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1.
Y Oka M Ogawa Y Matsuda A Murata J Nishijima K Miyauchi K Uda T Yasuda T Mori 《Enzyme》1990,43(2):80-88
The high incidence (40.6%) of elevated serum pancreatic phospholipase A2 (PLA2) was demonstrated in patients with various malignancies. Serum PLA2 was significantly increased in cancer patients compared with healthy sex- and age-matched blood donors (358.4 +/- 168.0 vs. 241.7 +/- 69.0 ng/dl; p less than 0.01). No correlation was observed between serum PLA2 and carcinoembryonic antigen (CEA) in these patients. Although patients with advanced and distantly metastatic cancer of the liver, gallbladder and pancreas showed higher PLA2 levels in serum than those with early cancer, patients with other cancers showed no correlation between serum PLA2 and clinical stage. A combined assay of PLA2 and CEA increased the sensitivity of detection of cancers to 60.8%. 相似文献
2.
The interaction between porcine pancreatic phospholipase A2 and low-molecular fragments of its substrate -- lecithine was studied using gel-diffusion of the enzyme in lecithin-agarose plates. When the inhibitor was added, a decrease in the magnitude of cleared areas (l/l0) around the depots filled with enzyme solution was observed. A marked decrease in l/l0 in the presence of alpha- and beta-glycerophosphates supported the statement that the cathionic center is a part of the enzyme active site SII. The potent inhibition of phospholipase activity in the presence of phosphocholine, choline, acetylcholine, thiocholine and acylthiocholines suggests the existence of an anionic center SIII in the active site. This suggestion is supported by intensive inhibition of phospholipase activity by certain, aliphatic amines. It was shown that the center is spaced in the direction of the cathionic center. SII. The main contribution to the binding of the cathionic lecithin part ("head") with the anionic center SIII is probably provided by the ion-ionic interactions. 相似文献
3.
4.
Porcine pancreatic phospholipase A2 (PLA2) was immobilized to Sepharose 4B and porcine serum was passed through this affinity column. Bound substances were eluted by an EDTA-containing buffer and fractionated in a Sepharose 6B column. A single protein peak of the eluate from the latter column was found to inhibit PLA2 activity in a dose-dependent manner in an assay system using radioactive lecithin as a substrate and porcine pancreatic PLA2 as the enzyme source. The serum fraction containing the PLA2 inhibitory protein(s) (PIP) appeared inhomogeneous on SDS-polyacrylamide gel electrophoresis with two major bands close to each other, corresponding to a molecular weight of approximately 60,000. It was concluded that PIP might act as a protective principle against autodigestion in acute pancreatitis and other inflammatory diseases as well as playing a regulatory role in prostaglandin metabolism. 相似文献
5.
The interaction of the lecithin molecule fragments and their analogues with phospholipase C Cl. perfringens was studied by gel-diffusion in agarose-lecithin gels. It was found intense inhibition of phospholipase C activity in the presence of cathionic compounds; this phenomenon shows the existence of anionic centre in the active site of enzyme. The esteric centre is probably hydrophobic nature and is not capable to bind the negatively charged groups. However, phosphoserine, phosphothreonine, gamma-aminobutyric, aspartic and glutamic acids can interact with an additional cathionic centre, whose location in phospholipase C differs from that in pancreatic phospholipase A2. 相似文献
6.
微孔比色法采用合成的磷脂类似物2-硫代十六酰乙基磷酸胆碱作底物,在多孔聚苯乙烯板的小孔中反应,并用酶联免疫检测器连续测定和记录吸收值.同时应用此法及滴定法检测酶活力,从猪胰腺中制备了一种分子量低(14.3kD),对热、酸稳定,活性依赖Ca2+的PLA2.两种方法检测结果具有可比性,而微孔比色法同时可测多个样品,有节约样品,灵敏度较高等优点.微孔比色法特别适用于大量的样品测定,如拮抗剂筛选、临床样品及制备酶时层析级分的检测等. 相似文献
7.
