Peroxidase, an alternate pathway to cytochrome P-450 for xenobiotic metabolism in skin: partial purification and properties of the enzyme from neonatal rat skin.
Peroxidase activity was partially purified from neonatal (3 to 6 days old) rat skin. The membrane-bound peroxidase activity was extracted with 0.5 M calcium chloride and was monitored spectrophotometrically at 470 nm with 2-methoxyphenol (guaiacol) and hydrogen peroxide as substrates. Subcellular distribution studies indicated the activity to be highest and comparable in nuclei and mitochondria, lowest in microsomes, and absent in cytosol. The peroxidase activity was partially purified by affinity chromatography on concanavalin A-sepharose 4B and by gel filtration using Bio-Gel P-150. Purification factors from these two steps were about 25 and 4, respectively. Peroxidase extraction in the presence of 2 mM N-ethylmaleimide increased activity about twofold. The combination of 2 mM N-ethylmaleimide and 10% (w/v) glycerol was found to be optimal for preservation of activity. Peroxidase activity increased linearly with increases in protein concentration, time, and guaiacol concentration. Activity was inhibited approximately 75% by 0.1 mM potassium cyanide or 0.05 mM sodium azide. Pyrogallol, hydroquinone, p-cresol, catechol, benzidine, 3,3'-dimethoxybenzidine, tetramethylbenzidine and p-phenylenediamine also acted as substrates for the rat cutaneous peroxidase.
Strohm
B H
BH
Department of Environmental and Industrial Health, School of Public Health, University of Michigan, Ann Arbor 48109.
Kulkarni
A P
AP
D000818:Animals; D000831:Animals, Newborn; D002467:Cell Nucleus / Q000201:enzymology / Q000378:metabolism; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D003577:Cytochrome P-450 Enzyme System / Q000302:isolation & purification*; D004789:Enzyme Activation / Q000187:drug effects; D006139:Guaiacol / Q000494:pharmacology; D006861:Hydrogen Peroxide / Q000494:pharmacology; D008861:Microsomes / Q000201:enzymology / Q000378:metabolism; D008928:Mitochondria / Q000201:enzymology / Q000378:metabolism; D009195:Peroxidase / Q000037:antagonists & inhibitors / Q000302:isolation & purification*; D051381:Rats; D012867:Skin / Q000201:enzymology* / Q000378:metabolism; D013379:Substrate Specificity; D013696:Temperature; D015262:Xenobiotics / Q000378:metabolism*
D015262:Xenobiotics; D006139:Guaiacol; D003577:Cytochrome P-450 Enzyme System; D006861:Hydrogen Peroxide; D009195:Peroxidase