Isolation and characteristics of bovine pituitary secretory granules

Cm, Moriarty; Dowd, Frank J.; Fontaine, Michael A. La

doi:10.1016/0304-4165(83)90067-3

Cited by 7 publications

(7 citation statements)

References 23 publications

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“…According to Table I, fractions B1 and B2, mainly membranes, displayed very low rPRL contents, which shows that granular lysis was minimal in the extraction buffer used. Moreover, according to Moriarty et al (9) pituitary secretory granules are resistant to osmotic stress. The low rPRL content of B3 is in good agreement with the fact that it corresponded in EM to only a few granules.…”

Section: Discussionmentioning

confidence: 99%

“…Contamination of P26,OOO with artifactually released hormone was minimal. Indeed, extractions were performed at room temperature, since the cold increases the fragility of the secretory granules (9). Moreover, in the case of artificially released hormone, glycosylated rPRL should also be observed in P26,OOO.…”

Section: Discussionmentioning

confidence: 99%

“…The function of glycosylated rPRL is not yet understood, but it has been shown (3, 4) that the carbohydrates of glycoproteins contribute to stabilization and secretion of proteins, and to recognition of receptor-mediated endocytosis sites. In earlier investigations (5)(6)(7)(8)(9) the location of mature ovine, bovine and rat prolactin (M, 23,000) in subcellular fractions and the association of anterior pituitary hormones with secretory granules has been demonstrated, but to our knowledge, almost nothing is known about the subcellular localization, identification and hormonal content of glycosylated rPRL. In this report we addressed these questions using subcellular fractionation methods, electron microscopy (EM), immunoblotting, radioimmunological assays, protease digestion and alkaline treatment.…”

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confidence: 99%

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Identification and Localization of 23,000 and Glycosylated Rat Prolactin in Subcellular Fractions of Rat Anterior Pituitary and Purified Secretory Granules

Bollengier

Geerts

Matton

et al. 1993

J Neuroendocrinology

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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.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

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Identification and Localization of 23,000 and Glycosylated Rat Prolactin in Subcellular Fractions of Rat Anterior Pituitary and Purified Secretory Granules

Bollengier

Geerts

Matton

et al. 1993

J Neuroendocrinology

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“…The following protein standards [2-5 ng each; obtained from Calbiochem or Sigma (St. Louis, MO)] were mixed with each sample immediately before gel electrophoresis to serve as internal mol wt markers: carbonic afthydrase (29,000), soybean trypsin inhibitor (20,000), myoglobin (17,000), and cytochrome c (12,000). Coomassie brilliant bluestained gels were slices into 1-or 2-mm sections, solubilized, and counted in 10 ml of a xylene-based detergent-containing solution (Scint A, Packard, Downers Grove, IL).…”

Section: Sds-gel Electrophoresismentioning

confidence: 99%

“…We sought to apply subcellular fractionation techniques to nontumorous mouse pituitary tissue. Although pituitary tissue from various species has been subjected to subcellular fractionation (12)(13)(14)(15)(16)(17)(18), biosynthetic labeling studies of pituitary tissue employing subcellular fractionation have not previously been performed for study of any of the glycoprotein hormones. Moreover, our study is the first to include exogenous radioactive proteins at the time of pituitary tissue homogenization, as described by Scheele et al (19), to assess the cross-contamination of pituitary subcellular fractions by the nonspecific adsorption of labeled proteins to organelles.…”

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confidence: 99%

Subcellular Localization of Fucose Incorporation into Mouse Thyrotropin and Freeα-Subunits: Studies Employing Subcellular Fractionation and Inhibitors of the Intracellular Translocation of Proteins^*

