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At least 721 records · Page 40Linked to original sources
PubMed1988

Identification of a G protein in rough endoplasmic reticulum of canine pancreas.

Employing [32P]ADP-ribosylation by pertussis toxin we have identified a G protein that is located in the rough endoplasmic reticulum of canine pancreas and therefore termed it GRER. Identification of GRER is based on the following data. A 41-kDa polypeptide was the only polypeptide that was [32P]ADP-ribosylated by pertussis toxin in pancreas rough microsomes. Guanosine 5'-(gamma-thio)triphosphate (GTP gamma S) and 1 mM ATP, 6 mM MgCl2, 10 mM NaF (AMF) inhibited ADP-ribosylation of this polypeptide. The [32P]ADP-ribosylated 41-kDa polypeptide was immunoprecipitated by antisera which specifically recognized the C-terminal residues of the alpha subunits of Gi and transducin, indicating that the 41-kDa polypeptide is immunologically related to the alpha subunits of heterotrimeric G proteins. Treatment with GTP gamma S resulted in a reduction in the sedimentation rate of the [32P]ADP-ribosylated, detergent-solubilized GRER. It also induced the release of the [32P]ADP-ribosylated 41-kDa polypeptide from rough microsomes in the absence of detergent, unlike ADP-ribosylated alpha subunits of plasma membrane-associated G proteins. These data are consistent with an oligomeric nature of GRER. The codistribution of GRER with an endoplasmic reticulum marker protein during subcellular fractionation and the lack of plasma membrane contamination of the rough microsomal fraction, combined with the isodensity of GRER with rough microsomes as well as the isodensity of GRER with "stripped" microsomes after extraction of rough microsomes with EDTA and 0.5 M KCl, localized GRER to the rough endoplasmic reticulum. Preliminary experiments suggest that GRER appears not to be involved in translocation of proteins across the rough endoplasmic reticulum membrane.

Adenosine Diphosphate Ribose↗
PubMed1977

Dependence of calcium permeability of sarcoplasmic reticulum vesicles on external and internal calcium ion concentrations.

The ability of sarcoplasmic reticulum vesicles to retain calcium following ATP-supported calcium uptake in the presence of the calcium-precipitating anions oxalate and phosphate depends on Cao (calcium ion concentration outside the vesicles) and Cai (calcium ion concentration within the vesicles). Calcium efflux rates at any level of Cai are accelerated when Cao is increased. Higher Cao at the time that calcium uptake reactions reach steady state is associated with a spontaneous calcium release that reflects this effect of increased Cao. Increasing Cai at any level of Cao causes little or no acceleration of calcium efflux rate so that calcium permeability coefficients, estimated by dividing calcium efflux rates by Cai, the "driving force", are inversely proportional to Cai. Calcium permability coefficients thus correlate, as a first approximation, with the ratio Cai/Cao, decreasing 1000-fold as this ratio increases over a 3000-fold range (Cao = 0.1 to 3.3 muM, Cai =4 to 750 muM). Oscillations in both the calcium content of the vesicles and Cao are seen as calcium uptake reactions approach steady state, suggesting that calcium permeability undergoes time-dependent variations. Sudden reduction of Cao to levels that markedly inhibit calcium influx via the calcium pump unmasks a calcium efflux that decreases slowly over 60 to 90 s. The maximal calcium permeability observed in the present study would allow the calcium efflux rate from the sarcoplasmic reticulum at a Cai of 100 muM to be approximately 10(-10) mol/cm2/s, which is about 1 order of magnitude less than that estimated for the sarcoplasmic reticulum of activated skeletal muscle in vivo. The release of most of the stored calcium in some experiments indicates that the observed permeability changes can occur over a large portion of the surface of the sarcoplasmic reticulum.

Animals↗
PubMed1974

Role of sarcoplasmic reticulum phospholipids in calcium ion binding activity.

