HEXOSAMINE AND HEPARIN IN HOMOGENATE AND SUBCELLULAR FRACTIONS FROM BOVINE NEUROHYPOPHYSES

1973 ◽  
Vol 74 (2) ◽  
pp. 209-214 ◽  
Author(s):  
Susanne Hvas ◽  
N. A. Thorn
Keyword(s):  
Frontal Cortex ◽  
Secretory Granule ◽  
Secretory Granules ◽  
Subcellular Fractions ◽  
Neurosecretory Granule ◽  
Granule Fraction ◽  
Detectable Concentration

ABSTRACT The concentration of hexosamine in a purified secretory granule fraction from ox neurohypophyses was 29 ± 3 (sem) μg/mg protein. This was significantly higher than the concentration in homogenate of whole neurohypophyses and the frontal cortex of ox brain. The hexosamine concentration in two lipoprotein fractions isolated from secretory granules was comparable to the concentration found in the granule fraction. The only glycosaminoglycan (GAG) found in detectable concentration in a homogenate from ox neurohypophyses was heparin. In the purified neurosecretory granule fraction no GAG was detectable.

scholarly journals ISOLATION AND PROPERTIES OF SECRETORY GRANULES FROM RAT ISLETS OF LANGERHANS

1969 ◽  
Vol 41 (1) ◽  
pp. 154-161 ◽  
Author(s):  
S. L. Howell ◽  
C. J. Fink ◽  
P. E. Lacy
Keyword(s):  
Islets Of Langerhans ◽  
Secretory Granule ◽  
Islet Cells ◽  
Secretory Granules ◽  
Morphological Characterization ◽  
Differential Centrifugation ◽  
Rat Islets ◽  
Granule Fraction ◽  
Final Yield ◽  
Rat Islets Of Langerhans

A method has been devised for the isolation of a secretory granule fraction from isolated rat islets of Langerhans. The islets were homogenized in buffered sucrose, and the homogenate was separated into nuclear, mitochondrial, secretory granule, and microsomal fractions by differential centrifugation. The secretory granule fraction was purified by differential centrifugation in discontinuous sucrose density gradients. A greater degree of purification could be achieved by the use of two successive gradients of this type, although the final yield was greatly reduced. Biochemical and morphological characterization of the fractions was obtained; the secretory granule fraction contained both insulin and glucagon. The limiting membranes of the granules remained intact and the general appearance of the granules was similar to that seen within the whole islet cells.

scholarly journals β-adrenoceptor activation increased VAMP-2 and syntaxin-4 in secretory granules are involved in protein secretion of submandibular gland through the PKA/F-actin pathway

2018 ◽  
Vol 38 (1) ◽  
Author(s):  
Chong Ding ◽  
Xin Cong ◽  
Yan Zhang ◽  
Sheng-Lin Li ◽  
Li-Ling Wu ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Submandibular Gland ◽  
Secretory Granule ◽  
Protein Secretion ◽  
Protein Fraction ◽  
Secretory Granules ◽  
Dependent Manner ◽  
Granule Fraction ◽  
Enhanced Expression ◽  
Syntaxin 4

Autologous submandibular gland transplantation is an effective treatment for severe dry eye syndrome. However, the protein secretion in transplanted gland is altered by a mechanism that remains to be elucidated. In the present study, we found that β1-adrenoceptor (β1-AR) and β2-AR expression and the phosphorylation of the downstream molecule protein kinase A (PKA) were elevated in transplanted submandibular glands obtained from epiphora patients. Synaptobrevin/vesicle-associated membrane protein 2 (VAMP-2) interacted with syntaxin-4 and actin in human submandibular gland. The contents of syntaxin-4 and actin interacting with VAMP-2 were increased in transplanted gland. Moreover, VAMP-2 and syntaxin-4 expression in the secretory granule fraction, and VAMP-2 expression in the membrane protein fraction were increased in isoproterenol-treated and transplanted glands. Isoproterenol increased F-actin polymerization in the apical and lateral regions of the cytoplasm in both control and transplanted glands. Inhibiting PKA activity and/or F-actin formation abolished the isoproterenol-enhanced expression of VAMP-2 and syntaxin-4 in the secretory granule fraction and the isoproterenol-enhanced expression of VAMP-2 in the membrane protein fraction. Taken together, these results indicate that the activation of β-ARs induces secretory granules and cell membrane fusion via the interaction of VAMP-2 and syntaxin-4 in a PKA- and F-actin-dependent manner in human submandibular gland. Up-regulated β-ARs might participate in altering protein secretion in transplanted submandibular gland by promoting the interaction of VAMP-2 with syntaxin-4.

