Hopkins Marine Station Student Paper

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(full paper is archived in the Miller Library)

Title: A biochemical analysis of granules from the venom duct of Conus californicus
Student Author(s): Sandoval, Brynnen
Faculty Advisor(s): Gilly, Wiliam
Pages: 30
Location: Final Papers Biology 176H
Date: June 2001
Abstract: Marine Conus snails produce a wide variety of neurotoxins ("conotoxins") that specifically bind to different ion channels and neuronal receptors. Much research has revolved around the physiological effects of the conotoxins without focus on how these snails produce their venom. For example, the temperate snail Conus californicus produces a novel conotoxin that targets voltage-sensitive sodium channels in cephalopod, but not gastropod neurons. The predicted molecular weight from the cDNA sequence encoding this peptide is 4680 Da. Numerous dense granules appear in the venom duct of Conus californicus, where venom production occurs. In an attempt to isolate the granules, as a first step towards biochemical characterization, crude venom was run on a Ficoll-sucrose gradient. Inspection of these granules with light microscopy and scanning electron microscopy confirmed the presence of dense granules of approximately 2-6 µm in diameter. Electrophoresis of the SDS solubilized granules revealed a prominent 5 kDa band on SDS-polyacrylamide tricine gels that matches a band in "milked" venom and duct venom. This 5 kDa granule peptide was not solubilized by nonionic detergent (NP40) suggesting that it is packed within a tightly associated granule complex. Observation with light microscopy indicated that application of heat disintegrated the granules and released soluble, bio-active peptide into the medium. Preliminary whole cell voltage-clamp recordings of disassociated cells from the stellate ganglion of Sepia officinalis showed that the granule contents block voltage-gated sodium channels. In conclusion, it appears that these dense granules contain a peptide resembling mature milked venom peptide in both mass and function.