Cumulative concentration–response Wortmannin molecular weight curves to exogenous ACh were obtained before and after incubation with purified toxin. The protocol consisted of first obtaining a concentration–response curve in the absence of toxin and then incubating indirectly stimulated preparations with toxin until complete blockade of the contractile responses, after which electrical stimulation was stopped and a new concentration–response curve
to ACh was obtained in the presence of toxin. Repeated curves without toxin were performed as control for tissue fatigue. The membrane resting potential was recorded from mouse diaphragm muscle (Bülbring, 1946) using conventional microelectrode techniques (Ling and Gerard, 1949; Fatt and Katz, 1951). The dissected muscle was mounted in a lucite chamber containing aerated (95% O2 + 5% CO2) Tyrode solution
(composition, in mM: NaCl 137; KCl 2.7; CaCl2 1.8; MgCl2 0.49; NaH2PO4 0.42; NaHCO3 11.9 and glicose 11.1, pH 7.0) at 37 °C. The resting potential of up to eight fibers in each muscle was recorded using glass microelectrodes filled with 3 M KCl (resistance 10–20 MΩ) and positioned within the muscle fiber. All recordings were displayed Sotrastaurin in vitro on a Tektronix oscilloscope. To examine the influence of the toxin on carbachol-induced membrane depolarization, the membrane resting potential was measured followed by the addition of carbachol (68 μM) and 15 min later the membrane potential was measured again. Subsequently, the preparation was washed, the resting potential was checked and toxin (110 μM) was added for 15 min. Acetophenone At the end of this incubation
carbachol was added (without washing the preparation) and the membrane potential was measured after 15 min. A low molecular mass fraction of the venom was initially obtained by filtering venom (10 mg dissolved in distilled water) through a 5 kDa nominal cut-off Amicon® filter (Millipore, Billerica, MA, USA) by centrifugation. The resulting fractions were referred to as the LM (low-mass; <5 kDa) and HM (high-mass; >5 kDa) fractions and both were tested for neuromuscular activity in biventer cervicis preparations. The LM fraction was subsequently fractionated by cation exchange HPLC on a Luna SCX column (Phenomenex, Torrance, CA, USA) equilibrated with 0.05 M potassium phosphate, pH 2.5, and eluted with a linear gradient of 0–1 M KCl as the mobile phase for 40 min. The resulting peaks were screened for neuromuscular activity and the active peak was chromatographed by reversed-phase HPLC on a C18 column (ACE, Aberdeen, Scotland) using aqueous 0.1% trifluoroacetic acid as the mobile phase and 90% acetonitrile in the mobile phase as the eluent with a gradient run from 40% to 50% over 15 min. The major peak obtained in this second step corresponded to purified toxin referred to as VdTX-1. In both chromatographic steps the elution profiles were monitored at 214 nm and 280 nm.
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