BINDING-PROTEINS ON SYNAPTIC-MEMBRANES FOR CROTOXIN AND TAIPOXIN, 2 PHOSPHOLIPASES A(2) WITH NEUROTOXICITY

Crotoxin and taipoxin are both neurotoxic phospholipases A(2) capable of affecting the presynaptic activity to bring about ultimate blockade of synaptic transmission. The enzymatic activity has generally been c onsidered to be necessary but not sufficient for the blockade. Since m any phospholipases A(2) with comparable or even higher enzymatic activ ity are not toxic, it has been postulated that the difference lies in the affinity of binding to the presynaptic membrane. In confirmation o f this proposition, we and others have previously shown that iodinated crotoxin and taipoxin bind specifically with high affinity to the iso lated synaptic membrane fraction from guinea-pig brain, whereas specif ic binding is not detected with the nontoxic pancreatic phospholipase A(2). Experiments based on photoaffinity labeling and simple chemical cross-linking techniques have led to the identification of three polyp eptides preferentially present in neuronal membranes as (subunits of) the binding protein(s) for crotoxin and/or taipoxin. Some, but not all , other toxic phospholipases A(2) also appear to be ligands for the th ree polypeptides. We now report studies on partial purification of the se polypeptides using affinity chromatography and other techniques. In order to learn the normal physiological roles played by the toxin-bin ding proteins, the phospholipase-independent effects of the toxins on the synaptosomes have been sought. We have found that under Ca2+-free condition, taipoxin or crotoxin inhibits with IC50 of 20-1000 nM the N a+-dependent uptake of norepinephrine, dopamine and serotonin by the s ynaptosomes contrast, choline uptake is not affected. Furthermore, the high-affinity site for [H-3]desipramine binding, known to be the nore pinephrine transporter, is inhibited with an IC50 of 14 nM by taipoxin independent of phospholipase activity. These results are strong indic ations that certain synaptosomal biogenic amine transporters are part of the binding proteins for taipoxin and crotoxin. Chemical modificati on at Tyr-21 of the phospholipase subunit of crotoxin greatly reduces the neurotoxicity and the binding affinity with little effect on the e nzymatic activity of the toxin. Hence, Tyr-21 may be an important resi due for the binding of crotoxin and perhaps other phospholipase A(2) n eurotoxins. Even stronger evidence comes from the finding that replace ment of the corresponding Phe residue of bovine pancreatic phospholipa se A(2) with Tyr by site-directed mutagenesis enables it to compete wi th an IC50 of 1 mu M for the binding of [I-125]crotoxin, contrasting s harply with the complete lack of such ability with the wild type even at 50 mu M. In addition, rat phospholipase A(2), which has a correspon ding Tyr residue, can inhibit the binding of [I-125]crotoxin.