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1.
Enhancing factor (EF), a growth factor modulator, recently identified as the mouse secretory phospholipase A2 (PLA2), has been isolated in our laboratory from the intestines of mice. EF modulates the action of epidermal growth factor (EGF)
by mediating an almost 2-fold increase in EGF binding in a radioreceptor assay. EF has been localized immunohistochemically
to the Paneth cells of the intestine, adjacent to the proliferating stem cell population. Although very weak staining was
observed in the intestines of ICRC mice (ICRC is an inbred strain of mouse developed at this Institute) as compared to Balb/c
mice, the enhancing activity was not detected in the partially purified, acid soluble intestinal proteins of the ICRC strain.
However, studies using polyclonal antibodies against purified EF demonstrated that EF from Balb/c and ICRC intestines are
either immunologically identical or closely related to each other although, quantitatively, EF was very low in ICRC mice.
RFLP studies indicated that ICRC mice carry a mutation in the coding region of the EF gene resulting in loss of the BamHI.
restriction site. On sequencing, a T insertion was found at position 166 from the ATG site thereby causing a disruption in
the ORF. This probably results in undetectable levels of enhancing activity. In this paper we report the molecular characterization
of the ICRC mouse with respect to theenhancing factor gene 相似文献
2.
Abhay R. Redkar Shilpa Kadam Madhav G. Deo Rita Mulherkar 《Journal of biosciences》1997,22(5):529-536
Enhancing factor (EF), a mouse phospholipase A2 (PLA2), has been purified from the small intestines, based on its ability to increase the binding of epidermal growth factor in
a radioreceptor assay. EF/PLA2 was found to be localized predominantly in the Paneth cells in the small intestines. Whether mouse intestinal EF/PLA2 is identical/similar to mouse secretory PLA2 was to be determined. Phospholipases are known to play a crucial role in the process of inflammation. This paper reports
the presence of trace amounts of EF/PLA2 in the peritoneal exudate cells. Western blot analysis of the acid extracts showed the presence of a 14 kDa immunologically
cross-reactive protein. RT-PCR analysis using EF specific primers amplified a ∼700 bp product which was further confirmed
to be EF-specific by nested PCR analysis and sequencing. Presence of EF in the peritoneal exudate cells could be a unique
mode of transport of growth factor modulator to the site of injury to aid in regeneration/cell proliferation of damaged tissue. 相似文献
3.
Archana S. Wagle Varsha Patki Shefali J. Desai Rita Mulherkar 《Journal of biosciences》1997,22(5):537-543
Enhancing factor (EF), a mouse intestinal phospholipase A2 (PLA2), has been isolated and characterized. EF increases the binding of epidermal growth factor (EGF) to A431 cells almost two-fold
by interacting with EGF. EF binds to a 100 kDa cell surface receptor and brings about an increase in the binding of EGF. In
the present study we demonstrate that EF is a heparin binding protein and at the time of iodination of EF, the heparin binding
site of EF has to be protected. Heparin inhibits the enhancing activity of EF as well as the binding of labelled EF to A431
cells. Inhibition of binding of EF to cells by heparin indicates that heparin binding region forms at least part of the receptor
binding domain. These data suggest that the receptor for EF on the cell surface could be a heparin sulphate proteoglycan. 相似文献
4.
The unicellular Tetrahymena enzymatically split the synthetic phosphodiester, 4-methylum-belliferyl phosphocoline substrate. The enzyme activity was completely blocked in vitro and drastically inhibited in vivo by G-protein activating fluorides (NaF; AlF4
– and BeF3
–). The phospholipase A2 inhibitor, quinacrine, and the protein phosphatase inhibitor, neomycin, inhibited the enzyme activity in vitro and activated it in vivo. Another phospholipase A2 inhibitor 4-bromo phenacyl bromide was ineffective in vivo and in vitro alike, as well as the cyclooxygenase inhibitor indomethacin. Results of these experiments indicate that some treatments could be specific for a well defined activity (e.g., phospholipase A2, G-protein) but subject to influence by other enzymes (e.g., phospholipase C, sphingomyelinase). The experiments call attention to the differences in the results of the in vivo and in vitro studies. 相似文献
5.
