On the other hand, free CO contents in the effluent from the isolated rat perfused liver (Kyokane et al., 2001, Suematsu et al., 1995 and Suematsu et al., 1994) and the cultured medium of the rat hepatocytes (Goda et al., 1998) were determined spectrophotometrically by measuring the formation of the ferrous–CO complex of myoglobin. The steady-state generation of CO was calculated to be ∼0.7 nmol/min/g of liver. When the differences in local flow rates between ex vivo and in vivo systems are considered, it appears that local concentrations of CO in the liver are approximately 1 μM ( Suematsu et al., 1995 and Suematsu et al., 1994). By contrast,
tissue concentrations of NO are likely to be in the range of 0.1–100 nM ( Bellamy et al., 2002, Buerk, 2001 and Buerk et al., 2003), much lower than those of CO (see review by Kajimura et al. (2010) for tissue concentrations of gases). Although Selleckchem Crizotinib the crystallographic structure of CO-ligated forms has yet to be determined, spectroscopic characterization of CO binding and dissociation kinetics to CBS suggest that disruption of a salt bridge between the Cys52 ligand to heme
and Arg266 of the enzyme by CO binding is communicated to the active site with concomitant inhibition of enzyme activity (Puranik et al., 2006). While such a regulatory role for the ferrous heme of CBS has been clearly demonstrated in vitro, the existence of the ferrous state, of which CO can bind, has been controversial Bosutinib chemical structure in vivo ( Singh et al., 2007). Recent study showed the evidence
for reversible inhibition of CBS by CO in the presence of a human flavoprotein and NADPH as redox partners ( Kabil et al., 2011). These results in vitro provide a mechanistic basis for interactions between CO and H2S in vivo discussed in Section 3.2. Differential display of metabolic footprint-profiling is designed to assess the control points by a specific intervention. Changes in patterns of metabolic fluxes on various pathways give a clue for a candidate enzyme responding to a gas. Shintani et al. Anacetrapib (2009) applied this method to identify the enzyme on which CO targets by comparing metabolic responses between livers from control mice and those treated with hemin to increase CO production. CO overproduction increases metabolites in the remethylation cycle and simultaneously decreases those in the transsulfuration pathway, which occurs in parallel with a decrease in hepatic H2S content. Subsequent in vivo pulse-chase analysis of 15N-methionine in livers of control mice and hemin-treated mice showed accumulation of 15N-homocysteine and suppression of 15N-cystathionine under the CO-overproducing conditions, suggesting that CO inhibits CBS in vivo. The ability of CO to limit CBS activity as a regulator of the transsulfuration pathway may have diverse impacts on biological systems.