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 phosphono analog of choline phosphoglyceride was used to examine the relative contributions of phospholipase C and D in the generation of diglycerides in fMLP- and A23187-stimulated human neutrophils. The phosphono analog, 1-O-[3H]alkyl-2-lyso-sn-glycero-3-phosphonocholine, contains a carbon-phosphorus bond adjacent to the base moiety and is resistant to phospholipase D hydrolysis, while remaining susceptible to phospholipase C hydrolysis. fMLP stimulated the production of [3H]phosphatidic acid and subsequently [3H]diglyceride from cells containing 1-O-[3H]alkyl-2-acyl-sn-glycero-3-phosphocholine, but not from cells prelabeled with the phosphono analog. Treatment with A23187 also resulted in the formation of these products from cells containing 1-O-[3H]alkyl-2-acyl-sn-glycero-3-phosphocholine. Additionally, A23187 stimulated the conversion of the phosphono analog to phosphodiester-containing choline phosphoglyceride which then resulted in the generation of [3H]phosphatidic acid and subsequently [3H]diglyceride. This study demonstrates the use of a phosphono analog in assessing phospholipase C and D activity in cells and provides evidence that in fMLP- and A23187-stimulated human neutrophils, diglyceride is generated indirectly from choline phosphoglycerides by the combined activities of phospholipase D and phosphatidate phosphohydrolase.
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PMID:Evaluation of phospholipase C and D activity in stimulated human neutrophils using a phosphono analog of choline phosphoglyceride. 833 46

It is well established that production of prostaglandins by ocular tissues is dependent upon the species. The rabbit iris-ciliary body produces greater amounts of prostaglandins than that of the bovine. To throw more light on the biochemical basis underlying these differences we have compared the fatty acid composition and phospholipases A2 and C activities in rabbit and bovine irides. When the concentration of arachidonic acid is expressed as % of total fatty acids, phosphatidylcholine, phosphatidylethanolamine and phosphatidylinositol, separated from rabbit iris phospholipids, contained 56, 38 and 18% more arachidonic acid, respectively, than those of the bovine. The total lipid phosphorus in rabbit and bovine iris-ciliary body were found to be 13.74 and 9.34 mumol g-1 wet tissue, respectively. Subcellular fractions, prepared from rabbit iris, contained 30-230% more phospholipase C activity than those of the bovine, and about 5-41 times higher phospholipase A2 activity than those of the bovine. In the rabbit iris microsomal fraction, phospholipase C activity is 33 times higher than that of phospholipase A2. However, the data presented suggest that phospholipase A2, rather than phospholipase C, is the enzyme which is more involved in arachidonic acid release for eicosanoid biosynthesis. These findings suggest that the high contents of arachidonic acid and phospholipases A2 and C in the rabbit iris could contribute to its unique capacity to synthesize and release prostaglandins in the anterior segment as compared to that of other mammalian species.
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PMID:Comparative studies on fatty acid composition and phospholipases A2 and C activities in rabbit and bovine iris-ciliary body. 846 48

For the first time a consistent catalytic mechanism of phospholipase C from Bacillus cereus is reported based on molecular mechanics calculations. We have identified the position of the nucleophilic water molecule, which is directly involved in the hydrolysis of the natural substrate phosphatidylcholine, in phospholipase C. This catalytically essential water molecule, after being activated by an acidic residue (Asp55), performs the nucleophilic attack on the phosphorus atom in the substrate, leading to a trigonal bipyramidal pentacoordinated intermediate (and structurally similar transition state). The subsequent collapse of the intermediate, regeneration of the enzyme, and release of the products has to involve a not yet identified second water molecule. The catalytic mechanism reported here is based on a series of molecular mechanics calculations. First, the x-ray structure of phospholipase C from B cereus including a docked substrate molecule was subjected to a stepwise molecular mechanics energy minimization. Second, the location of the nucleophilic water molecule in the active site of the fully relaxed enzyme-substrate complex was determined by evaluation of nonbonded interaction energies between the complex and a water molecule. The nucleophilic water molecule is positioned at a distance (3.8 A) from the phosphorus atom in the substrate, which is in good agreement with experimentally observed distances. Finally, the stability of the complex between phospholipase C, the substrate, and the nucleophilic water molecule was verified during a 100 ps molecular dynamics simulation. During the simulation the substrate undergoes a conformational change, but retains its localization in the active site. The contacts between the enzyme, the substrate, and the nucleophilic water molecule display some fluctuations, but remain within reasonable limits, thereby confirming the stability of the enzyme-substrate-water complex. The protocol developed for energy minimization of phospholipase C containing three zinc ions located closely together at the bottom of the active site cleft is reported in detail. In order to handle the strong electrostatic interactions in the active site realistically during energy minimization, delocalization of the charges from the three zinc ions was considered. Therefore, quantum mechanics calculations on the zinc ions and the zinc-coordinating residues were carried out prior to the molecular mechanics calculations, and two different sets of partial atomic charges (MNDO-Mulliken and AMI-ESP) were applied. After careful assignment of partial atomic charges, a complete energy minimization of the protein was carried out by a stepwise procedure without explicit solvent molecules. Energy minimization with either set of charges yielded structures, which were very similar both to the x-ray structure and to each other, although using AMI-ESP partial atomic charges and a dielectric constant of 4, yielded the best protein structure.
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PMID:Substrate binding and catalytic mechanism in phospholipase C from Bacillus cereus: a molecular mechanics and molecular dynamics study. 927 25

