Free radical reduction by thioredoxin reductase at the surface of normal and vitiliginous human keratinocytes.
Cell cultures of human keratinocytes contain membrane-associated thioredoxin reductase that is extremely active in reducing radicals on the outer plasma membrane. This enzyme activity was confirmed by its purification from cultures of stratified human keratinocytes by affinity column chromatography. The enzyme was assayed both in vivo and in vitro using a spin-labeled quaternary ammonium compound as the substrate, under saturating conditions in free radical substrate. Specific activities were determined by monitoring the sequential decrease in the amplitude of the electron spin resonance signal per unit of cell protein. The following properties were found: Cultures of adult stratified cells have approximately twice the thioredoxin reductase activity of neonatal cells. The enzyme is inhibited by thioprotein inhibitors (i.e., parachloromecuribenzoate and dinitrochlorobenzene). The activity is regulated by calcium concentrations of the cell culture medium. Stratified keratinocytes are half as active in medium containing 2 mM Ca++ compared with 0.1 mM Ca++ concentration. Product inhibition of the enzyme occurs with oxidized coenzyme NADP+ (i.e., 87% inhibition of enzyme activity over 30 min). The enzyme is heat stable at temperatures of 70 degrees C for 10 min. It is inactivated at 75 degrees C. A comparative study of thioredoxin reductase activity on stratified differentiated and undifferentiated rapidly growing cells was performed. Also, enzyme activity was quantitated for cultured keratinocytes isolated from vitiliginous and normal skin of the same donor. The results of this study, and the connection between this enzyme activity and UV-generated free radicals are reconciled in terms of the mechanism of action and metabolic activity of thioredoxin reductase.
D000328:Adult; D001706:Biopsy; D005609:Free Radicals; D006801:Humans; D007231:Infant, Newborn; D007633:Keratins / Q000378:metabolism*; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism*; D012016:Reference Values; D012867:Skin / Q000201:enzymology* / Q000473:pathology; D013113:Spin Labels; D013880:Thioredoxin-Disulfide Reductase / Q000378:metabolism*; D014820:Vitiligo / Q000201:enzymology* / Q000473:pathology
D005609:Free Radicals; D013113:Spin Labels; D007633:Keratins; D009247:NADH, NADPH Oxidoreductases; D013880:Thioredoxin-Disulfide Reductase