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1.
Phospholipase A 2 and acyltransferase were assayed and characterized in pure axoplasm and neural tissues of squid. Intracellular phospholipase A 2 activity was highest in giant fiber lobe and axoplasm, followed by homogenates from retinal fibers, optic lobe and fin nerve. In most preparations, exogenous calcium (5 mM) caused a slight stimulation of activity. EGTA (2 mM) was somewhat inhibitory, indicating that low levels of endogenous calcium may be required for optimum activity. Phospholipase A 2 was inhibited by 0.1 mM p-bromophenacylbromide, and was completely inactivated following heating. The level of acylCoA: lysophosphatidylcholine acyltransferase activity was higher in axoplasm and giant fiber lobe than in other neural tissues of the squid. Km (apparent) and Vmax (apparent) for oleoyl-CoA and lysophosphatidylcholine were quite similar for axoplasm and giant fiber lobe enzyme preparations. Acyltransferase activity was inactivated by heat treatment, and greatly inhibited by 0.2 mM p-chloromercuribenzoate, and to a lesser extent by 20 mM N-ethylmaleimide. Phospholipase A2 activity was present in fractions enriched in axolemmal membranes (separated from squid retinal fibers and garfish olfactory nerve) from both tissues, and it was also highly concentrated in vesicles derived from squid axoplasm. In all three preparations, phospholipase A2 activity was stimulated by Ca++ (5 mM) and inhibited by EGTA (2 mM). In addition, axoplasmic cytosol (114,000 g supernatant) retained a substantial portion of a Ca++-independent phospholipase A2, active in the presence of 2 mM EGTA. Acyltransferase activity was present at high content in both axolemma membrane rich fractions, and among subaxoplasmic fractions and axoplasmic vesicles. 相似文献
2.
Peroxiredoxins (Prx) are enzymes that catalyze the reduction of hydrogen peroxide and alkyl hydroperoxides. Prxs are ubiquitous enzymes with representatives found in Bacteria, Archaea and Eukarya. Many 1-cysteine peroxiredoxins (1-CysPrx) are dual-function enzyme with both peroxidase and acidic Ca 2+-independent phospholipase A 2 (aiPLA 2) activities. The functions proposed for 1-CysPrx/aiPLA 2 include the protection of cell membrane phospholipids against oxidative damage (peroxidation) and the metabolism (hydrolysis) of phospholipids, such as those of lung surfactant. The peroxidase active site motif PVCTTE of 1-CysPrx contains the conserved catalytic cysteine residue, and the esterase (lipase) motif GXSXG of the enzyme contains the conserved catalytic serine residue. In addition to the classic lipase motif GXSXG, various 1-CysPrx/aiPLA 2s have closely related variant putative lipase motifs containing the catalytic serine residue. The PLA 2 moieties are prevalent and highly homologous in vertebrate and bacterial 1-CysPrx/aiPLA 2s that is consistent with a high degree evolutional conservation of the enzyme. 相似文献
3.
Phospholipases A 2 are a heterogeneous class of enzymes that hydrolyse fatty acids from the sn-2 position of membrane phospholipids. Prolonged stimulation of phospholipase A 2 may damage membrane integrity, not only because of the loss of essential phospholipid from the lipid bilayer but also as a result of an uncontrollable Ca 2+ influx. The increased levels of intracellular Ca 2+ may be responsible for enhanced lipolysis, proteolysis and DNA fragmentation. This process along with the accumulation of lipid peroxidation products may be associated with neurodegeneration in acute neural trauma (ischemia, head and spinal cord injuries) and neurodegenerative diseases (Alzheimer's disease). 相似文献
4.
The polyamines spermine, spermidine, and putrescine inhibit the activity of phospholipase A 2 ( Naja naja) and phospholipase C ( Clostridium welchii) on phospholipid vesicles and mitochondrial membranes as sources of substrate phospholipids. The inhibitory effect is highest for spermine and lowest for putrescine. With both enzymes, inhibition is stronger when phospholipid vesicles rather than mitochondrial membranes are used as the substrate. No clear competition of polyamines with Ca 2+, which is required for the activity of both enzymes, has been observed. The inhibition appears to be due to steric hindrance of enzyme-substrate interaction due to the binding of the organic polycations to the phospholipid bilayer. 相似文献
5.
