Microsomal formation of S-nitrosoglutathione from organic nitrites: possible role of membrane-bound glutathione transferase

Abstract

The formation of S-nitrosoglutathione (GSNO) from amyl nitrite and n-butyl nitrite was studied in rat liver microsomes, employingN-ethylmaleimide (MalNEt) as an activator and indomethacin as an inhibitor of microsomal glutathione S-transferase (GST). Rates were compared with GST activity measured with 1-chloro-2,4-dinitrobenzene(CDNB) as a substrate. MalNEt stimulated GST activity and the formation of GSNO from amyl nitrite and n-butyl nitrite about 10-fold. Increasing concentrations of indomethacin inhibited both reactions in parallel. N-Acetyl-L-cysteine but not L-cysteine could substitute for GSH. It is concluded that rat liver microsomal GST catalyses the formation of GSNO from amyl nitrite and n-butyl nitrite. The activity of the MalNEt-stimulated microsomal GST is calculated to be about 17 units/mg of enzyme with the alkyl nitrites and about 16 units/mg of enzyme with CDNB as a substrate, assuming that 3% of microsomal protein is GST. These rates are comparable with those obtained for cytosolic GSTs. Thus microsomal GST may play a significant role in the metabolism of alkyl nitrites in biological membranes.

Biotransformation of Glyceryl Trinitrate by Rat Hepatic Microsomal GlutathioneS-Transferase 1

“…In contrast, in a previous study it was found that incubation of hepatic microsomes with the alkyl nitrites, amyl nitrite and n-butyl nitrite, resulted in the formation of GSNO, and that treatment of microsomes with NEM increased the rate of GSNO formation (Ji et al, 1996). These data suggest that subtle changes in substrate structure have profound effects on catalysis by MGST1 and that the interaction of organic nitrites and organic nitrates with MGST1 is fundamentally different.…”

“…GSNO was prepared by reacting equimolar concentrations of sodium nitrite and GSH as described previously (Ji et al, 1996). The concentration of GSNO was determined spectrophotometrically at 334 nm ( ϭ 767 M Ϫ1 cm Ϫ1 ).…”

“…Rat hepatic or aortic microsomes were prepared as described previously (McDonald and Bennett, 1993;Ji et al, 1996). Microsomes were washed twice with 100 mM Tris-HCl (pH 7.4) to decrease cytosolic contamination and then stored at Ϫ70°C.…”

Activation of Microsomal GlutathioneS-Transferase by Peroxynitrite

“…Male Sprague-Dawley rats (250-300 g) were fasted overnight, and hepatic microsomes were prepared as described previously (Ji et al, 1996). To remove cytosolic contamination, the microsomes were washed twice with 100 mM Tris-HCl, pH 7.4.…”

“…Samples (1.0 ml) contained 100 mM potassium phosphate, pH 6.5, 0.5% Triton X-100, 1 mM GSH, and 1 mM CDNB at 25°C. Enzyme activation by NEM was assessed after incubation of enzyme in 100 mM potassium phosphate, pH 7.0, for 1 min at room temperature with 1.0 mM NEM as described previously (Ji et al, 1996). For kinetic studies, enzyme activities were assayed with constant CDNB concentration (1.0 mM) and varying GSH concentrations (0.1-1.0 mM).…”