pates in the synthesis of glutathione (GSH) through the We investigated the modulation of rat liver S-adenotranssulfuration pathway. 2 sylmethionine (SAM) synthetase in a model of acute sep-Human and rat liver SAM synthetases exist in high-and sis. Our results show that animals treated with bacterial low-molecular weight forms, with values of 220 and 110 kd, lipopolysaccharide experience a marked decrease in respectively. 3,4 When both forms are analyzed by sodium doliver SAM synthetase activity. No changes were detected decyl sulphate-polyacrylamide gel electrophoresis (SDSin the hepatic levels of SAM synthetase protein, sug-PAGE), only a 48-kd polypeptide band is detected. 4 The gesting that inactivation of the existing enzyme was the expression in Escherichia coli of rat liver SAM synthetase cause of the observed activity loss. Lipopolysaccharide complementary DNA (cDNA) results in the appearance of two treatment resulted in the expression of calcium-indeactive forms of the same molecular weight as those present pendent/cytokine-inducible nitric oxide (NO) synthase in human and rat liver, indicating that the high-molecular in liver and the accumulation in plasma of the NO-deweight form is a tetramer and the low-molecular weight rived species nitrite and nitrate. NO implication in the form is a dimer of the same 48-kd subunit. 5 The human and in vivo regulation of SAM synthetase was evaluated in rat liver SAM synthetases have been cloned and sequenced. 6,7 animals treated with the NO donor molecule 3-morphol-
The deduced amino acid sequence of both enzymes shows the inosydnonimine. The analysis of liver enzymatic activpresence of 10 cysteine residues that are conserved in rodents ity, along with protein and messenger RNA levels and humans. 8 Sulphydryl groups may play a role in the reguyielded results similar to those obtained with lipopolylation of a number of enzymes, 9 including rat liver SAM synsaccharide treatment. To assess directly the sensitivity thetase. 10,11 In this sense, the exchange of certain cysteine of SAM synthetase to NO, the rat liver-purified highresidues in SAM synthetase for serine by site-directed mutaand low-molecular weight forms of the enzyme were genesis significantly inhibited the catalytic activity and alexposed to various doses of 3-morpholinosydnonimine tered the oligomeric state. 8 and other NO donors such as S-nitroso-N-acetylpenicil-Several liver disorders have been associated with an imlamine, resulting in a dose-dependent inhibition of enzypairment of methionine metabolism and the methylation cymatic activity. This effect was reversed by addition of cle. SAM synthesis and utilization are altered in conditions the reducing agents b-mercaptoethanol and glutathione.
such as alcoholic and posthepatic cirrhosis 12 and in response Finally, cysteine 121 was identified as the site of molecuto the administration of certain drugs and hepatotoxins, inlar interaction between NO and rat liver SAM synthetase cluding alcohol, carbon tetrachloride, galactosamine, and that is responsible for the inhibition of the enzyme. To acetaminophen (reviewed by Mato et al. 13 ). reach this conclusion, the 10 cysteine residues of the Alterations of hepatic function have been described during enzyme were changed to serine by site-directed mutaseptic shock, 14 a pathological condition triggered by the lipogenesis, and the effect of NO on the various recombinant polysaccharide (LPS) present in the outer membranes of enzymes was measured. (HEPATOLOGY 1997;25:391-396.) gram-negative bacteria. 15 In sepsis the liver is a target for LPS and a number of endotoxic shock effectors, such as proin-Liver methionine metabolism starts with the formation of flammatory cytokines 16 ; however, the mechanisms behind S-adenosylmethionine (SAM). This reaction involves the the hepatic changes elicited by endotoxemia are not comtransfer of the adenosyl moiety of adenosine triphosphate pletely understood. The expression in liver of the cytokine-(ATP) to methionine, and is catalyzed by the enzyme SAM inducible nitric oxide (NO) synthase (iNOS) after LPS adminsynthetase. 1 SAM serves as the methyl donor for essentially istration has been the focus of much attention (reviewed by all known biological methylation reactions, provides the pro-Freeswick et al. 17 ). NO has been implicated in the regulation pylamine group for the synthesis of polyamines, and particiof some of the metabolic changes taking place in the liver during sepsis. [18][19][20] Given the prominent role of SAM synthetase in maintaining hepatic function, we studied the regulation of this enzyme in a model of septic shock and evaluated Abbreviations: SAM, S-adenosylmethionine; ATP, adenosine triphosphate; GSH, glutathe hypothesis of NO as a reversible modulator of SAM synthione; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; cDNA, comthetase activity.