共查询到20条相似文献,搜索用时 296 毫秒
2.
Esterification of lysophosphatidylcholine (LPC) with conjugated linoleic acid (CLA) was carried out using porcine pancreatic phospholipase A 2 (PLA 2). PLA 2 only slightly synthesized phosphatidylcholine containing CLA (CLA-PC) at 2.6% by the addition of water. Addition of formamide in place of water markedly increased the yield of CLA-PC. In addition, synthesis of CLA-PC by PLA 2 was affected by the amount of substrate CLA and PLA 2 in the reaction system. Under optimal reaction conditions using 11 mg LPC, 18 mg CLA, 550 mg glycerol, 50 μL formamide, 3.3 × 10 4 U PLA 2, and 0.3 μmol CaCl 2 at 37 °C for 6 h, the reaction yield of CLA-PC reached 65 mol%. Furthermore, addition of protein such as albumin and casein suppressed the decrease of CLA-PC yield after 6 h. PLA 2 exhibited the highest activity for the 10 t,12 c-CLA isomer among four CLA isomers (9 c,11 t-CLA, 9 c,11 c-CLA, 9 t,11 t-CLA and 10 t,12 c-CLA), whereas that for 9 c,11 c-CLA was the lowest. These results showed that the present esterification system for LPC and CLA by PLA 2 is effective for producing CLA-PC. 相似文献
3.
The Group IV phospholipase A 2 family is comprised of six intracellular enzymes commonly called cytosolic phospholipase A 2 (cPLA 2) , cPLA 2β, cPLA 2γ, cPLA 2δ, cPLA 2ε and cPLA 2ζ. They are most homologous to phospholipase A and phospholipase B/lysophospholipases of filamentous fungi particularly in regions containing conserved residues involved in catalysis. However, a number of other serine acylhydrolases (patatin, Group VI PLA 2s, Pseudomonas aeruginosa ExoU and NTE) contain the Ser/Asp catalytic dyad characteristic of Group IV PLA 2s, and recent structural analysis of patatin has confirmed its structural similarity to cPLA 2. A characteristic of all these serine acylhydrolases is their ability to carry out multiple reactions to varying degrees (PLA 2, PLA 1, lysophospholipase and transacylase activities). cPLA 2, the most extensively studied Group IV PLA 2, is widely expressed in mammalian cells and mediates the production of functionally diverse lipid products in response to extracellular stimuli. It has PLA 2 and lysophospholipase activities and is the only PLA 2 that has specificity for phospholipid substrates containing arachidonic acid. Because of its role in initiating agonist-induced release of arachidonic acid for the production of eicosanoids, cPLA 2 activation is important in regulating normal and pathological processes in a variety of tissues. Current information available about the biochemical properties and tissue distribution of other Group IV PLA 2s suggests they may have distinct mechanisms of regulation and functional roles. 相似文献
4.
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
5.
Rat uterine stromal cells (U III) express pancreatic type PLA 2 (PLA 2-I) receptor and internalize the enzyme bound to receptors. Here, we investigate the proliferating effect and alterations in binding of PLA 2-I. There is a dramatic decline in PLA 2-I binding in U III cells as they progress from a nonconfluent proliferating state (40,000 sites/cell) to a confluent state (1300 sites/cell). Intracellular concentration of PLA 2-I changed with the alteration in binding, suggesting that regulation in the PLA 2 binding capacity may have important implications in growth control mechanisms. 相似文献
7.
Many plants are used in traditional medicine as active agents against various effects induced by snakebite. Few attempts have been made however to identify the nature of plant natural products with anti-ophidian properties. Baccharis trimera (Less) DC (Asteraceae), known in Brazil as carqueja, has been popularly used to treat liver diseases, rheumatism, diabetes, as well as digestive, hepatic and renal disorders. The active component was identified as 7-hydroxy-3,13-clerodadiene-16,15:18,19-diolide, C 20H 28O 5, (clerodane diterpenoid, Bt-CD). We report now the anti-proteolytic and anti-hemorrhagic properties against snake venoms of a Bt-CD inhibitor from B. trimera. Bt-CD exhibited full inhibition of hemorrhage and proteolytic activity caused by Bothrops snake venoms. The inhibitor was able to neutralize the hemorrhagic, fibrinogenolytic and caseinolytic activities of class P-I and III metalloproteases isolated from B. neuwiedi and B. jararacussu venoms. No inhibition of the coagulant activity was observed. Bt-CD also partially inhibited the edema induced by other crude venoms, metalloproteases, basic and acidic phospholipases A 2. To further elucidate the inhibitory specificity of Bt-CD against metalloproteases isolated from snake venoms, a deeper understanding of its structure and function is necessary. Furthermore, the potential use of these inhibitors to complement anti-venom as an alternative treatment of snakebite envenomations needs to be evaluated in future studies. 相似文献
8.