The relationship between sarcoplasmic reticulum phospholipid and Ca(2+) binding by sarcoplasmic reticulum membranes was explored. Ca(2+) bound in the absence of ATP was defined as "ATP-independent Ca(2+) binding," and the additional amount of Ca(2+) bound in the presence of ATP was defined as "ATP-dependent Ca(2+) binding." The latter was found to be very sensitive to the loss of sarcoplasmic reticulum phospholipid; the amount of Ca(2+) bound was reduced when as little as 3% of the phospholipid was destroyed by phospholipase C. Further destruction of membrane phospholipid up to a 40% loss caused little or no further reduction of this Ca(2+) binding. However, when the destruction of phospholipid exceeded 40%, further loss of this Ca(2+) binding occurred, and there was an almost complete loss of this function when more than 60% of the sarcoplasmic reticulum phospholipid was destroyed.

Adenosine Triphosphate↗
PubMed1979

[Ca2+-accumulating capacity of mitochondria, sarcolemma and sarcoplasmic reticulum of rat heart].

Using the isotope exchange technique including 45Ca, the Ca2+-binding and Ca2+-accumulating capacity of mitochondria, sarcolemma and sarcoplasmic reticulum of rat heart was studied. The ATP-independent binding of Ca2+ to isolated membrane fractions is by 1--2 orders of magnitude less than the ATP-dependent Ca2+-accumulating capacity of the fractions. The Ca2+-accumulating capacity of mitochondria is increased 6--8 fold after addition of physiological concentrations of succinate and Pi to the incubation medium. Under these conditions the ratio of Ca2+-accumulating capacity of mitochondria, sarcolemma and sarcoplasmic reticulum of the heart is 100:3,12:2,9. The initial rate of Ca2+-uptake by the sarcoplasmic reticulum is much higher in comparison with sarcolemma and mitochondria. A high Ca2+-accumulating capacity of heart mitochondria probably determines a long-term regulation of the concentration of "troponin-accessible" Ca2+ in the sarcoplasm, whereas the high initial rate of Ca2+ accumulation by the sarcoplasmic reticulum provides for a rapid decrease of Ca2+ concentration during rhythmic contractions of the heart.

Animals↗
PubMed1977

Formation of concentric whorls of rough endoplasmic reticulum in the adrenal gland of the Mongolian gerbil.

All cortical cells appeared alike in fetal gerbils of both sexes though glomerulosa cells were distinguishable at the time of birth. Fenestrated cisternae of rough endoplasmic reticulum were observed in all cortical cells of fetal gerbils, but smooth endoplasmic reticulum predominated. Concentric whorls of rough endoplasmic reticulum first appeared in adrenocortical cells at 42 days of age. The whorls developed within cells having abundant smooth endoplasmic reticulum and which were located in the region between the zona fasciculata and zona reticularis. Cells with whorls possibly serve as reserve cells which transform into actively secreting steroid cells in times of stress.

Adrenal Glands↗
PubMed1983

Biosynthesis of intrinsic sarcoplasmic reticulum proteins during differentiation of the myogenic cell line L6.

Temporal patterns of biosynthesis and the rates of turnover of the 53,000- and 160,000-Da glycoproteins from the sarcoplasmic reticulum membrane were determined and compared with the rates of biosynthesis of the (Ca2+ + Mg2+)-dependent ATPase in the differentiating rat myogenic continuous cell line, L6. Cells were labeled at various stages of differentiation with [35S]methionine and sarcoplasmic reticulum proteins were isolated from Triton extracts of the cells by protein A-mediated immunoprecipitation with specific antibodies. The immunoprecipitates were separated by polyacrylamide gel electrophoresis and the radioactivity incorporated into particular protein bands was recorded by autoradiography. The patterns of biosynthesis and the rates of degradation of the three intrinsic sarcoplasmic reticulum proteins were similar, even though two of them were glycosylated and one was not. The glycoproteins and the ATPase were found in the microsomal fraction obtained from the cell homogenate but not in the cytosol. This suggests that the synthesis and processing of these intrinsic sarcoplasmic reticulum proteins occurred in membrane systems.

Animals↗
PubMed1983

Isolation and characterization of endoplasmic reticulum and Golgi apparatus from hepatocyte nodules in male wistar rats.

