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At least 19 recordsLinked to original sources
PubMed1992

The endoplasmic reticulum-sarcoplasmic reticulum connection: distribution of endoplasmic reticulum markers in the sarcoplasmic reticulum of skeletal muscle fibers.

The skeletal muscle sarcoplasmic reticulum (SR) was investigated for the presence of well-known endoplasmic reticulum (ER) markers: the lumenal protein BiP and a group of membrane proteins recognized by an antibody raised against ER membrane vesicles. Western blots of SR fractions revealed the presence of BiP in fast- and slow-twitch muscles of the rabbit as well as in rat and chicken muscles. Analyses of purified SR subfractions, together with cryosection immunofluorescence and immunogold labeling, revealed BiP evenly distributed within the longitudinal SR and the terminal cisternae. Within the terminal cisternae BiP appeared not to be mixed with calsequestrin but to be distributed around the aggregates of the latter Ca2+ binding protein. Of the various membrane markers only calnexin (91 kDa) was found to be distributed within both SR subfractions, whereas the other markers (apparent molecular masses of 64 kDa and 58 kDa and a doublet around 28 kDa) were concentrated in the terminal cisternae. These results suggest that the SR is a specialized ER subcompartment in which general markers, such as the ones we have investigated, coexist with the major SR proteins specifically responsible for Ca2+ uptake, storage, and release. The differential distribution of the ER markers reveals new aspects of the SR molecular structure that might be of importance for the functioning of the endomembrane system.

Animals↗
PubMed1993

The endoplasmic reticulum-sarcoplasmic reticulum connection. II. Postnatal differentiation of the sarcoplasmic reticulum in skeletal muscle fibers.

The postnatal differentiation of sarcoplasmic reticulum (SR) of rabbit skeletal muscles (the slow-twitch soleus and the fast-twitch adductor muscles) was monitored between Days 1 and 12 by following on Western blots the expression and accumulation of molecular markers specific not only for the muscle endomembrane system, i.e., calsequestrin (CS) and the ryanodine-sensitive Ca2+ release channel, but also for the endoplasmic reticulum (ER) at large, i.e., BiP, calnexin (CN) and calreticulin. Our results demonstrate that SR development, documented by the increase of the SR fractional volume, terminal cisternae proliferation, and reorientation of triads, is accompanied by the accumulation of the SR-specific proteins and also of CN, with no change of the other ER general markers. Moreover, the distribution of two of the markers, BiP and CS, was investigated by immunocytochemistry at both the light and the electron microscope level. At Day 1 CS was found to be concentrated both within the few recognizable triad terminal cisternae and within the lumen of numerous, apparently discrete cisternae and tubules, widely scattered throughout both the contractile and the subplasmalemmal areas of the cytoplasm. These structures remain evident until Day 12, when most triad junctions have acquired proper configuration, composition and orientation. BiP, on the other hand, appears widely distributed within the ER/SR of the fibers. From the early stages of postnatal development it does colocalize with the Ca2+ binding protein in the lumen of the CS-rich structures and appears also within the longitudinal SR and the conventional ER cisternae.

Animals↗
PubMed1991

Calreticulin, and not calsequestrin, is the major calcium binding protein of smooth muscle sarcoplasmic reticulum and liver endoplasmic reticulum.

