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)

1 The effect of (+/-)-, (+)- and (-)-verapamil on the Ca2+-binding, Ca2+-transporting activity, and Ca2+-dependent adenosine triphosphatase (ATPase) activity of isolated cardiac sarcolemmal preparations was studied. Enzymatic treatment was used to establish the nature of the sites facilitating [14C]-(+/-)-verapamil binding. 2 (+/-)-Verapamil 1 microM inhibited the passive binding of 45Ca2+. The (+/-)- and (-)-isomers were equiactive. 3 (+/-)-Verapamil 1 microM inhibited the ATP-dependent transport of 45Ca2+ and the associated activation of the Ca2+-sensitive ATPase. The activity resided in the (-)-isomer. 4 Lineweaver-Burk plots for the initial rates of ATP-dependent transport showed that the inhibition induced by the (-)-isomer was accompanied by a reduced Km and Vmax. 5 Enzymatic removal of N-acetyl neuraminic acid and galactose residues increased [14C]-(+/-)-verapamil binding; removal of N-acetylglucosamine and treatment with phospholipase C and trypsin decreased the binding. 6 These results have been interpreted to mean that (-)-verapamil interferes with the ATP-dependent Ca2+-transporting properties of the sarcolemma, and that this effect is accompanied by an altered activity of the intrinsic Ca2+-sensitive ATPase. N-acetylneuramic acid and galactose residues do not provide binding sites for verapamil at the cell surface.
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PMID:The effect of verapamil on the Ca2+-transporting and Ca2+-ATPase activity of isolated cardiac sarcolemmal preparations. 645 Dec 52

The maturation-associated human B cell rosette receptor (MER) for mouse erythrocytes has been solubilized from B cells by mild trypsinization. It specifically agglutinates mouse red cells. Material with hemagglutinating activity partitioned into the lipid-soluble phase of a Folch partition of the trypsin extract was sensitive to phospholipase C and alkali, and on two-dimensional thin layer chromatography, it co-migrated principally with phosphatidylethanolamine (PE). Phosphatidylcholine, the major lipid present, was inactive. The relationship of phospholipid structure to hemagglutinating activity has been described. PE in the crude trypsin extract was associated with unidentified glycoprotein and albumin. Material containing hemagglutinating lipid bound to a wheat germ lectin-Sepharose column and was released by N-acetylglucosamine, indicating that the PE was complexed with glycoprotein. When the crude trypsin extract or eluate from the lectin column was extracted with aqueous phenol, hemagglutinin in the aqueous phase no longer bound to wheat germ lectin-Sepharose; however, albumin was greatly enriched, indicating that some of the PE exists in a complex with albumin. The molar ratio of PE to albumin was approximately 200:1. After delipidation, this albumin (in molar excess) inhibited hemagglutination by PE in the same way as a recently described subclass of serum albumin. Studies with phospholipase-treated B cells were also consistent with PE being the MER. We conclude that MER is PE, existing in a complex containing glycoprotein and a subclass of albumin. The capacity to form rosettes can be transferred to nonrosetting Raji B cells by the complex, but not pure PE, indicating that the proteins may be involved in orienting PE correctly for it to function as the MER.
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PMID:A phosphatidylethanolamine-containing complex on human B cells that mediates rosette formation with mouse erythrocytes. 660 1

The kinetic binding characteristics of four Bacillus thuringiensis CryI insecticidal crystal proteins to a Cry-binding protein, purified from Manduca sexta brush-border vesicles, were analyzed by an optical biosensor. This 120-kilodalton binding protein, previously determined to be aminopeptidase N, was converted to a 115-kilodalton water-soluble form by removing the attached glycosylphosphatidylinositol anchor with phospholipase C. The solubilized form recognized the three major subclasses of CryIA toxins but not CryIC even though all four CryI proteins are toxic to larvae of M. sexta. CryIA(a) and CryIA(b) toxins bound to a single site on the solubilized aminopeptidase N molecule whereas CryIA(c) bound to two distinct sites. Apparent kinetic rate constants were determined for each binding reaction. All three CryIA toxins exhibited moderately fast on rates (approximately 10(-5) M-1 s-1) and a slow reversible off rate (approximately 10(-3) s-1). Although the second CryIA(c)-binding site retained a moderately fast association rate, it was characterized by a rate of dissociation from the amino-peptidase an order of magnitude faster than observed for the other CryIA-binding sites. CryIA(c) binding to both sites was strongly inhibited in the presence of N-acetylgalactosamine (IC50 = 5 mM) but not N-acetylglucosamine, mannose, or glucose. CryIA(a) and CryIA(b) binding were unaffected in the presence of the same sugars. Our results serve to illustrate both the complexity and the diverse nature of toxin interactions with Cry-binding proteins.
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PMID:The CryIA(c) receptor purified from Manduca sexta displays multiple specificities. 765 2

