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)

Ecto-5'-nucleotidase (eNT) from mouse muscle has been purified after extraction with detergent followed by chromatography on concanavalin A- and AMP-Sepharose. Three fractions were recovered: UF was NT non-retained in immobilised AMP; F-I was bound enzyme eluted with beta-glycerophosphate, and F-II was bound NT released with AMP. eNT was 80000-fold purified in F-II, this fraction showing proteins of 74, 68 and 51 kDa after immunoblotting. NT in UF migrated at 6.7S after centrifugation in sucrose gradients with Triton X-100, the peak being split into two of 6.7S and 4.4S in gradients with Brij 96. Ecto-NT in F-I or F-II migrated at 5.8S in Triton X-100-, or 4.4S in Brij 96-containing gradients. The hydrodynamic behaviour, concentration in Triton X-114, binding to phenyl-agarose, and sensitivity to phosphatidylinositol-specific phospholipase C revealed that enzyme forms in F-I or F-II were amphiphilic dimers with linked phosphatidylinositol residues, whilst most of NT forms in UF were hydrophilic dimers. A zinc/protein molar ratio of 2.2 was determined for eNT in F-II. NT activity was decreased in assays made in imidazole buffer, and was partly restored with 10 microM Zn2+ or 100 microM Mn2+. In assays with Tris buffer, NT showed a Km for AMP of 12 microM, and was competitively inhibited by ATP or ADP.
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PMID:Biochemical properties of 5'-nucleotidase from mouse skeletal muscle. 967 34

Clostridium perfringens alpha-toxin is the key virulence determinant in gas gangrene and has also been implicated in the pathogenesis of sudden death syndrome in young animals. The toxin is a 370-residue, zinc metalloenzyme that has phospholipase C activity, and can bind to membranes in the presence of calcium. The crystal structure of the enzyme reveals a two-domain protein. The N-terminal domain shows an anticipated structural similarity to Bacillus cereus phosphatidylcholine-specific phospholipase C (PC-PLC). The C-terminal domain shows a strong structural analogy to eukaryotic calcium-binding C2 domains. We believe this is the first example of such a domain in prokaryotes. This type of domain has been found to act as a phospholipid and/or calcium-binding domain in intracellular second messenger proteins and, interestingly, these pathways are perturbed in cells treated with alpha-toxin. Finally, a possible mechanism for alpha-toxin attack on membrane-packed phospholipid is described, which rationalizes its toxicity when compared to other, non-haemolytic, but homologous phospholipases C.
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PMID:Structure of the key toxin in gas gangrene. 969 20

An aminopeptidase N (APN) with a molecular weight of 110kDa was released from the midgut membrane of Bombyx mori by phosphatidylinositol-specific phospholipase C (PI-PLC), and purified to a homogeneous state. This 110-kDa APN was different from the 100-kDa APN that we previously reported, in chromatographic behaviors, substrate specificity, and N-terminal and internal amino acid sequences. However, the N-terminal sequence of 110-kDa APN, DPAFRLPTTTRPRHYQVTLT, was highly homologous with those of Manduca sexta and Heliothis virescens APNs, which were identified as a receptor for an insecticidal toxin of Bacillus thuringiensis. From a B. mori midgut cDNA library, we cloned the 110-kDa APN cDNA that possessed a 2958-bp open reading frame encoding a 111573-Da polypeptide of 986 residues. The sequence of the eicosa-peptide Asp42Thr61 deduced from the cDNA was completely matched with the N-terminal sequence of the mature 110-kDa APN. One potential N-glycosylation site, HEXXHXW zinc-binding motif and characteristic proline-rich repeats were observed in the ORF. Moreover, the primary sequence contained two hydrophobic peptides on N- and C-termini. The N-terminal peptide sequence showed characteristics of leader peptide for secretion and the C-terminal peptide contained a possible glycosylphosphatidylinositol (GPI) anchoring site. Taken together, the deduced amino acid sequence suggests that the 110-kDa APN is a GPI-anchored protein and a specific receptor protein for B. thuringiensis CryIA delta-endotoxin.
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PMID:Molecular cloning of a GPI-anchored aminopeptidase N from Bombyx mori midgut: a putative receptor for Bacillus thuringiensis CryIA toxin. 972 21

