scholarly journals Isolation of a plasma-membrane fraction from gastric smooth muscle. Comparison of the calcium uptake with that in endoplasmic reticulum

1983 ◽  
Vol 210 (2) ◽  
pp. 315-322 ◽  
Author(s):  
L Raeymaekers ◽  
F Wuytack ◽  
J Eggermont ◽  
G De Schutter ◽  
R Casteels
Keyword(s):  
Endoplasmic Reticulum ◽  
Smooth Muscle ◽  
Plasma Membrane ◽  
Membrane Fraction ◽  
Microsomal Fraction ◽  
Membrane Vesicles ◽  
Cholesterol Content ◽  
Sucrose Density Gradient ◽  
Plasma Membrane Vesicles ◽  
Plasma Membrane Fraction

1. A plasma-membrane fraction was isolated from the smooth muscle of the pig stomach by using differential and sucrose-density-gradient centrifugations. When the centrifugation was carried out after preloading the crude microsomal fraction with Ca2+ in the presence of oxalate, the contamination of the plasma-membrane fraction by endoplasmic reticulum was decreased and a fraction enriched in endoplasmic reticulum vesicles filled with calcium oxalate crystals was obtained. 2. The plasmalemmal and endoplasmic-reticulum membranes could be distinguished by differences in the activity of marker enzymes and in the cholesterol content and by their different permeability to oxalate and phosphate. Oxalate and phosphate stimulated the Ca2+ uptake in the endoplasmic reticulum much more than in the plasmalemmal vesicles. In the plasma-membrane vesicles 40 mM-phosphate was more effective for stimulating the Ca2+ uptake than was 5 mM-oxalate, but the reverse was seen in the endoplasmic reticulum. 3. The high cholesterol/phospholipid ratio of the crude microsomal fraction are of the majority of the vesicles present in the crude microsomal fraction are of plasmalemmal origin. 4. The Ca2+ pump of the plasmalemmal and endoplasmic-reticulum vesicles could be differentiated by their different sensitivities to calmodulin. However, the two Ca2+-transport ATPases did not differ by their sensitivity to vanadate nor by the energization of the Ca2+ transport by different nucleoside triphosphates.

scholarly journals Phorbol ester-sensitive phospholipase D is mainly localized in the endoplasmic reticulum of BHK cells

1996 ◽  
Vol 320 (3) ◽  
pp. 885-890 ◽  
Author(s):  
Christina DECKER ◽  
Maria Jesus MIRO OBRADORS ◽  
Daniel J. SILLENCE ◽  
David ALLAN
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Phospholipase D ◽  
Phorbol Ester ◽  
Membrane Vesicles ◽  
Sucrose Density Gradient ◽  
Subcellular Fractionation ◽  
Plasma Membrane Vesicles ◽  
Intact Cells ◽  
Site Of Action

The localization of phorbol ester-sensitive phospholipase D (PLD) in baby hamster kidney cells has been investigated by determining the subcellular distribution of the phosphatidylbutanol produced when the cells are incubated with phorbol 12-myristate 13-acetate and n-butanol. Results derived by isolation of plasma membrane vesicles from intact cells or by subcellular fractionation on a sucrose density gradient suggest the PLD is specific for phosphatidylcholine and its primary site of action is not the plasma membrane but the endoplasmic reticulum.

Isolation and characterization of plasma membranes from bovine carotid arteries

1986 ◽  
Vol 250 (1) ◽  
pp. C65-C75 ◽  
Author(s):  
R. V. Sharma ◽  
R. C. Bhalla
Keyword(s):  
Plasma Membrane ◽  
Cytochrome C ◽  
Membrane Fraction ◽  
Plasma Membranes ◽  
Carotid Arteries ◽  
Membrane Vesicles ◽  
Plasma Membrane Vesicles ◽  
Cytochrome C Reductase ◽  
Plasma Membrane Fraction