Sajal Chakraborti Sandip K. Batabyal John R. Michael Tapati Sanyal 《Molecular and cellular biochemistry》1994,130(2):121-127
Exposure of rabbit pulmonary arterial smooth muscle cells to 10 M of the calcium ionophore A23187 dramatically stimulates cell membrane-associated phospholipase A2 activity and arachidonic acid release. In addition, A23187 also enhances cell membrane-associated serine esterase activity. Serine esterase inhibitors phenylmethylsulfonylfuoride and diisopropyl fluorophosphate prevent the increase in serine esterase and phospholipase A2 activities and arachidonic acid release caused by A23187. A23187 still stimulated serine esterase and phospholipase A2 activities and arachidonic acid release in cells pretreated with nominal Ca2+ free buffer. Treatment of the cell membrane with A23187 does not cause any appreciable change in serine esterase and phospholipase A2 activities. Pretreatment of the cells with actinomycin D or cycloheximide did not prevent the increase in the cell membrane associated serine esterase and phospholipase A2 activities, and arachidonic acid release caused by A23187. These results suggest that (i) a membrane-associated serine esterase plays an important role in stimulating the smooth muscle cell membrane associated phospholipase A2 activity (ii) in addition to the presence of extracellular Ca2+, release of Ca2+ from intracellular storage site(s) by A23187 also appears to play a role in stimulating the cell membrane-associated serine esterase and phospholipase A2 activities, and (iii) the increase in the cell membrane-associated serine esterase and phospholipase A2 activities does not appear to require new RNA or protein synthesis.Abbreviations A23187
calcium ionophore
- AA
arachidonic acid
- PMSF
phenylmethyl sulfonylfuoride
- DFP
diisopropyl-fluorophosphate
- DMEM
Dulbecco's modified Eagles medium
- FCS
fetal calf serum
- PBS
phosphate buffered saline
- HBPS
Hank's buffered physiological saline
- PLA2
phospholipase A2 相似文献
6.
A series of inhibitors of glucosylceramide synthesis, the PDMP based family of compounds, has been developed as a tool for
the study of sphingolipid biochemistry and biology. During the course of developing more active glucosylceramide synthase
inhibitors, we identified a second site of inhibitory activity for PDMP and its structural homologues that accounted for the
ability of the inhibitors to raise cell and tissue ceramide levels. This inhibitory activity was directed against a previously
unknown pathway for ceramide metabolism, viz. the formation of 1-O-acylceramide. In this pathway the addition of a fatty acyl group to the primary hydroxyl of ceramide occurs through a transacylation
with either phosphatidylethanolamine or phosphatidylcholine as a substrate. However, both in the absence and presence of ceramide,
water serves as an acceptor for the fatty acid. Thus the enzyme may be considered to be a phospholipase A2. The enzyme is unique in that it has an acidic pH optimum and is localized to lysosomes by cell fractionation. More recently,
the 1-O-acylceramide synthase has been purified, sequenced, and cloned. This phospholipase A2 was discovered to be structurally homologous to lecithin cholesterol acyltransferase (LCAT). However, this phospholipase
A2 does not recognize cholesterol and lacks the defined lipoprotein-binding domain present in LCAT. We now refer to this enzyme
as lysosomal phospholipase A2 (LPLA2). Although acidic phospholipase A2 activities have been previously identified, LPLA2 appears to be the first lysosomal PLA2 to have been sequenced. This new phospholipase A2 lacks an obvious and proven biological function. Published in 2004.
This revised version was published online in August 2006 with corrections to the Cover Date. 相似文献
7.