Sera of patients with chronic Chagas' disease (American trypanosomiasis) contain elevated levels of anti-alpha-galactosyl antibodies that are lytic to Trypanosoma cruzi. The T. cruzi trypomastigote F2/3 antigen complex recognized by these antibodies runs as a broad smear on SDS/PAGE [Almeida, Krautz, Krettli and Travassos (1993) J. Clin. Lab. Anal. 7, 307-316]. Treatment of T. cruzi trypomastigote cells with bacterial phosphatidylinositol-specific phospholipase C (PI-PLC) abolished most of their reactivity to chronic Chagas'-disease ((Chagasic, Ch) anti-alpha-galactosyl antibodies (anti-Gal). The F2/3 antigen complex, purified by solvent extraction and hydrophobic-interaction chromatography, contained 60% carbohydrate by weight and substantial amounts of Thr, Ser, Glx, Asx, Gly, Ala and Pro, but relatively few hydrophobic amino acids. The presence of myoinositol, ethanolamine and 1-O-hexadecylglycerol suggested the presence of glycosyl-phosphatidylinositol membrane anchors. This was confirmed by PI-PLC treatment, which rendered the F2/3 molecules hydrophilic and reactive to anti-(cross-reacting determinant) antibodies. The majority of the GlcNAc content of the F2/3 antigens was found at the reducing termini of oligosaccharides in O-glycosidic linkage to Thr residues. These O-linked oligosaccharides could be released by beta-elimination and by mild hydrazinolysis. The smallest released oligosaccharitol that was reactive with the Ch anti-Gal was Gal alpha 1-3Gal beta 1-4GlcNAcol (where GlcNAcol is N-acetyl-glucosaminitol). Several other Gal-containing oligosaccharitols were observed, most of which were branched and contained 4,6-di-O-substituted GlcNAcol at their reducing termini. About half of the total released oligosaccharitols could bind to immobilized Ch anti-Gal, but none of them bound to the anti-Gal isolated from normal human sera. These data suggest that the specificities of the Ch anti-Gal are quite different from the natural anti-Gal isolated from normal human sera. Therefore, these novel T. cruzi O-linked oligosaccharides are highly immunogenic under the conditions of natural infection and are the targets for lytic Ch anti-Gal.
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PMID:Lytic anti-alpha-galactosyl antibodies from patients with chronic Chagas' disease recognize novel O-linked oligosaccharides on mucin-like glycosyl-phosphatidylinositol-anchored glycoproteins of Trypanosoma cruzi. 781 83

Ascidian sperm bind to vitelline coat N-acetylglucosamine groups of the egg via sperm surface N-acetylglucosaminidase. This sperm surface egg receptor remains anchored throughout penetration. Localization to the sperm surface was verified by biotinylation of intact sperm followed by solubilization in Triton X-100 and binding to streptavidin agarose. The enzyme was determined to be an integral membrane protein as judged by resistance to release by Kl and high pH. Linkage of the enzyme to the sperm surface was probed through differential solubilization followed by measuring released enzymatic activity with a fluorogenic substrate. Nonionic detergents released 90% of the activity. Proteases released about 40%. No activity was released by a phosphatidylinositol specific phospholipase C. This finding, combined with the similarity of release level by all the detergents, including Triton X-100 and octylglucoside, argues against a phosphatidyl-inositol linkage. The release form enters the hydrophilic phase of a Triton X-114 phase separation experiment. This observation, coupled with the findings of release by nonionic detergents, suggests that the protein is hydrophilic once released from the membrane. Thus, although clearly an integral membrane protein, the enzyme has limited hydrophobicity such as would be present in a single transmembrane sequence or extensive glycosylation.
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PMID:Attachment of the ascidian sperm surface egg receptor N-acetylglucosaminidase to the cell membrane. 798 Sep 54

