Fluoroacetate-specific defluorinase (FSD) is a critical enzyme in the detoxication of fluoroacetate. This study investigated whether FSD can be classed as a glutathione S-transferase (GST) isoenzyme with a high specificity for fluoroacetate detoxication metabolism. The majority of FSD and GST activity, using 1-chloro-2,4-dinitrobenzene (CDNB) and 1,2-epoxy-3-(p-nitrophenoxy)propane (EPNP) as GST substrates, in rat liver was cytosolic. GSTT1 specific substrate, EPNP caused a slight non-competitive inhibition of FSD activity. CDNB, a general substrate of GST isoenzyme, was a more potent non-competitive inhibitor of FSD activity. The fluoroacetate defluorination activity by GST isoenzymes was determined in this study. The results showed that the GSTZ1C had the highest fluoroacetate defluorination activity of the various GST isoenzymes studied, while GSTA2 had a limited activity toward fluoroacetate. The human GSTZ1C recombinant protein then was purified from a human GSTZ1C cDNA clone. Our experiments showed that GSTZ1C catalysed fluoroacetate defluorination. GSTZ1 shares many of the characteristics of FSD; however, it accounts only for 3% of the total cytosolic FSD activity. GSTZ1C based enzyme kinetic studies has low affinity for fluoroacetate. The evidence suggests that GSTZ1 may not be the major enzyme defluorinating fluoroacetate, but it does detoxify the fluoroacetate. To clarify the identity of enzymes responsible for fluoroacetate detoxication, further studies of the overall FSD activity are needed.

Two forms of fluoroacetate-specific defluorinase (FSD) were purified from rat hepatic cytosol. The first form, FSD 1 (molecular weight 38 kDa), contained 81% of the total cytosolic fluoroacetate defluorination activity and did not bind to the glutathione-affinity, orange A or mono P columns used in the purification procedures. The second form, FSD2 (molecular weight 27 kDa), contained only 13% of the fluoroacetate defluorination activity, had a pI=7.8, and exhibited a high glutathione S-transferase (GST)-like activity towards dichloroacetic acid. The FSD1 proteins were identified from peptide mass data and best matched with rat sorbitol dehydrogenase (SDH) (short form), although pure sheep liver SDH enzyme did not possess defluorination activity when subsequently investigated. The FSD2 protein was identified from peptide mass data and best matched with the amino acid sequence of mouse and human Zeta 1 of glutathione S-transferase (GSTZ1) and showed a high GSTZ1 specific activity. This study suggests that the major FSD component (FSD1) represents a new and unique dehalogenating or dehydrogenating enzyme present in rat liver cytosol. The minor FSD component (FSD2) is due to the GSTZ1 present in rat liver cytosol. However, it is not yet clear that FSD1 is indeed SDH and FSD2 is indeed GSTZ1, due to sequence homology being less than 60 and 45%, respectively.

Defluorination activity in liver subcellular fractions was investigated in species of rodent, marsupials and monotremes. The defluorination activity in the mouse and rat was compared with that of two Australian marsupials, the brushtail possum and tamar wallaby to dtermine the species related differences in fluoroacetate sensitivity. The results indicated that there are clear species differences in the subcellular distribution of defluorination activity in all species, the marsupials possessed a certain amount of defluorination ability in their mitochondrial fractions. The nuclear fractions generally contained a consistent amount of defluorination activity across the four species, indicating the need for further investigation. The single playpus and echidna samples also exhibited a similar distribution of defluorination activity in the liver subcellular fractions. These results clearly localise the fluoroacetate defluorination activity in liver cytosol and provide further insight into the fluoroacetate detoxication ability of Austrlian marsupials and montoremes. The enzyme kinetic study revealed that the Australian marsupials living outside the range of fluoroacetate-containing plants appear to have a lower ability to detoxify fluoroacetate than rodents have. This suggests that marsupials may be more disadvantaged in there metabolic detoxication of some xenobiotics than previously thought.