Measurement of Human Phospholipase a in Arthritis Plasma Using a Newly Developed Sandwich Elisa

Smith, Gilbert M.; Ward, Robyn L.; McGuigan, L; Rajkovic, I. A.; Scott, Kieran F.

doi:10.1093/rheumatology/31.3.175

Cited by 68 publications

(41 citation statements)

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“…Construction by site-directed mutagenesis, expression, purification, and characterization of the sPLA 2 -IIA mutant enzyme H 48 Q will be described elsewhere. 8 H 48 Q was quantified by enzyme-linked immunosorbent assay (11). The mutant protein had no detectable enzyme activity in our E. coli membrane assay, but an activity of 2 to 4% of wild-type was reported in other studies (12).…”

Section: Methodsmentioning

confidence: 60%

“…sPLA 2 -IIA was purified from conditioned media produced by a Chinese hamster ovary cell line (5A2) stably transfected with the human sPLA 2 -IIA cDNA, as described previously (10), and quantified by enzymelinked immunosorbent assay (11). sPLA 2 -IIA contained Ͻ0.1 ng of endotoxin per milligram of protein (Limulus amebocyte lysate pyrochrome assay; Associates of Cape Cod, Falmouth, MA) and was enzymatically active in a [ 3 H]arachidonate-labeled Escherichia coli membrane assay (10).…”

Section: Methodsmentioning

confidence: 99%

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Oncogenic Action of Secreted Phospholipase A2 in Prostate Cancer

et al. 2004

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Mortality from prostate cancer is associated with progression of tumors to androgen-independent growth and metastasis. Eicosanoid products of both the cyclooxygenase (COX) and lipoxygenase (LOX) pathways are important mediators of the proliferation of prostate cancer cells in culture and regulate tumor vascularization and metastasis in animal models. Pharmacologic agents that block either COX or LOX products effectively reduce the size of prostate cancer xenografts. Phospholipase A 2 (PLA 2 ) enzymes regulate the provision of arachidonic acid to both COX-and LOX-derived eicosanoids, and a secreted form of the enzyme (sPLA 2 -IIA) is elevated in prostate cancer tissues. Here, we show by immunohistochemistry, in patients receiving androgen ablation therapy, that sPLA 2 -IIA remains elevated in remaining cancer cells relative to benign glands after treatment. Furthermore, sPLA 2 -IIA expression seen in benign glands is substantially decreased after androgen depletion, whereas cytosolic PLA 2 -␣ (cPLA 2 -␣) levels are unchanged. sPLA 2 -IIA mRNA expression is detectable and inducible by androgen (0.01-10 nmol

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Section: Methodsmentioning

confidence: 60%

Section: Methodsmentioning

confidence: 99%

Oncogenic Action of Secreted Phospholipase A2 in Prostate Cancer

et al. 2004

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“…Enzyme was stored at 4°C in 35% acetonitrile in 0.1% trifluoroacetic acid and diluted immediately prior to use. The enzyme was initially quantified by amino acid analysis, and the concentration was checked routinely using an enzyme-linked immunosorbent assay developed in our laboratory (8). Snake venom PLA 2 from Crotalus durissus and Crotalus atrox were purchased from Boehringer Mannheim and Sigma, respectively.…”

Section: Methodsmentioning

confidence: 99%

“…Type I PLA 2 of mammalian origin is mainly found in the pancreas (2), while the type II enzyme is stored in secretory granules in blood platelets, macrophages, and neutrophils (3, 4) and in tissues is localized in mast cells, Paneth cells, and chondrocytes (5, 6). It is also found in fluids derived from patients with inflammatory conditions (7,8) and is induced in several cell types in response to inflammatory stimuli (5). Despite differences in their primary sequences (ϳ30% homology only), crystal structures of PLA 2 from bovine pancreas (type I) and human synovial fluid (type II) are almost superimposable and, not surprisingly, the active sites from both types of enzymes are virtually identical (9).…”

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confidence: 99%

Native Peptide Inhibition

Tseng

Inglis

Scott

1996

Journal of Biological Chemistry

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The binding of low molecular weight type II phospholipase A 2 (EC 3.1.1.4) to membrane surfaces and hydrolysis of phospholipid are thought to involve the formation of a hydrophobic channel into which a single substrate molecule diffuses before cleavage. The floor and right side of the channel are provided by hydrophobic residues 2, 5, and 9 of an amphipathic amino-terminal helix. The channel is postulated to form via a conformational change in this helix and inward movement of a hydrophobic flap (residue 69 side chain). We show that the amino-terminal tryptic peptide of human type II phospholipase A 2 forms a noncovalent complex with the tryptic peptide from residues 70 -74 of the enzyme. Further, the 70 -74-peptide sequence (FLSYK) dose-dependently inhibits phospholipid hydrolysis in a mixed micelle assay. This native peptide inhibition also occurred with type II enzymes from Crotalus durissus and Crotalus atrox, which have different amino acid sequences at the amino terminus as well as different 70 -74 regions of the molecules. Despite significant conservation of tertiary structure among the enzymes, inhibition by each peptide is specific to the enzyme from which the peptide sequence is derived. We propose that these native peptides inhibit enzyme activity via a sequencespecific, noncovalent interaction with the amino-terminal residues of the enzyme, thereby preventing the conformational change on binding to the micelle interface. These experiments demonstrate a new method for specific inhibition of phospholipase A 2 which, in principle, would be applicable to other biologically active polypeptides and proteins.Secretory phospholipases A 2 (PLA 2 ) 1 are a family of calciumdependent 14-kDa enzymes that catalyze the hydrolysis of the S n 2 fatty acyl ester bond of phospholipids (1). These enzymes, which were first described as components of snake venoms and later in mammals, are classified into two major classes, type I and type II, based on their primary structures. Type I PLA 2 of mammalian origin is mainly found in the pancreas (2), while the type II enzyme is stored in secretory granules in blood platelets, macrophages, and neutrophils (3, 4) and in tissues is localized in mast cells, Paneth cells, and chondrocytes (5, 6). It is also found in fluids derived from patients with inflammatory conditions (7,8) and is induced in several cell types in response to inflammatory stimuli (5). Despite differences in their primary sequences (ϳ30% homology only), crystal structures of PLA 2 from bovine pancreas (type I) and human synovial fluid (type II) are almost superimposable and, not surprisingly, the active sites from both types of enzymes are virtually identical (9). Asp-99 and His-48 form an essential catalytic dyad in the fashion of serine proteases (10). Tyr-52 and Tyr-73 appear to be associated with these two residues via a hydrogen bonding network, but there is evidence (11) that Tyr-52 is not essential for the catalytic reaction.From structure-function studies of type I and II enzymes, the first eight residu...