scholarly journals Protein kinase C and an endogenous substrate associated with adenohypophyseal secretory granules

1986 ◽  
Vol 237 (1) ◽  
pp. 53-61 ◽  
Author(s):  
J L Turgeon ◽  
R H Cooper
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Secretory Granule ◽  
Anterior Pituitary ◽  
Secretory Granules ◽  
Dependent Protein Kinase ◽  
Kinase C ◽  
Protein Kinase C Activity ◽  
Granule Fraction ◽  
Dependent Protein

Secretory granules isolated from anterior pituitary glands were examined for Ca2+/phospholipid-dependent protein kinase (protein kinase C) activity as well as the occurrence of granule-associated substrate proteins. Sheep adenohypophyses were fractionated by differential and sucrose-density-gradient centrifugation to yield a granule fraction enriched for luteinizing-hormone (lutropin)-containing secretory granules. Marker-enzyme analysis showed no detectable cytosolic contamination, although there were small amounts of plasma membranes (2-4%) and lysosomes (4-6%) associated with the preparation. As determined by histone-H1 phosphorylation after DEAE-cellulose DE-52 chromatography, protein kinase C activity with a marked dependence on Ca2+ and lipid (4-fold increase in their presence) was evident in the secretory-granule fraction. Phosphorylation in vitro of the secretory-granule fraction by endogenous and exogenous protein kinase C revealed a protein of Mr 36,000, which by two-dimensional SDS/polyacrylamide-gel electrophoresis showed multiple sites of phosphorylation. The Mr-36,000 protein was not found in cytosolic or plasma-membrane fractions and was not phosphorylated by the catalytic subunit of cyclic AMP-dependent protein kinase. Several secretory-granule proteins served as substrates for the catalytic subunit, the most prominent of which were of Mr 63,000, 23,000 and 21,000. From these data, we suggest that phosphorylation of secretory-granule-associated proteins by protein kinase C and by cyclic AMP-dependent protein kinase may be important in secretion regulation in the anterior pituitary gland.

scholarly journals Studies on proinsulin and proglucagon biosynthesis and conversion at the subcellular level: II. Distribution of radioactive peptide hormones and hormone precursors in subcellular fractions after pulse and pulse- chase incubation of islet tissue

1977 ◽  
Vol 74 (2) ◽  
pp. 589-604 ◽  
Author(s):  
BD Noe ◽  
CA Baste ◽  
GE Bauer
Keyword(s):  
Secretory Granule ◽  
Short Pulse ◽  
Secretory Granules ◽  
Gel Filtration ◽  
Subcellular Fractionation ◽  
Bound State ◽  
Subcellular Fractions ◽  
Cell Of Origin ◽  
Islet Tissue ◽  
Microsome Fraction