尖吻蝮蛇毒碱性磷脂酶A2的表达及其生化特征 总被引:4,自引:0,他引:4
将尖吻蝮蛇毒碱性磷脂酶A2(A.aBPLA2)基因克隆至温敏表达载体pBLMVL2,在大肠杆菌RR1中成功诱导表达.表达产物A.aBPLA2约占细菌蛋白质总量的20%,并以包涵体的形式存在.纯化包涵体后,将产物变性、复性,然后用FPLC SuperoseTM12纯化,产物经过SDS-聚丙烯酰胺凝胶电泳检测只有单一条带.对纯化后的表达A.aBPLA2进行了酶活性、抑制血小板聚集活性和溶血活性的测定.结果显示,表达A.aBPLA2的酶活性与变性后复性江浙蝮蛇酸性磷脂酶A2酶活性相近,具有类似变性后复性江浙蝮蛇碱性磷脂酶A2的溶血活性,没有抑制血小板聚集活性.最后对磷脂酶A2的结构与这些活性的关系进行了讨论. 相似文献
8.
Chang WC Shyu WJ Shi GY Lin MT Jen CJ Wing LY Tang MJ Wu HL 《Journal of biomedical science》1996,3(1):59-66
Treatment of cultured bovine carotid artery endothelial cells with 0.1 µM human plasmin has been reported to induce a receptor-mediated short burst of arachidonate release, which is a pertussis toxin-sensitive and extracellular calcium-dependent reaction. Plasmin-induced calcium influx in cells was significantly inhibited by pretreatment with pertussis toxin, indicating that the former was coupled with a pertussis toxin-sensitive guanosine 5-triphosphate (GTP)-binding protein. Plasmin significantly induced the formation of lysophosphatidylcholine but not lysophosphatidylethanolamine. A cellular phospholipase A2 with an arachidonyl specificity at the sn-2 position of phosphatidylcholine, which required submicromolar calcium, was identified as a cytosolic phospholipase A2 by immunoblot analysis. By a cell-free enzyme activity assay and immunoblot analysis, plasmin was found to induce a translocation of the cytosolic phospholipase A2 from the cytosol to the membrane. Taken together, the results suggest that plasmin bound to its putative receptor and activated a GTP-binding protein coupled to calcium influx channel, followed by translocation and activation of cytosolic phospholipase A2 in endothelial cells. 相似文献
9.
Pascale Nègre-Aminou †Raphael A. Nemenoff Malcolm R. Wood B. A. de la Houssaye † K. H. Pfenninger 《Journal of neurochemistry》1996,67(6):2599-2608
Abstract: Nerve growth cones isolated from fetal rat brain exhibit in their cytosol a robust level of phospholipase A2 activity hydrolyzing phosphatidylinositol (PI) and phosphatidylethanolamine (PE) but not phosphatidylcholine (PC). Western blot analysis with an antibody to the well-characterized cytosolic phospholipase A2 (mol wt 85,000) reveals only trace amounts of this PC- and PE-selective enzyme in growth cones. By gel filtration on Superose 12, growth cone phospholipase A2 activity elutes essentially as two peaks of high molecular mass, at ~65 kDa and at well over 100 kDa. Anion exchange chromatography completely separates a PI-selective from a PE-selective activity, indicating the presence of two different, apparently monoselective phospholipase A2 species. The PI-selective enzyme, the predominant phospholipase A2 activity in whole growth cones, is enriched greatly in these structures relative to their parent fractions from fetal brain. This phospholipase A2 is resistant to reducing agents and is found in the cytosol as well as membrane-associated in the presence of Ca2+. However, its catalytic activity is Ca2+-independent regardless of whether the enzyme is associated with pure substrate or mixed-lipid growth cone vesicles. The PE-selective phospholipase A2 in growth cones was studied in less detail but shares with the PI-selective enzyme several properties, including intracellular localization, the existence of cytosolic and membrane-associated forms, and Ca2+ independence. Our data indicate growth cones contain two high-molecular-weight forms of phospholipase A2 that share many properties with known, Ca2+-independent cytosolic phospholipase A2 species but that appear to be monoselective for PI and PE, respectively. In particular, the PI-selective enzyme may represent a new member of the growing family of cytoplasmic phospholipase A2. The enrichment of the PI-selective phospholipase A2 in growth cones suggests it plays a major role in the regulation of growth cone function. 相似文献
10.