The mechanism of phosphatidylinositol-specific phospholipase C (PI-PLC) has been suggested to resemble that of ribonuclease A. The goal of this work is to rigorously evaluate the mechanism of PI-PLC from Bacillus thuringiensis by examining the functional and structural roles of His-32 and His-82, along with the two nearby residues Asp-274 and Asp-33 (which form a hydrogen bond with His-32 and His-82, respectively), using site-directed mutagenesis. In all, twelve mutants were constructed, which, except D274E, showed little structural perturbation on the basis of 1D NMR and 2D NOESY analyses. The H32A, H32N, H32Q, H82A, H82N, H82Q, H82D, and D274A mutants showed a 10(4)-10(5)-fold decrease in specific activity toward phosphatidylinositol; the D274N, D33A, and D33N mutants retained 0. 1-1% activity, whereas the D274E mutant retained 13% activity. Steady-state kinetic analysis of mutants using (2R)-1, 2-dipalmitoyloxypropane-3-(thiophospho-1d-myo-inositol) (DPsPI) as a substrate generally agreed well with the specific activity toward phosphatidylinositol. The results suggest a mechanism in which His-32 functions as a general base to abstract the proton from 2-OH and facilitates the attack of the deprotonated 2-oxygen on the phosphorus atom. This general base function is augmented by the carboxylate group of Asp-274 which forms a diad with His-32. The H82A and D33A mutants showed an unusually high activity with substrates featuring low pKa leaving groups, such as DPsPI and p-nitrophenyl inositol phosphate (NPIPs). These results suggest that His-82 functions as the general acid with assistance from Asp-33, facilitating the departure of the leaving group by protonation of the glycerol O3 oxygen. The Bronsted coefficients obtained for the WT and the D33N mutant indicate a high degree of proton transfer to the leaving group and further underscore the "helper" function of Asp-33. The complete mechanism also includes activation of the phosphate group toward nucleophilic attack by a hydrogen bond between Arg-69 and a nonbridging oxygen atom. The overall mechanism can be described as "complex" general acid-general base since three elements are required for efficient catalysis.
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PMID:Mechanism of phosphatidylinositol-specific phospholipase C: a unified view of the mechanism of catalysis. 952 77

Hexadecylphosphocholine (HePC) is known as antitumor agent but the mechanism has not yet been understood. In rat liver mitochondria its effect on phospholipid transformation has been studied by quantitative HPTLC and phosphorus determination. From the results it can be concluded that HePC influences the activities of phospholipase A2, phospholipase C, phospholipase D, and lysophospholipase A. The phospholipid transformation as well as the influence of HePC are affected by exogenous calcium ions. In the presence of calcium HePC has been found to inhibit enzyme activities, whereas in the absence of exogenous calcium ions enzymatic phospholipid transformations are activated or inhibited depending on HePC concentrations.
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PMID:The effect of hexadecylphosphocholine on the degradation of mitochondrial phospholipids. 965 93

