Identification and properties of very high affinity brain membrane-binding sites for a neurotoxic phospholipase from the taipan venom.
Four new monochain phospholipases were purified from the Oxyuranus scutellatus (taipan) venom. Three of them were highly toxic when injected into mice brain. One of these neurotoxic phospholipases, OS2, was iodinated and used in binding experiments to demonstrate the presence of two families of specific binding sites in rat brain synaptic membranes. The affinities were exceptionally high, Kd1 = 1.5 +/- 0.5 pM and Kd2 = 45 +/- 10 pM, and the maximal binding capacities were Bmax 1 = 1 +/- 0.4 and Bmax 2 = 3 +/- 0.5 pmol/mg of protein. Both binding sites were sensitive to proteolysis and demonstrated to be located on proteins of Mr 85,000-88,000 and 36,000-51,000 by cross-linking and photoaffinity labeling techniques. The binding of 125I-OS2 to synaptic membranes was dependent on Ca2+ ions and enhanced by Zn2+ ions which inhibit phospholipase activity. Competition experiments have shown that, except for beta-bungarotoxin, a number of known toxic snake or bee phospholipases have very high affinities for the newly identified binding sites. A good correlation (r = 0.80) was observed between toxicity and affinity but not between phospholipase activity and affinity.
Lambeau
G
G
Centre de Biochimie, Centre National de la Recherche Scientifique, Nice, France.
Barhanin
J
J
Schweitz
H
H
Qar
J
J
Lazdunski
M
M
D000345:Affinity Labels; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D001921:Brain / Q000378:metabolism*; D002118:Calcium / Q000494:pharmacology; D002414:Cations, Monovalent; D002845:Chromatography; D003432:Cross-Linking Reagents; D004546:Elapid Venoms / Q000032:analysis* / Q000633:toxicity; D004591:Electrophoresis, Polyacrylamide Gel; D019286:Endopeptidase K; D007457:Iodine Radioisotopes; D051379:Mice; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010740:Phospholipases / Q000302:isolation & purification / Q000378:metabolism*; D010777:Photochemistry; D015221:Potassium Channels / Q000378:metabolism; D012697:Serine Endopeptidases / Q000494:pharmacology; D015222:Sodium Channels / Q000378:metabolism; D013570:Synaptic Membranes / Q000378:metabolism*; D014357:Trypsin / Q000494:pharmacology; D015032:Zinc / Q000494:pharmacology
D000345:Affinity Labels; D002414:Cations, Monovalent; D003432:Cross-Linking Reagents; D004546:Elapid Venoms; D007457:Iodine Radioisotopes; D015221:Potassium Channels; D015222:Sodium Channels; D010740:Phospholipases; D012697:Serine Endopeptidases; D014357:Trypsin; D019286:Endopeptidase K; D015032:Zinc; D002118:Calcium