The secreted broad-range phosphatidylcholine (PC)-preferring phospholipase C (PC-PLC) of Listeria monocytogenes plays a role in the bacterium's ability to escape from phagosomes and spread from cell to cell. Based on comparisons with two orthologs, Clostridium perfringens alpha-toxin and Bacillus cereus PLC (PLCBc), we generated PC-PLC mutants with altered enzymatic activities and substrate specificities and analyzed them for biological function in tissue culture and mouse models of infection. Two of the conserved active-site zinc-coordinating histidines were confirmed by single amino acid substitutions H69G and H118G, which resulted in proteins inactive in broth culture and unstable intracellularly. Substitutions D4E and H56Y remodeled the PC-PLC active site to more closely resemble the PLCBc active site, while a gene replacement resulted in L. monocytogenes secreting PLCBc. All of these mutants yielded similar amounts of active enzyme as wild-type PC-PLC both in broth culture and intracellularly. D4E increased activity on and specificity for PC, while H56Y and D4E H56Y showed higher activity on both PC and sphingomyelin, with reduced specificity for PC. As expected, PLCBc expressed by L. monocytogenes was highly specific for PC. During early intracellular growth in human epithelial cells, the D4E mutant and the PLCBc-expressing strain performed significantly better than the wild type, while the H56Y and D4E H56Y mutants showed a significant defect. In assays for cell-to-cell spread, the H56Y and D4E mutants had close to wild-type characteristics, while the spreading efficiency of PLCBc was significantly lower. These studies emphasize the species-specific features of PC-PLC important for growth in mammalian cells.
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PMID:Modulation of enzymatic activity and biological function of Listeria monocytogenes broad-range phospholipase C by amino acid substitutions and by replacement with the Bacillus cereus ortholog. 974 85

Light triggers the phototransduction cascade by activating the visual pigment rhodopsin (Rho --> Rho*). Phosphorylation of Rho* by rhodopsin kinase (RK) is necessary for the fast recovery of sensitivity after intense illumination. Ca2+ ions, acting through Ca2+-binding proteins, have been implicated in the desensitization of phototransduction. One such protein, recoverin, has been proposed to regulate RK activity contributing to adaptation to background illumination in retinal photoreceptor cells. In this report, we describe an in vitro assay system using isolated retinas that is well suited for a variety of biochemical assays, including assessing Ca2+ effects on Rho* phosphorylation. Pieces of bovine retina with intact rod outer segments were treated with pore-forming staphylococcal alpha-toxin, including an alpha-toxin mutant that forms pores whose permeability is modulated by Zn2+. The pores formed through the plasma membranes of rod cells permit the diffusion of small molecules <2 kDa but prevent the loss of proteins, including recoverin (25 kDa). The selective permeability of these pores was confirmed by using the small intracellular tracer N-(2-aminoethyl) biotinamide hydrochloride. Application of [gamma-32P]ATP to alpha-toxin-treated, isolated retina allowed us to monitor and quantify phosphorylation of Rho*. Under various experimental conditions, including low and high [Ca2+]free, the same level of Rho* phosphorylation was measured. No differences were observed between low and high [Ca2+]free conditions, even when rods were loaded with ATP and the pores were closed by Zn2+. These results suggest that under physiological conditions, Rho* phosphorylation is insensitive to regulation by Ca2+ and Ca2+-binding proteins, including recoverin.
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PMID:Phosphorylation of photolyzed rhodopsin is calcium-insensitive in retina permeabilized by alpha-toxin. 984 7