Production of egg yolk lysolecithin was compared using free phospholipase A 2 (PLA 2) and immobilized PLA 2 in alginate-silicate sol-gel matrix. Choice of solvent, water content, calcium, and temperatures changed the activity of the free and immobilized PLA 2 a lot, owing to their effects on the catalytic properties of the enzyme as well as the conformational change of lecithin in ethanol-buffer mixture. Free PLA 2 shows typical microemulsion kinetics in ethanol-buffer system. The effect of the water content on the enzyme reaction was greatly influenced by the presence of calcium ion. In the absence of calcium ion, certain optimal water content for the production of lysolecithin always exists in the free PLA 2 reaction. However, with calcium ion, three distinctive regions were observed with free PLA 2 reactions. Initially, in the micro-aqueous region of the ethanol-buffer system with calcium ion, the hydrolysis activity of PLA 2 was proportional to the water content. Beyond the region, concave type of activity profiles were observed as the water content increases. As the water content increases further, the hydrolysis rate of the PLA 2 abruptly decreased by the phase separation. On the contrary, in case of immobilized enzyme, optimal water content for the production of lysolecithin exists regardless of the presence of calcium ion. The calcium ion was essential for achieving the maximum activity of both free and immobilized PLA 2. The addition of calcium ion not only affected the catalytic activity of the enzyme but also was necessary to improve the enzyme stability. As the immobilization of the enzyme remarkably increased thermal stability of the free enzyme, the immobilized PLA 2 is more desirable to be used in the production of various lysophospholipids. It was successfully reused over 250 h. 相似文献
9.
Venom of the Australian tiger snake, Notechis scutatus scutatus was fractionated by conventional ion-exchange chromatography. The fraction containing notexin, a well-known single-chain toxic phospholipase A2, was further purified by reverse-phase high-performance liquid chromatography. Two main components were isolated and the major one corresponded to notexin. The other component, designated as notechis Ns, was an isoform of notexin. Notechis Ns and notexin possessed similar in vitro esterase activity, in vitro neuromuscular activity and in vivo lethality. Amino acid composition and sequence of the Staphylococcus aureus V8-protease peptides demonstrated that primary structures of notechis Ns and notexin differed from each other by a single substitution amongst 119 amino acids: Lys----Arg at position 16. 相似文献
10.
Here we studied the role of phosphoinositide 3-kinase (PI 3-kinase) and mitogen activated protein (MAP) kinase in regulating bradykinin (BK) induced prostaglandin E 2 (PGE 2) production in human pulmonary artery smooth muscle cells (HPASMC). BK increased PGE 2 in a three step process involving phospholipase A 2 (PLA 2), cyclooxygenase (COX) and PGE synthase (PGES). BK stimulated PGE 2 release in cultured HPASMC was inhibited by the PI 3-kinase inhibitor LY294002 and the p38 MAP kinase inhibitor SB202190. The inhibitory mechanism used by LY294002 did not involve cytosolic PLA 2 activation or COX-1, COX-2 and PGES protein expression but rather a novel effect on COX enzymatic activity. SB202190 also inhibited COX activity. 相似文献
11.