A plasma membrane fraction from bovine carotid arteries has been isolated by extraction of a crude microsomal fraction with a low-ionic-strength buffer containing ATP and Ca2+. This step was followed by sucrose-density-gradient centrifugation in the presence of 0.6 M KCl. The plasma membrane vesicles were enriched 60- to 80-fold in Na+-K+-adenosinetriphosphatase, 5'-nucleotidase, and phosphodiesterase I activities. The final yields of these marker enzymes were 12-18% of the total activities in the postnuclear supernatant, and the protein yield was 100-120 micrograms/g wet wt of carotid arteries. Contamination of the plasma membrane fraction by mitochondria and sarcoplasmic reticulum was low as judged by low activities of succinate--cytochrome-c reductase and NADPH--cytochrome-c reductase, respectively. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoprecipitation with smooth muscle-specific actin antibodies showed that the plasma membrane fraction was substantially free from myosin and actin contamination. The plasma membrane vesicles accumulated Ca2+ in the presence of ATP, and the accumulation was increased by calmodulin. Ca2+ accumulated in the presence or absence of calmodulin could be released almost completely from the vesicles by the addition of the Ca2+ ionophore A23187 but not by ethyleneglycol-bis(beta-aminoethylether)-N,N'-tetraacetic acid, indicating that Ca2+ uptake in the presence of ATP is intravesicular. The effects of phosphate and oxalate on Ca2+ uptake in the plasma membranes were different from one another. Phosphate increased Ca2+ uptake in a concentration- and time-dependent manner, and the increase in Ca2+ uptake could be observed as early as 1 min. On the other hand, oxalate at concentrations up to 5 mM did not increase Ca2+ uptake significantly during the 30-min incubation. These plasma membranes can prove useful for the study of ion transport across plasma membranes, hormone binding, characterization of calcium channels, and preparation of antibodies against plasma membrane proteins.

The effects of acetylcholine and atropine on the 22Na+ permeability of intestinal smooth muscle vesicles

1978 ◽  
Vol 56 (6) ◽  
pp. 921-925
Author(s):  
L. Spero
Keyword(s):  
Smooth Muscle ◽  
Plasma Membrane ◽  
Membrane Vesicles ◽  
Intestinal Smooth Muscle ◽  
Erythrocyte Ghosts ◽  
Muscarinic Agonists ◽  
Plasma Membrane Vesicles ◽  
Muscle Plasma Membrane ◽  
Whole Muscle ◽  
Kinetics Of

A technique is described which has enabled us to measure changes in 22Na+ efflux from smooth muscle plasma membrane vesicles. The resting 22Na+ efflux from these sealed vesicles showed a concentration-dependent increase in response to acetylcholine and other muscarinic agonists, in similar concentrations to those which increased 42K+ efflux in whole muscle. The kinetics of this efflux were complex and could not be described by less than three exponential processes. The response to agonists has, therefore, been characterized by measurement of the half-life of 22Na+ efflux (t1/2). The acetylcholine effect was inhibited by atropine, but unlike the situation in the whole muscle, this inhibition was noncompetitive. Tubocuraine (a nicotinic antagonist) had no effect on this acetylcholine response. Atropine has no effect by itself on the resting 22Na+ efflux, neither did tetrodotoxin or ouabain. 22Na+ efflux from erythrocyte ghosts and liposomes, prepared from lipid extracts of the smooth muscle plasma membrane, was not modified by acetylcholine or atropine.

Preferential support of Ca 2+ uptake in smooth muscle plasma membrane vesicles by an endogenous glycolytic cascade

1989 ◽  
Vol 3 (11) ◽  
pp. 2298-2301 ◽  
Author(s):  
Richard J. Paul ◽  
Christopher D. Hardin ◽  
Luc Raeymaekers ◽  
Frank Wuytack ◽  
Rik Casteels
Keyword(s):  
Smooth Muscle ◽  
Plasma Membrane ◽  
Membrane Vesicles ◽  
Plasma Membrane Vesicles ◽  
Muscle Plasma Membrane

scholarly journals myo-Inositol 1,4,5-trisphosphate mobilizes Ca2+ from isolated adipocyte endoplasmic reticulum but not from plasma membranes

