Purification and characterization of rat uterine glycerylphosphorylcholine diesterase and its tissue-specific induction by 17 beta-estradiol.
The rat uterine enzyme glycerylphosphorylcholine (GPC) diesterase found in the proestrous secretions was purified and characterized biochemically with respect to its subunit mol wt, native size, pI, and amino acid and carbohydrate composition. The 30-kDa protein was assessed to have a native mol wt of 105 kDa, as determined by analytical gel filtration. It had a basic pI with an unusually high carbohydrate/protein ratio (0.83:1). The estrogen inducibility of the protein was identified by its de novo synthesis in vitro after in vivo 17 beta-estradiol administration. For this experiment, uteri from estradiol-treated immature rats were incubated in the presence of [14C]glucosamine, and the 14C-labeled total glycoconjugates were then subjected to the enzyme purification procedure. The peak enzyme fraction was observed to correspond to one of the 14C-labeled protein peaks in the elution profile of the glycoconjugates. Assay of GPC diesterase activity showed significant enhancement only in the uterus, not in other organ systems, after in vivo estradiol treatment, and this hormone-stimulated increase was inhibited by nafoxidine, a potent estrogen antagonist. The enzyme activity profile of the three main separated uterine cellular types showed that the enzyme was secreted by epithelial cells. No antigenic homology of the enzyme was observed with GPC diesterase from nonmammalian sources. The results suggested that GPC diesterase was one of the estrogen-regulated proteins of the preovulatory uterine fluid of rats, and the availability of a purified preparation of the enzyme could serve as a useful tool to study the mechanism of estrogen action.
Mitra
J
J
Indian Institute of Chemical Biology, Calcutta.
Chowdhury
M
M
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002241:Carbohydrates / Q000032:analysis; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D004790:Enzyme Induction; D004958:Estradiol / Q000494:pharmacology*; D005260:Female; D006031:Glycosylation; D007700:Kinetics; D037102:Lectins; D046911:Macromolecular Substances; D008970:Molecular Weight; D009256:Nafoxidine / Q000494:pharmacology; D010727:Phosphoric Diester Hydrolases / Q000096:biosynthesis / Q000302:isolation & purification* / Q000378:metabolism; D011359:Proestrus / Q000502:physiology; D051381:Rats; D011919:Rats, Inbred Strains; D014599:Uterus / Q000187:drug effects / Q000201:enzymology*
D000596:Amino Acids; D002241:Carbohydrates; D037102:Lectins; D046911:Macromolecular Substances; D009256:Nafoxidine; D004958:Estradiol; D010727:Phosphoric Diester Hydrolases; C020677:glycerophosphocholine phosphodiesterase