Rabbit placental relaxin: purification and immunohistochemical localization.
Rabbit placentas were extracted with 0.7 N HCl-acetone (3:5, vol/vol) containing protease inhibitors. Gel filtration (Sephadex G-50) followed by ion exchange chromatography (carboxymethyl cellulose) separated a bioactive relaxin-like fraction with a specific activity of 8.5 U/mg protein as determined by the in vitro mouse uterus bioassay. Column chromatography using Sepharose CL-4B in 6 M guanidine HCl was employed to purify the bioactive sample. The yield of the purified relaxin-like protein was 12 micrograms/g placenta and the specific activity was 23 U/mg protein. The bioactive sample was also immunoreactive after being electrophoretically transferred to nitrocellulose paper and stained using rabbit antiporcine relaxin serum and peroxidase-antiperoxidase immunochemistry. Isoelectrofocusing of the purified relaxin-like protein revealed one band with an isoelectric point of approximately 6.5. The apparent molecular weight of the rabbit relaxin was approximately 7200 as determined by sodium dodecyl sulfate-urea slab gel electrophoresis. Upon reduction with 5.0% mercaptoethanol and electrophoresis on sodium dodecyl sulfate-urea polyacrylamide gels, both the 7200 dalton rabbit immunoreactive relaxin-like polypeptide and porcine relaxin migrated as a lower molecular weight protein. These results suggest that rabbit relaxin, like pig, rat, shark, and human relaxin, consists of two chains linked by disulfide bonds. At the light microscopy level, immunohistochemical staining with guinea pig antiporcine relaxin serum indicated that relaxin was located in the syncytiotrophoblast cells of the placental labyrinth of day-23 and day-30 pregnant rabbits. The syncytiotrophoblast cells from day 16 of pregnancy did not stain for relaxin.
D000818:Animals; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003429:Cross Reactions; D004220:Disulfides / Q000032:analysis; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D006651:Histocytochemistry; D007158:Immunologic Techniques; D007525:Isoelectric Focusing; D008970:Molecular Weight; D010920:Placenta / Q000032:analysis*; D011247:Pregnancy; D011817:Rabbits; D012065:Relaxin / Q000276:immunology / Q000302:isolation & purification*
D004220:Disulfides; D012065:Relaxin