Purification and characterization of mouse decidual calcyclin: a novel stimulator of mouse placental lactogen-II secretion.
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.
Thordarson
G
G
Department of Biology, Sinsheimer Laboratories, University of California, Santa Cruz 95064.
Southard
J N
JN
Talamantes
F
F
D000595:Amino Acid Sequence; D000818:Animals; D002135:Calcium-Binding Proteins / Q000302:isolation & purification* / Q000494:pharmacology; D018797:Cell Cycle Proteins; D002478:Cells, Cultured; D003656:Decidua / Q000502:physiology*; D004815:Epidermal Growth Factor / Q000494:pharmacology; D005260:Female; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000302:isolation & purification; D010920:Placenta / Q000187:drug effects / Q000378:metabolism*; D010928:Placental Lactogen / Q000378:metabolism*; D011247:Pregnancy; D000075383:S100 Calcium Binding Protein A6; D009418:S100 Proteins; D012689:Sequence Homology, Nucleic Acid; D014327:Trophoblasts / Q000187:drug effects / Q000378:metabolism
D002135:Calcium-Binding Proteins; D018797:Cell Cycle Proteins; D010446:Peptide Fragments; D000075383:S100 Calcium Binding Protein A6; D009418:S100 Proteins; C489134:S100a6 protein, mouse; C055351:S100A6 protein, human; D004815:Epidermal Growth Factor; D010928:Placental Lactogen