AbstractsBiology & Animal Science

Glutathione-S-transferase (GST) is an important phase II detoxification enzyme, which fuses an electrophilic substrate with glutathione (Blanchette, Feng, & Singh, 2007). The conjugation products, with rare exceptions, are more polar, less toxic, and more readily excreted than their parent compounds (Hodgson & Rose, 2010). Most animals have several GST forms to act upon a variety of substrates (Blanchette et al., 2007). Tritonia diomedea is a marine gastropod which lives in sub-tidal waters along the Pacific West coast. This species is also known as Tritonia tetraquetra (Martynov, 2009). T. diomedea is an interesting invertebrate model due to its ability to feed on the toxic sea pen Ptilosarcus gurneyi. The compound ptilosarcenone has been isolated and characterized from this sea pen (Hendrickson & Cardellina, 1986). In order to handle the menace from toxic compounds, it is possible that the marine gastropod T. diomedea possesses a unique detoxification system to facilitate the excretion of toxic compounds such as ptilosarcenone. My master???s thesis project is about the isolation and purification of GST in the marine gastropod T. diomedea. The gastropods were collected via SCUBA close to Tofino (British Columbia, Canada) and Dash Point (Washington, USA). Digestive glands were chosen as tissue source as they house detoxification enzymes and accumulate heavy metals and other toxins. Ptilosarcenone was purified from sea pen tissue by Dr. Taro Amagata, a Chemistry Professor from San Francisco State University. After extraction and purification of GSTs via affinity chromatography, I quantified protein content and measured GST activity. Upon optimizing the assay conditions for the samples, I discovered that 2.4 mM 1-chloro-2,4-dinitrobenzene (CDNB) and 10 mM glutathione (GSH) yielded optimum activity at pH 7.5. Further kinetic analysis revealed substrate inhibition for CDNB, but not GSH. The Km values for the substrate GSH range from 1.10 mM to 1.73 mM when experiments were conducted with crude extracts. With the optimized assay conditions specific GST activity values for Dash Point samples were on average 4-times greater than for Tofino samples. SDS-PAGE analysis (Coomassie blue and silver staining) showed one major GST isoform with a molecular weight of ~25 kDa. Besides the universal substrate CDNB, other more specific alternative substrates such as 1,2-dichloro-4-nitrobenzne (DCNB), p-nitrobenzyl chloride (pNBC), and ethacrynic acid were tested. However, none of the alternative substrates showed any appreciable activity. The effect of ptilosarcenone on the NIH-3T3 L1 cells and on the GST activity remained unclear due to the limited solubility of ptilosarcenone. Advisors/Committee Members: Murray, Dr. James Alan (advisor), Sommerhalter, Dr. Monika (primaryAdvisor).