Growth plate chondrocytes from both male and female rats have nuclear receptors for 17β-estradiol (E 2); however, recent studies indicate that an alternative pathway involving a membrane receptor may also be involved in the female cell response. E 2 directly affects the fluidity of chondrocyte membranes derived from female, but not male, rats. In addition, E 2 activates PKC in a nongenomic manner in female cells, and chelerythrine, a specific inhibitor of PKC, inhibits E 2-dependent alkaline phosphatase activity in these cells, indicating PKC is involved in the signal transduction mechanism. The aims of this study were: (1) to examine if PKC mediates the effect of E 2 on chondrocyte proliferation, differentiation, and matrix synthesis; and (2) to determine the pathway that mediates the membrane effect of E 2 on PKC. Confluent, fourth passage resting zone (RC) and growth zone (GC) chondrocytes from female rat costochondral cartilage were treated with 10 −10 to 10 −7 M E 2 in the presence or absence of the PKC inhibitor chelerythrine, and changes in alkaline phosphatase specific activity, proteoglycan sulfation, and [ 3H]thymidine incorporation were measured. To examine the pathway of PKC activation, chondrocyte cultures were treated with E 2 in the presence or absence of genistein (an inhibitor of tyrosine kinases), U73122 or D609 (inhibitors of phospholipase C [PLC]), quinacrine (an inhibitor of phospholipase A 2 [PLA 2]), and melittin (an activator of PLA 2). Alkaline phosphatase specific activity and proteoglycan sulfation were increased and [ 3H]thymidine incorporation was decreased by E 2. The effects of E 2 on all parameters were blocked by chelerythrine. Treatment of the cultures with E 2 produced a significant dose-dependent increase in PKC. U73122 dose-dependently inhibited the activation of PKC in E 2-stimulated female chondrocyte cultures. However, the classical receptor antagonist ICI 182780 was unable to block the stimulatory effect of E 2 on PKC. Moreover, the classical receptor agonist diethylstilbestrol (DES) had no effect on PKC, nor did it alter the stimulatory effect of E 2. Inhibition of tyrosine kinase and PLA 2 had no effect on the activation of PKC by E 2. The PLA 2 activator also had no effect on PKC activation by E 2. E 2 stimulated PKC activity in membranes isolated from the chondrocytes, demonstrating a direct membrane effect for this steroid hormone. These data indicate that the rapid nongenomic effect of E 2 on PKC activity in chondrocytes from female rats is sex-specific and dependent upon a G-protein-coupled phospholipase C. 相似文献
12.
Notexin from Notechis scutatus scutatus snake venom was modified with trinitrobenzenesulfonic acid, and the major trinitrophenylated (TNP) derivative was separated by high-performance liquid chromatography. Modification resulted in the incorporation of only one TNP group on the N-terminal alpha-amino group. The TNP derivative showed a precipitous decrease in enzymatic activity and lethal toxicity, whereas the antigenicity remained unchanged. However, trinitrophenylation did not significantly affect the secondary structure of the toxin molecule as revealed by the CD spectra. The results, that the modification reaction was accelerated by the Ca2+ and that the TNP derivative retains its affinity for Ca2+, indicate that the N-terminal alpha-amino group did not participate in the Ca2(+)-binding. The TNP derivative could be regenerated with hydrazine hydrochloride. The biological activities of the regenerated notexin are almost the same as those of native notexin. These results suggest that the N-terminal alpha-amino group is essential for the phospholipase A2 activity and lethal toxicity of notexin, and that incorporation of the TNP group on the N-terminal alpha-amino group might give rise to a distortion of the active conformation of notexin. 相似文献
13.
Phospholipase A 2 (PLA 2) from cobra venom, which can hydrolyze the S N2 ester bond of 1,2-diacylphosphatides, was immobilized by covalent binding to porous chitosan beads. Immobilization has to be carried out by using the carboxylic groups instead of the amine groups of the enzyme to get reasonable activity retention (higher than 50%). The effects of amount of activating reagent EDC and enzyme loading during the immobilization step were investigated. Since EDC could modify important Asp groups in the enzyme, the EDC/enzyme weight ratio should be less than 10. Although the activity retention of immobilized enzyme increased with enzyme/bead weight ratio, this ratio should be kept to a minimum at 1×10 −3 to optimize coupling yield of enzyme activity and reduce internal diffusion resistance. The kinetic properties and stability of the immobilized enzyme were determined. The immobilized PLA 2 was packed into a column to hydrolyze phospholipid in a circulating packed-bed reactor. The flow rate of the substrate solution should be set at 37.5 cm/min (superficial velocity) to eliminate external diffusion resistance, under which condition the column reactor could be reused up to 10 times with less than 20% loss of activity. Since enzymatic hydrolysis of phospholipid on low density lipoprotein (LDL) particle surface with PLA 2 could result in faster plasma clearance of the modified LDL particles, an in vitro bioreactor containing immobilized PLA 2 should be able to lower serum cholesterol concentration. A significant decrease in total serum cholesterol concentration in hypercholesterolemic rabbits was observed after 90-min treatment. 相似文献
14.
