An Iron-containing Phosphatase Induced by Progesterone in the Uterine Fluids of Pigs

Abstract

A basic, progesterone-induced, intrauterine glycoprotein with a purple coloration can be purified from the uterine fluids of ovariectomized female pigs given daily doses of progesterone, by successive chromatography on columns of CM-cellulose and Sephadex G-100.The protein contains 1 atom of iron per 32,000 molecular weight polypeptide and shows phosphatase activity toward p-nitrophenylphosphate as substrate.The pH optimum of the enzyme is 4.9 and the apparent K, is 2.2 rnM at 30".Activity is enhanced 2-to 4-fold in presence of /3-mercaptoethanol, although the K,,, remains unchanged.Under such optimal conditions 1 molecule of enzyme can hydrolyze approximately 5 x lo3 molecules of substrate per min.Activity is inhibited by fluoride, arsenate, phosphate, and molybdate.To a lesser extent, the enzyme will catalyze the hydrolysis of ATP and pyrophosphate, but has very low activity toward /3-glycerophosphate and n-glucose 6-phosphate.The activation of the enzyme by P-mercaptoethanol is accompanied by a shift in the extinction maximum from 545 nm to around 510 nm.Sodium dithionite causes loss of color and inactivates the enzymatic activity completely.The precise role of the protein in the early pregnant uterus, however, remains unknown.A progesterone-induced, basic glycoprotein was recently purified from the uterine fluids of pigs by a simple two-step procedure (1).This glycoprotein, which had a natural purple color, was one of a group of at least eight proteins which were unique to the luteal phase of the estrous cycle when plasma progesterone levels were high (2-4).The same group of proteins could also be induced in ovariectomized animals given daily doses of progesterone (5) sufficient to maintain pregnancy in the fertilized pig (6).