Processing of receptor-bound somatostatin: internalization and degradation by pancreatic acini

1987 ◽  
Vol 252 (4) ◽  
pp. G535-G542 ◽  
Author(s):  
N. Viguerie ◽  
J. P. Esteve ◽  
C. Susini ◽  
N. Vaysse ◽  
A. Ribet
Keyword(s):  
Plasma Membrane ◽  
Plasma Membranes ◽  
Specific Binding ◽  
Specific Radioactivity ◽  
Pancreatic Acinar Cells ◽  
Nuclear Fraction ◽  
Pancreatic Acini ◽  
Specific Binding Sites ◽  
Membrane Bound ◽  
Membrane Level

We have previously demonstrated the presence of specific binding sites for somatostatin on plasma membranes from pancreatic acinar cells. In the present study we attempted to characterize the fate of receptor-bound 125I-[Tyr11]somatostatin. Internalization of somatostatin was rapid (reaching a plateau at 20% of the cell-associated specific radioactivity) and temperature dependent. To follow the processing of bound somatostatin, acini were incubated with 125I-[Tyr11]somatostatin at 5 degrees C during 16 h then, after washing, incubated at 37 degrees C for 90 min in fresh medium. Surface-bound somatostatin decreased rapidly, whereas radioactivity increased in the cell interior and the incubation medium. Intracellular and membrane-bound radioactivity was mainly intact 125I-[Tyr11]somatostatin. Degradation occurred at the plasma membrane level and led to iodotyrosine production. After 15 min of incubation, 15% of the initially surface-bound 125I-[Tyr11]somatostatin was compartmentalized within the cell, mainly in the microsomal fraction. After 30 min, a significant increase in radioactivity appeared in the nuclear fraction. These results indicate that the major part of somatostatin cellular degradation takes place at the plasma membrane level. Within the cell, somatostatin is routed to the nucleus via particular fractions sedimenting with microsomal vesicles.

scholarly journals Specific binding sites for inositol 1,3,4,5-tetrakisphosphate are located predominantly in the plasma membranes of human platelets

1994 ◽  
Vol 298 (3) ◽  
pp. 739-742 ◽  
Author(s):  
P J Cullen ◽  
Y Patel ◽  
V V Kakkar ◽  
R F Irvine ◽  
K S Authi
Keyword(s):  
Plasma Membrane ◽  
Binding Sites ◽  
Plasma Membranes ◽  
Human Platelet ◽  
Specific Binding ◽  
Human Platelets ◽  
Intracellular Membranes ◽  
Specific Binding Sites ◽  
Carrier Free ◽  
Membrane Location

In the present study we describe the characterization and localization of Ins(1,3,4,5)P4-binding sites in human platelet membranes. Specific binding sites for Ins(1,3,4,5)P4 have been identified on mixed, plasma and intracellular membranes from neuraminidase-treated platelets using highly purified carrier-free [32P]Ins(1,3,4,5)P4. The displacement of Ins(1,3,4,5)P4 from these sites by Ins(1,4,5)P3 and InsP6 occurs at greater than two orders of magnitude higher concentrations and with Ins(1,3,4,5,6)P5 at about 40-fold higher concentrations than with Ins(1,3,4,5)P4. The membranes were further separated by free-flow electrophoresis into plasma and intracellular membranes. The Ins(1,3,4,5)P4-binding sites separated with plasma membranes, and showed similar affinities and specificities as mixed membranes, whereas Ins(1,4,5)P3-binding sites were predominantly in the intracellular membranes. These results suggest a predominantly plasma membrane location for putative Ins(1,3,4,5)P4 receptors in human platelets.

scholarly journals Biogenesis of hepatocyte plasma-membrane domains. Incorporation of (3H)fucose into plasma-membrane and golgi-apparatus glycoproteins

1980 ◽  
Vol 192 (3) ◽  
pp. 903-910 ◽  
Author(s):  
W H Evans ◽  
N A Flint ◽  
P Vischer
Keyword(s):  
Plasma Membrane ◽  
Gel Electrophoresis ◽  
Plasma Membranes ◽  
Polyacrylamide Gel Electrophoresis ◽  
Specific Radioactivity ◽  
Direct Transfer ◽  
Membrane Domains ◽  
Secretory Glycoproteins ◽  
Membrane Bound ◽  
Kinetics Of