1986 ◽  
Vol 236 (1) ◽  
pp. 37-44 ◽  
Author(s):  
D M Delfert ◽  
S Hill ◽  
H A Pershadsingh ◽  
W R Sherman ◽  
J M McDonald
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Plasma Membranes ◽  
Initial Rate ◽  
Membrane Vesicles ◽  
Heavy Fraction ◽  
Differential Centrifugation ◽  
Plasma Membrane Vesicles ◽  
Inositol 1,4,5 Trisphosphate ◽  
Uptake And Release

The effects of myo-inositol 1,4,5-trisphosphate (IP3) on Ca2+ uptake and release from isolated adipocyte endoplasmic reticulum and plasma membrane vesicles were investigated. Effects of IP3 were initially characterized using an endoplasmic reticulum preparation with cytosol present (S1-ER). Maximal and half-maximal effects of IP3 on Ca2+ release from S1-ER vesicles occurred at 20 microM- and 7 microM-IP3, respectively, in the presence of vanadate which prevents the re-uptake of released Ca2+ via the endoplasmic reticulum Ca2+ pump. At saturating IP3 concentrations, Ca2+ release in the presence of vanadate was 20% of the exchangeable Ca2+ pool. IP3-induced release of Ca2+ from S1-ER was dependent on extravesicular free Ca2+ concentration with maximal release occurring at 0.13 microM free Ca2+. At 20 microM-IP3 there was no effect on the initial rate of Ca2+ uptake by S1-ER. IP3 promoted Ca2+ release from isolated endoplasmic reticulum vesicles (cytosol not present) to a similar level as compared with S1-ER. Addition of cytosol to isolated endoplasmic reticulum vesicles did not affect IP3-induced Ca2+ release. The endoplasmic reticulum preparation was further fractionated into heavy and light vesicles by differential centrifugation. Interestingly, the heavy fraction, but not the light fraction, released Ca2+ when challenged with IP3. IP3 (20 microM) did not promote Ca2+ release from plasma membrane vesicles and had no effect on the (Ca2+ + Mg2+)-ATPase activity or on the initial rate of ATP-dependent Ca2+ uptake by these vesicles. These results support the concept that IP3 acts exclusively at the endoplasmic reticulum to promote Ca2+ release.

scholarly journals Novel adipocyte aminopeptidases are selectively upregulated by insulin in healthy and obese rats

2015 ◽  
Vol 228 (2) ◽  
pp. 97-104 ◽  
Author(s):  
Rafaela Fadoni Alponti ◽  
Patricia Lucio Alves ◽  
Paulo Flavio Silveira
Keyword(s):  
Plasma Membrane ◽  
Energy Metabolism ◽  
Enzyme Activities ◽  
Metabolic Pathways ◽  
Membrane Fraction ◽  
Microsomal Fraction ◽  
Low Density ◽  
Plasma Membrane Fraction ◽  
Catalytic Activities ◽  
Obese Rats

The lack of a complete assembly of the sensitivity of subcellular aminopeptidase (AP) activities to insulin in different pathophysiological conditions has hampered the complete view of the adipocyte metabolic pathways and its implications in these conditions. Here we investigated the influence of insulin on basic AP (APB), neutral puromycin-sensitive AP (PSA), and neutral puromycin-insensitive AP (APM) in high and low density microsomal and plasma membrane fractions from adipocytes of healthy and obese rats. Catalytic activities of these enzymes were fluorometrically monitoring in these fractions with or without insulin stimulus. Canonical traffic such as insulin-regulated AP was not detected for these novel adipocyte APs in healthy and obese rats. However, insulin increased APM in low density microsomal and plasma membrane fractions from healthy rats, APB in high density microsomal fraction from obese rats and PSA in plasma membrane fraction from healthy rats. A new concept of intracellular compartment-dependent upregulation of AP enzyme activities by insulin emerges from these data. This relatively selective regulation has pathophysiological significance, since these enzymes are well known to act as catalysts and receptor of peptides directly related to energy metabolism. Overall, the regulation of each one of these enzyme activities reflects certain dysfunction in obese individuals.