Notexin from Notechis scutatus scutatus snake venom was subjected to Lys modification with pyridoxal 5′-phosphate (PLP), and one major modified derivative was purified on a cation-exchanger SP-8HR column. The results of amino acid analysis and sequence determination revealed that only 2 Lys residues at positions 82 and 115 out of 11 Lys residues in notexin were modified. The incorporation of PLP into the protein was accompanied by the loss of 53% lethal toxicity, but the modified notexin showed an about 1.2-fold increase in enzymatic activity. However, the secondary structure of the toxin molecule did not significantly change after modification with PLP as revealed by the CD spectra, and the antigenicity of PLP derivative remained unchanged. The modified derivative retained its affinity for Ca 2+, indicating that the modified Lys residues did not participate in Ca 2+ binding. These results indicate that modification of Lys residues causes a differential effect on the enzymatic activity and lethal toxicity of notexin, and suggest that notexin might possess two functional sites, one responsible for the catalytic activity and the other associated with its lethal effect. 相似文献
15.
Rabbit antibodies were prepared against both purified catalytic (component-B) and purified non-catalytic (component-A) subunits of crotoxin, the major phospholipase A2 neurotoxin from the South American rattlesnake. They cross-react with crotoxin-like toxins from the venom of several Crotalus species as well as with single-chain phospholipase A2 neurotoxins from Crotalid and Viperid venoms (agkistrodontoxin and ammodytoxin A) but not from Elapid venoms (notexin). Immunological cross-reactions of anti-component-A and anti-component-B sera with crotoxin and with its isolated components A and B showed that component-A exposes determinants of low immunogenicity which are present on component-B, whereas the major antigenic determinants of component-B are not present on component-A. Anti-component-B antibodies, but not anti-component-A antibodies, neutralize the lethal potency of crotoxin and inhibit its enzymatic activity. Furthermore, non-precipitating anti-component-B Fab fragments were as potent as antibodies, indicating that crotoxin neutralization results from the binding of the antibodies to the catalytic subunit, rather than the formation of an immunoprecipitate. 相似文献
16.
A series of diplatinum(III) complexes derived from cis-(NH 3) 2Pt II and the model nucleobase 1-methylcytosine (1-MeC) has been prepared and X-ray structurally characterized, all of which contain two anionic base ligands (1-MeC −) in a head–tail ( ht) arrangement: ht- cis-[( ONO 2)(NH 3) 2Pt(1-MeC −- N3, N4) 2Pt(NH 3) 2( ONO 2)](NO 3) 2·HNO 3·3H 2O (2b), ht- cis-[( NO 2) (NH 3) 2 Pt(1-MeC −- N3, N4) 2Pt(NH 3) 2( OH 2)](ClO 4) 3·3.5H 2O (3), ht- cis-[( OH 2)(NH 3) 2Pt(1-MeC −- N3, N4) 2Pt(NH 3) 2( OH 2)](ClO 4) 4·H 2O (4b), and ht- cis-[(9-EtGH- N7)(NH 3) 2Pt(1-MeC −- N3, N4) 2Pt (NH 3) 2(9-EtGH- N7)](NO 3) 4·9H 2O (7b) (9-EtGH=9-ethylguanine). Several other compounds, differing in the nature of the axial ligands, have been isolated and or observed in solution by 1H and 195Pt NMR spectroscopy. The chemistry of these diplatinum(III) compounds is dominated by facile substitution reactions of the axial ligands. Of particular interest in this context is the ready reaction of 2b or 3 with guanine nucleobases. Since similar compounds are not obtained with any of the other common nucleobases, 2b and 3 can be considered guanine-specific chemical probes. 相似文献
17.
Hybrid membrane particles from two mutants of Escherichia coli K 12, B v4 and K I1, defective in oxidative phosphorylation, have been prepared, in which ATP-driven membrane energization is restored. A soluble factor of mutant KI1 was found to have properties similar to parental crude coupling factor, ATPase (EC 3.6.1.3). Membrane particles of this mutant could not be reconstituted by parental coupling factor. Either parental coupling factor, or the soluble factor of mutant KI1 could reconstitute both respiration-driven and ATP-driven energization to membrane particles of mutant BV4 or to parental particles depleted of ATPase. Mutant BV4 was found to be devoid of coupling factor activity, while retaining the ability to hydrolyze ATP. Both mutants possess an ATPase with an altered binding to the membrane. Mutant KI1 is impaired in respiration-driven amino acid transport, in contrast to mutant BV4. The three major subunits of parental Escherichia coli ATPase have been isolated and antibodies have been prepared against these subunits. Antibodies against the largestsubunit ( component) or against the intact catalytic subunits ( + β components) inhibit both ATP-Pi exchange in the parent organism as well as ATP hydrolytic activity in parent and mutants. Antibodies against the two other subunits (β or γ components) also inhibit these two reactions, but were found to be less effective. Mutant NI44, which lacks ATPase activity, shows no precipitin lines with anti-, anti-β, anti-γ, or anti-( + β) preparations. In contrast, mutants BV4 and KI1, exhibit cross-reactivity with all of the antisera. 相似文献
18.