Using a specific polyclonal antibody raised against rat pancreatic phospholipase A2 (PLA2), we investigated the localization of the enzyme in the rat pancreas and stomach by light and electron microscopy. In the pancreas, the enzyme was localized in the acinar cells, whereas the pancreatic islets showed no immunoreaction. In the stomach, the PLA2 reactive with the anti-pancreatic PLA2 antibody was distributed exclusively in the gastric glands, but not in the gastric pits or the pyloric glands. On the section of the stomach subjected to immuno- and PAS-staining, immunopositive cells were not the PAS-positive cells located in the gastric pit and the neck region of the gastric gland. Immunopositive cells were present from the neck to the bottom of the gastric gland. Immunoelectron microscopic observation revealed that the immunogold-labeled cell had a highly-developed rough endoplasmic reticulum in the basal cytoplasm and characteristic zymogen granules in the apical cytoplasm. Taking into account the cell position in the gastric gland, the immunopositive cell could therefore be identified as a chief cell. Since no double stainability with PLA2 and PAS was observed in the same cell, it is suggested that PLA2 could be used cytochemically as a marker enzyme of the chief cell in the gastric gland at the light-microscopic level. From the immunoelectron microscopic findings, we believe that the PLA2 in the stomach is released into the lumen of the stomach by exocytosis and could function as a digestive enzyme in the alimentary tract, like the PLA2 secreted from the pancreas. Other possible roles of the PLA2 in the stomach are discussed. 相似文献
8.
The edema inducing activity of phospholipase A2 (PLA2) enzymes from snake venoms and porcine pancreas was investigated using mouse paw as experimental model. All ten PLA2 enzymes exhibited potent edema inducing activity. PLA2, however, is generally not the major edema inducing component of snake venom. Chemical modification studies indicated that enzymatic activity of PLA2 was required for its edema inducing activity. All PLA2 enzymes examined displayed a rapid onset edema which was suppressed by pretreatment of the mice with antihistamine. Dexamethasone pretreatment also inhibited edemas elicited by some PLA2 enzymes. 相似文献
9.
Purification of a soluble phospholipase A2 from synovial fluid in rheumatoid arthritis 总被引:2,自引:0,他引:2
A soluble phospholipase A2 (PLA2) was purified 4,500-fold from human rheumatoid synovial fluid. Preparative sodium dodecyl sulfate polyacrylamide gel electrophoresis yielded two bands of PLA2 activity of molecular weights 15,000 and 17,000 and pl 4.2-5.0. Purified PLA2 had absolute 2-acyl specificity, and hydrolyzed phosphatidylcholine with optimal activity at pH 7.5-8.0 and phosphatidylethanolamine with optimal activity at pH 7.0. Human synovial fluid PLA2 did not cross-react with anti-human pancreatic PLA2, as tested by radioimmunoassay. 相似文献
10.
We have developed a phospholipase A2(PLA2)-producing system using Saccharomyces cerevisiae. A 456-bp synthetic DNA fragment was constructed encoding bovine pancreatic phospholipase A2 (proPLA2; zymogen) along with the signal sequence of dog pancreatic PLA2. Yeast-preferred codons were chosen and unique restriction enzyme sites were incorporated. 22 oligodeoxynucleotides that varied in size from 33 to 48 nucleotides were chemically synthesized and assembled into the DNA fragment, which was then placed under the control of the yeast acid phosphatase repressible promoter. The resulting plasmid, transformed into S. cerevisiae, directed the synthesis of about 2.8 micrograms/ml of PLA2, most of which was secreted into the culture fluid. The secreted PLA2 comprised 18 to 26% of active enzyme, the remainder being proenzyme. Both had the expected N-terminal amino acid sequences, indicating that the yeast accurately released the signal peptide and the activation peptide (N-terminal heptapeptide of proPLA2). The specific activity of PLA2 thus produced is the same as that of the authentic bovine enzyme. 相似文献
11.