Phospholipase A 2, an enzyme which may regulate the formation of polyunsaturated fatty acids utilized for prostaglandin synthesis, was found to have significant higher activity in decidual than in myometrial tissue. The major part of phospholipase A 2 in the decidua had an acid pH optimum, which indicates that most of the enzyme is stored in the lysosomes of this tissue. These findings, together with previous observations, lend further support to the view that lysosomal phospholipase A 2 released within decidual cells might be a trigger of abortion and parturition. 相似文献
6.
Binding of phospholipase A 2 from porcine pancreas and from Naja melanoleuca venom to vesicles of 1,2-di(tetradecyl)- rac-glycero-3-phosphocholine (diether-PC 14) is studied in the presence and absence of 1-tetradecanoyl- sn-glycero-3-phosphocholine and myristic acid. The bound enzyme coelutes with the vesicles during gel filtration through a nonequilibrated Sephadex G-100 column, modifies the phase transition behavior of bilayers, and exhibits an increase in fluorescence intensity accompanied by a blue shift. Using these criteria it is demonstrated that the snake-venom enzyme binds to bilayers of the diether-PC 14 alone. In contrast, the porcine enzyme binds only to ternary codispersions of dialkyl (or diacyl) phosphatidylcholine, lysophosphatidylcholine and fatty acid. Binding of the pig-pancreatic enzyme to vesicles of the diether-PC 14 could not be detected even after long incubation (up to 24 h) below, at, or above the phase-transition temperature, whereas the binding in the presence of products is almost instantaneous and observed over a wide temperature range. Thus incorporation of the products in substrate dispersions increases the binding affinity rather than increase the rate of binding. The results are consistent with the hypothesis that the pancreatic enzyme binds to defect sites at the phase boundaries in substrate bilayers induced by the products. The spectroscopically obtained hyperbolic binding curves can be adequately described by a single equilibrium by assuming that the enzyme interacts with discrete sites. The binding experiments are supported by kinetic studies. 相似文献
8.
A novel phospholipase A 2, designated as Oh-DE-2, was isolated from the venom of Ophiophagus hannah (king cobra) by successive chromatography on SP-Sephadex C-25, DE-52, and Q-Sepharose columns. Oh-DE-2 with pI 5.1 showed an apparent molecular weight of 14 kD as revealed by SDS-PAGE and gel filtration. The amino acid sequence was homologous with those of PLA 2s from Elapidae venoms. Oh-DE-2 was effectively inactivated by p-bromophenacyl bromide, indicating that the conserved His-48 is essential for its enzymatic activity. However, modification of the conserved Trp-19 did not cause a precipitous drop in the enzymatic activity of Oh-DE-2 as observed with PLA 2s from Naja naja atra and Bungarus multicinctus venoms. A quenching study showed that the microenvironment of Trp in Oh-DE-2 was inaccessible to acrylamide, iodide, or cesium, a finding which was different from those observed with PLA 2s from N. naja atra and B. multicinctus venoms. These results might suggest that, unlike other PLA 2 enzymes, Trp-19 in Oh-DE-2 is not directly involved in its enzymatic mechanisms. 相似文献
9.
A phospholipase A 2 activity was characterized in adult rabbit lung. This activity was calcium- and deoxycholate-dependent and displayed an alkaline pH optimum. K m and V max were 0.176mM and 256.8 pmoles/min./mg protein respectively. The microsomal fraction displayed the highest enzymatic specific activity; the lowest activity was present in the cytosol. Yet this latter fraction accounted for the majority of the total activity. Although the specific activity was high within the lamellar body fraction this compartment contained only 2% of the total activity. Phospholipase A 2 activity was inhibited by bromophenacyl bromide, chlorpromazine and mepacrine in decreasing order of effectiveness. Treatment of the microsomes with increasing concentrations of NaCl indicated that the lung phospholipase A 2 activity was related loosely bound to the microsomal membranes and was maximally removed with salt at a concentration only slightly higher than physiological. Addition of calmodulin to the enzyme assay did not significantly alter hydrolysis of labelled phosphatidylcholine. 相似文献
10.
We have monitored the composition of supported phospholipid bilayers during phospholipase A 2 hydrolysis using specular neutron reflection and ellipsometry. Porcine pancreatic PLA 2 shows a long lag phase of several hours during which the enzyme binds to the bilayer surface, but only 5 ± 3% of the lipids react before the onset of rapid hydrolysis. The amount of PLA 2, which resides in a 21 ± 1 Å thick layer at the water-bilayer interface, as well as its depth of penetration into the membrane, increase during the lag phase, the length of which is also proportional to the enzyme concentration. Hydrolysis of a single-chain deuterium labelled d 31-POPC reveals for the first time that there is a significant asymmetry in the distribution of the reaction products between the membrane and the aqueous environment. The lyso-lipid leaves the membrane while the number of PLA 2 molecules bound to the interface increases with increasing fatty acid content. These results constitute the first direct measurement of the membrane structure and composition, including the location and amount of the enzyme during hydrolysis. These are discussed in terms of a model of fatty-acid mediated activation of PLA 2. 相似文献
11.