The distribution of calsequestrin and calreticulin in smooth muscle and non-muscle tissues was investigated. Immunoblots of endoplasmic reticulum proteins probed with anti-calreticulin and anti-calsequestrin antibodies revealed that only calreticulin is present in the rat liver endoplasmic reticulum. Membrane fractions isolated from uterine smooth muscle, which are enriched in sarcoplasmic reticulum, contain a protein band which is immunoreactive with anti-calreticulin but not with anti-calsequestrin antibodies. The presence of calreticulin in these membrane fractions was further confirmed by 45Ca2+ overlay and "Stains-All" techniques. Calreticulin was also localized to smooth muscle sarcoplasmic reticulum by the indirect immunofluorescence staining of smooth muscle cells with anti-calreticulin antibodies. Furthermore, both liver and uterine smooth muscle were found to contain high levels of mRNA encoding calreticulin, whereas no mRNA encoding calsequestrin was detected. We have employed an ammonium sulfate precipitation followed by Mono Q fast protein liquid chromatography, as a method by which calsequestrin and calreticulin can be isolated from whole tissue homogenates, and by which they can be clearly resolved from one another, even where present in the same tissue. Calreticulin was isolated from rabbit and bovine liver, rabbit brain, rabbit and porcine uterus, and bovine pancreas and was identified by its amino-terminal amino acid sequence. Calsequestrin cannot be detected in preparations from whole liver tissue, and only very small amounts of calsequestrin are detectable in ammonium sulfate extracts of uterine smooth muscle. We conclude that calreticulin, and not calsequestrin, is a major Ca2+ binding protein in liver endoplasmic reticulum and in uterine smooth muscle sarcoplasmic reticulum. Calsequestrin and calreticulin may perform parallel functions in the lumen of the sarcoplasmic and endoplasmic reticulum.

Amino Acid Sequence↗
PubMed1981

31P-NMR studies of oriented multilayers formed from isolated sarcoplasmic reticulum and reconstituted sarcoplasmic reticulum.

31P-NMR spectra were obtained from oriented multilayer preparations of normal sarcoplasmic reticulum and reconstituted sarcoplasmic reticulum with lipid to protein ratios varying between 41 : 1 and 110 : 1. The dependence of the 31P-NMr spectra on the alignment of the membranes with respect to the magnetic field was used to draw two conclusions about the motion of the phospholipid molecules that contribute to the observed spectra. First, the phosphate group and the two adjacent methylene groups are able to rapidly rotate (i.e., tau R much less than 10-5 S) around the normal to the plane of the membrane. Second, the restricted internal motion of the phosphate group and the glycerol CH2OP group is very similar to that found in liposomes formed from sarcoplasmic reticulum phospholipids. Calibration experiments showed that all (100 +/- 7%) of the phospholipid molecules in the membrane can be accounted for in the observed spectra. Thus, essentially all the phospholipid molecules in the sarcoplasmic reticulum and the reconstituted sarcoplasmic reticulum membranes have the same motion in the polar headgroup region as found in model bilayer membranes. Since a large fraction of the phospholipid molecules (between one-quarter and one-half, depending on ;the lipid to protein ratio) are immediately surrounding the calcium-pump protein, we conclude that the calcium-pump protein does not perturb the motion of these 'boundary-layer' lipids.

Animals↗
PubMed1982

The effect of pH on the transient-state kinetics of Ca2+-Mg2+-ATPase of cardiac sarcoplasmic reticulum. A comparison with skeletal sarcoplasmic reticulum.

The effect of pH on the Ca2+-Mg2+-dependent ATPase of sarcoplasmic reticulum (SR) was investigated with a rapid mixing quench-flow apparatus capable of measuring phosphorylation and dephosphorylation at times as rapid as 4 msec. The rates of formation and decomposition of the phosphorylated intermediate (E approximately P) of the Ca2+-Mg2+-ATPase were studied in the pH range between 7.6 and 6.0. At pH 6.8, the rates of formation of the phosphorylated intermediate of the Ca2+-Mg2+-ATPase of sarcoplasmic reticulum are the same (t1/2 = 10 msec) for cardiac and skeletal sarcoplasmic reticulum preloaded with calcium, but decrease as the pH is lowered. The effect of acid pH (6.0) is more pronounced for cardiac sarcoplasmic reticulum (t 1/2 = 47 msec) than for skeletal sarcoplasmic reticulum (t 1/2 = 17 msec), in agreement with studies showing that acidosis has a more pronounced effect on cardiac muscle than on skeletal muscle. In addition, a decrease in pH results in a decrease in the rate of the E approximately P decomposition step (the slowest step in the SR reaction sequence). The E approximately P decomposition half-lives were observed to be 97 and 77 msec, respectively for cardiac and skeletal SR at pH 6.8. At pH 6.0, the half-lives were increased to 136 and 178 msec for cardiac and skeletal SR, respectively.