scholarly journals A procedure for the rapid isolation from rat liver of plasma membrane vesicles exhibiting Ca2+-transport and Ca2+-ATPase activities

1984 ◽  
Vol 223 (3) ◽  
pp. 733-745 ◽  
Author(s):  
R J Epping ◽  
F L Bygrave
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Rat Liver ◽  
Gradient Centrifugation ◽  
Membrane Surface ◽  
Membrane Vesicles ◽  
Mitochondrial Fraction ◽  
Plasma Membrane Vesicles ◽  
Percoll Density Gradient Centrifugation ◽  
Percoll Density Gradient

A technique is described for the isolation of a plasma membrane-enriched preparation from a rat liver post-mitochondrial fraction by using discontinuous Percoll density-gradient centrifugation. The procedure is simple, of high reproducibility and yield and requires a total isolation time of only 90 min. The preparation consists almost exclusively of membrane vesicles and is enriched approx. 26-fold in plasma membrane-localized enzymes with minor contamination (less than 10%) with membranes derived mainly from the endoplasmic reticulum and Golgi apparatus. Approx. 20% of the fraction comprises tightly-sealed vesicles in the inverted orientation which are capable of accumulating calcium ions and exhibiting vanadate-insensitive Ca2+-ATPase activity. The properties of these activities, including insensitivity to vanadate, oxalate, and to p-chloromercuribenzoate as well as a lack of requirement for added Mg2+, contrast markedly with the reported properties of Ca2+ transport by the endoplasmic reticulum isolated from rat liver. The technique may have wide application in the study of plasma membrane-associated activities in rat liver, particularly in relation to sinusoidal membrane surface-related events.

scholarly journals Inhibitory antibodies to plasmalemmal Ca2+-transporting ATPases. Their use in subcellular localization of (Ca2+ + Mg2+)-dependent ATPase activity in smooth muscle

1985 ◽  
Vol 231 (3) ◽  
pp. 737-742 ◽  
Author(s):  
J Verbist ◽  
F Wuytack ◽  
L Raeymaekers ◽  
R Casteels
Keyword(s):  
Endoplasmic Reticulum ◽  
Smooth Muscle ◽  
Plasma Membrane ◽  
Atpase Activity ◽  
Membrane Fraction ◽  
Transport Atpase ◽  
Calmodulin Binding ◽  
Dependent Atpase ◽  
Inhibitory Antibodies ◽  
Stimulation Of

Antibodies directed against the purified calmodulin-binding (Ca2+ + Mg2+)-ATPase [(Ca2+ + Mg2+)-dependent ATPase] from pig erythrocytes and from smooth muscle of pig stomach (antral part) were raised in rabbits. Both the IgGs against the erythrocyte (Ca2+ + Mg2+)-ATPase and against the smooth-muscle (Ca2+ + Mg2+)-ATPase inhibited the activity of the purified calmodulin-binding (Ca2+ + Mg2+)-ATPase from smooth muscle. Up to 85% of the total (Ca2+ + Mg2+)-ATPase activity in a preparation of KCl-extracted smooth-muscle membranes was inhibited by these antibodies. The (Ca2+ + Mg2+)-ATPase activity and the Ca2+ uptake in a plasma-membrane-enriched fraction from this smooth muscle were inhibited to the same extent, whereas in an endoplasmic-reticulum-enriched membrane fraction the (Ca2+ + Mg2+)-ATPase activity was inhibited by only 25% and no effect was observed on the oxalate-stimulated Ca2+ uptake. This supports the hypothesis that, in pig stomach smooth muscle, two separate types of Ca2+-transport ATPase exist: a calmodulin-binding ATPase located in the plasma membrane and a calmodulin-independent one present in the endoplasmic reticulum. The antibodies did not affect the stimulation of the (Ca2+ + Mg2+)-ATPase activity by calmodulin.

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