An aminopeptidase N (APN) isozyme having the molecular weight of 90 kDa, was released by phosphatidylinositol-specific phospholipase C (PI-PLC) and purified homogeneously, from the brush border membrane of Bombyx mori. From the result of cDNA cloning, the primary structure of 90 kDa APN proved to consist of 948 amino acid residues, containing a typical metalloprotease-specific zinc-binding motif in the deduced sequence. Moreover, the primary sequence contained two hydrophobic segments on N- and C-termini. The N-terminal one showed characteristics of leader peptide for secretion and the C-terminal one contained a possible glycosylphosphatidylinositol (GPI) anchoring site, suggesting that the APN encoded by the cDNA is not only a zinc-binding enzyme, but also a GPI-anchored protein. The primary sequence is significantly homologous with those of insect and mammalian APNs, and contains four conserved segments around the zinc-binding motif, two potential N-glycosylation sites and four conserved Cys residues. The deduced primary sequence had 30.7% identity with that of B. mori 110 kDa APN, and did not contain the N-terminal and internal amino acid sequences of B. mori 100 kDa APN, revealing B. mori 90 kDa APN to be the third isozyme on the midgut brush border membrane. On the other hand, the primary sequence of 90 kDa APN showed high homology with Manduca sexta APN2 (65.1% identity) and Plutella xylostella APN2 (63.8% identity). It appears that the B. mori 90 kDa APN should be classified in the insect apn2 cluster and differentiated from insect apn1 and mammalian apn clusters by phylogenetic analysis. These results suggest that 90 kDa APN isozyme encoded by the cDNA is a product of B. mori apn2 gene.
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PMID:Cloning and sequence analysis of the aminopeptidase N isozyme (APN2) from Bombyx mori midgut. 997 96

The metabolism and synthesis of an important mycobacterial lipid component, phosphatidylinositol (PI), and its metabolites, was studied in Mycobacterium smegmatis and M. smegmatis subcellular fractions. Little is known about the synthesis of PI in prokaryotic cells. Only a cell wall fraction (P60) in M. smegmatis was shown to possess PI synthase activity. Product was identified as PI by migration on TLC, treatment with phospholipase C and ion exchange chromatography. PI was the only major product (92.3%) when both cells and P60 fraction were labeled with [3H]inositol. Also, a neutral lipid inositol-containing product (4.1% of the total label) was identified in the P60 preparations. Strangely, PI synthase substrates, CDP-dipalmitoyl-DAG and CDP-NBD-DAG, added to the assay did not stimulate [3H]PI and NBD-PI yield by M. smegmatis. At the same time, addition of both substrates to rat liver and Saccharomyces cerevisiae PI synthase assays resulted in an increase in the product yield. Upon addition of CHAPS to the mycobacterial PI synthase assay, both substrates were utilized in a dose-dependent manner for the synthesis of NBD-PI and [3H]PI. These results demonstrate a strict substrate specificity of mycobacterial PI synthase toward endogenous substrates. K(m) of the enzyme toward inositol was shown to be 25 microM; Mg2+ stimulated the enzyme to a greater degree than Mn2+. Structural analogs of myo-inositol, epi-inositol and scyllo-inositol and Zn2+ were shown to be more potent inhibitors of mycobacterial PI synthase than of mammalian analogs. Lack of sequence homology with mammalian PI synthases, different kinetic characteristics, existence of selective inhibitors and an important physiological role in mycobacteria, suggest that PI synthase may be a good potential target for antituberculosis therapy.
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PMID:Phosphatidylinositol synthesis in mycobacteria. 998 74

The phosphatidylcholine-preferring phospholipase C from Bacillus cereus (PLCBc) is a 28.5 kDa enzyme with three zinc ions in its active site. Although much is known about the roles that various PLCBc active site amino acids play in binding and catalysis, there is little information about the rate-determining step of the PLCBc-catalyzed hydrolysis of phospholipids and the catalytic cycle of the enzyme. To gain insight into these aspects of the hydrolysis, solvent viscosity variation experiments were conducted to determine whether an external step (substrate binding or product release) or an internal step (hydrolysis) is rate-limiting. The data indicate that the PLCBc-catalyzed reaction is unaffected by changes in solvent viscosity. This observation is inconsistent with the notion of substrate binding or product release being rate-determining and supports the hypothesis that a chemical step is rate-limiting. Furthermore, a deuterium isotope effect of 1.9 and a linear proton inventory plot indicate one proton is transferred in the rate-determining step. These data may be used to formulate a comprehensive catalytic cycle that is for the first time based on experimental evidence. In this mechanism, Asp55 of PLCBc activates an active site water molecule for attack on the phosphodiester bond, the hydrolysis of which is rate-limiting. The phosphorylcholine product is the first to leave the active site, followed by diacylglycerol.
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PMID:Catalytic cycle of the phosphatidylcholine-preferring phospholipase C from Bacillus cereus. Solvent viscosity, deuterium isotope effects, and proton inventory studies. 1019 60