1. Rats were injected intracaudally with [3H]fucose and its rate of incorporation into the fucoproteins of serum, Golgi and plasma-membrane subfractions was followed for up tp 2h. 2. Incorporation into the Golgi dictyosome and secretory-vesicular fractions reached a maximum at 15 min or less, but most of the radioactivity was associated with classes of secretory glycoproteins. Incorporation into sinusoidal plasma-membrane fractions reached a maximum at 30 min, coinciding with the maximum release of fucoproteins into the serum. Contiguous and canalicular plasma-membrane fractions were labelled slightly later and at a lower rate and specific radioactivity. 3. Fluorography of fucoproteins separated by polyacrylamide-gel electrophoresis helped to distinguish between the major secretory and membrane-bound glycoproteins. The results show that a major biogenetic sequence is probably from Golgi dictyosomes to Golgi secretory elements to a sinusoidal plasma membrane. 4. The kinetics of incorporation make it unlikely that there is rapid and direct insertion of glycoproteins into the bile-canalicular plasma membrane. A route involving direct transfer of glycoproteins via a membrane-mediated intracellular path from the blood sinusoidal to the bile-canalicular plasma membranes is proposed.

scholarly journals Large-scale purification of presynaptic plasma membranes from Torpedo marmorata electric organ.

1985 ◽  
Vol 101 (5) ◽  
pp. 1757-1762 ◽  
Author(s):  
N Morel ◽  
J Marsal ◽  
R Manaranche ◽  
S Lazereg ◽  
J C Mazie ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Large Scale ◽  
Plasma Membranes ◽  
Electric Organ ◽  
Acetylcholinesterase Activity ◽  
Cholinergic Nerve Terminals ◽  
Membrane Bound ◽  
Torpedo Electric Organ ◽  
Presynaptic Plasma Membrane ◽  
Glycera Convoluta

The presynaptic plasma membrane (PSPM) of cholinergic nerve terminals was purified from Torpedo electric organ using a large-scale procedure. Up to 500 g of frozen electric organ were fractioned in a single run, leading to the isolation of greater than 100 mg of PSPM proteins. The purity of the fraction is similar to that of the synaptosomal plasma membrane obtained after subfractionation of Torpedo synaptosomes as judged by its membrane-bound acetylcholinesterase activity, the number of Glycera convoluta neurotoxin binding sites, and the binding of two monoclonal antibodies directed against PSPM. The specificity of these antibodies for the PSPM is demonstrated by immunofluorescence microscopy.

scholarly journals 5′-Nucleotidases of chicken gizzard and human pancreatic adenocarcinoma cells are anchored to the plasma membrane via a phosphatidylinositol–glycan

1989 ◽  
Vol 262 (1) ◽  
pp. 33-40 ◽  
Author(s):  
U Stochaj ◽  
K Flocke ◽  
W Mathes ◽  
H G Mannherz
Keyword(s):  
Plasma Membrane ◽  
Pancreatic Adenocarcinoma ◽  
Plasma Membranes ◽  
Chicken Gizzard ◽  
Human Pancreatic Adenocarcinoma ◽  
Adenocarcinoma Cells ◽  
Increased Sensitivity ◽  
Membrane Bound ◽  
Membrane Anchoring ◽  
Pancreatic Adenocarcinoma Cell Line

We have analysed the membrane anchorage of plasma-membrane 5′-nucleotidase, an ectoenzyme which can mediate binding to components of the extracellular matrix. We demonstrated that the purified enzyme obtained from chicken gizzard and a human pancreatic adenocarcinoma cell line were both completely transformed into a hydrophilic form by treatment with phospholipases C and D, cleaving glycosylphosphatidylinositol (GPI). These data indicate the presence of a glycolipid linker employed for membrane anchoring of the 5′-nucleotidase obtained from both sources. Incubation of plasma membranes under identical conditions revealed that about half of the AMPase activity was resistant to GPI-hydrolysing phospholipases. Investigation of the enzymic properties of purified chicken gizzard 5′-nucleotidase revealed only minor changes after removal of the phosphatidylinositol linker. However, cleavage of the membrane anchor resulted in an increased sensitivity towards inhibition by concanavalin A. After tissue fractionation, chicken gizzard 5′-nucleotidase could be obtained as either a membrane-bound or a soluble protein; the latter is suspected to be released from the plasma membrane by endogenous phospholipases. Higher-molecular-mass proteins immuno-cross-reactive with the purified chicken gizzard 5′-nucleotidase were detected as both soluble and membrane-bound forms.

scholarly journals Plasma membrane association of Acanthamoeba myosin I.