A pancreatic-type phospholipase A2 in rat gastric mucosa 总被引:1,自引:0,他引:1
A phospholipase A2, which is immuno-crossreactive with the anti-rat pancreatic phospholipase A2 antibody, is present in rat gastric mucosa. The content of the enzyme in the gastric mucosa was comparable to that in the pancreas, but the specific activity in the gastric mucosa homogenate (60.7 +/- 19.5 nmol/min/mg) was higher than that in the pancreas homogenate (3.16 +/- 0.77 nmol/min/mg). A greater proportion of the enzyme was found in the particulate fraction. The gastric enzyme and its proenzyme were purified from the supernatant. The amino acid sequence of the N-terminal 15 residues of the gastric enzyme was determined and found to be identical with that of rat pancreatic phospholipase A2. Like the pancreatic proenzyme, the gastric proenzyme was activated on trypsin treatment. 相似文献
12.
A phospholipase A2 in the supernatant fraction of rat spleen. Its similarity to rat pancreatic phospholipase A2 总被引:2,自引:0,他引:2
H Tojo T Ono S Kuramitsu H Kagamiyama M Okamoto 《The Journal of biological chemistry》1988,263(12):5724-5731
Rat spleen supernatant contained two forms of calcium-dependent cellular phospholipase A2 which could be separated from each other by TEAE-cellulose chromatography. The phospholipase A2, named PLA2 S-1, present in the major flow-through fraction was purified to homogeneity. The structural and catalytic properties of splenic PLA2 S-1 were systematically compared with those of rat pancreatic phospholipase A2. Structural evidence, including the sequence of the N-terminal 32 residues, peptide maps obtained on Achromobacter protease I digestion and cyanogen bromide cleavage, and the amino acid composition, showed the close similarity of the two enzymes. Their catalytic and immunochemical properties were also similar. These results demonstrated the existence of a pancreatic type phospholipase A2 in a non-pancreatic organ as a member of the cellular phospholipases A2 and suggest the potential functional involvement of pancreatic type phospholipase A2 in cellular phospholipid metabolism. 相似文献
13.
We have isolated, from canine pancreatic juice, two 14-kDa proteins with secretin-releasing activity that had N-terminal sequence homology with canine pancreatic phospholipase A2 (PLA2). In this study we have obtained evidence that secretin-releasing activity is an intrinsic property of pancreatic PLA2. Porcine pancreatic PLA2 from Sigma or Boehringer Mannheim was fractionated into several peaks by reverse phase high performance liquid chromatography. They were tested for stimulation of secretin release from murine neuroendocrine intestinal tumor cell line STC-1 and secretin cells enriched mucosal cell preparations isolated from rat upper small intestine. Each enzyme preparation was found to contain several components of secretin-releasing activity. Each bioactive fraction was purified to homogeneity by rechromatography and then subjected to mass spectral analysis and assays of PLA2 and secretin-releasing activities. It was found that the fraction with highest enzymatic activity also had the highest secretin-releasing activity and the same Mr as porcine pancreatic PLA2. Moreover, it also had the same N-terminal amino acid sequence (up to 30 residues determined) as that of porcine pancreatic PLA2, suggesting that it was identical to the enzyme. Purified porcine pancreatic PLA2 also stimulated secretin release concentration-dependently from both STC-1 cells and a mucosal cell preparation enriched in secretin-containing endocrine cells isolated from rat duodenum. Abolishment of the enzymatic activity by pretreatment with bromophenacyl bromide did not affect its secretin-releasing activity. The stimulatory effect of purified pancreatic PLA2 on secretin secretion from STC-1 cells was inhibited by an L-type Ca2+ channel blocker, by down-regulation of protein kinase C or by pretreatment of the cell with pertussis toxin. It is concluded that porcine pancreatic PLA2 possesses an intrinsic secretin-releasing activity that was independent of its enzymatic activity. This action is pertussis toxin-sensitive and is in part dependent on Ca2+ influx through the L-type channel and activation of protein kinase C. 相似文献
14.
