Thordarson G, Southard J N, Talamantes F
Department of Biology, Sinsheimer Laboratories, University of California, Santa Cruz 95064.
Endocrinology. 1991 Sep;129(3):1257-65. doi: 10.1210/endo-129-3-1257.
The effects of secretagogue(s) from mouse decidual tissue on the release of mouse placental lactogen-II (mPL-II) were studied. Decidual tissue was obtained from 10- and 11-day-pregnant mice. The tissue was homogenized, extracted, and the tissue extract was made 50% saturated with ammonium sulfate. Both the precipitate and supernatant were tested for their ability to stimulate mPL-II release from cultured trophoblasts. The supernatant contained an activity to stimulate the release of mPL-II. This activity was further purified using column chromatography. The purification resulted in isolation of a protein with a mol wt of 20 K as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions and 6 K under reducing conditions. Further characterization of this protein showed that it binds calcium and has an amino acid sequence that is highly homologous with calcyclin expressed in mouse embryonic fibroblast cells and with calcyclin from other species. This protein was designated mouse decidual calcyclin. Antiserum was raised against the purified decidual calcyclin for development of an RIA and for immunoblots. Western blots of various mouse tissue extracts and mouse serum from different physiological stages showed that the concentration of calcyclin was highest in decidual tissue. Detectable levels were found in extracts from trophoblast, lung, and stomach, but the concentrations in these tissues were about 100 times lower than in decidua. Decidual calcyclin was not detectable in mouse serum. Cultured decidual cells released calcyclin into the medium. On average, this release was about 7.8 ng/micrograms DNA.24 h. The rate of release did not change significantly during 4 days of culture. The ratio of calcyclin in cells per calcyclin released during 24 h averaged 2.3 and did not change significantly during the culture period. The purified decidual calcyclin stimulated the release of mPL-II from cultured trophoblasts in a dose-dependent manner at concentrations from 0.01 to 1 microgram/ml. The maximum stimulation averaged about 1.5 times above control. It is concluded that decidual calcyclin may be of physiological importance for the regulation of mPL-II secretion.
研究了来自小鼠蜕膜组织的促分泌素对小鼠胎盘催乳素-II（mPL-II）释放的影响。蜕膜组织取自妊娠10天和11天的小鼠。将组织匀浆、提取，然后用硫酸铵使其50%饱和。检测沉淀和上清液刺激培养的滋养层细胞释放mPL-II的能力。上清液含有刺激mPL-II释放的活性。使用柱色谱法对该活性进行进一步纯化。纯化后通过非还原条件下的十二烷基硫酸钠-聚丙烯酰胺凝胶电泳估计分离出一种分子量为20K的蛋白质，还原条件下为6K。对该蛋白质的进一步表征表明它能结合钙，并且其氨基酸序列与在小鼠胚胎成纤维细胞中表达的钙周期蛋白以及其他物种的钙周期蛋白高度同源。该蛋白质被命名为小鼠蜕膜钙周期蛋白。制备了针对纯化的蜕膜钙周期蛋白的抗血清，用于开发放射免疫分析和免疫印迹。对来自不同生理阶段的各种小鼠组织提取物和小鼠血清进行的蛋白质免疫印迹显示，钙周期蛋白的浓度在蜕膜组织中最高。在滋养层、肺和胃的提取物中可检测到该蛋白，但这些组织中的浓度比蜕膜中的低约100倍。在小鼠血清中未检测到蜕膜钙周期蛋白。培养的蜕膜细胞将钙周期蛋白释放到培养基中。平均而言，这种释放量约为7.8 ng/μg DNA·24 h。在培养4天期间，释放速率没有显著变化。每24小时释放的钙周期蛋白与细胞内钙周期蛋白的比率平均为2.3，在培养期间没有显著变化。纯化的蜕膜钙周期蛋白以剂量依赖方式刺激培养的滋养层细胞释放mPL-II，浓度范围为0.01至1 μg/ml。最大刺激平均比对照高约1.5倍。得出的结论是，蜕膜钙周期蛋白可能对mPL-II分泌的调节具有生理重要性。
