Identification and localization of 23,000 and glycosylated rat prolactin in subcellular fractions of rat anterior pituitary and purified secretory granules.
Rat pituitary homogenates were submitted to differential and density gradient centrifugation. Subcellular fractions as well as the purified secretory granules were examined in electron microscopy, radioimmunological techniques, protease digestion, alkaline treatment and immunoblotting. The global outcome of these experiments was that: 1) the glycosylated rPRL was foremost recorded in the crude secretory granular fraction, also in the microsomal fraction and the cytosol, but virtually not in the plasma membrane fraction; 2) in purified secretory granules glycosylated rPRL appeared as an array of near Mr, such as was formerly obtained by enzymatic deglycosylation; 3) protease digestion and ice-cold alkaline treatment of the secretory granules showed that 23,000 rPRL appears in three different physicochemical states in these organelles: unsequestered within a closed system, membrane-bounded and bound state; 4) likewise treatment of microsomal vesicles showed that 23,000 and glycosylated rPRL are sequestered in these bodies, but apparently 23,000 rPRL appears as both integral membrane-bound and released from the lumen, whereas glycosylated rPRL is chiefly retained as an integral membrane protein. 5) dopamine alters the pattern of glycosylation as well in Mr as in relative percentages of the molecular variants. The systematical occurrence of the array of near Mr glycosylated rPRL is biosynthesized as a pool of proteins with a different degree of glycosylation. On the basis of our data, we speculate that selection of definite molecular variants from this pool could play an important role in the biological function of 23,000 rPRL and that oligosaccharides could perhaps target the glycosylated forms of rPRL to specific sites of action.
Bollengier
F
F
Laboratorium voor Farmacologie, Faculteit Geneeskunde en Farmacie, Vrije Universiteit Brussel, Belgium.
Geerts
A
A
Matton
A
A
Mahler
A
A
Velkeniers
B
B
Hooghe-Peters
E
E
Vanhaeist
L
L
D000818:Animals; D001345:Autoradiography; D002499:Centrifugation, Density Gradient; D003594:Cytoplasmic Granules / Q000378:metabolism* / Q000648:ultrastructure; D004298:Dopamine / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D005947:Glucose / Q000378:metabolism; D006868:Hydrolysis; D015151:Immunoblotting; D008854:Microscopy, Electron; D010903:Pituitary Gland, Anterior / Q000378:metabolism* / Q000648:ultrastructure; D011388:Prolactin / Q000378:metabolism*; D011863:Radioimmunoassay; D051381:Rats; D017208:Rats, Wistar; D013347:Subcellular Fractions / Q000378:metabolism* / Q000648:ultrastructure
D011388:Prolactin; D005947:Glucose; D004298:Dopamine