Adenosine Triphosphatases↗
PubMed1992

Studies on the mechanism of different collagen glucosyltransferase reactions (Golgi apparatus, smooth endoplasmic reticulum, rough endoplasmic reticulum) in chick embryo liver.

1. The galactosylhydroxylysylglucosyltransferase (GGT) specific to collagen is located in the RER (rough endoplasmic reticulum), SER (smooth endoplasmic reticulum) and Golgi apparatus for the chick embryo liver. 2. The UDP-glucose collagen glucosyltransferase activities in chick embryo liver were solubilized by Nonidet P-40. 3. The mechanism of collagen glucosyltransferase reaction was studied with enzyme preparation of Golgi apparatus CF2, smooth endoplasmic reticulum CF4 and rough endoplasmic reticulum CF8. 4. For the three fractions, data obtained in experiments were consistent with a sequential ordered mechanism in which the substrates are bound to the enzyme in the following order: Mn2+, collagen and UDP-glucose substrate, with different values for Km and Vmax.

Animals↗
PubMed1985

Structure and assembly of the endoplasmic reticulum. Biosynthetic sorting of endoplasmic reticulum proteins.

We have studied the post-translational processing and the biosynthetic sorting of three protein components of murine endoplasmic reticulum (ER), ERp60, ERp72, and ERp99. In pulse-labeled MOPC-315 (where MOPC-315 represents mineral oil-induced plasmacytoma cells) plasmacytoma cells, no precursor forms of these proteins were detected and only ERp99 was sensitive to endoglycosidase H. The ERp99 oligosaccharide remained endoglycosidase H sensitive during a 3-h chase, and analysis by high performance liquid chromatography showed the predominant structure to be Man8GlcNAc2. We have used a sucrose gradient analysis of pulse-labeled MOPC-315 plasmacytoma cells in order to directly study the biosynthetic sorting of both glycosylated and nonglycosylated ERps and have found no strong evidence to suggest these proteins ever leave the endoplasmic reticulum. In spite of their common sorting pathway, these proteins differ in their membrane orientation. Both ERp60 and ERp72 are entirely protected by the endoplasmic reticulum membrane while ERp99 appears to have a large domain exposed on the cytoplasmic face of the endoplasmic reticulum.

Animals↗
PubMed1989

Monoclonal antibodies to dog heart sarcoplasmic reticulum as markers of endoplasmic reticulum.

A monoclonal antibody (mAb 4B4) was raised against purified sarcoplasmic reticulum vesicles from canine myocardium, and shown to inhibit Ca2+ uptake by microsomes isolated from cardiac, skeletal, and smooth muscle. The amount of mAb 4B4 needed to inhibit the Ca2+ uptake 50% at a given membrane concentration correlated with the amount of Ca2+ pump protein in the microsomal preparation. This is consistent with the observation the mAb 4B4 binds specifically to the sarcoplasmic/endoplasmic reticulum Ca2+ pump (Mr 100 kDa), but has no effect on the T-tubule Mg2+-ATPase. Changes in the binding of mAb 4B4 to crude microsomes isolated from dog heart after various durations of global ischemia showed that the decrease in microsomal Ca2+ transport during the first 15 min of ischemia correlated with a loss of active Ca2+ pump molecules. The monoclonal antibody mAb 4B4 may therefore serve as a specific marker for the sarcoplasmic/endoplasmic reticulum Ca2+ pump system in various cells, and can provide quantitative information about the loss of active Ca2+ pump proteins under pathological conditions.

Animals↗
PubMed1982

Smooth endoplasmic reticulum and other agranular reticulum in frog retinal photoreceptors.