Patients with cystic fibrosis are severely affected by an infection with Pseudomonas aeruginosa, a microbe known to synthesize phospholipase C. This study was designed to determine whether that enzyme would affect the function of pulmonary surfactant phospholipids. Mucoid and nonmucoid strains of P. aeruginosa, freshly obtained from patients with cystic fibrosis, were cultured for 12 h on agar plates. The bacteria were suspended in saline solution and then pelleted by centrifugation. The supernatant was used to dilute the surfactant preparation, calf lung surfactant extract, from 35 to 2 mg/mL. Surfactant function, before and after incubation, was examined with a capillary surfactometer, an instrument specifically developed for an evaluation of the ability of surfactant to maintain patency of a narrow glass tube, simulating a terminal conducting airway. Phospholipid hydrolysis was also evaluated biochemically by determining the total content of phospholipids in surfactant before and after incubation. In five experiments, the lipids were separated with thin-layer chromatography, and the phosphorus content was determined in the diacylphosphatidylcholine band before and after incubation for 6, 24, and 48 h. Capillary openness and phospholipid concentration decreased as enzyme concentration and time of incubation increased (p<0.0001). Linear regression showed a significant correlation between time of capillary openness and phospholipid concentration (r = 0.957; p<0.0001). Calf lung surfactant extract hydrolysis was catalyzed by extracts of the bacteria, particularly the nonmucoid, analogous to the catalysis observed with phospholipase C. Surfactant hydrolysis catalyzed by enzymes from P. aeruginosa might severely affect surfactant function provided enzyme concentration is high and time of incubation is long.
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PMID:Pseudomonas aeruginosa from patients with cystic fibrosis affects function of pulmonary surfactant. 1062 92

Phosphatidylinositol-specific phospholipase C (PI-PLC) catalyzes the cleavage of the P-O bond in phosphatidylinositol via intramolecular nucleophilic attack of the 2-hydroxyl group of inositol on the phosphorus atom. Our earlier stereochemical and site-directed mutagenesis studies indicated that this reaction proceeds by a mechanism similar to that of RNase A, and that the catalytic site of PI-PLC consists of three major components analogous to those observed in RNase A, the His32 general base, the His82 general acid, and Arg69 acting as a phosphate-activating residue. In addition, His32 is associated with Asp274 in forming a catalytic triad with inositol 2-hydroxyl, and His82 is associated with Asp33 in forming a catalytic diad. The focus of this work is to provide a global view of the mechanism, assess cooperation between various catalytic residues, and determine the origin of enzyme activation by the hydrophobic leaving group. To this end, we have investigated kinetic properties of Arg69, Asp33, and His82 mutants with phosphorothioate substrate analogues which feature leaving groups of varying hydrophobicity and pK(a). Our results indicate that interaction of the nonbridging pro-S oxygen atom of the phosphate group with Arg69 is strongly affected by Asp33, and to a smaller extent by His82. This result in conjunction with those obtained earlier can be rationalized in terms of a novel, dual-function triad comprised of Arg69, Asp33, and His82 residues. The function of this triad is to both activate the phosphate group toward the nucleophilic attack and to protonate the leaving group. In addition, Asp33 and His82 mutants displayed much smaller degrees of activation by the fatty acid-containing leaving group as compared to the wild-type (WT) enzyme, and the level of activation was significantly reduced for substrates featuring the leaving group with low pK(a) values. These results strongly suggest that the assembly of the above three residues into the fully catalytically competent triad is controlled by the hydrophobic interactions of the enzyme with the substrate leaving group.
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PMID:Involvement of the Arg-Asp-His catalytic triad in enzymatic cleavage of the phosphodiester bond. 1133 Oct 6

Metallothionein (MT) is a low-molecular-weight, cysteine-rich protein that contains heavy metals such as cadmium and zinc. The biological function of MT in platelets is not yet understood. Therefore, the aim of this study was to systematically examine the inhibitory mechanisms of metallothionein in platelet aggregation. In this study, metallothionein concentration-dependently (1-8 microM) inhibited platelet aggregation in human platelets stimulated by agonists. Metallothionein (4 and 8 microM) inhibited phosphoinositide breakdown in [3H]-inositol-labeled platelets, intracellular Ca+2 mobilization in Fura-2 AM-loaded platelets, and thromboxane A2 formation stimulated by collagen. In addition, metallothionein (4 and 8 microM) significantly increased the formation of cyclic GMP but not cyclic AMP in human platelets. Rapid phosphorylation of a protein of Mr 47,000 (P47), a marker of protein kinase C activation, was triggered by PDBu (100 nM). This phosphorylation was markedly inhibited by metallothionein (4 and 8 microM) in phosphorus-32-labeled platelets. In an in vivo thrombotic study, platelet thrombus formation was induced by irradiation of mesenteric venules in mice pretreated with fluorescein sodium. Metallothionein (6 microg/g) significantly prolonged the latency period for inducing platelet plug formation in mesenteric venules. These results indicate that the antiplatelet activity of metallothionein may involve the following pathways: (1) metallothionein may inhibit the activation of phospholipase C, followed by inhibition of phosphoinositide breakdown and thromboxane A2 formation, thereby leading to inhibition of intracellular Ca+2 mobilization; (ii) Metallothionein also activated the formation of cyclic GMP in human platelets, resulting in inhibition of platelet aggregation. The results strongly indicate that metallothionein provides protection against thromboembolism.
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PMID:Inhibitory mechanisms of metallothionein on platelet aggregation in in vitro and platelet plug formation in in vivo experiments. 1468 47