The lipophosphoglycan (LPG)-like glycoconjugate expressed on the cell surface of Trypanosoma cruzi epimastigotes was isolated, purified, and partially characterized. The glycoconjugate migrated as a homogeneous band (42 kDa) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Matrix-assisted laser desorption ionization mass spectral analysis of the native molecule indicated the presence of two major components whose molecular masses were about 18.4 and 22.5 kDa. The LPG could be metabolically labeled with [3H]galactose, [3H]mannose, [14C]glucose, or [3H]palmitic acid. Monosaccharide compositional analysis of the LPG indicated that galactose, glucosamine, and sialic acid predominate over mannose, galactosamine, and inositol. A peptide associated with the LPG molecule contained about 40 amino acid residues per inositol and had threonine as the predominant amino acid. The LPG showed strong binding to Ricinus communis agglutinin-1 and Tritium vulgare wheat germ agglutinin, indicating the presence of terminal beta 1,4-linked galactosyl residue(s) and N-acetylglucosamine, respectively. Lectin binding studies also suggested the presence of a terminal beta-galactose and GlcNAc in the glycan-inositol lipid core of LPG. Virtually all of the sialic acids appeared to be located in the saccharide portion of the molecule. Treatment of the LPG with phosphatidylinositol-specific phospholipase C liberated an alkylacylglycerol. Structural analysis of the alkylacylglycerol and its acidic methanolysis products by gas-liquid chromatography/mass spectrometry indicated that the glycerol substituents were primarily the C16 1-alkyl group and C16 2-acyl group. The ratio of inositol to 1-O-alkyl-2-O-acylglycerol was 1:1. Treatment of the glycoconjugate with nitrous acid released a major phospholipid product that migrated close to the phosphatidylinositol standard on thin layer chromatography. This result implied that phosphatidylinositol was glycosidically linked to the nonacetylated amino sugar. Furthermore, the LPG was found to contain phosphate and was labile to mild acid hydrolysis, strongly suggesting that the intact molecule is related to Leishmania LPG. The most striking and unique feature of T. cruzi LPG is the presence of large amounts of glucosamine and sialic acid as well as galactosamine. These results indicate that the glycoconjugate expressed on the T. cruzi cell surface is a new type of LPG-like molecule anchored on the cell surface via an alkylacylphosphatidylinositol.
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PMID:Expression of a novel cell surface lipophosphoglycan-like glycoconjugate in Trypanosoma cruzi epimastigotes. 807 17

Activities of nucleotide-sugar:dolichyl phosphate glycosyltransferases (UDP-N-acetylglucosamine:dolichyl phosphate N-acetylglucosaminyl 1-phosphotransferase, UDP-glucose:dolichyl phosphate glucosyltransferase and GDP-mannose:dolichyl phosphate mannosyltransferase) are not fully expressed in native microsomes and can be enhanced by pretreatment of the microsomes with detergent. To examine whether the latency of dolichyl phosphate glycosyltransferases in native microsomes reflects a lumenal orientation of the catalytic centre, we examined the effect of proteinase treatment of native microsomes on enzymic activity and investigated the relationship between enzymic activity and alteration of the permeability of the microsomal membrane barrier. The enzymic activities catalysing transfer of N-acetylglucosamine and glucose to lipid acceptors were proteinase-sensitive in native sealed microsomes. When various detergents were used to disrupt the membrane barrier, we found no relationship between activity of dolichyl phosphate glycosyltransferases and the latency of mannose-6-phosphatase, which is a marker of the permeability properties of the microsomal membrane. Permeabilization of the endoplasmic reticulum membrane by the pore-forming Staphylococcus aureus alpha-toxin did not affect glycosyltransferase activities. These results do not support the hypothesis that latency of the transferase activities is dependent on the permeability properties of the endoplasmic-reticulum membrane. Collectively our findings can best be explained by postulating that the active centres of the transferases are cytoplasmically oriented, while activation by detergent may be conformation-dependent.
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PMID:Topology of nucleotide-sugar:dolichyl phosphate glycosyltransferases involved in the dolichol pathway for protein glycosylation in native rat liver microsomes. 828 60