《Archives of biochemistry and biophysics》1985,241(2):413-417
Calmodulin interacted with phospholipase A2 from two different sources, as established by affinity chromatography, dimethylsuberimidate protein crosslinking, and phospholipase A2 assays. Calmodulin was covalently crosslinked to pancreatic and bee venom phospholipases A2 in a calcium-dependent manner, and enhanced the enzymatic activities of these phospholipases. Pancreatic phospholipase A2 was separated into two species of identical molecular weight by calmodulin affinity chromatography; the species that bound to immobilized calmodulin in a calcium-dependent manner was stimulated by calmodulin. This presents further evidence that phospholipase A2 is directly activated by calmodulin. 相似文献
11.
The presence of phospholipase A activity was detected in three dermatophytes:Microsporum cookei, Trichophyton mentagrophytes, andEpidermophyton floccosum. The activity was always higher inT. mentagrophytes than inM. cookei andE. floccosum. All exhibited phospholipase A1 and A2 activities, but the activity was largely A2 inM. cookei and A1 inE. floccosum. T. mentagrophytes possessed almost equal activities of phospholipase A1 and A2. 相似文献
12.
This paper described the effect of Fluosol® on phospholipase A2 activity both vitro and in vivo. This compound inhibited both postheparin plasma and hepatic phospholipase A2 activities. Furthermore, in Fluosol transfussed rats the postheparin plasma phospholipase A2 activity was 41% below that observed in control animals. The inhibition of phospholipase A2 activity was due to the micellar forms of pluoronic, F-68, a detergent present in Fluosol. It is postulated that the inhibition of phospholipase A2 activity by Fluosol might interfere with the release of arachidonic acid, a substrate used in biosynthesis of prostaglandin. 相似文献
13.
Summary A simple and rapid screening method for microorganisms with phospholipase A1, A2 and C activities using agar plate and gas chromatography (GC) method was successfully carried out. In agar plate method, soy bean lecithin and taurocholic acid were used as carbon source and emulsifier, respectively. In this agar plate method, microorganisms with phospholipase A1 and A2 or C activity produce a halo around the colony and two kinds(A's and C) of microorganisms are clearly distinguished by turbidity of the halo. Microorganisms with phospholipase A1 and A2 activity is simply distinguished by GC using a synthetic phospholipid containing different fatty acid at sn-1 and sn-2 position. 相似文献
14.
Raquel Gomes Fonseca Tatiana Lopes Ferreira Richard J. Ward 《Protein expression and purification》2009,67(2):82-87
The secreted phospholipases A2 (sPLA2s) are water-soluble enzymes that bind to the surface of both artificial and biological lipid bilayers and hydrolyze the membrane phospholipids. The tissue expression pattern of the human group IID secretory phospholipase A2 (hsPLA2-IID) suggests that the enzyme is involved in the regulation of the immune and inflammatory responses. With an aim to establish an expression system for the hsPLA2-IID in Escherichia coli, the DNA-coding sequence for hsPLA2-IID was subcloned into the vector pET3a, and expressed as inclusion bodies in E. coli (BL21). A protocol has been developed to refold the recombinant protein in the presence of guanidinium hydrochloride, using a size-exclusion chromatography matrix followed by dilution and dialysis to remove the excess denaturant. After purification by cation-exchange chromatography, far ultraviolet circular dichroism spectra of the recombinant hsPLA2-IID indicated protein secondary structure content similar to the homologous human group IIA secretory phospholipase A2. The refolded recombinant hsPLA2-IID demonstrated Ca2+-dependent hydrolytic activity, as measuring the release free fatty acid from phospholipid liposomes. This protein expression and purification system may be useful for site-directed mutagenesis experiments of the hsPLA2-IID which will advance our understanding of the structure–function relationship and biological effects of the protein. 相似文献
15.
