Abstract
S-nitro-N-acetyl-DL-penicillamine (SNAP), a nitric oxide (NO) donor, inactivated bovine glutathione peroxidase (GPx) in a dose- and time-dependent manner. The IC50 of SNAP for GPx was 2 microM at 1 h of incubation and was 20% of the IC50 for another thiol enzyme, glyceraldehyde-3-phosphate dehydrogenase, in which a specific cysteine residue is known to be nitrosylated. Incubation of the inactivated GPx with 5 mM dithiothreitol within 1 h restored about 50% of activity of the start of the SNAP incubation. A longer exposure to NO donors, however, irreversibly inactivated the enzyme. The similarity of the inactivation with SNAP and reactivation with dithiothreitol of GPx to that of glyceraldehyde-3-phosphate dehydrogenase, suggested that NO released from SNAP modified a cysteine-like essential residue on GPx. When U937 cells were incubated with 100 microM SNAP for 1 h, a significant decrease in GPx activity was observed although the change was less dramatic than that with the purified enzyme, and intracellular peroxide levels increased as judged by flow cytometric analysis using a peroxide-sensitive dye. Other major antioxidative enzymes, copper/zinc superoxide dismutase, manganese superoxide dismutase, and catalase, were not affected by SNAP, which suggested that the increased accumulation of peroxides in SNAP-treated cells was due to inhibition of GPx activity by NO. Moreover, stimulation with lipopolysaccharide significantly decreased intracellular GPx activity in RAW 264.7 cells, and this effect was blocked by NO synthase inhibitor N omega-methyl-L-arginine. This indicated that GPx was also inactivated by endogenous NO. This mechanism may at least in part explain the cytotoxic effects of NO on cells and NO-induced apoptotic cell death.
Highlights
S-nitro-N-acetyl-DL-penicillamine (SNAP), a nitric oxide (NO) donor, inactivated bovine glutathione peroxidase (GPx) in a dose- and time-dependent manner
Inactivation of Purified Bovine Cytosolic GPx by SNAP— Because NO modifies the activities of several enzymes in which thiol groups are essential for catalytic function (4 – 6), it was of interest to determine whether NO could affect the activity of GPx, which contains a seleno-cysteine in its catalytic center
Several enzymes that catalyze reactions essential to metabolism are known to be inactivated or modified by NO [22]. The modifications of these enzymes are classified into two groups. (i) In proteins containing heme or non-heme irons as their cofactors, such as a soluble guanylate cyclase [1], aconitase [22], cytochrome c oxidase [23], and cyclooxygenase [24], NO binds to the iron molecule of these cofactors. (ii) In enzymes that contain a catalytically essential sulfhydryl group, such as GAPDH (4 – 6), the N-methyl-D-aspartic acid subtype of glutamate receptor [25], and protein kinase C [7], NO interacts with the thiol group to form a nitrosocompound, thereby inactivating the enzyme
Summary
Michio Asahi‡§, Junichi Fujii‡, Keiichiro Suzuki‡, Han Geuk Seo‡, Tsunehiko Kuzuya§¶, Masatsugu Hori§, Michihiko Tada§¶, Shigeru Fujiiʈ, and Naoyuki Taniguchi‡**. The similarity of the inactivation with SNAP and reactivation with dithiothreitol of GPx to that of glyceraldehyde-3-phosphate dehydrogenase, suggested that NO released from SNAP modified a cysteine-like essential residue on GPx. When U937 cells were incubated with 100 M SNAP for 1 h, a significant decrease in GPx activity was observed the change was less dramatic than that with the purified enzyme, and intracellular peroxide levels increased as judged by flow cytometric analysis using a peroxide-sensitive dye. Stimulation with lipopolysaccharide significantly decreased intracellular GPx activity in RAW 264.7 cells, and this effect was blocked by NO synthase inhibitor N-methyl-L-arginine. This indicated that GPx was inactivated by endogenous NO. We described here the inactivation of GPx by NO in a rather specific manner compared with another NO-sensitive enzyme, GAPDH, and discussed its physiological relevance
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