Routing, processing and export of rat pituitary prolactin: identification of a 36 kDa disulphide-bridged oligomeric preprolactin.
To gain an insight in the routing, processing and export of rat prolactin, rat pituitary cells were cultured in serum-free medium in the presence of cycloheximide, carbonyl cyanide m-chlorophenylhydrazone, Brefeldin A and monensin. The potential influence of these perturbants, whose well documented effects are the altering of protein synthesis and transport, was studied on rat prolactin molecular size isoforms appearing in cellular extracts and in culture medium. The outcome of the culture experiments as recorded in vertical SDS-PAGE, thiol gradient electrophoresis and sequential SDS-PAGE followed by prolactin specific immunoblotting and densitometry, was as follows: (1) at the cellular level we were able to characterize a novel 36 kDa protein as a disulphide-bridged oligomeric precursor prolactin, which is presumably rapidly transformed in the cis/medial Golgi; to designate monomeric rat prolactin as an early Golgi protein and t o advance evidence that the main processing of the glycosylated rat prolactin is a cis/medial Golgi event; (2) in release none of the perturbants disturbed the relative distribution of monomeric and glycosylated rat prolactin, the main molecular size isoforms currently secreted by untreated pituitary cells, or induced the appearance of transformed molecular size isoforms; (3) the secretion mode indicates that rat prolactin is released via the regulated pathway in the presence of the perturbants used.
Bollengier
F
F
Laboratorium voor Farmacologie, Faculteit Geneeskunde en Farmacie, Vrije Universiteit Brussel, Brussel, Belgium.
Mahler
A
A
Vanhaelst
L
L
D000818:Animals; D020126:Brefeldin A / Q000494:pharmacology; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone / Q000494:pharmacology; D002478:Cells, Cultured; D016895:Culture Media, Serum-Free; D003513:Cycloheximide / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D006056:Golgi Apparatus / Q000378:metabolism; D007476:Ionophores / Q000494:pharmacology; D008985:Monensin / Q000494:pharmacology; D010902:Pituitary Gland / Q000187:drug effects / Q000378:metabolism*; D011388:Prolactin / Q000096:biosynthesis* / Q000378:metabolism*; D011498:Protein Precursors / Q000096:biosynthesis* / Q000378:metabolism*; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D051381:Rats; D017208:Rats, Wistar; D014475:Uncoupling Agents / Q000494:pharmacology
D016895:Culture Media, Serum-Free; D007476:Ionophores; D011498:Protein Precursors; D011500:Protein Synthesis Inhibitors; D014475:Uncoupling Agents; D020126:Brefeldin A; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone; C017671:preprolactin; D011388:Prolactin; D008985:Monensin; D003513:Cycloheximide