Frog retinal photoreceptors are favourable material for studying a number of unresolved issues concerning the interconnections, three-dimensional organization and functions of intracellular membrane systems in neurons. At least two distinct regions of smooth endoplasmic reticulum (SER) are present in these cells. One region, the subellipsoid SER, is located in rod cells at the base of the mitochondria-rich ellipsoid region, and is comprised of arrays of stacked tubules which exhibit frequent continuities with the rough endoplasmic reticulum (RER). The subellipsoid SER is also present throughout the ellipsoid region and at the apex of the inner segment. The second region of SER, the axonal SER, is comprised of agranular sacs and tubules present in the axons of rod cells, the perinuclear and Golgi regions of rod and cone cells and the synaptic terminals of rod and cone cells. There sacs and tubules exhibit continuities with cisternae of RER and with the nuclear envelope. Serial section analyses indicate that this SER can extend as a continuous networking along the entire length of the rod axons and throughout synaptic terminals. The axonal SER is distinct from the subellipsoid SER not only in location and morphology but also in its ability to bind divalent lead ions, a property it shares with synaptic vesicles, with agranular sacs at one face to the Golgi apparatus and with sacs extending from the Golgi apparatus toward the axons hillock. These latter sacs may serve in transport from the Golgi region to the axon. The axons SER in the axon, terminals, and the perinuculear and Golgi regions appear to be a source of synaptic vesicles as evidenced by this lead binding capacity and by the observation of vesicles, with the size (50-75 nm) and appearance of synaptic vesicles, budding from SER in direct continuity, with RER. The endoplasmic reticulum (ER) in synaptic terminals of frog photoreceptors is not continuous with endocytic structures found in the same region, such as blunt-ended tubules or anastomosing networks of tubules. Nor does the ER acquire exogenous horseradish peroxidase. These observations suggest that the ER does not play a direct role in membrane recycling in photoreceptors.

Animals↗
PubMed1986

Structure and assembly of the endoplasmic reticulum: biosynthesis and intracellular sorting of ERp61, ERp59, and ERp49, three protein components of murine endoplasmic reticulum.

Rabbit antibodies have been prepared against ERp61, ERp59, and ERp49, three protein components of rough endoplasmic reticulum (RER) purified from mineral oil-induced plasmacytoma 315 (MOPC-315) tissue. Analysis of subcellular fractions of MOPC-315 tissue by an immunoprecipitation procedure demonstrated that all three endoplasmic reticulum proteins (ERps) were most enriched in the RER. Immunologically cross-reacting proteins of similar molecular weight have been detected in other eucaryotic cell lines. We have used these antibodies to study the post-translational processing and biosynthetic sorting of the three ERps in pulse-labeled MOPC-315 cells. No larger precursor forms of the ERps were detected and none of the ERps were found to possess asparagine-linked oligosaccharide moieties. We have used a sucrose gradient analysis of pulse-labeled MOPC-315 cells to study the biosynthetic sorting of ERp61, ERp59 and ERp49 and have found no evidence to suggest that these proteins ever leave the endoplasmic reticulum. In addition, all three ERps appeared to have luminally exposed domains. ERp61 and ERp59 were entirely protected by the ER membrane in the absence of detergent, while ERp49 was a transmembrane protein that also possesses a cytoplasmically exposed domain. We have used the anti-ERp antibodies to quantitate the synthesis and accumulation of the three ERps during lipopolysaccharide (LPS)-induced lymphocyte differentiation. After 48 h of culture in the presence of LPS, the synthesis of ERp49 increased sixfold relative to that in control cells. The synthesis and membrane accumulation of ERp61 and ERp59 were less affected by the LPS treatment. Thus, membranes isolated from LPS-treated cells were enriched in ERp49 relative to those isolated from control cells.

Animals↗
PubMed1977

Biogenesis of endoplasmic reticulum membrane in rat liver cells. II. Discharge of the nascent peptides of NADPH-cytochrome c reductase and cytochrome b5 on the cytoplasmic side of the endoplasmic reticulum membrane.

The direction of discharge of the nascent peptides of NADPH-cytochrome c reductase and cytochrome b5 from bound polyribosomes of rough microsomes was investigated in order to elucidate the mechanism of separation of these membrane proteins from secretory proteins, which are also synthesized by the same class of ribosomes of rough endoplasmic reticulum. The nascent peptides of NADPH-cytochrome c reductase and cytochrome b5 in intact rough microsomes were accessible to externally added 125I-Fab's against these proteins, and were susceptible to trypsin digestion, whereas the nascent peptides of serum albumin were not. The nascent peptides of these two microsomal proteins were released into the cytoplasm by puromycin treatment of intact rough microsomes, while the nascent peptides of serum albumin were retained in the microsomal lumen. These observations suggest that the nascent peptides of microsomal proteins, which are present on the cytoplasmic surface of the endoplasmic reticulum membrane, are exposed on the surface of microsomal vesicles, while those of secretory proteins are enclosed inside the vesicles. Therefore, the topographical separation of microsomal membrane proteins from secretory proteins is accomplished at the step of their synthesis by the bound polyribosomes of rough endoplasmic reticulum.

