Isolation of cleaved prolactin variants that stimulate DNA synthesis in specific cell types in rat pituitary cell aggregates in culture.
Using an affinity column of monoclonal antibodies against rat prolactin (PRL), PRL immunoreactive material secreted by rat pituitary aggregate cell cultures was purified in milligram amounts from the culture medium. Further purification of the PRL immunoreactive molecules was achieved by anion-exchange and reversed-phase h.p.l.c. At least four variants could be distinguished which showed PRL-like bioactivity in the Nb2 lymphoma bioassay. On SDS/PAGE two variants migrated as single bands under both reducing and non-reducing conditions. The two other variants migrated as single bands under non-reducing conditions, but yielded under reducing conditions two fragments, the larger of which had molecular masses of approx. 16 and 17 kDa respectively. These variants were therefore considered to be cleaved PRL (clPRL) variants, and were designated clPRL-1 and clPRL-2 respectively. These variants were not artefactually produced at low pH during the h.p.l.c. purification step. Each of the clPRLs represented about 0.6-1.0% of total PRL. The cleavage site of clPRL-1, determined by N-terminal and C-terminal amino acid sequence analysis, was in the large disulphide loop between Tyr-145 and Leu-146. Purified clPRL-1 added to pituitary aggregate cell cultures from 14-day-old female rats in the presence of the dopamine agonist bromocriptine increased [3H]thymidine incorporation into DNA in gonadotrophs and thyrotrophs, but not in other pituitary cell types. Under the same experimental conditions the main forms of PRL and clPRL-2 were inactive. These data demonstrate that PRL is cleaved within the pituitary gland between specific amino acid residues, and that PRL cleaved between Tyr-145 and Leu-146 may have a specific biological role as a paracrine growth regulator in this tissue.
Andries
M
M
Laboratory of Cell Pharmacology, University of Leuven School of Medicine, Belgium.
Tilemans
D
D
Denef
C
C
D000595:Amino Acid Sequence; D000818:Animals; D002449:Cell Aggregation; D002455:Cell Division; D002478:Cells, Cultured; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D004247:DNA / Q000096:biosynthesis*; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D010903:Pituitary Gland, Anterior / Q000378:metabolism*; D011388:Prolactin / Q000302:isolation & purification / Q000378:metabolism / Q000502:physiology*; D051381:Rats; D011919:Rats, Inbred Strains; D013936:Thymidine / Q000378:metabolism; D014407:Tumor Cells, Cultured
D011388:Prolactin; D004247:DNA; D013936:Thymidine