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 Inhibitory effect of colchicines on amylase secretion by rat parotid glands: possible localization in the golgi area

1977 ◽  
Vol 73 (3) ◽  
pp. 578-593 ◽  
Author(s):  
C Patzelt ◽  
D Brown ◽  
B Jeanrenaud
Keyword(s):  
De Novo ◽  
Secretory Granules ◽  
Inhibitory Effect ◽  
Binding Activity ◽  
Amylase Secretion ◽  
Parotid Glands ◽  
Secretory Material ◽  
Delayed Effect ◽  
Rat Parotid

Colchicine inhibited amylase secretion by isolated rat parotid glands only 6 h after administration of the drug in vivo. This delayed effect was not the result of the inability of the drug to reach its reaction site. When parotid glands were emptied of their secretory granules by isoproterenol treatment, the subsequent replenishment of cells with granules was inhibited by colchicines. Colchicine concomitantly produced alterations of the Golgi complexes, the cisternae of which were reduced in size and surrounded by clusters of microvesicles. Incubation of parotid glands with colchicines for prolonged durations failed to alter stored amylase secretion as stimulated by isoproterenol, but it inhibited the release of de novo synthesized enzyme. Another colchicines-binding activity, firmly bound to the particular fraction of homogenates, was found, of which a part may represent membrane located microtubular protein. An assembly-disassembly cycle of microtubules appears to exist in the parotid gland, as in the liver. However, only 14 percent of tubulin was found to be polymerized as microtubules in parotid glands as opposed to 40 percent in the liver. The present data suggest that colchicine primarily inhibits the transfer of secretory material towards or away from the Golgi complexes but not the hormone-stimulated secretion of stored amylase.

The adrenal paraneurone: tubulin organization

1984 ◽  
Vol 62 (5) ◽  
pp. 502-511 ◽  
Author(s):  
M. F. Bader ◽  
F. Bernier-Valentin ◽  
B. Rousset ◽  
D. Aunis
Keyword(s):  
Cell Body ◽  
Intracellular Transport ◽  
Cultured Cells ◽  
Specific Binding ◽  
Secretory Granules ◽  
Immunofluorescent Staining ◽  
Chromaffin Granules ◽  
Two Dimensional Gel Electrophoresis ◽  
Granule Membrane

When chromaffin cells from the bovine adrenal medulla are maintained in culture, they develop neuritelike processes which end with growth-cone-like structures. Chromaffin granules were found to migrate from the cell body to the neurite endings. Thus, the intracellular transport of secretory granules, existing in vivo, seems to occur in an exaggerated way in the cultured cells. These cells offer an excellent model for studying the mechanism of transport, particularly the role of microtubules. By immunofluorescent staining, we observed that tubulin antibodies decorate a complex network visible along the neurites. Colchicine treatment induced the disappearance of this network followed by a return of granules in the cell body and a retraction of neurites. To test the presence of tubulin in the chromaffin granule membrane, we used two-dimensional gel electrophoresis and a radioimmunoassay. Our results indicate that tubulin is not a significant component of chromaffin granules. However, binding experiments show that granule membranes are able to bind tubulin through high affinity binding sites. These results show that microtubules appear involved in neurite formation and probably in granule transport. Tubulin is not an integral constituent of the granule membrane, but is present as a result of a reversible specific binding. This insertion of tubulin into the membrane might represent a step in the association between microtubules and secretory granules.

Intracellular Elemental Concentrations in Resting and Secreting Rat Parotid Glands

1987 ◽  
Vol 66 (2) ◽  
pp. 537-540 ◽  
Author(s):  
K.T. Izutsu ◽  
D.E. Johnson ◽  
M. Goddard
Keyword(s):  
Secretory Granules ◽  
Dry Weight ◽  
Parotid Glands ◽  
X Ray ◽  
Elemental Concentrations ◽  
Saliva Secretion ◽  
Concentration Changes ◽  
Rat Parotid ◽  
Micro Analysis

Electron probe x-ray micro-analysis was used to study the elemental concentration changes that occur during pilocarpine-stimulated saliva secretion. Quantitative x-ray micro-analysis of elemental concentrations in intracellular compartments of rat parotid glands stimulated in vivo with pilocarpine showed that Na concentration was significantly increased, while K concentration was significantly reduced. The magnitude of these changes was consistent with values obtained in other tissues with the x-ray micro-analysis method, and in the same tissue with other experimental methods. Comparisons with results from studies utilizing dispersed acini suggest that acinar dispersion procedures may affect intracellular elemental concentrations. Total electrolyte concentrations in cytoplasm and secretory granules were estimated to increase on a dry-weight basis following pilocarpine stimulation. The former change is consistent with the notion of a trans-cellular route of salivary fluid flow, while the latter change may be important in the exocytosis of secretory granules.

