SELENITE INCUBATED WITH NADPH AND MAMMALIAN THIOREDOXIN REDUCTASE YIELDS SELENIDE, WHICH INHIBITS LIPOXYGENASE AND CHANGES THE ELECTRON-SPIN-RESONANCE SPECTRUM OF THE ACTIVE-SITE IRON

Selenite and selenodiglutathione (GS-Se-SG) efficiently inhibited 5-li poxygenase activity in sonicates of human monoclonal B-lymphocytes. Th e apparent IC50 of GS-Se-SG was 0.5 mu M. The inhibitory effect of the se compounds was observed within 10 min of incubation. In order to elu cidate if the mechanism of inhibition by these compounds was result of direct interference with lipoxygenase or indirectly mediated by cellu lar factors, pure 15-lipoxygenase from soybeans was used as a model sy stem for enzyme assays and electron spin resonance (ESR) measurements. Incubation of 15-lipoxygenase with a mixture of human placenta thiore doxin reductase (TR) or calf-thymus TR, selenite, and NADPH blocked th e activity of the enzyme. Neither TR and NADPH nor selenite inhibited soybean lipoxygenase when incubated separately. These results suggest that selenite must be reduced to selenide in order to inhibit 5- and 1 5-lipoxygenase activities. Preincubation anaerobically of 15-lipoxygen ase with chemically generated selenide (6 mu M) resulted in a strong i nhibition of activity, in assays with arachidonic acid in the presence of oxygen. In contrast, selenide exposed to air prior to preincubatio n did not inhibit the enzyme. Since selenide is known to be efficientl y oxidized by oxygen and to form elemental selenium the results eviden ce that selenide was the inhibitor of lipoxygenase activity in the ana erobic preincubations. After incubation with TR, NADPH, and selenite o r with chemically generated selenide, the ESR spectrum of 15-lipoxygen ase changed: the dominant axial component with a peak at g = 6.1 decre ased, and a rhombic form with a feature at g = 4.28 grew. The results suggest that selenide produced by the reduction of selenite reduces th e active site iron to the ESR invisible state and changes the ligation geometry of the oxidized form.