Purification and structural characterization of ovine placental lactogen.
Discrepancies exist in the reported purity and biological activity of ovine placental lactogen (oPL), and little structural characterization has been reported. Ovine PL was purified from fetal cotyledonary tissue (day 100 of gestation) by monitoring activity with a recombinant bovine GH (bGH) liver radioreceptor assay. Two hundred grams of tissue yielded 4.2 mg of oPL, with an approximately 1000-fold purification of oPL specific activity following initial tissue extraction. The oPL was radioiodinated and used in an ovine fetal liver (day 100 of gestation) radioreceptor assay to examine competitive displacement of oPL, ovine GH (oGH) and ovine prolactin (oPRL). The potency of oPL (ED50 = 0.18 nmol/l; ED50 is the quantity of ligand necessary to displace 50% of specifically bound 125I-labelled oPL) was greater than that of oGH (ED50 = 4.1 nmol/l) and oPRL (ED50 = 1.1 mumol/l) in competing for 125I-labelled oPL-binding sites. Attempts to sequence the NH2 terminus of oPL by vapour-phase sequencing indicated that the NH2 terminus was blocked. Purified oPL was subjected to trypsin and CnBr digestion, and two CnBr and six tryptic peptides were sequenced. The peptide sequences were compared with other PLs, oPRL and bGH for sequence similarity, and found to be most similar to bovine PL (bPL; 68% overall identity) and oPRL (47% overall identity). Complementary DNA (cDNA) clones were isolated for oPL by screening a lambda ZAP cDNA library with a cDNA coding for bPL. Three cDNAs were nucleotide sequenced, and their combined sequence included 41 nucleotides of 5'-untranslated region, the complete coding region of pre-oPL (708 nucleotides) and a portion of the 3' untranslated region (158 nucleotides).(ABSTRACT TRUNCATED AT 250 WORDS)
Warren
W C
WC
Department of Animal Sciences, University of Missouri, Columbia 65211.
Liang
R
R
Krivi
G G
GG
Siegel
N R
NR
Anthony
R V
RV
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D004247:DNA / Q000032:analysis; D005260:Female; D005333:Fetus; D008099:Liver / Q000032:analysis; D008969:Molecular Sequence Data; D010928:Placental Lactogen / Q000302:isolation & purification*; D012333:RNA, Messenger / Q000032:analysis; D011869:Radioligand Assay; D012756:Sheep; D013329:Structure-Activity Relationship
D012333:RNA, Messenger; D004247:DNA; D010928:Placental Lactogen