scholarly journals Some biochemical effects of triamcinolone acetonide on rat liver and muscle

1970 ◽  
Vol 116 (3) ◽  
pp. 349-355 ◽  
Author(s):  
R. F. Peters ◽  
M. C. Richardson ◽  
Margaret Small ◽  
A. M. White
Keyword(s):  
Amino Acids ◽  
Triamcinolone Acetonide ◽  
Time Course ◽  
Muscle Protein ◽  
Specific Radioactivity ◽  
Synthetic Activity ◽  
Tissue Homogenate ◽  
Glycogen Concentration

1. The powerful anti-inflammatory glucocorticoid triamcinolone acetonide, administered to rats at 20 and 2.5mg/kg, leads to a decrease in the incorporation in vivo of [3H]uridine and [32P]orthophosphate into hind-limb skeletal muscle. 2. At the higher dose, this decrease in the rate of incorporation of precursors into RNA precedes a decrease in the incorporating ability of muscle ribosomes, which commences about 4–5h after drug administration, but is unaccompanied by any changes in the concentration of tissue ATP or free amino acids. 3. The ribosomal dysfunction extends to polyribosomes, which can only be successfully isolated from the muscle of triamcinolone-treated animals after the addition of α-amylase to the tissue homogenate to remove glycogen. 4. The specific radioactivity of muscle protein labelled in vivo with 14C-labelled amino acids does not decrease progressively after triamcinolone administration. After 2h there is an apparent stimulation of incorporation which leads to an overall discrepancy between measurements of protein-synthetic activity made in vivo and in vitro. 5. There is a significant increase in muscle-glycogen concentration between 8 and 12h after the administration of triamcinolone acetonide (20mg/kg), although a significant decrease occurs after 4h. The fall in glycogen concentration may be due to a decrease in the rate of synthesis of protein essential for glucose uptake into the tissues. 6. As judged by (a) incorporation of 14C-labelled amino acids into protein, (b) [3H]uridine and [32P]-orthophosphate incorporation into RNA, (c) the rate of induction of tryptophan pyrrolase and (d) changes in the pool sizes of taurine and tryptophan, the responses in liver followed the same time-course as those in muscle after administration of the drug.

Chloride transport across the membrane of parotid secretory granules

1990 ◽  
Vol 259 (3) ◽  
pp. C413-C420 ◽  
Author(s):  
K. W. Gasser ◽  
U. Hopfer
Keyword(s):  
Chloride Transport ◽  
Secretory Granules ◽  
Disulfonic Acid ◽  
Parotid Glands ◽  
Transport Pathways ◽  
Anion Selectivity ◽  
Granule Membrane ◽  
Cytoplasmic Side ◽  
Cl Conductance

The Cl- transport pathways in secretory granules isolated from the parotid glands of rats were characterized by the technique of ionophore-induced lysis in defined salt solutions. The granules were shown to possess a Cl- conductance that exhibited a distinct anion selectivity with a sequence I- greater than Br- greater than Cl- greater than F- greater than SO4(2-) much greater than gluconate-. This conductance could be reduced approximately 40% by the stilbene 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS) from the cytoplasmic side; the half-maximal concentration for inhibition was 50 microM. Furthermore, the apparent Cl- conductance was reduced by outwardly directed granule H+ gradients and stimulated by inwardly directed gradients. An outwardly directed H+ gradient mimics the in vivo environment and may serve in a regulatory capacity, providing for a tonic inhibition of transport until the granule fuses with the luminal membrane. The granules also possessed a Cl(-)-HCO3- exchange based on electroneutrality of Cl- uptake and stimulation of this uptake by HCO3-. This pathway displayed a different anion selectivity, I- greater than Br- greater than F- greater than Cl- much greater than SO4(2-) much greater than gluconate-, and was not inhibited by SITS on the cytoplasmic side. The presence of these electrolyte transport pathways in the granule membrane is consistent with the production of primary fluid by parotid acinar cells after fusion of granules with the luminal plasma membrane.

scholarly journals Ribonuclease-Gold Labels Heparin in Human Mast Cell Granules: New Use for an Ultrastructural Enzyme Affinity Technique

1998 ◽  
Vol 46 (6) ◽  
pp. 695-706 ◽  
Author(s):  
Ann M. Dvorak ◽  
Ellen S. Morgan
Keyword(s):  
Mast Cell ◽  
Cell Biology ◽  
Enzyme Inhibitors ◽  
De Novo ◽  
Secretory Granules ◽  
Human Mast Cell ◽  
Electron Microscopic ◽  
Binding Characteristics ◽  
Cord Blood Cell

