2B). These data indicate that the LM fraction preferentially affects neurotransmission by affecting cholinergic transmission while the venom and HM fraction affect

neurotransmission and muscle fiber contractility, the latter independently of nicotinic receptor involvement ( Harvey et al., 1994). Purification of the LM fraction component with neuromuscular activity was done in two HPLC steps. After initial experiments with standard reversed-phase HPLC (RP-HPLC) failed to provide adequate separation of the LM fraction components (data not shown) the system was substituted for strong cation exchange HPLC which yielded

seven major peaks and several minor peaks (Fig. 3A). Peaks 1 to 7 check details were desalted on a Resource RPC column and tested on chick biventer cervicis preparations. The activity was found only in the last major peak 7, but it was weaker than in LM fraction at same concentration, what lead us to assume the possibility of a degrading process during fractionation. Since the LM fractions contains low molecular mass components, possibly including polyamines, and knowing that polyamines are light-sensitive, we examined whether excessive exposure to light, i.e., photoinactivation, could account for the lack of neuromuscular activity in these peaks. When appropriate click here measures to protect against photoinactivation during purification and pharmacological testing were taken, the peaks corresponding to 3 mg of LM were tested (n = 2) and intense neuromuscular activity

was again detected only in the peak 7 of the elution profile ( Fig. 3A). Chromatography of this peak by RP-HPLC on a C18 column yielded pure toxin referred to as VdTX-1, the first toxin purified from V. dubius venom Sucrase ( Fig. 3B). The molecular mass of VdTX-1 as determined by MALDI-TOF mass spectrometry was 728 Da ( Fig. 3C). Comparison of the neuromuscular activity of the venom, the LM fraction and VdTX-1 (all tested at 20 μg/mL) showed that VdTX-1 (20 μg/mL = 27.4 μM toxin) reproduced the neuromuscular blockade caused by the LM fraction. As with the LM fraction, reversal of the blockade was also seen with VdTX-1. The decrease in twitch-tension caused by the LM fraction and VdTX-1 was greatest at 30 min, with a reduction to 38 ± 6% and 68 ± 6% of the control (n = 4), respectively. After 2 h incubation the twitch-tension had returned to 76 ± 5% and 63 ± 2% of pre-incubation levels for the LM fraction and VdTX-1, respectively ( Fig. 4).