1989 ◽  
Vol 109 (4) ◽  
pp. 1519-1528 ◽  
Author(s):  
H Miyata ◽  
B Bowers ◽  
E D Korn
Keyword(s):  
Plasma Membrane ◽  
Binding Site ◽  
Heavy Chain ◽  
Membrane Fraction ◽  
Plasma Membranes ◽  
Actin Binding ◽  
Plasma Membrane Fraction ◽  
Myosin I ◽  
Membrane Bound ◽  
Actin Binding Site

Myosin I accounted for approximately 2% of the protein of highly purified plasma membranes, which represents about a tenfold enrichment over its concentration in the total cell homogenate. This localization is consistent with immunofluorescence analysis of cells that shows myosin I at or near the plasma membrane as well as diffusely distributed in the cytoplasm with no apparent association with cytoplasmic organelles or vesicles identifiable at the level of light microscopy. Myosin II was not detected in the purified plasma membrane fraction. Although actin was present in about a tenfold molar excess relative to myosin I, several lines of evidence suggest that the principal linkage of myosin I with the plasma membrane is not through F-actin: (a) KI extracted much more actin than myosin I from the plasma membrane fraction; (b) higher ionic strength was required to solubilize the membrane-bound myosin I than to dissociate a complex of purified myosin I and F-actin; and (c) added purified myosin I bound to KI-extracted plasma membranes in a saturable manner with maximum binding four- to fivefold greater than the actin content and with much greater affinity than for pure F-actin (apparent KD of 30-50 nM vs. 10-40 microM in 0.1 M KCl plus 2 mM MgATP). Thus, neither the MgATP-sensitive actin-binding site in the NH2-terminal end of the myosin I heavy chain nor the MgATP-insensitive actin-binding site in the COOH-terminal end of the heavy chain appeared to be the principal mechanism of binding of myosin I to plasma membranes through F-actin. Furthermore, the MgATP-sensitive actin-binding site of membrane-bound myosin I was still available to bind added F-actin. However, the MgATP-insensitive actin-binding site appeared to be unable to bind added F-actin, suggesting that the membrane-binding site is near enough to this site to block sterically its interaction with actin.

Insulin action in pancreatic acini from streptozotocin-treated rats. III. Electron microscope autoradiography of 125I-insulin

1981 ◽  
Vol 240 (1) ◽  
pp. G69-G75
Author(s):  
I. D. Goldfine ◽  
B. M. Kriz ◽  
K. Y. Wong ◽  
G. Hradek ◽  
A. L. Jones ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Electron Microscope ◽  
Relative Concentration ◽  
Pancreatic Acinar Cells ◽  
Diabetic Rat ◽  
Dependent Manner ◽  
Electron Microscope Autoradiography ◽  
Intracellular Location ◽  
Pancreatic Acini ◽  
Grain Distribution

Electron microscope autoradiographs were prepared from diabetic rat pancreatic acini that had been incubated with 125I-insulin. Distribution histograms of the distance of the 125I-insulin silver grains from the nearest plasma membrane were prepared and compared with a histogram of an 125I line source. After 3 min of incubation, insulin was located predominately on the plasma membrane, but even at this early time 15% of the grains had an intracellular location. After 30 min of incubation, there was a decrease in grains on the plasma membrane and an increase (to 45%) in grains localized in the cell. At both times of incubation, a comparison of the distribution of the insulin grains overlying subcellular organelles to a theoretical random grain distribution pattern indicated that the intracellular distribution of insulin grains was nonrandom. At 3 min, there was a relative concentration of grains over the plasma membrane and vesicles with an average diameter of 100 nm. At 30 min, there was a concentration of grains over the plasma membrane, 100-nm vesicle, and Golgi. These studies suggest that 125I-insulin is internalized into pancreatic acinar cells in a time-dependent manner and then is nonrandomly distributed inside the cell.

Incorporation in vivo of [32P]orthophosphate and [Me-3H]choline into rough microsomal, Golgi, and plasma membranes of rat liver

1977 ◽  
Vol 55 (8) ◽  
pp. 876-885 ◽  
Author(s):  
Patricia L. Chang ◽  
John R. Riordan ◽  
Mario A. Moscarello ◽  
Jennifer M. Sturgess
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Rat Liver ◽  
Plasma Membranes ◽  
Specific Activity ◽  
Specific Radioactivity ◽  
Marker Enzyme ◽  
Golgi Membranes ◽  
Endomembrane Flow