A method is described for the isolation of endoplasmic reticulum and Golgi apparatus from hyperplastic liver nodules produced by discontinuous feeding of 2-acetylaminofluorene to male Wistar rats. The procedure involves three centrifugation steps and permits the separation of these cell components and their subfractions from the same sample of liver tissue as little as 1 g, wet weight. The fractions have been characterized by chemical, enzymatic, and morphological techniques and were found to be as pure as preparations from normal tissue. Furthermore, some of the characteristic histochemical features of hyperplastic liver nodules have been quantitated by biochemical methods in the fractions. Glucose-6-phosphatase activity in the endoplasmic reticulum subfractions of nodules is approximately 15% of the corresponding value in normal livers, whereas the activity of reduced nicotinamide adenine dinucleotide phosphate: cytochrome c reductase is reduced to 85% of the normal activity. The amount of cytochrome P-450 in nodular membranes as measured by differential spectroscopy is 25% of the control, indicating a decreased Phase I activity in drug metabolism. A 5-fold increase in cytosolic glutathione S-transferase activity without change in the corresponding microsomal activity was detected in hepatocyte nodules in rat liver. The activity of gamma-glutamyltransferase is increased more than 20-fold in all membrane fractions prepared from nodular tissue. The cytosolic activity, which is very low in the normal liver, is similarly increased more than 20-fold. The membrane-associated gamma-glutamyltransferase seems to be an integral membrane protein which cannot be washed away from the membranes. Chemically, membranes from nodules have phospholipid and cholesterol:protein ratios as found in membranes from normal liver tissue. However, the composition of individual phospholipids is changed with a 2-fold increase in nodular phosphatidylinositol and a slight decrease in phosphatidylcholine content in nodular membranes. The amount of endoplasmic reticulum membranes is of the same magnitude as in normal liver, although the smooth-surfaced component constitutes almost 60% of the isolated endoplasmic reticulum marker enzymes in nodules, compared with only 32% in preparations from normal tissue. The albumin contents of nodular and normal microsomal and Golgi membrane preparations are similar, indicating a normal synthesis of albumin by nodular tissue.

2-Acetylaminofluorene↗
PubMed1984

Target size of calcium pump protein from skeletal muscle sarcoplasmic reticulum.

The oligomeric size of calcium pump protein (CPP) in fast skeletal muscle sarcoplasmic reticulum membrane was determined using target theory analysis of radiation inactivation data. There was a parallel decrease of Ca2+-ATPase and calcium pumping activities with increasing radiation dose. The loss of staining intensity of the CPP band, observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, also correlated directly with the loss of activity. The target size molecular weight of the CPP in the normal sarcoplasmic reticulum membrane ranged between 210,000 and 250,000, which is consistent with a dimeric structure. Essentially the same size is obtained for the non-phosphorylated CPP or for the phosphoenzyme form generated from either ATP (E1 state) or inorganic phosphate (E2 state). Hence, the oligomeric state of the pump does not appear to change during the catalytic cycle. Similar results were obtained with reconstituted sarcoplasmic reticulum membrane vesicles with different lipid to protein ratios. We conclude that the CPP is a dimer in both native and reconstituted sarcoplasmic reticulum membranes. The target size of the calcium-binding protein (calsequestrin) was found to be 50,000 daltons, approximating a monomer.

Animals↗
PubMed1983

Characterization of Ca2+ release from the cardiac sarcoplasmic reticulum.

