Requirement of an essential thiol group and ferric iron for the activity of the progesterone-induced porcine uterine purple phosphatase.
The progesterone-induced purple phosphatase isolated from the uterine flushings of pigs is activated by a variety of reagents that cleave disulfide bonds, including 2-mercaptoethanol, dithiothreitol, L-ascorbate, L-cysteine, sulfite, and cyanide. It is inhibited by various mercurials, iodoacetamide, O-iodosobenzoate, and hydrogen peroxide. Thiols increase the specific phosphatase activity from 25 to about 300 units per mg of enzyme. This activation is accompanied by a shift in the extinction maximum to higher energy to yield a protein with a pink coloration. Following maximum activation there is a gradual decrease in enzyme activity and protein color which is accompanied by loss of ferrous iron from the protein. Sodium dithionite at 10 mM or higher causes an immediate inhibition of phosphatase activity and bleaching of color, and can be used to prepare the iron-free apoprotein. The latter can be partially reactivated by Fe3+ salts but not by Fe2+. The Fe3+ restores the pink form of the enzyme with a specific activity of about 200 units/mg of protein. Cu2+ also causes some reactivation, but other metal ions were ineffective. ESR studies showed that the pink form of phosphatase contains approximately 1 atom of high spin ferric iron per molecule. It is concluded that the phosphatase requires a free thiol and Fe3+ for activity. Reduction of the iron leads to complete loss of both color and enzyme activity. The color change from purple to pink represents disulfide reduction and is not due to reduction of iron.
D000135:Acid Phosphatase / Q000096:biosynthesis / Q000378:metabolism*; D000818:Animals; D003116:Color; D003486:Cyanides / Q000494:pharmacology; D003545:Cysteine / Q000494:pharmacology; D004227:Dithionite / Q000494:pharmacology; D004229:Dithiothreitol / Q000494:pharmacology; D004578:Electron Spin Resonance Spectroscopy; D004789:Enzyme Activation; D004790:Enzyme Induction; D005260:Female; D005290:Ferric Compounds / Q000494:pharmacology*; D005978:Glutathione / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007501:Iron / Q000494:pharmacology*; D007700:Kinetics; D008623:Mercaptoethanol / Q000494:pharmacology; D011374:Progesterone / Q000494:pharmacology*; D013438:Sulfhydryl Compounds / Q000494:pharmacology*; D013447:Sulfites / Q000494:pharmacology; D014599:Uterus / Q000201:enzymology*
D003486:Cyanides; D005290:Ferric Compounds; D013438:Sulfhydryl Compounds; D013447:Sulfites; D004227:Dithionite; D011374:Progesterone; D008623:Mercaptoethanol; D007501:Iron; D000135:Acid Phosphatase; D005978:Glutathione; D003545:Cysteine; D004229:Dithiothreitol