Animals↗
PubMed1982

Ca2+ release from sarcoplasmic reticulum vesicles derived from longitudinal reticulum and terminal cisternae of frog skeletal muscle.

Fragmented sarcoplasmic reticulum (FSR) of bullfrog skeletal muscle was fractionated into light and heavy sarcoplasmic reticulum (LSR and HSR) by sucrose density gradient centrifugation. Morphological and biochemical studies revealed that large parts of LSR and HSR were derived from longitudinal reticulum and terminal cisternae of SR, respectively. The Ca2+ uptake ability and ATPase activity of LSR were higher than those of HSR. Ca2+ release from Ca2+ preloaded SR vesicles by changing the medium from K-gluconate to KCl was suppressed by addition of 0.3 M sucrose or glucose; there was no correlation between Ca2+ release and membrane potential change either in LSR or HSR vesicles. Dantrolene sodium (DAN, 20 microM) had no effect on Ca2+ release. It is concluded that ion-induced Ca2+ release from SR (both HSR and LSR) in the isolated system is due to an osmotic effect.

Animals↗
PubMed1985

Structure and assembly of the endoplasmic reticulum. The synthesis of three major endoplasmic reticulum proteins during lipopolysaccharide-induced differentiation of murine lymphocytes.

Monospecific rabbit antibodies have been prepared against ERp72, ERp99, and ERp60, major protein components of a detergent-solubilized extract of endoplasmic reticulum purified from mineral oil-induced plasmacytoma 315 tissue. When subcellular fractions of mineral oil-induced plasmacytoma 315 tissue were assayed by an immunoprecipitation procedure, all three endoplasmic reticulum proteins (ERps) were found to be enriched in the rough endoplasmic reticulum. In murine lymphoid cells, the three ERps represent two major structural classes of protein. Both ERp72 and ERp60 contain no endoglycosidase H-sensitive, N-linked oligosaccharides. On the other hand, ERp99 is glycoprotein containing, in all likelihood, one endoglycosidase H-sensitive oligosaccharide. Immunologically cross-reacting proteins of similar molecular weight have also been detected in other eukaryotic cell lines. The anti-ERp antibodies were used to quantitate the synthesis and accumulation of the three ERps in splenic lymphocytes cultured in the presence and absence of bacterial lipopolysaccharide (Escherichia coli serotype B5:055) (LPS). In the presence of LPS, lymphocytes differentiate from resting cells into actively secreting cells. The synthesis of ERp72 and ERp99 increased 3- and 10-fold, respectively, in response to LPS. The synthesis of ERp60 does not change significantly. The turnover rates for these three proteins are similar in both control and LPS-treated lymphocytes. As a result, membranes isolated from LPS-treated cells are enriched in ERp72 and ERp99.

Animals↗
PubMed1986

Electron probe analysis of calcium content and movements in sarcoplasmic reticulum, endoplasmic reticulum, mitochondria, and cytoplasm.

Electron probe microanalysis (EPMA) of a variety of rapidly frozen nonmuscle (e.g., liver and retinal rods) and muscle cell systems indicates that the endoplasmic reticulum (ER) [in muscle, the sarcoplasmic reticulum (SR)] is the major intracellular store of Ca. In vascular smooth muscle, Ca stored in the SR can be released and recycled, and it is sufficient to activate maximal contractions even in those smooth muscles in which the volume of the SR is relatively small. The Ca content of mitochondria in situ in vascular smooth and striated muscles, in liver, and in retinal rods is low, indicating that mitochondria do not function as physiological regulators of cytoplasmic Ca2+ in any of the muscle or nonmuscle cells critically examined with EPMA. Mitochondria themselves may be regulated metabolically by small fluctuations in matrix free Ca2+. Massive accumulation of mitochondrial Ca occurs under pathological conditions, when mitochondria are exposed to abnormally high free Ca2+. In frog skeletal muscle, the return of Ca to the SR is characterized by two processes: a fast one (25% of the Ca released) associated with relaxation due to pumping by the SR, and a slow process (0.4/s) that occurs after relaxation and appears to be rate-limited by the removal of Ca from parvalbumin. Illumination in retinal rods causes no detectable change in the low endogenous Ca content of the outer segment.