The characteristics of Ca2+ release in relation to Ca2+ binding were studied in sarcoplasmic reticulum vesicles isolated from canine myocardium. The Ca2+ binding appeared to be dependent on ATP as a 4 fold increase in Ca2+ binding was observed upon the addition of ATP. In the presence of a suboptimal ATP concentration (20 mumol/l; without ATP regenerating system) a rapid release of Ca2+ started within 2 min. The rate of Ca2+ release was increased by increasing the concentration of Ca2+ in the preincubation medium when studied by diluting preloaded vesicles in medium free of Ca2+ and ATP; an apparent saturation was reached at 5 mmol/l Ca2+ but Ca2+ release again increased between 5 and 10 mmol/l Ca2+. High pH (8.0) enhanced the Ca2+ release process. When Ca2+ loaded vesicles were treated with various phospholipases and proteases, an enhanced Ca2+ release was observed in comparison to the control values. The release of Ca2+ was also increased by pharmacological agents like caffeine, ether and halothane. The Ca2+ release rate was stimulated by the p-chloromercurybenzoate treatment, which decreased ATP dependent Ca2+ binding and Ca2+-stimulated ATPase activities of the sarcoplasmic reticulum vesicles. The effect of temperature when evaluated by Arrhenius plots showed a higher energy of activation of Ca2+ release (66.15 kJ/mol) in comparison to that for Ca2+ binding (41.03 kJ/mol). These results indicate that, although Ca2+ release and Ca2+ binding activities of the cardiac sarcoplasmic reticulum appears to be related, Ca2+ release is probably a distinct process and is controlled differently. It seems that the Ca2+ release site in sarcoplasmic reticulum membranes is lipoprotein in nature.

Adenosine Triphosphatases↗
PubMed1984

[Effect of caffeine and glycerin on the Ca transport system of sarcoplasmic reticulum fragments from frog skeletal muscles].

Parameters of the Ca2+-ion transport system by a fragmented sarcoplasmic reticulum isolated from phasic and tonic frog skeletal muscles were investigated under the action of caffeine or caffeine in combination with glycerol. No changes were observed in the Ca-transport system of a light fraction of the sarcoplasmic reticulum under the influence of caffeine and caffeine-glycerol combination. Caffeine reduced the value of Ca/ATP and enhanced the outflux of Ca2+-ions from membrane fragments of the caffeine-sensitive sarcoplasmic reticulum fraction of both the muscles; the combined effect of caffeine and glycerol was analogous to the action of caffeine applied alone. It is concluded that the potentiation of muscle contraction in the presence of glycerol is not due to the excess of Ca-release from the sarcoplasmic reticulum caused by this agent.

Absorption↗
PubMed1980

[Cause of increase in the efficiency of Ca2+ transport by fragments of sarcoplasmic reticulum from fast skeletal muscles induced by protein kinase].

The effect of cAMP-dependent protein kinases from rabbit skeletal muscles on Ca2+ uptake by fragments of skeletal muscle sarcoplasmic reticulum was studied. It was shown that incubation of the sarcoplasmic reticulum fragments with protein kinase increases the rate of Ca2+ uptake without changing the activity of Ca2+-dependent ATPase. This phenomenon is not accompanied by phosphorus incorporation into the protein components of the reticulum membranes. The protein kinase preparation subjected to "self-phosphorylation" is also capable to increase the rate of Ca2+ uptake. Using (14C) -oleic acid, it was shown that the increase of the rate of Ca2+ transport under effects of the "self-phosphorylated" protein kinase occurs due to the binding of free fatty acids present in the sarcoplasmic reticulum membranes. It was found that the effect observed is due to phosphofructokinase (ATP : D-fructose-6-phosphate-1-phosphotransferase) present in the protein kinase preparation.

Animals↗
PubMed1981

Specialized formations of the endoplasmic reticulum in cultures of murine neuroblastoma cells.

Ultrastructural features of cytodifferentiation in monolayer cultures of mouse neuroblastoma cells (clone neuro-2A) was further characterized by the presence of annulate lamellar arrays with up to 10 lamellae. The lamellae were made up of fused smooth surfaced cisternae forming pores or annuli and were surrounded by a dense filamentous to granular material. Stacks of nonfenestrated, parallel, regularly spaced cisternae, designated as lamellar bodies, also appeared in the cytoplasm in association with the endoplasmic reticulum and the nucleus. Such flattened cisternae were also seen to be formed from transformed endoplasmic reticulum. Furthermore a relatively large number of concentric whorled lamellar bodies were presented as well as dense whorled structures reminiscent of the cytoplasmic laminar bodies. All of these structures are derived from the rough endoplasmic reticulum, the tubules of which are coexistent with those of the latter. In addition there is a topographic relationship of these bodies with mitochondria and with the nucleus. There is a relative persistence of most of these organelles in old or atropic cells which show marked changes including loss of endoplasmic reticulum. The significance of all of these cytoplasmic inclusions is discussed.