We evaluated an enzyme affinity-gold ultrastructural technique designed to identify RNA-rich structures, based on an RNase-gold (R-G) probe in human mast cells (HMCs). As expected, the R-G technique labeled RNA-containing ribosomes and nucleoli in HMCs. The heparin-rich secretory granules in HMCs were also labeled. Extensive studies revealed that HMCs isolated from lung or skin and sustained in short-term cultures, derived de novo in growth factor-supplemented cord blood cell cultures, or present in vivo in multiple sites all shared this property. We performed a large number of controls designed to examine the HMC granule binding characteristics of gold alone, of irrelevant protein- or enzyme-gold reagents, of the role of charge and enzyme activity after various enzyme digestions, after blocking with macromolecules, after exposure to inhibitors of RNase, of heparin, or to irrelevant enzyme inhibitors, including staining of macromolecule-containing test agar blocks and a variety of combined absorption and digestion experiments of the binding of R-G to HMC granules. These studies established that the R-G method detected heparin in this site in conventionally prepared, well-preserved electron microscopic samples. These findings demonstrate a new use for this enzyme affinity-gold technique in mast cell biology, based on the known property of heparin as an inhibitor of RNase.

scholarly journals Evidence for G proteins in rat parotid plasma membranes and secretory granule membranes

1992 ◽  
Vol 285 (2) ◽  
pp. 441-449 ◽  
Author(s):  
E L Watson ◽  
D DiJulio ◽  
D Kauffman ◽  
J Iversen ◽  
M R Robinovitch ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
G Proteins ◽  
Secretory Granule ◽  
Membrane Fraction ◽  
Immunoblot Analysis ◽  
Plasma Membrane Fraction ◽  
Adp Ribosylation ◽  
Granule Membrane ◽  
Rat Parotid ◽  
Gs Alpha

G proteins were identified in rat parotid plasma membrane-enriched fractions and in two populations of isolated secretory granule membrane fractions. Both [32P]ADP-ribosylation analysis with bacterial toxins and immunoblot analysis with crude and affinity-purified antisera specific for alpha subunits of G proteins were utilized. Pertussis toxin catalysed the ADP-ribosylation of a 41 kDa substrate in the plasma membrane fraction and both secretory granule membrane fractions. Cholera toxin catalysed the ADP-ribosylation of two substrates with molecular masses of 44 kDa and 48 kDa in the plasma membrane fraction but not in the secretory granule fractions. However, these substrates were detected in the secretory granule fractions when recombinant ADP-ribosylating factor was present in the assay medium. Immunoblot analysis of rat parotid membrane fractions using both affinity-purified and crude antisera revealed strong immunoreactivity of these membranes with anti-Gs alpha, -Gi alpha 1/alpha 2 and -Gi alpha 3 sera. In contrast Gs alpha was the major substrate found in both of the secretory granule fractions. Granule membrane fractions also reacted moderately with anti-Gi alpha 3 antiserum, and weakly with anti-Gi alpha 1/alpha 2 and -G(o) alpha sera. The results demonstrate that the parotid gland membranes express a number of G proteins. The presence of G proteins in secretory granule membranes suggests that they may play a direct role in regulating exocytosis in exocrine glands.

scholarly journals Sequential in vivo labeling of insulin secretory granule pools in INS-SNAP transgenic pigs

2021 ◽  
Vol 118 (37) ◽  
pp. e2107665118
Author(s):  
Elisabeth Kemter ◽  
Andreas Müller ◽  
Martin Neukam ◽  
Anna Ivanova ◽  
Nikolai Klymiuk ◽  
...  
Keyword(s):  
Secretory Granule ◽  
Cell Function ◽  
Ex Vivo ◽  
Secretory Granules ◽  
Pig Model ◽  
Transgenic Pigs ◽  
Glucose Levels ◽  
Molecular Features ◽  
Insulin Secretory Granule

β cells produce, store, and secrete insulin upon elevated blood glucose levels. Insulin secretion is a highly regulated process. The probability for insulin secretory granules to undergo fusion with the plasma membrane or being degraded is correlated with their age. However, the molecular features and stimuli connected to this behavior have not yet been fully understood. Furthermore, our understanding of β cell function is mostly derived from studies of ex vivo isolated islets in rodent models. To overcome this translational gap and study insulin secretory granule turnover in vivo, we have generated a transgenic pig model with the SNAP-tag fused to insulin. We demonstrate the correct targeting and processing of the tagged insulin and normal glycemic control of the pig model. Furthermore, we show specific single- and dual-color granular labeling of in vivo–labeled pig pancreas. This model may provide unprecedented insights into the in vivo insulin secretory granule behavior in an animal close to humans.

