HUMAN PLACENTAL BETA-ENDORPHIN (HP-BETA-EP)

The previous finding that the placenta is capable of synthesising an ACTH-like peptide, human chorionic corticotrophin (hCC)(l) and the observate that both ACTH andP. EP are part of the same glycoprotein of 31K daltons in t~ p i tui tary (2) led us to investigate the presence of" EP in placental e x t r ac cs Freshly collected, sliced ~nd cold saline Ivashed placenta was extractei with acid acetone (l) to obtain a placental powder (PP)(5.8mg PP/g placenta' protein content 460ug/mg) which was then tested EorfiEP in a radioreceptor assay (RRA). For this assay 3H-D-ala-enkephalin was used a s t r a c e r and)lEP i. Tris-HCl 50mM pH 7.4+Bacitracin 50ug/ml as standard. Membranes were obtaip.=~ by homogenisation of rat forebrains in 25 volumes of Tris-HCl (Polytron set = for 15sec) a nd c e n t-r i f ug a t i o n at 15,000rpm x 15min (JA 20 ro t o r ) . The memb:-- anes were diluted with 5ml MnC12 60mM (frozen), 125ul Bacitracln stock solu:- ion and 24.875ml 50Mrn Tris-HCl pH 7.4 buffer. Fo r the assay, 500).11. membrane suspension, 100ul tracer, 100).1m standard or unknown sample and buffer up to ::. mi were used. After 2hr incubation and shaking at 26°C, filtration was per:- ormed on Millipore with cold buffer washing solution. PP prepared as above gave a dilution curve p a r a l I e I to that oEPEP and a potency o f 5.15ngpEP/mb PP protein. These re~ult$ re~ain qp~n çq criticis~ due to the lack of spec- ificity of the RRA.