Animals↗
PubMedDate not supplied

[Structural and functional characteristics of the sarcoplasmic reticulum membranes of the skeletal muscles in thyrotoxicosis].

A rate of CA2+ accumulation and efficiency of Ca2+-pump were decreased in the fractions of skeletal muscle sarcoplasmic reticulum isolated from rabbits with thyrotoxicosis. Temperature of phase transition in membranes of sarcoplasmic reticulum was changed by 4-5 degrees towards low temperatures. Under conditions of thyrotoxicosis fatty acid composition of total lipids from the sarcoplasmic reticulum fractions was altered as follows: amount of palmitic acid was increased 1.4-fold but oleic and arachidonic acids were decreased 21.8% and 40.5%, respectively. Modification of Ca2+-transport properties in sarcoplasmic reticulum caused by long-term administration of thyroid hormones into animals appears to alter the membrane protein-lipid composition.

Animals↗
PubMed1982

[Interaction of spin-labelled phosphatidyl choline with Ca-dependent ATPase from sarcoplasmic reticulum].

The interaction between 3-acyl-2-(6-doxylpalmitoyl) phosphatidyl choline used as a hydrophobic spin probe and Ca2+-dependent ATPase from sarcoplasmic reticulum membranes of rabbit and carp white skeletal muscles was studied. The spin label incorporation into ATPase preparations was performed at initial steps of ATPase isolation by incubating reticulum membranes with the spin probe in the presence of cholic acid. A comparison of the molecular mobility of the probe incorporated into ATPase preparations and into liposomes formed from ATPase phospholipids demonstrated that the presence of the protein in the membrane produces the same effect on the probe mobility as does the decrease of temperature by 10-15 degrees C. The molecular mobility of the probe in the ATPase preparation is increased during protein thermal denaturation. The breaks on the Arrhenius plots for the probe molecular mobility are revealed at the same temperatures (25 degrees for rabbit reticulum and 16 degrees for carp reticulum) as those for the ATPase activity.

Animals↗
PubMed1981

Reactivity of sarcoplasmic reticulum from rabbit cardiac muscle with 1-fluoro- and 1,5-difluoro- 2,4-dinitrobenzene.

Sarcoplasmic reticulum preparations from rabbit cardiac and fast skeletal muscle react differentially with low concentrations of 1-fluoro- and 1,5-difluoro-2,4-dinitrobenzene. Dinitrophenylation of cardiac sarcoplasmic reticulum by 1-fluoro-2,4-dinitrobenzene is not affected by Ca2+ and is limited to the lipoprotein-lipid region. This contrasts sharply with the predominant Ca2+-dependent dinitrophenylation of the ATPase protein of rabbit skeletal sarcoplasmic reticulum by this reagent. Formation of non-serial high mol. wt. oligomers by 1,5-difluoro-2,4-dinitrobenzene is significantly greater in cardiac than in skeletal vesicles. Substrate MgATP2- does not protect rabbit cardiac sarcoplasmic reticulum ATPase activity or Ca2+ uptake from dinitrophenylation when monofunctional and bifunctional reagents are used. Chemical differences in the overall structure of the two kinds of membrane preparations can be ascertained from a comparison of the effects of Ca2+ and MgATP2- on the reactivity of these reagents.

Adenosine Triphosphate↗
PubMed1982

Uncoupling of Ca2+ transport in sarcoplasmic reticulum as a result of labeling lipid amino groups and inhibition of Ca2+-ATPase activity by modification of lysine residues of the Ca2+-ATPase polypeptide.

