Oviduct dolichyl phosphate phosphatase: estrogen effects and a possible biosynthetic role.

Estrogen-induced chick oviduct differentiation is accompanied by an increased capacity for protein glycosylation. A portion of this increase has been attributed to increased levels of dolichyl phosphate (Dol-P). Hormone withdrawal leads to an apparent decrease in Dol-P. Dol-P metabolism in the oviduct has been studied, and one of the enzymes having a direct effect on Dol-P, Dol-P phosphatase is herein described. Dol-P phosphatase has a pH optimum of 6.0, does not require a metal ion, and is inhibited by Mn2+ at concentrations greater than 5 mM. Inhibitor studies indicate that Dol-P hydrolysis is inhibited by polyprenyl phosphates having both saturated and unsaturated alpha-isoprene residues, but not by the corresponding alcohols. The enzyme is also inhibited by phosphatidic acid unless 2 mM Mn2+ is included in the incubations. Under these conditions Dol-P hydrolysis is only slightly inhibited (less than 10%), but phosphatidate inhibition is totally eliminated. Oviduct membranes also possess phosphatidate phosphatase, but this enzyme is distinct from Dol-P phosphatase based on thermolability, metal ion sensitivity, and sulfhydryl reagent sensitivity. Studies of enzyme activity in response to estrogen treatment reveal that both Dol-P phosphatase and phosphatidate phosphatase have maximal specific activity early in the differentiation process (peaking after 3 days of treatment), and low specific activity in fully differentiated oviducts, including laying hen oviduct. Hormone withdrawal elicits a small increase in specific activity of both phosphatases. The hormone effects suggest that Dol-P phosphatase may be a biosynthetic enzyme.

Estrogen-induced changes in chick oviduct membrane glycoproteins.

Membrane protein and glycoprotein changes during estrogen-induced differentiation of the chick oviduct were studied. Membrane preparations from chicks treated with diethylstilbestrol for 3, 6, 10, and 20 days; from chicks treated with hormone for 10 days and then withdrawn for 2 days; and from mature hens were incubated with either GDP-[14C]Man or GDP-[3H]Man. Double label analysis of proteins using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) indicates that labeling of proteins with apparent molecular weights of 91,000 and 57,000 is enhanced upon diethylstilbestrol treatment. The effect is reversed upon hormone withdrawal. Analysis of [14C]Man-proteins by SDS-PAGE and fluorography reveals at least 18 labeled proteins, confirms the double label gel results, and shows changes in three additional proteins. Estrogen-induced changes in membrane proteins and glycoproteins were also studied by Coomassie blue staining, periodic acid-Schiff staining, and autoradiography of 125I-concanavalin A binding following SDS-PAGE. Changes are observed in at least 9 Coomassie blue, 7 periodic acid-Schiff and 13 125I-concanavalin A bands. Some proteins are enhanced by estrogen treatment and are diminished upon withdrawal, whereas other proteins are diminished by estrogen treatment and are enhanced by withdrawal. Hen oviduct membrane preparations were labeled with combinations of UDP-GlcNAc, GDP-Man, and UDP-Glc. Tunicamycin inhibits incorporation of [3H]Man and [14C]GlcNAc into protein by 84% and 65%, respectively. SDS-PAGE reveals that transfer of GlcNAc to a 36,000 protein is the least sensitive to tunicamycin. SDS-PAGE of proteins labeled by incubation with GDP-[14C]Man and UDP-[3H]Glc indicate that the presence of Glc does not alter the typical Man-protein-labeling pattern and processing of Glc from individual acceptors uniform.