Purification and properties of m1-toxin, a specific antagonist of m1 muscarinic receptors.
The venom of the Eastern green mamba from Africa, Dendroaspis angusticeps, was found to block the binding of 3H-quinuclidinyl benzilate to pure m1 and m4 muscarinic ACh receptors expressed in Chinese hamster ovary cells. The principal toxin in the venom with anti-m1 muscarinic activity was purified by gel filtration and reversed-phase HPLC. This toxin has 64 amino acids, a molecular mass of 7361 Da, and an isoelectric point of 7.04. Its cysteine residues are homologous with those in curare-mimetic alpha-neurotoxins, and with those in fasciculin, which inhibits AChE. At low concentrations the toxin blocked m1 receptors fully and pseudoirreversibly while having no antagonist activity on m2-m5 receptors; the toxin is therefore named "m1-toxin." At higher concentrations m1-toxin interacted reversibly with m4 receptors, and half of the toxin dissociated in 20 min at 25 degrees C. The affinity of m1-toxin is therefore much higher for m1 than for m4 receptors. By comparison with m1-toxin, pirenzepine has sixfold higher affinity for m1 than for m4 receptors. Autoradiographs of muscarinic receptors in the rat brain demonstrated that m1-toxin blocked the binding of 2 nM 3H-pirenzepine only in regions known to bind m1-specific antibodies. Thus, m1-toxin is a much more selective ligand than pirenzepine for functional and binding studies of m1 muscarinic receptors.
Max
S I
SI
Department of Molecular and Cellular Pharmacology, University of Miami School of Medicine, Florida 33101.
Liang
J S
JS
Potter
L T
LT
D000595:Amino Acid Sequence; D000818:Animals; D001345:Autoradiography; D016466:CHO Cells; D002462:Cell Membrane / Q000378:metabolism; D002540:Cerebral Cortex / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D006224:Cricetinae; D004546:Elapid Venoms / Q000737:chemistry / Q000302:isolation & purification* / Q000494:pharmacology*; D017815:Elapidae; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D018727:Muscarinic Antagonists; D010890:Pirenzepine / Q000378:metabolism; D017433:Protein Structure, Secondary; D011712:Pyramidal Tracts / Q000378:metabolism; D011813:Quinuclidinyl Benzilate / Q000378:metabolism; D051381:Rats; D011976:Receptors, Muscarinic / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014162:Transfection; D014316:Tritium
D004546:Elapid Venoms; D018727:Muscarinic Antagonists; D011976:Receptors, Muscarinic; D011994:Recombinant Proteins; C083667:m1-toxin; D014316:Tritium; D010890:Pirenzepine; D011813:Quinuclidinyl Benzilate