Peptidases from pig reproductive tract: purification and properties of aminopeptidases from uterine secretions, allantoic fluid, and amniotic fluid.
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In ovariectomized sows, aminopeptidase is secreted into the uterine lumen under the influence of progesterone. The enzyme also accumulates in allantoic and amniotic fluids of pregnant animals. We have purified the predominant form of this enzyme from uterine flushings, allantoic fluid, and amniotic fluid by the following steps: ammonium sulfate precipitation, Sepharose 6B chromatography, ion-exhange chromatography on diethylaminoethyl cellulose, and affinity chromatography using l-leucylglycine immobilized on agarose. The overall procedure gave approximately 974-, 110-, and 230-fold purifications of the allantoic, uterine, and amniotic enzymes, respectively. The enzymes from all three sources are glycoproteins with pI's around 4 and molecular weights of about 480,000. They may be dissociated into six apparently identical subunits of molecular weight 80,000 as judged by sodium dodecyl sulfate gel electrophoresis. With l-leucyl-β-naphthylamide as substrate the pH optimum and apparent K m value for each enzyme were 7.1 and 14 μm, respectively. However, the uterine and allantoic aminopeptidases exhibited V values of 0.35 μmol of substrate hydrolyzed/min/mg of protein, whereas the V for the amniotic enzyme was at least sixfold greater. The amniotic enzyme also differed from the other two in pH and temperature stability. The activity of all three enzymes was stimulated by Co 2+ and inhibited by Cu 2+ , Fe 3+ , and chelating agents, while iodoacetate and mercaptoethanol had no effect on catalysis. The effect of Co 2+ on the allantoic enzyme was investigated in further detail. The stimulation of peptidase activity by Co 2+ was shown to be a complex process but consistent with Co 2+ replacing another metal at the active site and at some other additional site on the enzyme. The function of the aminopeptidases in the pregnant uterus is unknown. © 1978.
author list (cited authors)
Basha, S. M., Horst, M. N., Bazer, F. W., & Roberts, R. M
complete list of authors
Basha, SM||Horst, MN||Bazer, FW||Roberts, RM