Specific binding of crotoxin to brain synaptosomes and synaptosomal membranes
| Autor: | L.L. Degn, Ivan I. Kaiser, Corrine Seebart |
|---|---|
| Rok vydání: | 1991 |
| Předmět: |
Protein subunit
Guinea Pigs Synaptic Membranes Dendrotoxin In Vitro Techniques Biology Toxicology Iodine Radioisotopes Mice chemistry.chemical_compound Phospholipase A2 medicine Animals Neurotoxin Binding site Synaptosome Taipoxin Serine Endopeptidases Brain Crotoxin Trypsin Molecular Weight Kinetics chemistry Biochemistry biology.protein Female Endopeptidase K Synaptosomes medicine.drug |
| Zdroj: | Toxicon. 29:973-988 |
| ISSN: | 0041-0101 |
| DOI: | 10.1016/0041-0101(91)90080-b |
| Popis: | Specific binding of crotoxin to brain synaptosomes and synaptosomal membranes. Toxicon 29, 973–988 1991.—Crotoxin, the presynaptic neurotoxin from Crotalus durissus terrificus , was iodinated and used to demonstrate high affinity, specific binding to guineapig ( Cavia porcellus ) brain synaptosomes and synaptosomal membrane fragments. 125 I-crotoxin binding to the membrane fragments displays two binding plateaus, ( K d1 = 4 nM and K d2 = 87 nM, B max1 = 2 and B max2 = 4 pmoles/mg membrane protein), but binding to whole synaptosomes revealed only one plateau ( K d = 2 nM and B max = 5 pmoles/mg membrane protein). Rosenthal analyses of Scatchard plots yielded similar binding constants in the presence or absence of 0.025% Triton X-100. In addition to equilibrium analyses, kinetic analyses of 125 I-crotoxin binding to synaptosomal membrane fragments gave a K d -value of 3 nM. The K d value was not significantly changed by the exclusion of added calcium, but the binding site number was lowered. Crotoxin binding was inhibited by the acidic subunit of crotoxin and several presynaptic neurotoxins, which were classified according to their inhibitory properties as, strong (acidic subunit of crotoxin, Mojave toxin, concolor toxin, taipoxin and pseudexin), moderate (ammodytoxin A and textilotoxin), weak (notexin and scutoxin A), very weak (notechis II-5) and non-inhibitory (basic subunit of crotoxin, β-bungarotoxin, Crotalus atrox and porcine pancreatic phospholipases A 2 , dendrotoxin, and notechis III-4). Purified acidic subunit of crotoxin, the most potent competitor of crotoxin binding, was somewhat more competitive than intact crotoxin and the other strong inhibitors on a molar basis. Strong, moderate and weak inhibitor groups each differed from the preceding group by requiring about a ten fold increase in concentration to effect a 50% inhibition of crotoxin binding. The weak group was therefore at least two-orders of magnitude less effective than the strong inhibition shown by the acidic subunit of crotoxin. Treatment of synaptosomal membranes with protease K lowered 125 I-crotoxin binding, whereas treatment with trypsin did not. Iodinated, phospholipase A 2 from C. atrox venom showed no specific binding to whole synaptosomes. Our results demonstrate the presence and describe some of the properties of high affinity, specific binding sites in brain tissue for crotoxin and related presynaptic neurotoxins. |
| Databáze: | OpenAIRE |
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