Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Two crystal forms (P6(3) and R3) of human annexin V have been crystallographically refined at 2.3 A and 2.0 A resolution to R-values of 0.184 and 0.174, respectively, applying very tight stereochemical restraints with deviations from ideal geometry of 0.01 A and 2 degrees. The three independent molecules (2 in P6(3), 1 in R3) are similar, with deviations in C alpha positions of 0.6 A. The polypeptide chain of 320 amino acid residues is folded into a planar cyclic arrangement of four repeats. The repeats have similar structures of five alpha-helical segments wound into a right-handed compact superhelix. Three calcium ion sites in repeats I, II and IV and two lanthanum ion sites in repeat I have been found in the R3 crystals. They are located at the convex face of the molecule opposite the N terminus. Repeat III has a different conformation at this site and no calcium bound. The calcium sites are similar to the phospholipase A2 calcium-binding site, suggesting analogy also in phospholipid interaction. The center of the molecule is formed by a channel of polar charged residues, which also harbors a chain of ordered water molecules conserved in the different crystal forms. Comparison with amino acid sequences of other annexins shows a high degree of similarity between them. Long insertions are found only at the N termini. Most conserved are the residues forming the metal-binding sites and the polar channel. Annexins V and VII form voltage-gated calcium ion channels when bound to membranes in vitro. We suggest that annexins bind with their convex face to membranes, causing local disorder and permeability of the phospholipid bilayers. Annexins are Janus-faced proteins that face phospholipid and water and mediate calcium transport.
J Mol Biol 1992 Feb 05
PMID:Crystal and molecular structure of human annexin V after refinement. Implications for structure, membrane binding and ion channel formation of the annexin family of proteins. 131 70

HeLa cells attach to a variety of substrata but spread only on collagen or gelatin. Spreading is dependent on collagen-receptor upregulation, clustering, and binding to the cytoskeleton. This study examines whether second messengers are involved in initiating the spreading process on gelatin. The levels of cytosolic free calcium ([Ca++]i), cAMP, and cytoplasmic pH (pHi) do not change during cell attachment and spreading. However, a basal level of [Ca++]i and an alkaline pH(i) are required for spreading. There is an activation of protein kinase C (PKC) and a release of arachidonic acid (AA) on attachment and before cell spreading. Inhibition of PKC does not block cell spreading, indicating that PKC activation is not essential for spreading. Inhibition of phospholipase A2 blocks cell spreading, whereas addition of exogeneous AA overcomes this inhibitory effect. Among AA metabolic pathways, inhibitors of lipoxygenase (LOX) block cell spreading, suggesting that a LOX product(s) formed from AA initiates spreading. Clustering receptors for collagen with polyclonal antibodies, or with anti-collagen-receptor antigen-binding fragments (Fab) in combination with a secondary antibody, induce AA release. Also, AA is released when cells attach to either immobilized gelatin or immobilized Arg-Gly-Asp (RGD) peptide. Thus, AA is released whenever receptor clustering is observed. Receptor occupancy is not sufficient to release AA; when cells are treated with gelatin or RGD peptide in solution or anti-collagen-receptor Fab fragments without secondary antibody, conditions where receptor clustering is not observed, AA is not released. Thus, a LOX metabolite(s) of AA formed by collagen-receptor clustering is a second messenger(s) that initiates HeLa cell spreading. LOX inhibitors also block the spreading of bovine aortic endothelial cells, chicken embryo fibroblasts, and CV-1 fibroblasts on gelatin or fibronectin, indicating that other cells might use the same second messenger system in initiating cell-substratum adhesion.
Mol Biol Cell 1992 May
PMID:Spreading of HeLa cells on a collagen substratum requires a second messenger formed by the lipoxygenase metabolism of arachidonic acid released by collagen receptor clustering. 131 41