Anglerfish proinsulin and insulin were selectively labeled with [(14)C]isoleucine, while proglucagon, conversion intermediate(s), and glucagon were selectively labeled with[(3)H]tryptophan. After various periods of continuous or pulse-chase incubation, islet tissue was subjected to subcellular fractionation. Fraction extracts were analyzed by gel filtration for their content of precursor, conversion intermediate(s), and product peptides. Of the seven subcellular fractions prepared after each incubation, only the microsome and secretory granule fractions yielded significant amounts of labeled insulin-related and glucagon-related peptides. After short-pulse incubations, levels of both [(14)C]proinsulin and [(3)H]proglucagon (mol wt approximately 12,000) were highest in the microsome fraction. This fraction is therefore identified as the site of synthesis. With increasing duration of continuous incubation or during chase incubation in the absence of isotopes, proinsulin, proglucagon, and conversion intermediate(s) are transported to secretory granules. Conversion of proinsulin to insulin and proglucagon to a approximately 4,900 mol wt conversion intermediate and 3,500 mol wt glucagon occurs in the secretory granules. Converting activity also was observed in the microsome fraction. The recovery of most of the incorporated radioactivity in microsome and secretory granule fractions indicates that the newly synthesized islet peptides are relegated to a membrane-bound state soon after synthesis at the RER is completed. This finding supports the concept of intracisternal sequestration and intragranular maintenance of peptides synthesized for export from the cell of origin.

scholarly journals HID-1 is required for homotypic fusion of immature secretory granules during maturation

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Wen Du ◽  
Maoge Zhou ◽  
Wei Zhao ◽  
Dongwan Cheng ◽  
Lifen Wang ◽  
...  
Keyword(s):  
Secretory Granule ◽  
Molecular Mechanisms ◽  
Secretory Granules ◽  
Three Dimensional ◽  
Conditional Knockout ◽  
Active Peptides ◽  
Early Maturation ◽  
Proinsulin Processing ◽  
Granule Maturation ◽  
Golgi Network

Secretory granules, also known as dense core vesicles, are generated at the trans-Golgi network and undergo several maturation steps, including homotypic fusion of immature secretory granules (ISGs) and processing of prehormones to yield active peptides. The molecular mechanisms governing secretory granule maturation are largely unknown. Here, we investigate a highly conserved protein named HID-1 in a mouse model. A conditional knockout of HID-1 in pancreatic β cells leads to glucose intolerance and a remarkable increase in the serum proinsulin/insulin ratio caused by defective proinsulin processing. Large volume three-dimensional electron microscopy and immunofluorescence imaging reveal that ISGs are much more abundant in the absence of HID-1. We further demonstrate that HID-1 deficiency prevented secretory granule maturation by blocking homotypic fusion of immature secretory granules. Our data identify a novel player during the early maturation of immature secretory granules.

scholarly journals Secretory granule protein chromogranin B (CHGB) forms an anion channel in membranes

2018 ◽  
Vol 1 (5) ◽  
pp. e201800139 ◽  
Author(s):  
Gaya P Yadav ◽  
Hui Zheng ◽  
Qing Yang ◽  
Lauren G Douma ◽  
Linda B Bloom ◽  
...  
Keyword(s):  
Secretory Granule ◽  
Chloride Channels ◽  
Molecular Mechanisms ◽  
Single Channel ◽  
Secretory Granules ◽  
Anion Channel ◽  
Local Concentration ◽  
Chromogranin B ◽  
Fast Kinetics ◽  
Granule Protein

Regulated secretion is an intracellular pathway that is highly conserved from protists to humans. Granin family proteins were proposed to participate in the biogenesis, maturation and release of secretory granules in this pathway. However, the exact molecular mechanisms underlying the intracellular functions of the granin family proteins remain unclear. Here, we show that chromogranin B (CHGB), a secretory granule protein, inserts itself into membrane and forms a chloride-conducting channel. CHGB interacts strongly with phospholipid membranes through two amphipathic α helices. At a high local concentration, CHGB insertion in membrane causes significant bilayer remodeling, producing protein-coated nanoparticles and nanotubules. Fast kinetics and high cooperativity for anion efflux from CHGB vesicles suggest that CHGB tetramerizes to form a functional channel with a single-channel conductance of ∼125 pS (150/150 mM Cl−). The CHGB channel is sensitive to an anion channel blocker and exhibits higher anion selectivity than the other six known families of Cl−channels. Our data suggest that the CHGB subfamily of granin proteins forms a new family of organelle chloride channels.

