Regulation of Rat Liver S –Adenosylmethionine Synthetase During Septic Shock: Role of Nitric Oxide

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Abstract
: We investigated the modulation of rat liver S–adenosylmethionine (SAM) synthetase in a model of acute sepsis. Our results show that animals treated with bacterial lipopolysaccharide experience a marked decrease in liver SAM synthetase activity. No changes were detected in the hepatic levels of SAM synthetase protein, suggesting that inactivation of the existing enzyme was the cause of the observed activity loss. Lipopolysaccharide treatment resulted in the expression of calcium–independent/cytokine–inducible nitric oxide (NO) synthase in liver and the accumulation in plasma of the NO–derived species nitrite and nitrate. NO implication in the in vivo regulation of SAM synthetase was evaluated in animals treated with the NO donor molecule 3–morpholinosydnonimine. The analysis of liver enzymatic activity, along with protein and messenger RNA levels yielded results similar to those obtained with lipopolysaccharide treatment. To assess directly the sensitivity of SAM synthetase to NO, the rat liver–purified high– and low–molecular weight forms of the enzyme were exposed to various doses of 3–morpholinosydnonimine and other NO donors such as S –nitroso– N –acetylpenicillamine, resulting in a dose–dependent inhibition of enzymatic activity. This effect was reversed by addition of the reducing agents beta–mercaptoethanol and glutathione. Finally, cysteine 121 was identified as the site of molecular interaction between NO and rat liver SAM synthetase that is responsible for the inhibition of the enzyme. To reach this conclusion, the 10 cysteine residues of the enzyme were changed to serine by site–directed mutagenesis, and the effect of NO on the various recombinant enzymes was measured.