule damage. The mechanisms contributing to the myonecrosis induced by these peptides were evaluated. Two cardiotoxins and two Lys49 PLA2 myotoxins lysed primary cultures of human skeletal muscle within 24 hr at a concentration of 0.25 microM, while melittin, crotamine, and myotoxin a, and an Asp49 PLA2 myotoxin were non-cytolytic at concentrations up to 5.0 microM, suggesting that cytolysis is not a good measure of myotoxicity. Crotamine and the Lys49 PLA2 myotoxin altered Ca2+ ion flux in human heavy sarcoplasmic reticulum by opening the ryanocine receptor. Whole-cell patch-clamp studies demonstrated that administrating crotamine intracellularly increased Na+ currents. Free fatty acids, liberated by activation of tissue phospholipase C or by the PLA2 activity of the myotoxins, were monitored for crotamine, myotoxin a and a Lys49 PLA2 myotoxin in cell cultures in which the lipids had been radiolabeled. Only the Lys49 myotoxin produced significant amounts of fatty acid in cell cultures, supporting a potential role for fatty acid production only in the mechanism of sarcolemma-destroying myotoxins. These findings, coupled with those in the literature, support a hypothesis in which the myotoxins and/or products of lipase activity (e.g. fatty acids) are acting at a site existing on both the Na+ channel and a protein involved in Ca2+ release and probably serving a modulatory function for ion regulation. Based on the similarities in mechanisms between the toxins and fatty acids, the most likely site would be a fatty acid binding site on the protein (either similar to that on fatty acid binding proteins, or an acylated cysteine residue) or in the membrane.
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PMID:Similarities and differences in mechanisms of cardiotoxins, melittin and other myotoxins. 902 86

Staphylococcus aureus alpha-toxin forms heptameric pores on eukaryotic cell membranes. Assembly of the heptamer precedes formation of the transmembrane pore. The latter event depends on a conformational change that drives a centrally located stretch of 15 amino acid residues into the lipid bilayer. A second region of the molecule that has been implicated in the pre-pore to pore transition is the far N-terminus. Here, we used fluorescently labeled single cysteine replacement mutants to analyze the functional role of the far N-terminus of alpha-toxin. Pyrene attached to mutants S3C, I5C and 17C forms excimers within the toxin pore complex. This indicates that the distance of adjacent N-termini is less than 10-12 Angstrom. By labeling with the polarity-sensitive fluorophore acrylodan, pore formation is shown to cause distinct environmental changes in the N-terminus. Removal of membrane lipids from the labeled heptamers has no effect upon the acrylodan spectrum, indicating lack of direct contact of the N-terminus with the target membrane. The environmental alterations to the N-terminus are thus due to altered protein structure only. Both acrylodan emission shifts and pyrene excimers were shown to be absent in toxin heptamers that were arrested at the pre-pore stage. Therefore, while not being directly involved in membrane penetration, the N-termini of the alpha-toxin heptamer subunits move into immediate mutual proximity concomitantly with transmembrane pore formation.
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PMID:Staphylococcal alpha-toxin: the role of the N-terminus in formation of the heptameric pore -- a fluorescence study. 916 53

The composition, structure and immunogenicity of hepatitis B surface antigen (HBsAg) particles derived from Chinese hamster ovary (CHO) cells and from cells of the yeast Hansenula polymorpha were compared. The particles were similar in size distribution (mean 20-33 nm), in shape (spherical), in gross composition (protein to lipid weight ratio of 60:40), and in types of lipids (phospholipids > > sterols = sterol esters = triacylglycerols). Differences related to genetic engineering and type of host cells were found in peptide and lipid compositions. CHO-HBsAg has three peptides: S, M and L, each in two forms of glycosylation, while the Hansenula-HBsAg has only the nonglycosylated S peptide. The electrical surface potential at the lipid/water interface of HBsAg derived from Hansenula is more negative than that of HBsAg derived from CHO, which was close to neutrality. Although the numbers of cysteine residues (all in the S peptides) are identical (14), 11 of them are free thiols in the CHO-HBsAg, compared with three to four in the Hansenula-HBsAg. The fact that 85% of the phospholipids are hydrolyzed by phospholipase C and that all the aminophospholipids react with trinitrobenzenesulfate suggests that the particles derived from both cell types are either leaky vesicles or have a lipoprotein-like structure. Subcutaneous injection into mice of fluorescein-isothiocyanate-labeled HBsAg particles from both sources resulted in their accumulation in the marginal sinus of lymph nodes. The humoral responses to subcutaneous injection into mice of CHO- and Hansenula-HBsAg were similar: however, the cytotoxic T lymphocyte response to CHO-HBsAg was lower.
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PMID:Comparison between hepatitis B surface antigen (HBsAg) particles derived from mammalian cells (CHO) and yeast cells (Hansenula polymorpha): composition, structure and immunogenicity. 917 64


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