Abstract
A specific phospholipase A(2) receptor from porcine cerebral cortex has been characterized (K(d) = 145 nM, B(max) = 0.4 pmol/mg membrane protein) by using a radioiodinated derivative of ammodytoxin C (AtxC), a snake venom presynaptically neurotoxic group IIA phospholipase A(2). After the receptor was solubilized in a ligand-binding form, it was approximately 14,000-fold enriched by chromatography on wheat germ lectin-Sepharose and AtxC-Affi-Gel 10. The receptor is a single chain glycoprotein with an apparent molecular mass of 180 kDa and binds toxic and non-toxic phospholipases A(2) of either group I or II. It also recognizes conjugates of bovine serum albumin with mannose, N-acetylglucosamine, and galactose. In its molecular mass and pharmacological profile, the AtxC receptor resembles the M-type receptor for secretory phospholipases A(2) from rabbit skeletal muscle (a C-type multilectin, homologous to macrophage mannose receptor), yet in terms of relative abundance in brain and antigenicity, these two receptors are completely different. A further AtxC receptor of approximately 200 kDa discovered in porcine liver was, however, recognized by anti-rabbit M-type phospholipase A(2) receptor antibodies. There are, therefore, two immunologically distinct secretory phospholipase A(2) receptors of about 200 kDa in the same species. Although the liver receptor is related to the M-type secretory phospholipase A(2) receptors, the brain receptor is not and belongs to a novel group of secretory phospholipase A(2) receptors.
Highlights
A specific phospholipase A2 receptor from porcine cerebral cortex has been characterized (Kd ؍145 nM, Bmax ؍ 0.4 pmol/mg membrane protein) by using a radioiodinated derivative of ammodytoxin C (AtxC), a snake venom presynaptically neurotoxic group IIA phospholipase A2
The extract was incubated with wheat germ lectin-Sepharose since we have shown that R180 binds with high affinity and reversibly to this lectin. 125I-AtxC affinity labeling was used to follow R180 during the isolation
An M-type sPLA2 Receptor Is Present in Pig—We examined porcine liver as a non-neuronal tissue source for potential AtxC receptors. 125I-AtxC affinity labeled a specific receptor with a slightly higher molecular mass than that of R180 (Fig. 3B)
Summary
Materials—Ammodytoxins, ammodytin I2 and AtnL, were purified from V. ammodytes ammodytes venom as described previously [33, 35]. Solubilization of AtxC-binding Proteins—Membranes from porcine cerebral cortex or liver (2.6 mg of membrane protein/ml) were extracted for 1 h by gentle agitation at 4 °C in 75 mM Hepes, pH 8.2, containing 150 mM NaCl, 10 mM SrCl2, 0.5 mM EGTA, and 4% (w/v) Triton X-100 and afterward centrifuged at 106,200 ϫ g for 1 h. Cross-linking of 125I-AtxC to the Solubilized AtxC-binding Proteins— The membrane extract or the fractions containing solubilized receptor were incubated for 30 min at room temperature with 125I-AtxC (10 nM final concentration) in the presence or absence of an unlabeled competitor. The resin was washed with 100 ml of 50 mM Hepes, pH 8.2, containing 140 mM NaCl, 2 mM CaCl2, and 0.1% (w/v) Triton X-100 and stored in the same buffer at 4 °C. After extensive washing in PBS containing 0.1% (w/v) Tween 20, the secondary antibodies were detected by the BM chemiluminescent Western blotting system (Roche Molecular Biochemicals) following the manufacturer’s instructions
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