It is shown how 1D nOe and 2D COSY 1H NMR spectroscopy can be used to assign the stereochemistry of Co(III) amine complexes. By using d 6-DMSO as solvent together with a small quantity of DCl all non-equivalent N--- H hydrogens can be distinguished at 300 MHz. Through-space (nOe), and through-bond (COSY), associations with other N--- H and C--- H hydrogens can then be determined. This leads to a complete assignment of structure in solution. The technique is applied to the complexes syn(N), anti(N)-[Co(cyclen) (NH 3) 2] (ClO 4) 3, syn(N), anti(Cl)-[Co(cyclen) (NH 3)Cl] (ClO 4) 2, anti(N), syn(Cl)-[Co(cyclen) (NH 3)Cl](ClO 4) 2, syn(N), anti(O)-[Co(Mecyclen)-(GlyO)](ClO 4) 2 and Δ- cis-[Co(δ-en) 2(NO 2) 2](NO 2). 相似文献
19.
We described previously the in vivo immunoneutralization effects of a high affinity anti-oestradiol antibody clone 15 in blocking ovulation and synaptic remodeling in cycling female rats. In the present study we report the enhancing effects of this antibody. Treatment of ovariectomized female rats or female derived skeletal cell cultures in vitro with anti-E 2 15 plus oestrogen (E 2) potentiated the specific activity of the brain type creatine kinase (CK) response to E 2 in the rat tissues or skeletal cells. The enhancing CK response of anti E 2 15 plus E 2 was time- and dose-dependent in the uterus, thymus, epiphysis and diaphysis of ovariectomized female rats. In the pituitary, on the other hand, anti-E 2 15 blocked the stimulatory CK response to E 2. Two other high affinity anti-E 2 anti-bodies, clones 8D 9 and 11B 6, had no effect in augmenting the response of CK to E 2 in rat tissues. Moreover, the enhancing CK response in rat tissues was specific to anti-E 2 15 plus E 2 since the intact anti-E 2 in the presence of other oestrogen mimetics, such as oestriol or stilbestrol or tamoxifen did not potentiate the CK response in rat tissues. In this model system the Fab' monomer of anti-E 2 15 abolished the CK response to E 2 in rat tissues and not to anti-E 2 15 plus E 2 whereas tamoxifen completely blocked the CK response to anti E 2 plus E 2. Anti E 2 15 may therefore serve as a specific carrier in delivering E 2 to oestrogen sensitive rat tissues or cells containing functional oestrogen receptors and thereby increasing the magnitude of E 2 effects in vivo and in vitro. 相似文献
20.
The relationship between O 2 and an active oxygen scavenging system in Chlorella vulgaris var. vulgaris (IAM C-534) was investigated. When Chlorella vulgaris was exposed to 2% O 2, only traces of active oxygen scavenging enzymes were found. When the Chlorella vulgaris was treated with 20% or 50% O 2, it was shown that the level of enzyme activity increased as the O 2 concentration increased. An increase in enzyme activity was not found in any specific enzyme but in all of the enzymes, but the level of glutathione and ascorbate remained the same in all the cases. In addition, the photosynthetic efficiency also decreased as the concentration of O 2 was increased. These results suggest that an O 2 enriched environment can lead to an increase in the production of active oxygen species such as O bullet2 and H 2O 2 and to a decrease in the photosynthetic efficiency in Chlorella vulgaris. The hydroxyl radical ( bulletOH) was detected directly in the Chlorella vulgaris suspension with a spin trapping reagent. It was also clear that the increase in the bulletOH intensity as the visible light intensity increased was unrelated to the O 2 concentration. It was suggested that the conditions for producing bulletOH and the other active oxygen species were different, and that two types of oxygen stress should exist in the Chlorella vulgaris. 相似文献
|