Human non-pancreatic secretory phospholipase A2 (hnpsPLA2) is a group IIA phospholipase A2 which plays an important role in the innate immune response. This enzyme was found to exhibit bactericidal activity toward Gram-positive bacteria, but not Gram-negative ones. Though native hnpsPLA2 is active over a broad pH range, it is only highly active at alkaline conditions with the optimum activity pH of about 8.5. In order to make it highly active at neutral pH, we have obtained two hnpsPLA2 mutants, Glu89Lys and Arg100Glu that work better at neutral pH in a previous study. In the present study, we tested the bactericidal effects of the native hnpsPLA2 and the two mutants. Both native hnpsPLA2 and the two mutants exhibit bactericidal activity toward Gram-positive bacteria. Furthermore, they can also kill Escherichia coli, a Gram-negative bacterium. The two mutants showed better bactericidal activity for E. coli at neutral pH than the native enzyme, which is consistent with the enzyme activities. As hnpsPLA2 is highly stable and biocompatible, it may provide a promising therapy for bacteria infection treatment or other bactericidal applications. 相似文献
16.
Treatment of U937 cells with a sublethal concentration of tert-butylhydroperoxide generates DNA single strand breakage in U937 cells and this response is increased by caffeine, ATP, pyruvate or antimycin A. As we previously reported (Guidarelli, Clementi, Brambilla and Cantoni, (1997) Biochem. J. 328, 801–806), the enhancing effects of antimycin A are mediated by inhibition of complex III and the ensuing formation of superoxides and hydrogen peroxide in a reaction in which ubisemiquinone serves as an electron donor. Active electron transport was required in pyruvate-supplemented cells since the increased genotoxic response occurred as a consequence of enforced mitochondrial Ca2+ accumulation, a process driven by the increased electrochemical gradient. The enhancing effects of caffeine or ATP were also the consequence of mitochondrial Ca2+ accumulation but these responses were independent on electron transport. The increased formation of DNA lesions resulting from exposure to tert-butylhydroperoxide associated with the Ca2+-mobilizing agents or the respiratory substrate was mediated by arachidonic acid generated by Ca2+-dependent activation of phospholipase A2. Melittin, a potent phospholipase A2 activator, and reagent arachidonic acid mimicked the effects of caffeine, ATP or pyruvate on the tert-butylhydroperoxide-induced DNA single strand breakage. 相似文献
17.
Ji-Fu Wei Xiao-long Wei Qiu-Yu Chen Tian Huang Li-Ya Qiao Wan-Yu Wang Yu-Liang Xiong Shao-Heng He 《Biochimica et Biophysica Acta (BBA)/General Subjects》2006
A novel phospholipase A2 (PLA2) with Asn at its site 49 was purified from the snake venom of Protobothrops mucrosquamatus by using SP-Sephadex C25, Superdex 75, Heparin-Sepharose (FF) and HPLC reverse-phage C18 chromatography and designated as TM-N49. It showed a molecular mass of 13.875 kDa on MALDI-TOF. TM-N49 does not possess enzymatic, hemolytic and hemorrhagic activities. It fails to induce platelet aggregation by itself, and does not inhibit the platelet aggregation induced by ADP. However, it exhibits potent myotoxic activity causing inflammatory cell infiltration, severe myoedema, myonecrosis and myolysis in the gastrocnemius muscles of BALB/c mice. Phylogenetic analysis found that that TM-N49 combined with two phospholipase A2s from Trimeresurus stejnegeri, TsR6 and CTs-R6 cluster into one group. Structural and functional analysis indicated that these phospholipase A2s are distinct from the other subgroups (D49 PLA2, S49 PLA2 and K49 PLA2) and represent a unique subgroup of snake venom group II PLA2, named N49 PLA2 subgroup. 相似文献
18.