The role of the phosphorylation and dephosphorylation of sarcolemma and that of the alteration of membrane lipids in the endotoxin-induced impairment of the ATP-dependent Ca2+ transport in canine cardiac sarcolemma were investigated. The results indicate that the ATP-dependent Ca2+ transport in canine cardiac sarcolemma was decreased by 30-35% 4 h after endotoxin administration. Phosphorylation of sarcolemma by the catalytic subunit of the cAMP-dependent protein kinase or calmodulin stimulated ATP-dependent Ca2+ transport in both groups, however, the phosphorylation-stimulated activities remained significantly lower in endotoxic animals. Dephosphorylation of sarcolemma decreased ATP-dependent Ca2+ transport in both groups, yet, the time required to reach maximal dephosphorylation was reduced from 120 to 90 min 4 h post-endotoxin. Analysis of sarcolemmal membranes reveals that phosphatidylcholine and phosphatidylethanolamine contents were decreased while their respective lysophosphatide levels were increased significantly after endotoxin injection. Digestion of control heart sarcolemma with phospholipase A2 inhibited Ca2+ transport and the inhibition was reversible by phosphatidylcholine. The inhibition caused by the in vivo administration of endotoxin was completely reversible by the addition of phosphatidylcholine. Based on these data, it is concluded that endotoxin administration impairs ATP-dependent Ca2+ transport in canine cardiac sarcolemma and that the impairment may be due to i) a defective phosphorylation of sarcolemma; ii) a reduced number of Ca2+ pumps; iii) an accelerated dephosphorylation of sarcolemma; and iv) an alteration in membrane phospholipid profile in response to phospholipase A activation.
Mol Cell Biochem 1992 Jun 26
PMID:Heart sarcolemmal Ca2+ transport in endotoxin shock: II. Mechanism of impairment in ATP-dependent Ca2+ transport. 132 89

We have characterized the ANF-R2 receptor-mediated inhibition of adenylate cyclase with respect to its modulation by several regulators. ANF (99-126) inhibits adenylate cyclase activity only in the presence of guanine nucleotides. The maximal inhibition (approximately 45%) was observed in the presence of 10-30 microM GTP gamma S, and at higher concentrations, the inhibitory effect of ANF was completely abolished. ANF-mediated inhibition was not dependent on the presence of monovalent cations, however Na+ enhanced the degree of inhibition by about 60%, whereas K+ and Li+ suppressed the extent of inhibition by about 50%. On the other hand, divalent cation, such as Mn2+ decreased the degree of inhibition in a concentration dependent manner, with an apparent Ki of about 0.7 mM, and at 2 mM; the inhibition was completely abolished. In addition, proteolytic digestion of the membranes with trypsin (40 ng/ml) resulted in the attenuation of ANF-mediated inhibition of adenylate cyclase. Other membrane disrupting agents such as neuraminidase and phospholipase A2 treatments also inhibited completely, the ANF-mediated inhibition of enzyme activity. N-Ethylmaleimide (NEM), phorbol ester and Ca(2+)-phospholipid dependent protein kinase (C-kinase) which have been shown to interact with inhibitory guanine nucleotide regulating protein (Gi) also resulted in the attenuation of ANF-mediated inhibition of adenylate cyclase activity. These results indicate that in addition to the Gi, the phospholipids and glycoproteins may also play an important role in the expression of ANF-R2 receptor-mediated inhibition of adenylate cyclase.
Mol Cell Biochem 1992 Jul 06
PMID:Characterization of ANF-R2 receptor-mediated inhibition of adenylate cyclase. 132 94

A secreted form of phospholipase A2 (PLA2) is thought to play an important role in inflammatory diseases. To characterize this enzyme the cDNA encoding a low molecular weight PLA2 was cloned from a human placental cDNA library. The cDNA encoding the human PLA2 was subcloned into an expression vector and subsequently transfected into Chinese hamster ovary (CHO) cells. A stable CHO cell clone, secreting ca 1 mg/L of recombinant PLA2 into the medium, was scaled up in culture to 180 L. The recombinant enzyme was purified from the cell supernatant to apparent homogeneity by a novel procedure combining adsorption to poly(vinylidene difluoride) membranes, ion exchange chromatography and size exclusion chromatography. The final recovery of PLA2 activity was 58%. A direct comparison between the purified recombinant human PLA2 and PLA2 purified from human synovial fluid, including molecular weight, antigenicity, ionic dependence, substrate specificity and sensitivity to known PLA2 inhibitors, indicated that the two enzymes exhibit identical biochemical properties. These results show that the recombinant PLA2 can be efficiently expressed and purified in sufficient quantities to characterize the enzyme active site, to aid in the rational development of PLA2 inhibitors as potential anti-inflammatory drugs, and to investigate further the role of PLA2 in inflammatory disease.
J Mol Recognit 1992 Dec
PMID:Recombinant human secretory phospholipase A2: purification and characterization of the enzyme for active site studies. 133 83

