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 ou...

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Bibliographic Details
Published in:Journal of neuroendocrinology 1993-12, Vol.5 (6), p.669-676
Main Authors: Bollengier, F., Geerts, A., Matton, A., Mahler, A., Velkeniers, B., Hooghe-Peters, E., Vanhaelst, L.
Format: Article
Language:English
Subjects:
Aminoacids, peptides. Hormones. Neuropeptides
Analytical, structural and metabolic biochemistry
Animals
Autoradiography
Biological and medical sciences
Centrifugation, Density Gradient
Cytoplasmic Granules - metabolism
Cytoplasmic Granules - ultrastructure
Dopamine - metabolism
Electrophoresis, Polyacrylamide Gel
Female
Fundamental and applied biological sciences. Psychology
Glucose - metabolism
glycosylated rat prolactin
Hydrolysis
Immunoblotting
Microscopy, Electron
microsomal vesicle
Pituitary Gland, Anterior - metabolism
Pituitary Gland, Anterior - ultrastructure
Prolactin - metabolism
protease digestion
Proteins
Radioimmunoassay
Rats
Rats, Wistar
subcellular fractionation
Subcellular Fractions - metabolism
Subcellular Fractions - ultrastructure
translocation
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Summary: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 membrane‐bound, whereas glycosylated rPRL preferentially assumed the 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 variants in secretory granules and microsomal vesicles strongly indicates that 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.
ISSN:0953-8194
1365-2826
DOI:10.1111/j.1365-2826.1993.tb00538.x