Some aspects of rat platelet and serum phospholipase A2 activities

Aarsman, A.J.; Roosenboom, C.F.P.; Geffen, G.E.W. van; Bosch, H. van den

doi:10.1016/0005-2760(85)90052-9

Cited by 42 publications

(15 citation statements)

References 41 publications

(26 reference statements)

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“…Although our culture medium contains 5% calf bovine serum (v/v), which is known to contain PLA2 activity (Aarsman et al 1985;Nishijima et al 1983), our results from PLA2 assay revealed that its contribution did not exceed 4% of total PLA2 activity measured in hRPE growth medium (data not shown). We can therefore conclude that the PLA2 activity detected in hRPE growth medium was attributable to active enzyme secretion by hRPE cells.…”

Section: Cultured Hrpe Cells Contain Different Pla2-active Fractionsmentioning

confidence: 85%

Human retinal pigment epithelium secretes a phospholipase A2 and contains two novel intracellular phospholipases A2

Themsche¹,

Jacob²,

Salesse³

2001

Biochem. Cell Biol.

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The sensitivity of different phospholipase A2 (PLA2)-active fractions eluted from cation-exchange chromatography to para-bromophenacylbromide (pBPB), Ca2+-EGTA, DTT, heat, and H2SO4 indicates that human cultured retinal pigment epithelial (hRPE) cells probably contain two different intracellular PLA2 enzymes. Control experiments using "back-and-forth" thin-layer chromatography confirmed that, in our assay conditions, the generation of free fatty acids originated solely from PLA2 activity. Together with immunoblot experiments where no cross-reactivity was observed between the hRPE cytosolic PLA2 enzymes and several antisera directed against secretory PLA2s (sPLA2s) and cytosolic PLA2 (cPLA2), these findings suggest that intracellular hRPE PLA2s are different from well-known sPLA2s, cPLA2, and Ca2+-independent PLA2s. We also report an additional hRPE-PLA2 enzyme that is secreted and that exhibits sensitivity to pBPB, Ca2+-EGTA, DTT, heat, and H2SO4, which is characteristic of sPLA2 enzymes. This approximately 22-kDa PLA2 cross-reacted weakly with an antiserum directed against porcine pancreatic group I sPLA2 but strongly with an antiserum directed against N-terminal residues 1-14 of human synovial group II sPLA2, suggesting that this extracellular enzyme is a member of the sPLA2 class of enzymes. We thus conclude that there are three distinct PLA2 enzymes in cultured hRPE cells, including two novel intracellular PLA2s and a 22-kDa secreted sPLA2 enzyme.

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Section: Cultured Hrpe Cells Contain Different Pla2-active Fractionsmentioning

confidence: 85%

Human retinal pigment epithelium secretes a phospholipase A2 and contains two novel intracellular phospholipases A2

Themsche¹,

Jacob²,

Salesse³

2001

Biochem. Cell Biol.

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“…Preparation of a platelet suspension from I0 ml rat blood and extraction of phospholipase A2 from the platelets was performed as described earlier [17].…”

Section: Isolation Of Rat Platelet Phospholipase Amentioning

confidence: 99%

Monoclonal antibodies against an intracellular phospholipase A2 from rat liver and their cross-reactivity with other phospholipases A2