Jiménez M Cabanes J Gandía-Herrero F Escribano J García-Carmona F Pérez-Gilabert M 《Analytical biochemistry》2003,319(1):131-137
This paper describes a simple continuous spectrophotometric method for assaying phospholipase A(2) (PLA(2)) activity. The procedure is based on a coupled enzymatic assay, using dilinoleoyl phosphatidylcholine as phospholipase substrate and lipoxygenase as coupling enzyme. The linoleic acid released by phospholipase was oxidized by lipoxygenase and then phospholipase activity was followed spectrophotometrically by measuring the increase in absorbance at 234 nm due to the formation of the corresponding hydroperoxide from the linoleic acid. The optimal assay concentrations of hog pancreatic phospholipase A(2) and lipoxygenase were established. PLA(2) activity varied with pH, reaching its optimal value at pH 8.5. Scans of the deoxycholate concentration pointed to an optimal detergent concentration of 3mM. Phospholipid hydrolysis followed classical Michaelis-Menten kinetics (V(m)=1.8 microM/min, K(m)=4.5 microM, V(m)/K(m)=0.4 min(-1)). This assay also allows PLA(2) inhibitors, such as p-bromophenacyl bromide or dehydroabietylamine acetate, to be studied. This method was proved to be specific since there was no activity in the absence of phospholipase A(2). It also has the advantages of a short analysis time and the use of commercially nonradiolabeled and inexpensive substrates, which are, furthermore, natural substrates of phospholipase A(2). 相似文献
15.
The crystal structure of an acidic phospholipase A(2) from Ophiophagus hannah (king cobra) has been determined by molecular replacement at 2.6-A resolution to a crystallographic R factor of 20.5% (R(free)=23.3%) with reasonable stereochemistry. The venom enzyme contains an unusual "pancreatic loop." The conformation of the loop is well defined and different from those in pancreas PLA(2), showing its structural variability. This analysis provides the first structure of a PLA(2)-type cardiotoxin. The sites related to the cardiotoxic and myotoxic activities are explored and the oligomer observed in the crystalline state is described. 相似文献
16.
J Dimberg C Gustafson-Sv?rd B Westr?m C Tagesson P S?derkvist 《Biochimica et biophysica acta》1992,1130(1):47-51
The postnatal development of group I phospholipase A2 (group I PLA2) in the glandular stomach and pancreas of neonatal rats was investigated. The amounts of group I PLA2 mRNA (and also the PLA2 enzymatic activity) in the glandular stomach mucosa increased with age in 3-60-day-old animals. This postnatal development of rat stomach group I PLA2 mRNA agreed with that of group I PLA2 mRNA of the rat pancreas, and thus seems to follow the general development of the gastrointestinal tract during the neonatal period. The latter was further supported by the finding that maturation of group I PLA2 in both the stomach and pancreas was induced precociously in rats treated with cortisone acetate. It is suggested that the stomach group I PLA2 is involved in mucosal eicosanoid production. 相似文献
17.
The cellular localization of phospholipase A2 (PLA2) was examined in normal and ras-transformed rat fibroblasts using immunohistochemical techniques. Polyclonal antibodies were generated against porcine pancreatic PLA2 and were affinity purified for use in this study. The antibodies detected a 16-kD band on immunoblots of total cellular proteins from fibroblasts. In cell-free assays of phospholipase A2 activity, the purified antibodies inhibited the bulk of the enzyme activity whereas control IgG preparations had no effect. Immunofluorescence microscopy indicated that PLA2 was diffusely distributed throughout the cell. Increased concentration of PLA2 was detected under membrane ruffles in normal and ras-transformed cells. Specific immunofluorescence staining was also detected on the outer surface of the normal cells. Immunoelectron microscopy demonstrated the increased accumulation of PLA2 in membrane ruffles and also revealed the presence of the enzyme in microvilli and its association with intracellular vesicles. Ultrastructural localization of PLA2 and the ras oncogene protein, using a double immunogold labeling technique, indicated a spatial proximity between PLA2 and ras proteins in the ruffles of ras-transformed cells. The possible role of PLA2 in the structural rearrangements that underlie membrane ruffling is discussed. 相似文献
18.
