Human alcohol dehydrogenase 3/glutathione-dependent formaldehyde dehydrogenase was shown to rapidly and irreversibly catalyse the reductive breakdown of S-nitrosoglutathione. The steady-state kinetics of S-nitrosoglutathione reduction was studied for the wild-type and two mutated forms of human alcohol dehydrogenase 3, mutations that have previously been shown to affect the oxidative efficiency for the substrate S-hydroxymethylglutathione. Wild-type enzyme readily reduces S-nitrosoglutathione with a k cat /K m approximately twice the k cat /K m for S-hydroxymethylglutathione oxidation, resulting in the highest catalytic efficiency yet identified for a human alcohol dehydrogenase. In a similar manner as for S-hydroxymethylglutathione oxidation, the catalytic efficiency of S-nitrosoglutathione reduction was significantly decreased by replacement of Arg115 by Ser or Lys, supporting similar substrate binding. NADH was by far a better coenzyme than NADPH, something that previously has been suggested to prevent reductive reactions catalysed by alcohol dehydrogenases through the low cytolsolic NADH/NAD + ratio. However, the major products of S-nitrosoglutathione reduction were identified by electrospray tandem mass spectrometry as glutathione sulfinamide and oxidized glutathione neither of which, in their purified form, served as substrate or inhibitor for the enzyme. Hence, the reaction products are not substrates for alcohol dehydrogenase 3 and the overall reaction is therefore irreversible. We propose that alcohol dehydrogenase 3 catalysed S-nitrosoglutathione reduction is of physiological relevance in the metabolism of NO in humans.

Adhesion by means of b1-integrins induces the phosphorylation of Akt, an event strictly dependent on the activity of the phosphatidylinositol 3-kinase (PI3K). Binding of the p85 regulatory subunit of PI3K to phosphorylated tyrosine 397 in focal adhesion kinase (FAK) is considered to be the mechanism of cell adhesion-induced activation of class Ia PI3K. Here we show that PI3K-dependent phosphorylation of Akt in response to ligation of b1-integrins occurs efficiently in the absence of FAK tyrosine phosphorylation. Akt S473 phosphorylation was strongly promoted both in cells expressing the integrin subunit splice variant b1B, which is unable to activate FAK, and in FAK knockout cells. In addition, we found this phosphorylation to be independent of the Src family kinases Src, Fyn and Yes. These results indicate that a major pathway for adhesion-dependent activation of PI3K/Akt is triggered by the membrane proximal part of the b1 subunit in a FAK and Src-independent manner.

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