Crotoxin is the major neurotoxic component of the venom of the South American rattlesnake, Crotalus durissus terrificus. The crotoxin molecule is composed of two subunits: a basic and weakly toxic phospholipase A2 (PLA2) called component-B (CB), and an acidic, nonenzymatic and nontoxic subunit called component-A (CA). Crotoxin exists as a mixture of several isoforms (or variants) resulting from the association of several subunit isoforms. We prepared monoclonal antibodies (MAbs) against each isolated subunit. Six anti-CA MAbs and eight anti-CB MAbs were tested for their cross-reactivities with each subunit and with other toxic and nontoxic PLA2s. Four of the six anti-CA MAbs cross-reacted with CB, whereas only one of the eight anti-CB MAbs cross-reacted with CA. Two anti-CB MAbs were found to cross-react with agkistrodotoxin, a single chain neurotoxic PLA2 purified from the venom of Agkistrodon blomhoffii brevicaudus. We determined the dissociation constants of each MAb for CA and CB isoforms and their capacities to neutralize the lethality and to inhibit the catalytic activity of crotoxin. We defined three epitopic regions on CA and four on CB, and used a schematic representation of the two subunits to characterize these epitopic regions with respect to: (1) the "toxic" and the "catalytic" sites of CB, and (2) the zone of interaction between the two subunits. We propose three-dimensional structures of the crotoxin subunits in which we localize amino acid residues that might be involved in the epitopic regions described here.
Mol Immunol
PMID:Immunochemical analysis of a snake venom phospholipase A2 neurotoxin, crotoxin, with monoclonal antibodies. 137 31

In this study, the release of lysophospholipids (to depict phospholipase A2 activity) and diacylglycerols (DG) (to depict stimulated hydrolysis of polyphosphoinositides) was related to the decapitation-induced release of free fatty acid (FFA) in the mouse brain. To assay for lysophospholipids, Balb/c mice were injected intracerebrally with either [3H]choline or [3H]inositol for 16 h in order to label their respective phospholipids. These lipids were examined at various times (30 s to 30.5 min) after decapitation. Between 30 s and 1.5 min after decapitation, the rate of FFA release (3 micrograms FA/mg FA in phospholipids/min) was three times more rapid than that between 10 and 15 min (0.8 microgram FA/mg FA in phospholipids/min). FFA released during the initial phase were enriched in 20:4 and 18:0 whereas those released during the latter phase were nonspecific. The DG fatty acids are enriched in 18:0 and 20:4. Ischemia induced a rapid release of DG as measured by its fatty acid content (3.2 micrograms FA/mg FA in phospholipids/min). Unlike FFA, the level of DG reached a plateau after 1.5 min and remained elevated for the entire 30.5 min. In agreement with previous notions indicating the involvement of phospholipase A2 in ischemic insult, steady increases in radioactivity of both lysophosphatidylcholines and lysophosphatidylinositols were observed with time after decapitation. Based on the preferential increase in both 18:0 and 20:4 during the initial time period, the results suggest that poly-PI hydrolysis coupled to DG-lipase may contribute to the initial release of FFA, whereas the FFA released subsequent to the initial phase may be mainly a result of activation of phospholipase A2 acting on phosphatidylcholines and phosphatidylinositols.
Mol Chem Neuropathol 1992 Aug
PMID:Decapitation ischemia-induced release of free fatty acids in mouse brain. Relationship with diacylglycerols and lysophospholipids. 138 50

Our object was to obtain information about the regulatory mechanism which modulates the effect of basic fibroblast growth factor (bFGF) on commitment to growth in human umbilical vein endothelial (HUVE) cells. Firstly, phorbol ester PMA, a known activator of protein kinase C (PKC), was found to be able to act synergistically with bFGF to stimulate 3H thymidine incorporation in HUVE cells. Secondly, bFGF and PMA induced a stimulated phospholipase A2 (PLA2)-catalyzed release of 14C arachidonate. Thirdly, inhibitors of PLA2, PKC and HETE, but not an inhibitor of cyclooxygenase metabolites, inhibited FGF/PMA-stimulated DNA synthesis. Fourth, the stable cyclooxygenase metabolite of prostacyclin was not found to be changed when cells were treated with bFGF plus PMA. The present data suggest that PKC is able of acting synergistically with bFGF in order to stimulate DNA-primary initiation activity in HUVE cells via the PLA2-dependent generation of lipoxygenase metabolites such as HETE.
Cell Mol Biol 1992 Jul
PMID:Possible involvement of arachidonic acid metabolites in the synergistic action of endothelial mitogenesis by basic fibroblast growth factor and phorbol ester. 149 42