Four ergosterol derivatives (1–4) have been isolated for the first time from the fruiting bodies of a basidiomycete fungus, Lactarius hatsudake, through activity-guided fractionation. Their structures were determined, using spectroscopic analysis, as: (22 E,24 R)-ergosta-5,7,22-dien-3 β-ol (ergosterol, 1); 5 ,8 -epidioxy-(22 E,24 R)-ergosta-6,22-dien-3 β-ol (ergosterol peroxide, 2); 5 ,8 -epidioxy-(24 S)-ergosta-6-en-3 β-ol (3); and (22 E,24 R)-ergosta-7,22-dien-3 β,5 ,6 β-triol (cerevisterol, 4). Compounds 2 and 3 showed selective inhibitory activity against Crotalus adamenteus venom phospholipase A 2 (PLA 2) enzyme, but not against Apis mellifcra bee venom PLA 2. The antiphospholipase A 2 activity of compounds 2 and 3 are reported here for the first time. 相似文献
12.
Phospholipase A 2 (PLA 2) enzymes (EC3.1.4.4) regulate the release of biologically active fatty acids and lysophospholipids from membrane phospholipid pools. These lipids are also substrates for intracellular biochemical pathways that generate potent autocrine and paracrine lipid mediators such as the eicosanoids and platelet activating factor. These factors, in turn, regulate cell proliferation, survival, differentiation, motility, tissue vascularisation, and immune surveillance in virtually all tissues, functions that are subverted by cancer cells for tumour growth and metastasis. Thus the relevance of PLA 2-dependent pathways to the genesis and progression of cancer has been of interest since their discovery and with recent technological advances, their role in tumourigenesis has become more tractable experimentally. Limited human genetic studies have not yet identified PLA 2 enzymes as classical mutated oncogenes or tumour suppressor genes. However, there is strong evidence that of the 22 identified human PLA 2 enzymes, ten of which have been studied in cancer to date, most are aberrantly expressed in a proportion of tumours derived from diverse organs. Correlative and functional studies implicate the expression of some secreted enzymes (sPLA 2s), particularly the best studied enzyme Group IIA sPLA 2 in either tumour promotion or inhibition, depending on the organ involved and the biochemical microenvironment of tumours. As in immune-mediated inflammatory pathologies, genetic deletion studies in mice, supported by limited studies with human cells and tissues, have identified an important role for Group IVA PLA 2 in regulating certain cancers. Pharmacological intervention studies in prostate cancer suggest that hGIIA-dependent tumour growth is dependent on indirect regulation of Group IVA PLA 2. Group VI calcium-independent PLA 2 enzymes have also been recently implicated in tumourigenesis with in vitro studies suggesting multiple possible roles for these enzymes. Though apparently complex, further characterization of the regulatory relationships amongst PLA 2 enzymes, lipid mediator biosynthetic enzymes and the lipid mediators they produce during tumour progression is required to define the biochemical context in which the enzymes modulate cancer growth and development. 相似文献
13.
通过丙酮沉淀、DEAE-纤维素离子交换柱层析和Sephadex G-100凝胶过滤柱层析等分离纯化技术,对巴西橡胶树胶乳C-乳清磷脂酶A2进行分离纯化。用SDS-PAGE测定其亚基的相对分子量。测定该酶最适温度和pH,动力学常数Km和Vmax。并测定Ca2+和La3+对酶活性的影响。结果显示:该酶被纯化了49.47倍,产率为5.12%。SDS-PAGE检测为单一条带,其亚基相对分子量约43kDa。最适反应温度为37℃,最适反应pH为8.0,Km为0.44mmol·L-1,Vmax为7.22μmol.(mL.min)-1。最适Ca2+浓度为50μmol·L-1,稀土元素La3+离子对磷脂酶A2活性有抑制作用,但加入Ca2+后可缓解La3+对磷脂酶A2活性的抑制作用。胶乳C-乳清磷脂酶A2与其他植物磷脂酶A2在Ca2+的依赖性上存在差异。研究结果为今后探索橡胶树胶乳磷脂酶A2的催化机理、调节机理及生理功能等奠定了基础。 相似文献
14.