1986

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To determine the subcellular sites of fucose incorporation into TSH subunits, pituitaries from hypothyroid mice were incubated with [3H]fucose and fractionated by sucrose gradient centrifugation. To assess potential molecular cross-contamination between subcellular fractions enriched in rough endoplasmic reticulum (RER) or Golgi elements, trace amounts of exogenous [35S]methionine-labeled proteins or [125I]rat TSH were added before tissue homogenization. Particulate contamination of fractions was monitored by electron microscopy. TSH subunits were immunoprecipitated from fractions and analyzed by gel electrophoresis. After both a 2-h pulse incubation and a 2-h pulse, 3-h chase incubation, about half (range, 44-71%) of the [3H]fucose-labeled TSH subunit precursors present in microsomes were in the RER (amounts in excess of estimated contamination by nonspecific readsorption of molecules to vesicles or the presence of Golgi vesicles in the RER fractions); [3H]fucose-labeled free alpha-subunits were also detected in RER as well as in Golgi fractions. During chase incubations, both monensin and carboxyl cyanide m-chlorophenylhydrazone inhibited the appearance of [35S]methionine- or [3H]fucose-labeled TSH subunits in medium in a dose-dependent manner, suggesting that [3H] fucose was added to subunits, in part, early in the secretory pathway. Free alpha-subunits were more fucosylated than was TSH; in TSH heterodimers, beta-subunits were richer in fucose than were alpha-subunits. Thus, the fucosylation of TSH and free alpha-subunits in pituitaries of hypothyroid mice appears to begin at an unusually early stage of intracellular transport and may represent an adaptation to special posttranslational processing requirements.

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Morphological characterisation of lactotrophs separated from the bovine pituitary by a rapid enrichment technique

Ingram¹,

Keefe

Wooding

et al. 1988

Cell Tissue Res.

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The morphological characteristics of bovine pituitary cells separated by a rapid enrichment procedure are described. Single-cell suspensions were prepared from pituitary glands of steers by use of a collagenase technique and separated by discontinuous gradient centrifugation. The separation of prolactin- and growth hormone-containing cells was assessed by radioimmunoassay of hormone content and immunocytochemistry, and the distribution of fibroblasts assessed after establishing cell cultures. Morphometric analysis of the fine structure of two fractions respectively enriched and depleted in the proportion of immuno-cytochemically-identified lactotrophs was performed after labelling with anti-prolactin antiserum coupled to immunogold complex. Cells recovered from the higher-density fraction were more highly granulated, suggesting that this was a major characteristic determining separation. Cells labelled for prolactin could not be distinguished from unlabelled cells on the basis of their granule size range, but unlabelled cells had a significantly greater coefficient of variation. These data suggest that granule density and distribution, but not granule size per se, are useful characteristics for the identification of bovine lactotrophs.

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Isolation and characteristics of bovine pituitary secretory granules

Cited by 7 publications

References 23 publications

Identification and Localization of 23,000 and Glycosylated Rat Prolactin in Subcellular Fractions of Rat Anterior Pituitary and Purified Secretory Granules

Identification and Localization of 23,000 and Glycosylated Rat Prolactin in Subcellular Fractions of Rat Anterior Pituitary and Purified Secretory Granules

Subcellular Localization of Fucose Incorporation into Mouse Thyrotropin and Freeα-Subunits: Studies Employing Subcellular Fractionation and Inhibitors of the Intracellular Translocation of Proteins^*

Morphological characterisation of lactotrophs separated from the bovine pituitary by a rapid enrichment technique

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Isolation and characteristics of bovine pituitary secretory granules

Cited by 7 publications

References 23 publications

Identification and Localization of 23,000 and Glycosylated Rat Prolactin in Subcellular Fractions of Rat Anterior Pituitary and Purified Secretory Granules

Identification and Localization of 23,000 and Glycosylated Rat Prolactin in Subcellular Fractions of Rat Anterior Pituitary and Purified Secretory Granules

Subcellular Localization of Fucose Incorporation into Mouse Thyrotropin and Freeα-Subunits: Studies Employing Subcellular Fractionation and Inhibitors of the Intracellular Translocation of Proteins*

Morphological characterisation of lactotrophs separated from the bovine pituitary by a rapid enrichment technique

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Subcellular Localization of Fucose Incorporation into Mouse Thyrotropin and Freeα-Subunits: Studies Employing Subcellular Fractionation and Inhibitors of the Intracellular Translocation of Proteins^*