Reactive oxygen species stimulate metabolism of arachidonic acid (AA) to eicosanoids in a variety of cells and tissues, yet the pathway(s) by which oxidants increase the availability of AA for oxidative metabolism are not known. Thus, we explored the effects of hydrogen peroxide (H2O2) on deacylation and reacylation of AA to determine the enzymatic mechanism(s) by which this oxidant increases levels of free, unesterified AA, and thereby its oxidative metabolism to eicosanoids, in the rat alveolar macrophage (AM). Over the range from 0.1 to 0.5 mM, H2O2 caused marked time- and dose-dependent inhibition of incorporation of [3H]AA into macrophage phospholipids, whereas calcium ionophore A23187 and zymosan particles did not cause such inhibition. Within this concentration range, there was an almost exact reciprocal correlation between inhibition of [3H]AA acylation and H2O2-stimulated accumulation of free [3H]AA in prelabeled AM cultures. Thimerosal, which blocks AA reacylation but spares deacylation via phospholipase A2 (PLA2), did not affect accumulation of free [3H]AA in prelabeled cells stimulated with H2O2, while markedly augmenting [3H]AA release in response to A23187 and to zymosan. Despite its ability to block AA acylation almost completely, H2O2 did not directly inhibit arachidonoyl CoA synthetase or arachidonoyl CoA:lysophosphatide acyltransferase, which catalyze AA incorporation into phospholipids. However, H2O2 (0.1 to 0.5 mM) markedly depleted AMs of ATP, required for synthesis of the acylation intermediate arachidonoyl CoA, suggesting that this was the means by which H2O2 inhibited acylation. Notably, H2O2 (0.03 to 3 mM) failed to stimulate macrophage PLA2 activity. We conclude that H2O2, in contrast to A23187 and zymosan, inhibits incorporation of AA into phospholipids, and that this represents the major mechanism by which the oxidant increases the availability of free AA for oxidative metabolism in the AM. This may be an important basis for release of eicosanoids in oxidant-induced inflammation and injury of the lung.
Am J Respir Cell Mol Biol 1992 Sep
PMID:Hydrogen peroxide increases the availability of arachidonic acid for oxidative metabolism by inhibiting acylation into phospholipids in the alveolar macrophage. 152 Apr 93

In contrast to previous studies, Parker et al. (Diabetes (1989) 38, 1123) have recently found that isolated rat adipocytes alone were unable to synthesize prostaglandins (PG) and that the PG measured in adipocyte suspensions were due to contaminating non-adipocyte cells. In the present study the capacity of adipocytes to produce PGE2 has further been explored. Preparations of isolated rat adipocytes were extensively washed in order to get rid of contaminating cells. The released PGE2 was measured by radioimmunoassay (RIA) after high-performance liquid chromatography (HPLC) separation. We found that after repetitive washing (up to 20 times) the isolated adipocytes were still able to synthesize PGE2 and this process was fully activatable by epinephrine, which indicates that pure adipocytes, themselves, are able to produce PGE2. However, addition of non-adipocyte material (from the adipose tissue) to 'pure' adipocytes (washed 10 times) enhanced the PGE2 synthesis significantly (P less than 0.001) as compared to 'pure' adipocytes alone. Thus, some kind of synergy exists between adipocytes and non-adipocyte cells in the adipose tissue in respect to PG formation. Some regulatory aspects of PG synthesis in 'pure' adipocytes were also investigated. Phospholipase A2 (2 U/ml) enhanced PGE2 synthesis significantly (119 +/- 21 to 658 +/- 85 pg/10(6) cells, P less than 0.001) without affecting lipolysis (glycerol release). The combined effect of epinephrine (5 microM) and phospholipase A2 (2 U/ml) on PGE2 formation was almost additive. Insulin inhibited the epinephrine-induced PG formation (P less than 0.01) but had no effects on the action induced by phospholipase A2.(ABSTRACT TRUNCATED AT 250 WORDS)
Mol Cell Endocrinol 1992 May
PMID:Biosynthetic capacity and regulatory aspects of prostaglandin E2 formation in adipocytes. 152 16


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