G Lin J Noel W Loffredo H Z Stable M D Tsai 《The Journal of biological chemistry》1988,263(26):13208-13214
A great mystery in the mechanism of phospholipase A2 (PLA2) and many other lipolytic enzymes is the "interfacial activation" induced by micellar but not monomeric substrates. Equally mysterious is the lack of interfacial activation in bee venom PLA2, as opposed to PLA2s from pancreas and other sources. We have probed these problems using the conformationally restricted short-chain cyclopentano-analogues of diacylphosphatidylcholine (Cp-DCnPC, all-trans isomer). In the reaction catalyzed by bovine pancreatic PLA2, Cp-DC8PC behaved differently from DC8PC in that its monomers and micelles showed comparable activities (but lower than the activity of DC8PC). This result suggests that the activity of PLA2 can be regulated by substrate conformation and supports the "substrate conformation model" (Wells, M. A. (1974) Biochemistry 13, 2248-2257), but raises a question as to whether Cp-DC8PC mimics monomers or micelles of DC8PC. Conformational analysis by 1H NMR revealed that monomeric Cp-DC8PC was conformationally restricted near the carbonyl region, a property characteristic of micelles. Thus, monomeric CP-DC8PC can be considered as a conformational analogue of micelles, but the important structural feature lies in the CH2COO region instead of the glycerol backbone. CP-DC8PC was then used to test a previous proposal that the bee venom PLA2 hydrolyzes monomers but not micelles (which would predict little or no activity for Cp-DC8PC since its conformation is micelle-like whether below or above its critical micelle concentration). The results showed that Cp-DC8PC is a relatively good substrate for the bee venom PLA2 in comparison with the pancreatic PLA2. This and other evidence together suggest that the bee venom PLA2 is not sensitive to the conformation of monomeric and micellar substrates and hydrolyzes both monomers and micelles. The results in both PLA2s demonstrate the usefulness of cyclopentano-phospholipids in probing the mechanism of phospholipases and the roles of substrate conformation in the catalysis of PLA2. 相似文献
19.
A gene coding for the (pro)phospholipase A2 (PLA2) from bovine pancreas has been designed, synthesized, and expressed in Escherichia coli. The gene was designed with a variety of restriction sites that will facilitate future mutagenesis studies. Codons occurring frequently in prokaryotic systems were chosen whenever possible. The total gene spans 404 base pairs and was divided into 33 oligonucleotides. The gene was constructed in two halves of 224 and 180 base pairs from the oligonucleotides by the shotgun ligation technique using pBSM13- as the cloning vehicle. The two fragments were then ligated and cloned into pBSM13- to complete the gene. The (pro)PLA2 gene was then verified by restriction site mapping and dideoxy sequencing. The gene was expressed to high levels from a high copy number vector, designated as pJPN, derived from the E. coli secretion vector pIN-III-ompA3. Although the protein failed to be excreted and was in the form of insoluble inclusion body, active PLA2 could be obtained by renaturation of the inclusion body pellet followed by tryptic activation, which removes the signal sequence and the pro-peptide of proPLA2. The PLA2 thus obtained reacted with the antisera raised against the natural PLA2 purified from bovine pancreas, and the specific activity of the expressed PLA2 was identical to that of the natural PLA2. The shotgun ligation and synthetic gene approaches are simple and inexpensive and can be adapted to express most of the enzymes in the phospholipase A2 family. 相似文献
20.
Antibodies raised against porcine pancreatic phospholipase A2 (PLA2) react in immunoblottings with both the antigen as well as with one protein band of about 14 kDa from hamster spermatozoa extracts. Immunoblottings of proteins extracted from spermatozoon head and tail fractions also show similar results. Anti-PLA2 purified IgGs were employed for light and electron microscopic immunocytochemistry in order to detect PLA2 in hamster cauda epididymal spermatozoa. When whole mount spread spermatozoa were used under light (employing the PAP complex) or electron microscopy (using anti-rabbit gold conjugated), the acrosomal area of the gametes shows a noticeable labelling; a characteristic which is not observed in samples treated with the pre-immune serum. Immunocytochemistry undertaken in ultrathin sections from spermatozoon samples embedded in Lowicryl, demonstrates that the antigen appears preferentially distributed in the acrosome. Besides, sperm tails showed a scattered distribution of gold granules in the mitochondria of the midpiece. Results suggest that the antibody used recognizes a PLA2 which is preferentially located in the acrosome and mitochondria. On the other hand, the presence of a surface PLA2 in the plasma membrane covering the acrosome is suggested. This surface PLA2 would be probably related to the acrosome reaction phenomenon that occurs in the spermatozoon before penetrating the oocyte. 相似文献