Skin sulfhydryl oxidase. Purification and some properties.
A sulfhydryl-oxidizing enzyme has been found in skin of young rats and a method for purifying the enzyme over 600-fold has been developed. Enzymatic activity was assayed either by its ability to oxidize dithiothreitol of by measuring its ability to renature reductively denatured ribonuclease A. Skin sulfhydryl oxidase catalyzed the oxidation of various thiols: dithiothreitol, dithioerythritol, D-penicillamine, and L-cysteine. Glutathione and 2-mercaptoethanol were very poor substrates for the enzyme. The enzyme also reactivated reductively denatured ribonuclease A, with neither the presence of a thiol nor prior reduction of the enzyme being necessary. The molecular weight of the enzyme was estimated to be 66 000 +/- 2000, and the isoelectric point was determined to be at pH 4.65. Alkylating reagents alone had some inhibiting effect  on skin sulfhydryl oxidase; when the enzyme was preincubated with thiols which were substrates, inhibition by alkylating reagents was greatly increased. After preincubation with dithiothreitol, treatment of the enzyme with alkylating reagents or N-ethylmaleimide caused significant inhibition; preincubation with a poor substrate, reduced glutathione, did not enhance inhibition by alkylating reagents or N-ethylmaleimide.
D000818:Animals; D002852:Chromatography, Ion Exchange; D004492:Edetic Acid / Q000494:pharmacology; D004720:Endonucleases / Q000378:metabolism; D006358:Hot Temperature; D008297:Male; D017830:Octoxynol; D010088:Oxidoreductases / Q000378:metabolism*; D011092:Polyethylene Glycols / Q000494:pharmacology; D051381:Rats; D012259:Ribonuclease, Pancreatic; D012260:Ribonucleases / Q000378:metabolism; D012867:Skin / Q000201:enzymology*; D013379:Substrate Specificity; D013438:Sulfhydryl Compounds / Q000378:metabolism; D050862:Oxidoreductases Acting on Sulfur Group Donors
D004492:Edetic Acid; D004720:Endonucleases; D017830:Octoxynol; D010088:Oxidoreductases; D011092:Polyethylene Glycols; D012259:Ribonuclease, Pancreatic; D012260:Ribonucleases; D013438:Sulfhydryl Compounds; D050862:Oxidoreductases Acting on Sulfur Group Donors