Glutathione-S-Crotonyl-N-Acetylthioethanolamine Alkenetransferase: Properties and Comparison with Other Glutathione-S-Transferases

<p>A mercapturic acid is an N-acetylated-S-substituted -L-cysteine in which the substitutive group is generally inert to further enzymic reaction (Wood, 1970). Detoxication of foreign compounds to mercapturic acids in mammals has been known for almost 100 years, however the source of cysteine for conjugation has only recently been positively determined.</p>

The role of the liver in the disposition of circulating mercapturic acids was examined in anesthetized rats and in the isolated perfused rat liver using S-2,4-dinitrophenyl- N-acetylcysteine (DNP-NAC) as the model compound. When DNP-NAC was infused into the jugular vein (150 or 600 nmol over 60 min) it was rapidly and nearly quantitatively excreted as DNP-NAC into bile (42–36% of the dose) and urine (48–62% of dose). Some minor metabolites were detected in bile (<4%), with the major metabolite coeluting on HPLC with the DNP conjugate of glutathione (DNP-SG). Isolated rat livers perfused single pass with 3 μM DNP-NAC removed 72 ± 9% of this mercapturic acid from perfusate. This rapid DNP-NAC uptake was unaffected by sodium omission, or byl-cysteine,l-glutamate,l-cystine, or N-acetylated amino acids, but was decreased by inhibitors of hepatic sinusoidal organic anion transporters (oatp), indicating that DNP-NAC is a substrate for these transporters. The DNP-NAC removed from perfusate was promptly excreted into bile, eliciting a dose-dependent choleresis. DNP-NAC itself constituted ∼75% of the total dose recovered in bile, reaching a concentration of 9 mM when livers were perfused in a recirculating mode with an initial DNP-NAC concentration of 250 μM. Other biliary metabolites included DNP-SG, DNP-cysteinylglycine, and DNP-cysteine. DNP-SG was likely formed by a spontaneous retro-Michael reaction between glutathione and DNP-NAC. Subsequent degradation of DNP-SG by biliary γ-glutamyltranspeptidase and dipeptidase activities accounts for the cysteinylglycine and cysteine conjugates, respectively. These findings indicate the presence of efficient hepatic mechanisms for sinusoidal uptake and biliary excretion of circulating mercapturic acids in rat liver and demonstrate that the liver plays a role in their whole body elimination.

SummaryThrombin acts on several coagulant proteins to produce products with physiologic, pharmacologic and pathologic potential. The most sensitive thrombin substrate seems to be factor VIII. Some thrombin dependent reactions studied in vitro and proposed as control reactions seem too insensitive to the action of thrombin to be of in vivo significance.The only enzymic reaction the thrombin-like venom enzymes, Ancrod and Batroxobin, have in common with thrombin is the removal of fibrinopeptide A.