To study membrane biogenesis and to test the validity of the endomembrane flow hypothesis, incorporation of 32P and [Me-3H]choline in vivo into membranes of the rat liver was followed. Rough microsomal, Golgi-rich, and plasma membrane fractions were monitored with marker enzyme assays and shown with morphometric analysis to contain 82% rough microsomes, at least 70% Golgi complexes, and 88% plasma membranes, respectively. Membrane subfractions from the rough microsomal and Golgi-rich fractions were prepared by sonic disruption.At 5 to 30 min after 32P injection, the specific radioactivity of phosphatidylcholine was higher in the rough microsomal membranes than in the Golgi membranes. From 1 to 3 h, the specific activity of phosphatidylcholine in Golgi membranes became higher and reached the maximum at about 3 h. Although the plasma membrane had the lowest specific radioactivity throughout 0.25–3 h, it increased rapidly thereafter to attain the highest specific activity at 5 h. Both rough microsomal and plasma membranes reached their maxima at 5 h.The specific radioactivity of [32P]phosphatidylethanolamine in the three membrane fractions was similar to that of [32P]phosphatidylcholine except from 5 to 30 min, when the specific radioactivity of phosphatidylethanolamine in the Golgi membranes was similar to the rough microsomal membranes.At 15 min to 5 h after [Me-3H]choline injection, more than 90% of the radioactivity in all the membranes was acid-precipitable. The specific radioactivities of the acid-precipitated membranes, expressed as dpm per milligram protein, reached the maximum at 3 h. After [Me-3H]choline injection, the specific radioactivity of phosphatidylcholine separated from the lipid extract of the acid-precipitated membranes (dpm per micromole phosphorus) did not differ significantly in the three membrane fractions. The results indicated rapid incorporation of choline into membrane phosphatidylcholine by the rough endoplasmic reticulum, Golgi, and plasma membranes simultaneously.The data with both 32P and [Me-3H]choline precursors did not support the endomembrane flow hypothesis. The Golgi complexes apparently synthesized phosphatidylethanolamine and incorporated choline into phosphatidylcholine as well as the endoplasmic reticulum. The results are discussed with relevance to current hypotheses on the biogenesis and transfer of membrane phospholipids.

scholarly journals Partial purification and characterization of oestrogen receptors in subfractions of hepatocyte plasma membranes

1980 ◽  
Vol 191 (3) ◽  
pp. 743-760 ◽  
Author(s):  
Richard J. Pietras ◽  
Clara M. Szego
Keyword(s):  
Plasma Membrane ◽  
Binding Sites ◽  
Wheat Germ Agglutinin ◽  
Wheat Germ ◽  
Plasma Membranes ◽  
Specific Binding ◽  
Low Density ◽  
Marker Enzymes ◽  
Low Density Plasma ◽  
Density Plasma

To assess the subcellular distribution of oestrogen-binding components in their native state, plasma membrane and other cell fractions were prepared from hepatocytes in the absence of [3H]oestradiol-17β. Cells from livers of ovariectomized rats were disrupted, with submaximal homogenization in buffered isotonic sucrose with CaCl2 and proteinase inhibitor, and fractionated by using isotonic media. Fractions were characterized by determinations of enzyme activities, biochemical constituents and ligand binding. Specific binding of 2nm-[3H]oestradiol-17β to intact cells and their fractions was detemined after equilibration for 1.5h at 4°C. More than 92% of the radioactivity from representative preparations was verified as authentic oestradiol by thin-layer chromatography. Activities of plasma-membrane marker enzymes as well as binding sites for oestrogen and for wheat germ agglutinin were present principally in particulate fractions, rather than in 105000g-supernatant fractions. However, by using alternative homogenization procedures (i.e. hypotonic media), known to fragment and strip structural components, oestradiol-binding sites and activities of plasma-membrane marker enzymes were distributed predominantly into cytosol. By using the more conservative procedures, plasma membranes of low (ρ=1.13–1.16) and high (ρ=1.16–1.18) density were purified from crude nuclear fractions. A second low-density subfraction of plasma membrane was prepared from microsome-rich fractions. Activities of plasma-membrane marker enzymes were enriched to about 28 and four times that of the homogenate in plasma membranes of low and high density respectively. Binding sites for wheat germ agglutinin and oestradiol were concentrated in low-density plasma membranes to 46–63 times that of the homogenate. Specific binding of oestrogen in low-density plasma membranes purified from crude nuclei was saturable, with an apparent association constant of 3.5nm. At saturation, such oestradiol receptors corresponded to 526fmol/mg of membrane protein. A Hill plot showed a moderate degree of positive co-operativity in the interaction of hormone with plasma membranes. Specific binding of [3H]oestradiol-17β was reduced by a 200-fold molar excess of unlabelled oestradiol-17β, oestriol or diethylstilbestrol, but not by oestradiol-17α, cortisol, testosterone or progesterone. Binding was also blocked by prior exposure of membranes to trypsin or to 60°C, but remained essentially undiminished by extraction of membranes with either hypotonic or high-salt buffers. Extraction with 0.1% (v/v) Triton X-100 partially solubilized the oestrogen-binding component(s) of plasma membranes. Particle-free extracts were resolved on 5–20% (w/v) sucrose density gradients with either 0.01m- or 0.4m-KCl, and the fractions were analysed by adsorption to hydroxyapatite. In low-salt gradients macromolecule-bound oestrogen sedimented at predominantly 7.4S and binding was 1560 times that of the homogenate. Under high-salt conditions oestradiol-binding activity occurred at both 3.6S and 4.9S.

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