title: Biophysical, histopathological and pharmacological characterization of crotamine isoforms F22 and F32.
abstract: Two major crotamine isoforms (F22 and F32) were obtained after three chromatographic steps and were assayed in mouse phrenic nerve-diaphragm preparations. F32 and F22 (0.5 microg/ml, n=4) produced a facilitatory effect, which increased isometric twitch-tension by 300 and 230%, respectively, after a 120 min incubation. At a concentration of 0.1 microg/ml, both isoforms increased the twitch-tension by about 160%. However, when the isoforms were co-incubated (final concentration, 0.5 microg/ml) for 30 min prior to testing, they did not cause the facilitation seen with > or =0.1 microg/ml of each isoform alone. Histologically, F32 and F22 at 0.5 and 1 microg/ml were quantitatively alike in inducing tissue myonecrosis. However, a mixture of the two isoforms (final concentration, 0.5 microg/ml) significantly attenuated the damage seen with either toxin alone. Mass spectrometry analysis showed that the isoforms had the same molecular mass (4.8 kDa) and that they existed as monomers with a highly stable structure. These results indicate that F22 and F32 acted on muscle cells of the mouse phrenic-nerve diaphragm preparation through similar mechanisms. Since the isoforms did not produce the expected summation in the increase in muscle twitch-tension, it is possible that they may have different affinities for the sodium channel subunits.
authors.affiliation: Department of Physiology and Biophysics, Institute of Biology, P.O. Box 6109, CEP 13083-970, Campinas, SP, Brazil.
authors.forename: Marcos H
authors.initials: MH
authors.lastname: Toyama
authors.forename: Sérgio
authors.initials: S
authors.lastname: Marangoni
authors.forename: José C
authors.initials: JC
authors.lastname: Novello
authors.forename: Gildo B
authors.initials: GB
authors.lastname: Leite
authors.forename: Julia
authors.initials: J
authors.lastname: Prado-Franceschi
authors.forename: Maria Alice
authors.initials: MA
authors.lastname: da Cruz-Höfling
authors.forename: Léa
authors.initials: L
authors.lastname: Rodrigues-Simioni
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002851:Chromatography, High Pressure Liquid; D003435:Crotalid Venoms / Q000737:chemistry* / Q000494:pharmacology*; D003964:Diaphragm / Q000187:drug effects / Q000294:innervation / Q000473:pathology; D013058:Mass Spectrometry; D051379:Mice; D008969:Molecular Sequence Data; D009119:Muscle Contraction / Q000187:drug effects; D018482:Muscle, Skeletal / Q000187:drug effects; D010791:Phrenic Nerve / Q000187:drug effects; D020033:Protein Isoforms / Q000737:chemistry / Q000494:pharmacology; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013997:Time Factors
chemical_list: D003435:Crotalid Venoms; D020033:Protein Isoforms; C010563:crotamine