The phosphatidylcholine-preferring phospholipase C from Bacillus cereus (PLC(Bc)) is a 28.5 kDa enzyme with three zinc ions in its active site. The roles that a number of amino acid residues play as zinc ligands and in binding and catalysis have been elucidated. Recent mechanistic studies indicate that the rate of the reaction is limited by a proton-transfer step during chemical hydrolysis and not substrate binding or product release. An X-ray structure of PLC(Bc) complexed with a phosphonate inhibitor related to phosphatidylcholine revealed that the three amino acid residues Glu4, Tyr56, and Phe66 comprise the choline binding pocket. However, because the contributions that these three residues make to substrate recognition and specificity were unknown, a series of site-specific mutants for Glu4, Tyr56, and Phe66 were constructed by PCR mutagenesis. On the basis of a comparison of their respective CD spectra and melting temperatures, it appears that the mutants adopt folded structures in solution that are virtually identical to that of wild-type PLC(Bc). The kinetic parameters k(cat) and K(m) for the hydrolysis of the three soluble substrates 1, 2-dihexanoyl-sn-glycero-3-phosphocholine (C6PC), 1, 2-dihexanoyl-sn-glycero-3-phosphoethanolamine (C6PE), and 1, 2-dihexanoyl-sn-glycero-3-phospho-L-serine (C6PS) at concentrations below their corresponding critical micelle concentration (cmc) values were determined for each mutant. Replacement of Phe66 with a nonaromatic residue dramatically decreased k(cat) (approximately 200-fold) and reduced PLC(Bc) activity toward C6PC, C6PE, and C6PS, whereas changes to Glu4 and Tyr56 typically led to much more modest losses in catalytic efficiencies. Mutations of Glu4 had relatively little effect upon k(cat) and K(m) for C6PS, but they significantly influenced K(m) for C6PC and C6PE. Replacing Tyr56 with nonaromatic residues also affects catalytic efficiency, albeit to a much lesser degree than the corresponding changes at position 66. However, the presence of an aromatic residue at position 56 seems to confer some substrate selectivity for C6PC and C6PE, which bear a positive charge on the headgroup, relative to C6PS, which has no net charge on the headgroup; this increase in specificity arises largely from a reduced k(cat) for C6PS.
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PMID:The choline binding site of phospholipase C (Bacillus cereus): insights into substrate specificity. 1072 35

Listeria monocytogenes, a facultative intracellular pathogen, synthesizes an extracellular protease which is responsible for the maturation of phosphatidylcholine phospholipase C (lecithinase), a virulence factor involved in cell-to-cell spread. This work describes the environmental parameters necessary for increased production of mature, 35-kDa active protease in strains of L. monocytogenes, and its detection using polyclonal antibodies raised against Bacillus subtilis neutral protease. High performance liquid affinity chromatography was exploited to isolate the biologically active form of the mature protease, which was then subjected to biochemical characterization using casein as a substrate. The protease is a zinc-dependent metalloprotease which degrades casein over a wide range of temperatures and pH values. It can also degrade actin, the most abundant protein in many eukaryotic cells. The Listeria protease was shown to exhibit a high thermal stability and a relatively narrow substrate specificity. A three-dimensional model built on the basis of the homology with thermolysin was used to understand the structural basis of these characteristics.
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PMID:Characteristics of the biologically active 35-kDa metalloprotease virulence factor from Listeria monocytogenes. 1073 52


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