scholarly journals Evidence for a Holin-Like Protein Gene Fully Embedded Out of Frame in the Endolysin Gene of Staphylococcus aureusBacteriophage 187

1999 ◽  
Vol 181 (15) ◽  
pp. 4452-4460 ◽  
Author(s):  
Martin J. Loessner ◽  
Susanne Gaeng ◽  
Siegfried Scherer
Keyword(s):  
Amino Acids ◽  
Cytoplasmic Membrane ◽  
Transmembrane Domain ◽  
De Novo ◽  
Atp Synthesis ◽  
Large Cell ◽  
Lytic Enzyme ◽  
Reading Frame ◽  
C Terminus

ABSTRACT We have cloned, sequenced, and characterized the genes encoding the lytic system of the unique Staphylococcus aureus phage 187. The endolysin gene ply187 encodes a large cell wall-lytic enzyme (71.6 kDa). The catalytic site, responsible for the hydrolysis of staphylococcal peptidoglycan, was mapped to the N-terminal domain of the protein by the expression of defined ply187 domains. This enzymatically active N terminus showed convincing amino acid sequence homology to anN-acetylmuramoyl-l-alanine amidase, whereas the C-terminal part, whose function is unknown, revealed striking relatedness to major staphylococcal autolysins. An additional reading frame was identified entirely embedded out of frame (+1) within the 5′ region of ply187 and was shown to encode a small, hydrophobic protein of holin-like function. The hol187 gene features a dual-start motif, possibly enabling the synthesis of two products of different lengths (57 and 55 amino acids, respectively). Overproduction of Hol187 in Escherichia coli resulted in growth retardation, leakiness of the cytoplasmic membrane, and loss of de novo ATP synthesis. Compared to other holins identified to date, Hol187 completely lacks the highly charged C terminus. The secondary structure of the polypeptide is predicted to consist of two small, antiparallel, hydrophobic, transmembrane helices. These are supposed to be essential for integration into the membrane, since site-specific introduction of negatively charged amino acids into the first transmembrane domain (V7D G8D) completely abolished the function of the Hol187 polypeptide. With antibodies raised against a synthetic 18-mer peptide representing a central part of the protein, it was possible to detect Hol187 in the cytoplasmic membrane of phage-infected S. aureus cells. An important indication that the protein actually functions as a holin in vivo was that the gene (but not the V7D G8D mutation) was able to complement a phage λ Sam mutation in a nonsuppressing E. coli HB101 background. Plaque formation by λgt11::hol187 indicated that both phage genes have analogous functions. The data presented here indicate that a putative holin is encoded on a different reading frame within the enzymatically active domain of ply187 and that the holin is synthesized during the late stage of phage infection and found in the cytoplasmic membrane, where it causes membrane lesions which are thought to enable access of Ply187 to the peptidoglycan of phage-infected Staphylococcus cells.

Recycling of a secretory granule membrane protein after stimulated secretion

1993 ◽  
Vol 106 (2) ◽  
pp. 649-655 ◽  
Author(s):  
S.M. Hurtley
Keyword(s):  
Plasma Membrane ◽  
Membrane Protein ◽  
Secretory Granule ◽  
Chromaffin Cells ◽  
Secretory Granules ◽  
Primary Cultures ◽  
Dopamine Beta Hydroxylase ◽  
Granule Membrane ◽  
Antibody Complex ◽  
Adrenal Chromaffin

Recycling of a secretory granule membrane protein, dopamine-beta-hydroxylase, was examined in primary cultures of bovine adrenal chromaffin cells. Cells were stimulated to secrete in the presence of antibodies directed against the luminal domain of dopamine-beta-hydroxylase. The location of the antibodies after various times of reincubation and after a second secretory stimulus was assessed using immunofluorescence microscopy. Stimulation led to the exposure of dopamine-beta-hydroxylase at the plasma membrane, which could be detected by a polyclonal antibody in living and fixed cells. The plasma membrane dopamine-beta-hydroxylase, either alone or complexed with antibody, was rapidly internalized after removal of the secretagogue. Internalized protein-antibody complex remained stable for at least 24 hours of reculture. Twenty four hours after stimulation the cells with internalized antibody could respond to further stimulation and some of the antibody was re-exposed at the plasma membrane. These findings were confirmed using FACS analysis. This suggests that the antibody-protein complex had returned to secretory granules that could respond to further secretagogue stimulation.

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