Three phospholipases A2 purified from cobra venoms and two presynaptically acting neurotoxins that exhibit phospholipase A2 activity were subjected to tryptophan modification with 2-hydroxy-5-nitrobenzyl bromide. Associated with the modification of an increasing number of Trp residues were marked decreases in enzymatic activity and lethality, whereas antigenicity remained unchanged. The degree of exposure of tryptophanyl groups as determined by acrylamide quenching was consistent with the relative reactivity toward 2-hydroxy-5-nitrobenzyl bromide, except for Hemachatushaemachatus phospholipase A2, which showed unusually high reactivity due to its characteristic dimeric conformation. Difference spectra of Trp-modified derivatives differed from those of their native enzymes by the presence of a new positive perturbation between 350 and 500 nm, with a maximum at 415 nm. Scatchard plots revealed only one type of binding site for Ca2+, and the binding abilities of the modified enzymes were not impaired. At pH 8.0, all native enzymes enhanced the emission intensity of 8-anilinonaphthalene sulfonate (ANS) dramatically, and the emission intensity of the ANS-enzyme complex increased or decreased in parallel with increasing concentration of Ca2+ for the respective enzyme. The Trp-modified derivatives did not enhance the emission intensity of ANS at all either in the presence or absence of Ca2+. By means of tryptophan modification, we were able to infer that the tryptophan residues are in the vicinity of the Ca2+ binding site and are directly involved in the binding with ANS. This, together with the suggestion that the hydrophobic pocket that interacts with ANS might be the site of binding of the phospholipase A2 enzyme with the substrate, suggests that the Trp residues in phospholipase A2 enzymes and presynaptic toxins are involved in substrate binding. 相似文献
19.
S. Bhargava Periwal A. Farooq V.L. Bhargava N. Bhatla U. Vij K. Murugesan 《Prostaglandins & other lipid mediators》1996,51(3):191-201
Phospholipase A2 activity was studied in isolated human endometrial predecidual cells, and in human endometrium collected from day 19–23 of the menstrual cycle, by performing a radiochemical assay. Phospholipase A2 activity on day 20 was significantly higher than other days (P < 0.001), and the activity was found to gradually decrease after day 20 of the menstrual cycle. The effects of the hormones estradiol and progesterone, and antihormones tamoxifen and RU 486, were studied on the phospholipase A2 activity in isolated predecidual stromal cells. Estradiol produced a significant stimulatory effect (P < 0.001) on phospholipase A2 activity in predecidual cells, and this effect was antagonized by tamoxifen. The combination of estradiol and tamoxifen was significantly different from estradiol alone (P < 0.001), but not from tamoxifen alone. RU 486 alone significantly increased (P < 0.001) phospholipase A2 activity in predecidual stromal cells. However, progesterone had no effect on phospholipase A2 activity in predecidual stromal cells. 相似文献
20.
Shiho Morisada Ikuhisa Nishida Makoto Kawamukai Hiroyuki Horiuchi 《Bioscience, biotechnology, and biochemistry》2018,82(9):1633-1639
DDL1 encodes a mitochondrial phospholipase A1 involved in acyl chain remodeling of mitochondrial phospholipids and degradation of cardiolipin in Saccharomyces cerevisiae. The deletion of DDL1 leads to respiratory growth defects. To elucidate the physiological role of DDL1, we screened for genes that, when overexpressed, suppress the respiratory growth defect of the DDL1 deletion mutant. Introduction of COQ8, COQ9, or COQ5, which are involved in coenzyme Q (CoQ) synthesis, using a multicopy vector suppressed the respiratory growth defect of the DDL1 deletion mutant. In contrast, introduction of COQ8 using a multicopy vector did not accelerate the growth of the deletion mutants of TAZ1 or CLD1, which encode an acyltransferase or phospholipase A2, respectively, involved in the remodeling of cardiolipin. These results suggest genetic interactions between the mitochondrial phospholipase A1 gene and the genes involved in CoQ synthesis. 相似文献