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

Abstract

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 ...

Interleukin‐1β and transforming growth factor‐β2 enhance cytosolic high‐molecular‐mass phospholipase A2 activity and induce prostaglandin E2 formation in rat mesangial cells

“…When treated with antibodies against group-I1 PLA2, the cytosolic fractions were diluted with 20 mM Tris pH 7.4 containing 1 mM EDTA, 10% glycerol, 2 M NaCl and 2 mg/ml bovine serum albumin. Monoclonal antibodies against a group-I1 PLAZ, i. e. rat liver mitochondria1 PLA2, wcre prepared, purified and coupled to CNBr-activated Sepharose 4B as described before [49,50]. The diluted cytosolic fractions were incubated with an excess of this mAbSepharose suspension for 1 h at 4°C.…”

PDGF suppresses the activation of group II phospholipase A2gene expression by interleukin I and forskolin in mesangial cells

“…on PEA2 secretion In contrast, PDGF-AA at 100 n&ml had no effect on both basal and cytokme-mduced PL& release To study the blosynthesls of PLA2, mesanglal cells were stimulated with IL-l/I and forskolm (10 @I) For 22 h and ['%Jmethlonme was added for the I& 6 h of the stlmulatlon periods. A combmatlon of IL-lb and forskolm has been shown to synerglstrcally stimulate PLA, secretion from mesanglal cells 116,171 PLA, was lmmunoprecipltated from the medium with a monoclonal group II PEA, antlbody [25,26] and subJected to SDS-PAGE As shown m Fig 1, no 14 kDa group II PLA? could be detected m the culture supernatants of unstlmulated cells Addltlon of IL-l/?…”

Lipocortin inhibition of extracellular and intracellular phospholipases A2 is substrate concentration dependent

“…After centrifugation for 1 h at 100000 × g, the clear supernatant containing over 85°70 of the platelet phospholipase A2 activity, was chromatographed over a Sepharose 4B column to which a monoclonal antibody against rat liver mitochondrial phospholipase A2 had been coupled. As shown [22] this monoclonal antibody cross-reacted with rat platelet phospholipase Az. After more than 95°70 of the applied proteins had been eluted with the application buffer, phospholipase A2 activity was eluted with a glycine buffer (pH 2.5) in yields of 150-200°70.…”

“…After more than 95°70 of the applied proteins had been eluted with the application buffer, phospholipase A2 activity was eluted with a glycine buffer (pH 2.5) in yields of 150-200°70. The high yields are probably due to the removal of inhibiting compounds present in the platelet extract [22]. Details of this purification will be published elsewhere.…”

“…Rat liver mitochondrial phospholipase A2 was purified as described [22] by AcA 54 chromatography of delipidated mitochondrial extracts. The specific activity of this preparation amounted to 350 nmol-min-l.mg protein -1. to initiate the reaction which was terminated 10 min later by adding 100/A of 2N HC1.…”