Utkin YN , Weise C , Kasheverov IE , Andreeva TV , Kryukova EV , Zhmak MN , Starkov VG , Hoang NA , Bertrand D , Ramerstorfer J , Sieghart W , Thompson AJ , Lummis SC , Tsetlin VI .

01925 Wellcome Trust

	FIGURE 1. Isolation of azemiopsin. A, separation of crude A. feae venom by gel filtration on Superdex HR75 column (10 × 300 mm) in 0.1 m ammonium acetate buffer, pH 6.2, at a flow rate of 30 ml/h. B, isolation of azemiopsin by reverse-phase HPLC on a Jupiter C18 (4.6 × 250 mm) column in a linear gradient of acetonitrile in water (0.1% TFA). Flow rate is 1 ml/min.
	FIGURE 2. Comparison of the amino acid sequences of azemiopsin and waglerins. Identical residues are underlined, conservative substitutions are italicized. The residues essential for biological activity are indicated by gray hatching. The first 5 N-terminal residues in waglerin, the removal of which results in loss of activity (LD50 > 10 μg/g), are shown in outline.
	FIGURE 3. CD spectrum of azemiopsin at pH 7.0.
	FIGURE 4. Inhibition by azemiopsin of human muscle-type nAChRs expressed in Xenopus oocytes. To evaluate effects of the toxin, cells were challenged at regular intervals (2 min) with brief ACh test pulses (5 μm, 5 s). Following a stabilization period, toxin was applied at different concentrations for at least 1 min. The response to ACh (5 μm) was tested again, and recovery from inhibition was monitored over several minutes. Results obtained with adult α1β1ϵδ nAChR are illustrated in A whereas effects on fetal α1β1γδ form are shown in B. Plot of the peak current as a function of the logarithm of the azemiopsin concentration yielded typical inhibition curves that were fitted with a single Hill equation (continuous curves). Values for the best fit are indicated on the figure.
	FIGURE 5. Biological activity of Ala analogues of azemiopsin. Abscissa indicates azemiopsin amino acid residues replaced by alanine. The binding of 125I-labeled α-bungarotoxin (Bgt) was determined after incubation of Torpedo membranes with 2 μm analogue for 1 h. 125I-Labeled α-bungarotoxin binding in the absence of peptide was taken as control (no inhibition).

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