Incubation of native rat liver microsomes with GTP resulted in enhanced incorporation of N-acetylglucosamine (GlcNAc) from UDP-GlcNAc into lipid acceptors. The stimulation of GlcNAc transfer by GTP was specific for GTP; ATP exerted no effect. The GTP effect was blocked by a non-hydrolysable GTP analogue guanosine 5'-[beta gamma-imido]triphosphate, indicating that GTP hydrolysis was crucial. Though dolichyl pyrophosphate NN'-diacetylchitobiose [Dol-PP-(GlcNAc)2] was the main radiolabelled product formed upon incubation of GTP-treated microsomes with UDP-GlcNAc, GTP selectively stimulated UDP-GlcNAc:dolichyl phosphate (Dol-P) N-acetylglucosaminyl 1-phosphotransferase (N-acetylglucosaminyl 1-phosphotransferase). This conclusion was reached on the basis of experiments in which tunicamycin was used to selectively inhibit N-acetylglucosaminyl 1-phosphotransferase. The enhanced transformation of Dol-P to dolichyl pyrophosphate N-acetylglucosamine (Dol-PP-GlcNAc) by GTP ultimately led to enhanced protein glycosylation. GTP-induced stimulation of GlcNAc incorporation in lipid and protein by GTP was observed also in microsomes fully permeabilized with Staph. aureus alpha-toxin. These findings refute the previous proposal [Godelaine, Beaufay, Wibo and Ravoet (1983) J. Cell Biol. 97, 340-350] that increased membrane permeability constitutes the mechanism whereby GTP activates the reactions of the dolichol pathway.
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PMID:Effect of GTP on the dolichol pathway for protein glycosylation in rat liver microsomes. 828 61

Glycosylated phosphoinositides serve as membrane anchors for numerous eukaryotic cell surface glycoproteins. Recent biochemical and genetic studies indicate that the glycolipids are assembled by sequential addition of components (monosaccharides and phosphoethanolamine) to phosphatidylinositol. The biosynthetic steps are presumed to occur in the ER, but formal proof of this is lacking. We describe experiments designed to establish the subcellular location of the initial steps in glycosyl-phosphatidylinositol (GPI) anchor biosynthesis and to define the transmembrane distribution of early biosynthetic lipid intermediates. The experiments were performed with the thymoma cell line BW5147.3. A subcellular fractionation protocol was used to show that early biosynthetic steps in GPI assembly, i.e., synthesis and deacetylation of N-acetylglucosaminyl phosphatidylinositol, occur in the ER. GPI biosynthetic intermediates were synthesized by incubating the microsomes with UDP-[3H]GlcNAc, and the transmembrane distribution of the labeled lipids was probed with phosphatidylinositol-specific phospholipase C (PI-PLC). Treatment of the radiolabeled microsomes with PI-PLC showed that > 70% of the N-acetylglucosaminyl phosphatidylinositol and glucosaminyl phosphatidylinositol could be hydrolyzed, indicating that the two lipids were primarily distributed in the cytoplasmic (outer) leaflet of the microsomes. Similar cleavage results were obtained using Streptolysin O-permeabilized thymoma cells. When permeabilized cells were incubated with UDP-[3H]GlcNAc and treated with PI-PLC, approximately 85% of the radiolabeled N-acetylglucosaminyl phosphatidylinositol and glucosaminyl phosphatidylinositol could be cleaved, indicating that they were accessible to the enzyme. The cumulative data indicate that early GPI intermediates are primarily located in the cytoplasmic leaflet of the ER, and are probably synthesized from PI located in the cytoplasmic leaflet and UDP-GlcNAc synthesized in the cytosol.
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PMID:Early lipid intermediates in glycosyl-phosphatidylinositol anchor assembly are synthesized in the ER and located in the cytoplasmic leaflet of the ER membrane bilayer. 850 Nov 24

Previous studies have identified beta-N-acetylglucosaminidase (GlcNAc'ase) and (alpha-mannosidase activities on the Drosophila melanogaster sperm surface which may have a role in fertilization. The aim of this study was to investigate their linkage to the sperm plasma membrane. We verified that glycosidases are not peripherally adsorbed to the cell surface by evaluating their resistance to release by KI, by buffered salt solutions of high ionic strength or alkaline buffers. Glycosidases were released from the sperm surface by detergents and, only to a minor extent, by mild proteolysis. Differential detergent solubilization pointed out that Triton X-114 was the most effective releasing agent for GlcNAc'ase and CHAPS for mannosidase. No activity was released from the membrane by a phosphatidylinositol-specific phospholipase C (PI-PLC). The released forms were quite hydrophilic in phase separation experiments with Triton X-114. This finding indicates the presence of a hydrophobic domain limited to a single transmembrane helix or/and the presence of an extensive glycosylation. The use of a Con-A binding assay demonstrated that both the enzymes are glycosylated. The molecular weight of the released glycosidases estimated by gel filtration was 158 kDa for GlcNAc'ase and 317 kDa for mannosidase. These results suggest that Drosophila melanogaster GlcNAc'ase and mannosidase are mannosylated integral membrane proteins that would function as exoenzymes with their active sites accessible in the extracellular space.
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PMID:Plasma membrane association and preliminary characterization of Drosophila sperm surface glycosidases. 989 Jul 47


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