scholarly journals A novel function for Rab proteins during maturation of secretory granules

2021 ◽  
Author(s):  
Sarah D Neuman ◽  
Annika R Lee ◽  
Jane E Selegue ◽  
Amy T Cavanagh ◽  
Arash Bashirullah
Keyword(s):  
Salivary Glands ◽  
Secretory Granule ◽  
Secretory Granules ◽  
Secretory Cells ◽  
Rab Proteins ◽  
Dependent Manner ◽  
Rab Gtpases ◽  
Cargo Proteins ◽  
Granule Maturation ◽  
Granule Growth

Regulated exocytosis is an essential process whereby professional secretory cells synthesize and secrete specific cargo proteins in a stimulus-dependent manner. Cargo-containing secretory granules are synthesized in the trans-Golgi Network (TGN); after budding from the TGN, granules undergo many modifications, including a dramatic increase in size. These changes occur during a poorly understood process called secretory granule maturation. Here we leverage the professional secretory cells of the Drosophila larval salivary glands as a model system to characterize a novel and unexpected role for Rab GTPases during secretory granule maturation. We find that secretory granules in the larval salivary glands increase in size ~300-fold between biogenesis and release, and loss of Rab1 or Rab11 dramatically reduces granule size. Surprisingly, we find that Rab1 and Rab11 protein localize to secretory granule membranes. Rab11 associates with granule membranes throughout the maturation process, and Rab11 is required for recruitment of Rab1. In turn, Rab1 associates specifically with immature secretory granules and drives granule growth. In addition to their roles in granule growth, both Rab1 and Rab11 appear to have additional roles during exocytosis; Rab11 function is necessary for exocytosis, while the presence of Rab1 on immature granules may prevent precocious exocytosis. Overall, these results highlight a new and unexpected role for Rab GTPases in secretory granule maturation.

scholarly journals Golgi apparatus, GERL, and secretory granule formation within neurons of the hypothalamo-neurohypophysial system of control and hyperosmotically stressed mice.

1981 ◽  
Vol 90 (2) ◽  
pp. 474-484 ◽  
Author(s):  
R D Broadwell ◽  
C Oliver
Keyword(s):  
Golgi Apparatus ◽  
Secretory Granule ◽  
Supraoptic Nucleus ◽  
Salt Water ◽  
Secretory Granules ◽  
Hyperosmotic Stress ◽  
Granule Formation ◽  
Smooth Membrane ◽  
Secondary Lysosomes ◽  
The Relationship

The vasopressin-producing neurons of the hypothalamo-neurohypophysial system are a particularly good model with which to consider the relationship between the Golgi apparatus nd GERL and their roles in secretory granule production because these neurons increase their synthesis and secretion of vasopressin in response to hyperosmotic stress. Enzyme cytochemical techniques for acid phosphatase (AcPase) and thiamine pyrophosphatase (TPPase) activities were used to distinguish GERL from the Golgi apparatus in cell bodies of the supraoptic nucleus from normal mice, mice hyperosmotically stressed by drinking 2% salt water, and mice allowed to recover for 5-10 d from hyperosmotic stress. In nonincubated preparations of control supraoptic perikarya, immature secretory granules at the trans face of the Golgi apparatus were frequently attached to a narrow, smooth membrane cisterna identified as GERL. Secretory granules were occasionally seen attached to Golgi saccules. TPPase activity was present in one or two of the trans Golgi saccules; AcPase activity appeared in GERL and attached immature secretory granules, rarely in the trans Golgi saccules, and in secondary lysosomes. As a result of hyperosmotic stress, the Golgi apparatus hypertrophied, and secretory granules formed from all Golgi saccules and GERL. Little or no AcPase activity could be demonstrated in GERL, whereas all Golgi saccules and GERL-like cisternae were TPPase positive. During recovery, AcPase activity in GERL returned to normal; however, the elevated TPPase activity and secretory granule formation seen in GERL-like cisternae and all Golgi saccules during hyperosmotic stress persisted. These results suggest that under normal conditions GERL is the predominant site for the secretory granule formation, but during hyperosmotic stress, the Golgi saccules assume increased importance in this function. The observed cytochemical modulations in Golgi saccules and GERL suggest that GERL is structurally and functionally related to the Golgi saccules.