Animals↗
PubMed1992

The sarcoplasmic reticulum Ca(2+)-ATPase, SERCA1a, contains endoplasmic reticulum targeting information.

The fast-twitch skeletal muscle Ca(2+)-ATPase isoenzyme, SERCA1a, is localized in chick skeletal myotubes to both the sarcoplasmic reticulum (SR) and to the nuclear envelope, an extension of the endoplasmic reticulum (ER). The ER labeling remained after cycloheximide treatment, indicating that it did not represent newly synthesized SERCA1a in transit to the SR. Expression of the cDNA encoding SERCA1a in cultured non-muscle cells led to the localization of the enzyme in the ER, as indicated by organelle morphology and the co-localization of SERCA1a with the endogenous ER luminal protein, BiP. Immunopurification analysis showed that SERCA1a was not bound to BiP, nor was any degradation apparent. Thus, the SR Ca(2+)-ATPase appears to contain ER targeting information.

Animals↗
PubMedDate not supplied

[Peroxide modification of skeletal muscle sarcoplasmic reticulum in antioxidant deficiency and under the effect of ionol. II. Physico- chemical properties of the sarcoplasmic reticulum membrane].

Physico-chemical parameters of membranes of skeletal muscles' sarcoplasmic reticulum in antioxidant insufficiency, which was modelled by excluding alpha-tocopherol from the animals ration, and after treatment with phenol antioxidant ionol were studied. It was shown that activation of lipid peroxidation in vitamin E insufficiency results in a significant lowering of microviscosity of lipid bilayer membranes of sarcoplasmic reticulum. Using polarography significant changes in membrane protein conformation were revealed, which were characterized by lowering of integrity and by disorganization of protein globules. Treatment of animals with antioxidant insufficiency with ionol led to certain normalization of changes of physico-chemical characteristics of the learned membrane structures caused by lipid peroxidation.

Animals↗
PubMed1988

Endoplasmic reticulum of rat liver contains two proteins closely related to skeletal sarcoplasmic reticulum Ca-ATPase and calsequestrin.

Rat liver endoplasmic reticulum (ER) membranes were investigated for the presence of proteins having structural relationships with sarcoplasmic reticulum (SR) proteins. Western immunoblots of ER proteins probed with polyclonal antibodies raised against the 100-kDa SR Ca-ATPase of rabbit skeletal muscle identified a single reactive protein of 100 kDa. Also, the antibody inhibited up to 50% the Ca-ATPase activity of isolated ER membranes. Antisera raised against the major intraluminal calcium binding protein of rabbit skeletal muscle SR, calsequestrin (CS), cross-reacted with an ER peptide of about 63 kDa, by the blotting technique. Stains-All treatment of slab gels showed that the cross-reactive peptide stained metachromatically blue, similarly to SR CS. Two-dimensional electrophoresis (Michalak, M., Campbell, K. P., and MacLennan, D. H. (1980) J. Biol. Chem. 255, 1317-1326) of ER proteins showed that the CS-like component of liver ER, similarly to skeletal CS, fell off the diagonal line, as expected from the characteristic pH dependence of the rate of mobility of mammalian CS. In addition, the CS-like component of liver ER was released from the vesicles by alkaline treatment and was found to be able to bind calcium, by a 45Ca overlay technique. From these findings, we conclude that a 100-kDa membrane protein of liver ER is the Ca-ATPase, and that the peripheral protein in the 63-kDa range is closely structurally and functionally related to skeletal CS.

Animals↗