Jong

Amesz²,

Aarsman

et al. 1987

Eur J Biochem

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The membrane-associated phospholipase A2 from rat liver mitochondria was solubilized and partially purified by AcA 54 gel filtration and Matrex gel blue A chromatography. The approximately 2500-fold purified preparation was injected into mice to prepare monoclonal antibodies against phospholipase A2 after fusion of spleen cells and mouse SP2/0 myeloma cells. Hybridoma supernatants were assayed for antibody production in enzymelinked immunosorbent assay with partially purified phospholipase A2 as antigen. Positive clones were tested for their ability to bind phospholipase A2 in a specific immunoprecipitation assay involving protein-A -Sepharose to which rabbit anti-(mouse immunoglobulins) and monoclonal antibodies from hybridoma supernatants were complexed. Twelve clones producing antibodies that bound mitochondrial phospholipase A2 were identified. The binding of all of these antibodies to protein fractions eluted from AcA 54 and Matrex gel blue A columns coincided with the phospholipase A2 activity in these fractions. All monoclonal antibodies showed cross-reactivity with rat liver cytosolic and solubilized rat platelet phospholipase A2. Extracellular phospholipase A2 from rat and pig pancreas or Crotalus atrox were not recognized by the anti-(mitochondria1 phospholipase A2) antibodies.Phospholipases A2 are widespread in nature. The digestive enzyme from pancreas has been purified and analyzed for many mammalian species and much is known about the molecular mechanisms of its enzyme action 111. Also the other secreted phospholipases A2 from bee, snake, wasp and scorpion venoms are easily accessible for investigation as all these enzymes are soluble proteins and can be purified in reasonable quantities.Intracellular phospholipases A2 have been described for many cell types and within the cell their occurrence is often not restricted to one compartment. Investigation of these phospholipases is not only hampered by the low concentration within the cell and the low specific activity of the enzyme, but also by the diversity of their localization. The enzyme can be found in soluble form in the cytosol and membrane-bound in many of the subcellular compartments [2, 31. Little is known about the structural relationship between the enzymes with different subcellular localization and between the intracellular phospholipases A2 from the various tissues. It is even not clear to what extent these intracellular phospholipases from the various organs can be compared with the secreted enzymes from pancreas, bee, snake, wasp and scorpion venoms. Only a few of the intracellular phospholipases A2 have been purified to homogeneity [3]. With some exceptions most of the purified intracellular phospholipases A2 have similar molecular masses (about 14 kDa). They all need calcium and have a basic or neutral pH optimum. They share these properties with secreted phospholipases A2, although their biological Enzyme. Phospholipase A2 (EC 3.1.1.4).function is completely different. The specific activities measured for the intracellular enzymes ...

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“…These analyses raise the question, what is the in vivo target substrate of platelet phospholipase A2? Aarsman et al [44] showed that the phospholipase A, from platelet Table 1. Effect of inhibitors on phospholipase A2.…”

Section: Resultsmentioning

confidence: 99%

Rat platelet phospholipase A₂

Ransac¹,

Aarsman²,

Bosch³

et al. 1992

European Journal of Biochemistry

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We have determined some kinetic parameters of rat platelet phospholipase A2, such as surface pressure dependency and substrate specificity, using the monomolecular film technique. We found that rat platelet phospholipase A2 is very specific for phospholipids having a negatively charged headgroup, no activity was detected when using zwitterionic phospholipids such as phosphatidylcholine. Furthermore, the interfacial pressure window which permits enzyme activity is very narrow as compared to pancreatic phospholipase A2. Maximal enzyme activity is found at 22 mN/m when using 1,2-dilauroylphosphatidylglycerol as substrate. Studies of the competitive inhibition of mixed films containing 2-acylaminophosphatidylglycol show that platelet phospholipase A2 is less sensitive than pancreatic and intestinal phospholipase A2. These results imply that, despite the high degree of sequence similarity, one must be very cautious in extrapolating inhibition data from one phospholipase A2 to similar enzymes from other origins.Platelet phospholipids are rich in arachidonic acid [l -31, the precursor of thromboxanes [4], hydroxylated-polyunsaturated fatty acids [S] and prostacyclin 161, whosc role in stimulating and inhibiting platelet activation is now well established [7 -91. Platelets contain two major possible pathways for arachidonate release. The most direct of these involves the action of phospholipase A2 towards all major platelet glycerophospholipids. The alternative pathway is initiated by phospholipase C action on inositolphospholipids followed by diacylglycerol lipases and monoacylgl ycerol lipases producing free arachidonate (for reviews see [lo, 111). A quantitative evaluation of the contribution of these pathways in human platelets upon stimulation by thrombin indicates that the phospholipase Az route is of major importance [I2 -361. Furthermore, rat platelets secrete an extracellular phospholipase

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Some aspects of rat platelet and serum phospholipase A2 activities

Cited by 42 publications

References 41 publications

Human retinal pigment epithelium secretes a phospholipase A2 and contains two novel intracellular phospholipases A2

Human retinal pigment epithelium secretes a phospholipase A2 and contains two novel intracellular phospholipases A2

Monoclonal antibodies against an intracellular phospholipase A2 from rat liver and their cross-reactivity with other phospholipases A2

Rat platelet phospholipase A₂

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Some aspects of rat platelet and serum phospholipase A2 activities

Cited by 42 publications

References 41 publications

Human retinal pigment epithelium secretes a phospholipase A2 and contains two novel intracellular phospholipases A2

Human retinal pigment epithelium secretes a phospholipase A2 and contains two novel intracellular phospholipases A2

Monoclonal antibodies against an intracellular phospholipase A2 from rat liver and their cross-reactivity with other phospholipases A2

Rat platelet phospholipase A2

Contact Info

Product

Resources

About

Rat platelet phospholipase A₂