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“…PLA 2 s of mammalian origin were found in the pancreas (2), blood platelets, macrophages, and neutrophils (3,4). These are also found in fluids derived from patients with inflammatory conditions (5,6) and are induced in several cell types in response to inflammatory stimuli (4). It is understood that the concentrations of PLA 2 enzymes in serum and tissues correlate with the disease severity in several immune-mediated inflammatory pathologies in humans.…”

mentioning

confidence: 99%

Crystal Structure of a Complex Formed between a Snake Venom Phospholipase A2 and a Potent Peptide Inhibitor Phe-Leu-Ser-Tyr-Lys at 1.8 Å Resolution

Chandra

Jasti

Kaur

et al. 2002

Journal of Biological Chemistry

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Phospholipase A 2 is an important enzyme involved in the production of prostaglandins and their related compounds causing inflammatory disorders. Among the several peptides tested, the peptide Phe-Leu-Ser-Tyr-Lys (FLSYK) showed the highest inhibition. The dissociation constant (K d ) for this peptide was calculated to be 3.57 ؎ 0.05 ؋ 10 ؊9 M. In order to further improve the degree of inhibition of phospholipase A 2 , a complex between Russells viper snake venom phospholipase A 2 and a peptide inhibitor FLSYK was crystallized, and its structure was determined by crystallographic methods and refined to an R-factor of 0.205 at 1.8 Å resolution. Phospolipase A 2 (EC 3.1.1.4, PLA 2 ) 1 enzyme specifically cleaves the sn-2 acyl ester bond of phospholipids (1). These enzymes were first described as components of snake venoms and were later identified in mammals. PLA 2 s of mammalian origin were found in the pancreas (2), blood platelets, macrophages, and neutrophils (3, 4). These are also found in fluids derived from patients with inflammatory conditions (5, 6) and are induced in several cell types in response to inflammatory stimuli (4). It is understood that the concentrations of PLA 2 enzymes in serum and tissues correlate with the disease severity in several immune-mediated inflammatory pathologies in humans. These are also associated with the onset of rheumatoid arthritis (7, 8) and septic shock (5, 9). Therefore, it is of great interest to develop specific inhibitors for each of these enzymes. Several crystal structures of the tetrahedral mimic inhibitors with an sn-2-phosphate, substituent, L-1-(O-octyl-2-(heptyl phosphonyl)-sn-glycero-3-phosphoethanolamine, complexed with secreted PLA 2 from Chinese cobra venom (10), bee venom (11), and inflammatory exudates (12) have been reported. Also the crystal structure of an sn-2-acyl-amino analogue of phospholipids complexed with the engineered (without the residues of the surface 62-66) porcine PLA 2 (13) has been determined. The crystal structures of the complexes of bovine pancreatic PLA 2 with an inhibitor n-dodecyl-phosphorylcholine (14) and human synovial PLA 2 with an acyl amino analogue of phospholipids (15) are also of some interest. Recently, a series of indole inhibitors of human secretory PLA 2 were developed (16). Further, there is an example of a natural inhibition (17) where specific interactions from one molecule of a heterodimer acts as a natural inhibitor for the other. The synthetic peptides derived from the primary sequence of the PLA 2 itself were also studied as specific inhibitors (18,19). The kinetic studies (18, 19) support their high inhibitory potencies. These peptide inhibitors were stable against acyl esterase as well as phospholipase A 1 , C, and D. Therefore, they were suited for in vivo studies where phospholipids may be more readily metabolized. As part of the program on structure-based rational design of specific potent inhibitors using a model snake venom phospholipase A 2 , we have synthesized a number of peptides (20). One of these ...

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Measurement of Human Phospholipase a in Arthritis Plasma Using a Newly Developed Sandwich Elisa

Cited by 68 publications

References 0 publications

Oncogenic Action of Secreted Phospholipase A2 in Prostate Cancer

Oncogenic Action of Secreted Phospholipase A2 in Prostate Cancer

Native Peptide Inhibition

Crystal Structure of a Complex Formed between a Snake Venom Phospholipase A2 and a Potent Peptide Inhibitor Phe-Leu-Ser-Tyr-Lys at 1.8 Å Resolution

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