The synergistic effect of exogenous cobra phospholipase A 2 on the hemolysis rate of guinea pig erythrocytes by highly purified snake venom cardiotoxins was investigated. In the presence of phospholipase A 2 the reaction was not only faster and had a lower activation energy but followed a sigmoidal instead of a linear time course. Similar results were obtained using porcine pancreatic phospholipase A 2. Significantly, addition of even a trace of cobra phospholipase A 2 (approx. 0.1%, w/w) was sufficient to bring about the full synergistic effect, emphasizing the stringent purity requirements for any meaningful investigation of cardiotoxin's own action. The possibility that the action of cardiotoxin on its own may involve the stimulation of an endogenous phospholipase is discussed in the light of the results obtained with exogenous cobra enzyme. 相似文献
15.
A marine snail digestive phospholipase A 2 (mSDPL) was purified from delipidated hepatopancreas. Unlike known digestive phospholipases A 2, which are 14 kDa proteins, the purified mSDPL has a molecular mass of about 30 kDa. It has a specific activity of about 180 U/mg measured at 50 °C and pH 8.5 using phosphatidylcholine liposomes as a substrate in the presence of 4 mM NaTDC and 6 mM CaCl 2. The N-terminal amino-acid of the purified mSDPL does not share any homology with known phospholipases.Moreover, the mSDPL exhibits hemolytic activity in intact erythrocytes and can penetrate phospholipid monolayers at high surface pressure, comparable to snake venom PLA 2. These observations suggest that mSDPL could be toxic to mammal cells. However, mSDPL can be classified as a member of a new family of enzymes. It should be situated between the class of toxic phospholipase A 2 from venoms and another class of non toxic pancreatic phospholipase A 2 from mammals. 相似文献
16.
Venomous snakes such as Gloydius brevicaudus have three distinct types of phospholipase A 2 inhibitors (PLIα, PLIβ, and PLIγ) in their blood so as to protect themselves from their own venom phospholipases A 2 (PLA 2s). Expressions of these PLIs in G. brevicaudus liver were found to be enhanced by the intramuscular injection of its own venom. The enhancement of gene expressions of PLIα and PLIβ in the liver was also found to be induced by acidic PLA 2 contained in this venom. Furthermore, these effects of acidic PLA 2 on gene expression of PLIs were shown to be unrelated to its enzymatic activity. These results suggest that these venomous snakes have developed the self-protective system against their own venom, by which the venom components up-regulate the expression of anti-venom proteins in their liver. 相似文献
17.
Secretory phospholipase A 2 (sPLA 2) hydrolyzes phosphatidylcholines (PC) within lipid bilayers to produce lyso-PC and a fatty acid, which can act as signaling molecule in biological membranes. The activity of sPLA 2 depends on the membrane structure. Bilayer defects, curvature, and gel-fluid micro-heterogeneity are known to activate sPLA 2. Here, we investigate if liquid-liquid immiscibility within model membranes is sufficient for sPLA 2 activation. The onset of the hydrolytic activity of cobra-venom sPLA 2 towards mixed monolayers of dimyristoyl-PC (DMPC)/cholesterol 2:1 (mol/mol) has been determined using infrared reflection-absorption spectroscopy (IRRAS) and polarization-modulated (PM-) IRRAS. The lag phase of sPLA 2 activity increases exponentially with rising surface pressures starting at 12 mN/m. This indicates that enzyme activation is hampered at higher surface pressures. Below 12 mN/m, no lag phase is observed, and sPLA 2 is efficiently activated. The surface pressure that is critical for sPLA 2 activation correlates with the critical miscibility pressure according to the phase diagram of DMPC and cholesterol. Thus, coexisting, liquid-phase domains provide sufficient boundaries to activate sPLA 2. Moreover, liquid-liquid immiscibility is an activating mechanism for sPLA 2 that also applies to biological membranes under physiological conditions because the corresponding bilayer structure is associated with that of membrane rafts. 相似文献
20.
Phospholipase A 2 was purified from the pyloric ceca of the starfish Asterina pectinifera. The final enzyme preparation was nearly homogeneous in sodium dodecyl sulfate-polyacrylamide gel electrophoresis and its molecular weight was estimated as approximately 20,000. The optimum pH and temperature of the enzyme were at around pH 9.0 and 50°C, respectively, and the activity was enhanced by sodium deoxycholate and 1 mM or higher concentration of Ca 2+. The enzyme had no fatty acid specificity. Starfish phospholipase A 2 hydrolyzed phosphatidylcholine more effectively than phosphatidylethanolamine. 相似文献
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