Multiple forms of rat prolactin and growth hormone in pituitary cell subpopulations separated using a Percoll gradient system: disulphide-bridged dimers and glycosylated variants.
Prolactin and GH cells from rat pituitary glands were separated into three main fractions on discontinuous Percoll gradient layers. SDS-PAGE and subsequent immunoblotting of these fractions revealed that: (1) multiple rat prolactin (rPRL) molecular variants were present in total culture, Percoll layer 1 and 2; four variants were clear-cut: Mr approximately 23,000, Mr doublet approximately 25,000-26,000, Mr approximately 40,000 and Mr approximately 42,000; (2) cell cytosol from Percoll gradient layer 1 was particularly enriched in prolactin; (3) cells from gradient layer 1 secreted into the culture medium only prolactin in detectable amounts; (4) three distinct molecular forms of rat growth hormone (rGH) were recorded in layer 3: Mr approximately 36,000, 24,000 and 20,000; the 20,000 variant was paramount; and (5) cells from layer 3 secreted both rPRL and rGH into the culture medium. Reduction experiments showed that, on the one hand, 42,000 and 40,000 rPRL variants and, on the other hand, 36,000 rGH variants are disulphide-bridged dimers. An important finding was the presence of glycosylated rPRL and rGH: indeed Concanavalin A-Sepharose 4B affinity chromatography indicated that 26,000 rPRL and 24,000 rGH display a very strong affinity for lectin. Competitive inhibition tests showed that this affinity is specific and not due to hydrophobic binding. When rPRL was submitted to deglycosylation in conditions specific for O-linked glycoproteins, the 26,000 rPRL variant disappeared. The biological role of glycosylated rPRL is as yet unknown.
Bollengier
F
F
Laboratory of Pharmacology, Faculty of Medicine and Pharmacy, Free University of Brussels, Belgium.
Velkeniers
B
B
Hooghe-Peters
E
E
Mahler
A
A
Vanhaelst
L
L
D000818:Animals; D002469:Cell Separation; D002478:Cells, Cultured; D004220:Disulfides; D005260:Female; D013006:Growth Hormone / Q000031:analogs & derivatives / Q000302:isolation & purification*; D007536:Isomerism; D008970:Molecular Weight; D010903:Pituitary Gland, Anterior / Q000378:metabolism*; D011388:Prolactin / Q000031:analogs & derivatives / Q000302:isolation & purification*; D011487:Protein Conformation; D051381:Rats
D004220:Disulfides; C043928:prolactin, glycosylated; C050621:somatotropin, glycosylated; D011388:Prolactin; D013006:Growth Hormone