Limited labeling of amino groups with fluorescamine in fragmented sarcoplasmic reticulum vesicles inhibits Ca2+-ATPase activity and Ca2+ transport. Under the labeling conditions used, 80% of the label reacts with phosphatidylethanolamine and 20% with the Ca2+-ATPase polypeptide. This degree of labeling does not result in vesicular disruption or in loss of vesicular proteins and does not increase the membrane permeability to Ca2+. Fluorescamine labeling of a purified Ca2+-ATPase devoid of aminophospholipids also inhibits Ca2+-ATPase activity, suggesting that labeling of lysine residues of the enzyme polypeptide is responsible for the inhibition of Ca2+-ATPase activity in sarcoplasmic reticulum. Fluorescamine labeling interferes with phosphoenzyme formation and decomposition in both the native vesicles and the purified enzyme; addition of ATP during labeling, and with less effectiveness ADP or AMP, protects both partial reaction steps. Addition of a nonhydrolyzable ATP analog protects phosphoenzyme formation but not decomposition. The inhibition of Ca2+ transport but not of Ca2+-ATPase occurs in sarcoplasmic reticulum vesicles labeled in the presence of ATP, indicating that the transport reaction is uncoupled from the Ca2+-ATPase reaction. The inhibition of Ca2+ transport but not of Ca2+-ATPase activity is also found in sarcoplasmic reticulum vesicles in which only phosphatidylethanolamine has reacted with fluorescamine. Furthermore, the extent of labeling of phosphatidylethanolamine is correlated with the inhibition of Ca2+ transport rates. The inhibition of Ca2+ transport is a reflection of the inhibition of Ca2+ translocation and is not due to an increase in Ca2+ efflux. We propose that labeling of phosphatidylethanolamine perturbs the lipid environment around the enzyme, producing a specific defect in the Ca2+ translocation reaction.

Animals↗
PubMed1984

Quantitative analysis of smooth endoplasmic reticulum proliferation in hepatocytes of early postnatal and adult mice treated with phenobarbital.

The smooth endoplasmic reticulum in hepatocytes of early postnatal and adult ddY male mice injected with phenobarbital was analyzed stereologically. The animals received daily intraperitoneal injections of phenobarbital and were killed about 24 h after the last injection. In early postnatal animals (injected with 35 mg/kg of the drug at days 1 and 2 after birth, or injected with 35 mg/kg at day 2 and with 50 mg/kg at days 3 and 4 after birth), the smooth endoplasmic reticulum proliferated both in perivenular and periportal hepatocytes; there was no significant difference in amount of smooth endoplasmic reticulum between the cells of the two zones. In adult animals, however, proliferation occurred only in perivenular cells after the administration of 35 mg/kg of phenobarbital for 2 days, and predominantly in perivenular cells even after the administration of 150 mg/kg of the drug for 7 days. These results suggest that the conspicuous proliferation of the smooth endoplasmic reticulum in perivenular hepatocytes in response to phenobarbital administration, which is characteristic of adult liver, does not occur in early postnatal mice but becomes manifest during postnatal development.

Aging↗
PubMed1983

Evidence for an alpha-mannosidase in endoplasmic reticulum of rat liver.

An alpha-mannosidase activity has been identified in a preparation of rat liver endoplasmic reticulum and shown to be distinct from the previously described Golgi alpha-mannosidases I and II and the lysosomal alpha-mannosidase. The enzyme was solubilized with deoxycholate and separated from other alpha-mannosidases by passage over concanavalin A-Sepharose to which it does not bind. The endoplasmic reticulum alpha-mannosidase cleaves alpha-1,2-linked mannoses from high mannose oligosaccharides and, unlike Golgi alpha-mannosidase I, is active against p-nitrophenyl-alpha-D-mannoside (Km = 0.17 mM). It has no activity toward GlcNAc-Man5GlcNAc2 peptide, the specific substrate of the Golgi alpha-mannosidase II. The endoplasmic reticulum alpha-mannosidase activity toward p-nitrophenyl-alpha-D-mannoside is relatively insensitive to swainsonine, an inhibitor of both the lysosomal alpha-mannosidase and Golgi alpha-mannosidase II. We propose that the endoplasmic reticulum alpha-mannosidase is responsible for the removal of mannose residues from asparagine-linked high mannose type oligosaccharides prior to their entry into the Golgi.

Animals↗