Phospholipid hydroperoxidase are substrates for non-selenium glutathione peroxidase

This study investigated phospholipid hydroperoxides as substrates for non-s elenium GSH peroxidase (NSGPx), an enzyme also called 1-Cys peroxiredoxin. Recombinant human NSGPx expressed in Escherichia coli from a human cDNA clo ne (HA0683) showed GSH peroxidase activity with sn-2-linolenoyl- or sn-2-ar achidonoyl-phosphatidylcholine hydroperoxides as substrate; NADPH or thiore doxin could not substitute for GSH, Activity did not saturate with GSH, and kinetics were compatible with a ping-pong mechanism; kinetic constants (mM (-1) min(-1)) were k(1) = 1-3 x 10(5) and k(2) = 4-11 x 10(4). In the prese nce of 0.36 mM GSH, apparent K-m was 120-130 mu M and apparent V-max was 1. 5-1.6 mu mol/min/mg of protein. Assays with H2O2 and organic hydroperoxides as substrate indicated activity similar to that with phospholipid hydroper oxides, Maximal enzymatic activity was at pH 7-8. Activity with phospholipi d hydroperoxide substrate was inhibited noncompetitively by mercaptosuccina te with K-i 4 mu M. The enzyme had no GSH S-transferase activity. Bovine cD NA encoding NSGPx, isolated from a lung expression library using a polymera se chain reaction probe, showed >95% similarity to previously published hum an, rat, and mouse sequences and does not contain the TGA stop codon, which is translated as selenocysteine in selenium-containing peroxidases, The mo lecular mass of bovine NSGPx deduced from the cDNA is 25,047 Da, These resu lts identify a new GSH peroxidase that is not a selenoenzyme and can reduce phospholipid hydroperoxides, Thus, this enzyme may be an important compone nt of cellular antioxidant defense systems.