Phosphatidylinositol-specific phospholipase C from Bacillus thuringiensis catalyzes the cleavage of the phosphorus-oxygen bond in phosphatidylinositol. The focus of this work is to dissect the roles of the carboxylate side chain of Asp(274) in the Asp(274)-His(32) dyad, where a short strong hydrogen bond (SSHB) was shown to exist based on NMR criteria. A regular hydrogen bond (HB) was observed in D274N, and no low field proton resonance was detected for D274E and D274A. Comparison of the activity of wild type, D274N, and D274A suggested that the regular HB contributes significantly (approximately 4 kcal/mol) to catalysis, whereas the SSHB contributes only an additional 2 kcal/mol. The mutant D274E displays high activity similar to wild type, suggesting that the negative charge is sufficient for the catalytic role of Asp(274). To further support this interpretation and rule out possible contribution of regular HB or SSHB in D274E, we showed that the activity of D274G can be rescued by exogenous chloride ions to a level comparable with that of D274E. Comparison between different anions suggested that the ability of an anion to rescue the activity is due to the size and the charge of the anion not the property as a HB acceptor. In conclusion, a major fraction of the functional role of Asp(274) in the Asp(274)-His(32) dyad can be attributed to a negative charge (as in D274E and D274G-Cl(-)), and the SSHB in the wild type enzyme provides minimal contribution to catalysis. These results represent novel insight for an Asp-His catalytic dyad and for the mechanism of phosphatidylinositol-specific phospholipase C.
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PMID:The catalytic role of aspartate in a short strong hydrogen bond of the Asp274-His32 catalytic dyad in phosphatidylinositol-specific phospholipase C can be substituted by a chloride ion. 1515 21

Lycopene is a natural carotenoid antioxidant that is present in tomatoes and tomato products. The pharmacologic function of lycopene in platelets is not yet understood. Therefore, in this study we sought to systematically examine the effects of lycopene in the prevention of platelet aggregation and thrombus formation. We found that lycopene concentration-dependently (2-12 micromol/L) inhibited platelet aggregation in human platelets stimulated by agonists. Lycopene (6 and 12 micromol/L) inhibited phosphoinositide breakdown in platelets labeled with tritiated inositol, intracellular Ca+2 mobilization in Fura-2 AM-loaded platelets, and thromboxane B2 formation stimulated by collagen. In addition, lycopene (6 and 12 micromol/L) significantly increased the formations of cyclic GMP and nitrate but not cyclic AMP in human platelets. Rapid phosphorylation of a protein of 47,000 Da (P47), a marker of protein kinase C activation, was triggered by PDBu (60 nmol/L). This phosphorylation was markedly inhibited by lycopene (12 micromol/L) in phosphorus-32-labeled platelets. In an in vivo study, thrombus formation was induced by irradiation of mesenteric venules in mice pretreated with fluorescein sodium. Lycopene (5, 10, and 20 mg/kg) significantly prolonged the latency period for the induction of platelet-plug formation in mesenteric venules. These results indicate that the antiplatelet activity of lycopene may involve the following pathways: (1) Lycopene may inhibit the activation of phospholipase C, followed by inhibition of phosphoinositide breakdown and thromboxane B2 formation, thereby leading to inhibition of intracellular Ca+2 mobilization. (2) Lycopene also activated the formations of cyclic GMP/nitrate in human platelets, resulting in the inhibition of platelet aggregation. The results may imply that tomato-based foods are especially beneficial in the prevention of platelet aggregation and thrombosis.
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PMID:Inhibitory effects of lycopene on in vitro platelet activation and in vivo prevention of thrombus formation. 1619 83


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