scholarly journals CONCOMITANT SYNTHESIS OF MEMBRANE PROTEIN AND EXPORTABLE PROTEIN OF THE SECRETORY GRANULE IN RAT PAROTID GLAND

1971 ◽  
Vol 50 (1) ◽  
pp. 187-200 ◽  
Author(s):  
Abraham Amsterdam ◽  
Michael Schramm ◽  
Itzhak Ohad ◽  
Yoram Salomon ◽  
Zvi Selinger
Keyword(s):  
Amino Acids ◽  
Secretory Granule ◽  
De Novo ◽  
Secretory Granules ◽  
Specific Radioactivity ◽  
Staining Intensity ◽  
Granule Membrane ◽  
Rat Parotid ◽  
Cell Fractions

After enzyme secretion the membrane of the secretory granule, which had been fused to the cell membrane, was resorbed into the cell. Experiments were therefore carried out to test whether formation of new secretory granules involves reutilization of the resorbed membrane or synthesis of a new membrane, de novo, from amino acids. Incorporation of amino acids-14C into proteins of various cell fractions was measured in vivo, 30, 120, and. 300 min after labeling. At all times the specific radioactivity of the secretory granule membrane was about equal to that of the granule's exportable content. At 120 and 300 min the specific radioactivity of the granule membrane and of the granule content was much higher than that of any other subcellular fraction. It is therefore concluded that the protein of the membrane is synthesized de novo concomitantly with the exportable protein. The proteins of the granule membrane could be distinguished from those of the granule content by gel electrophoresis. All major bands were labeled proportionately to their staining intensity. The amino acid composition of the secretory granule membrane was markedly different from that of the granule's content and also from that of the mitochondrial membrane. The granule membrane showed a high proline content, 30 moles/100 moles amino acids. The analyses show that the radioactivity of the granule membrane is indeed inherent in its proteins and is not due to contamination by other fractions. The possibility is considered that the exportable protein leaves the endoplasmic reticulum already enveloped by the newly synthesized membrane.

scholarly journals Cell Fractionation of Anterior Pituitary Glands from Beef and Pig

1959 ◽  
Vol 5 (1) ◽  
pp. 17-23 ◽  
Author(s):  
Frank S. Labella ◽  
J. H. U. Brown
Keyword(s):  
Alkaline Phosphatase ◽  
Anterior Pituitary ◽  
Secretory Granules ◽  
Succinic Dehydrogenase ◽  
Subcellular Fractions ◽  
Acid Proteinase ◽  
Cell Fractionation ◽  
Ph Optimum ◽  
Pituitary Glands ◽  
The Individual

Fresh anterior pituitary glands from beef and pig were separated by differential centrifugation into subcellular fractions. Nuclei and debris were obtained at 700 g for 15 minutes, secretory granules at 7000 g for 20 minutes, mitochondria at 34,000 g for 15 minutes, and microsomes at 78,000 g for 3 hours. Electron micrographs were taken of the individual fractions. Each fraction was analyzed for nitrogen, pentosenucleic acid (PNA), and phospholipide. Beef and pig anterior lobes were quite similar in their intracellular composition as seen in the subcellular fractions. Succinic dehydrogenase was localized in mitochondria, while alkaline phosphatase was concentrated in the microsomes. A proteinase with pH optimum at 8.2 was exclusively localized. in microsomal and supernatant fractions. Acid phosphatase, acid ribonuclease, and acid proteinase were distributed among the subcellular fractions in another pattern, indicating the presence of a particle type distinct from mitochondria and microsomes. The distribution of cytoplasmic PNA paralleled that of alkaline phosphatase.

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