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E Leberer

Publications and source records attributed to E Leberer.

At least 55 records · Page 3Linked to original sources
PubMed1989

Molecular cloning and expression of cDNA encoding the 53,000-dalton glycoprotein of rabbit skeletal muscle sarcoplasmic reticulum.

The 53-kDa glycoprotein of rabbit skeletal muscle sarcoplasmic reticulum was purified by lentil lectin affinity chromatography and preparative polyacrylamide gel electrophoresis and partially sequenced. Polyclonal and monoclonal antibodies were raised against the 53-kDa glycoprotein and found to cross-react with the 160-kDa glycoprotein. A combination of antibody and synthetic oligonucleotide screening was used to isolate a cDNA encoding the 53-kDa glycoprotein of rabbit fast-twitch skeletal muscle sarcoplasmic reticulum. The cDNA encodes a protein of 453 amino acids with Mr of 52,421 and a 19-residue amino-terminal signal sequence. The deduced sequence contains two potential glycosylation sites and is largely hydrophilic. The presence of a glycine-rich sequence in the glycoprotein with homology to mononucleotide binding domains supports earlier observations that the glycoprotein binds ATP with high affinity. Although two sequences appear to be hydrophobic on a hydropathy plot, they are not sufficiently long nor sufficiently hydrophobic to qualify unambiguously as transmembrane sequences. The glycoprotein, like calsequestrin, was shown to be inaccessible to trypsin in intact sarcoplasmic reticulum. It can be eluted from the sarcoplasmic reticulum by extraction with [ethylenebis(oxyethylenenitrilo)]tetraacetic acid under hypotonic conditions. Thus, the glycoprotein appears to be localized entirely in the lumen of the sarcoplasmic reticulum and to be associated with the inner membrane surface through Ca2+-dependent mechanisms. Cotransfection of COS-1 cells with cDNAs encoding the glycoprotein and the Ca2+-ATPase led to expression of both proteins with a common localization in the microsomal fraction. The Ca2+ pumping activity of the microsomes isolated from transfected cells was unaltered by the presence of the glycoprotein. Thus the glycoprotein does not appear to modulate Ca2+-ATPase function.

Adenosine Triphosphate↗
PubMed1989

The fast-twitch muscle calsequestrin isoform predominates in rabbit slow-twitch soleus muscle.

The major form of calsequestrin in rabbit slow-twitch soleus muscle is shown to be identical to that isolated and cloned from rabbit fast-twitch muscle on the following bases: identity of cDNAs cloned from mRNAs from the two muscle sources; equivalent hybridization of a fast-twitch calsequestrin cDNA probe to mRNAs isolated from fast-twitch and slow-twitch muscles; identity of the 23 amino-terminal amino acids; strong binding of 45Ca2+ in a gel overlay of slow muscle sarcoplasmic reticulum protein to a band at the level of the fast-twitch calsequestrin isoform and only weak binding at the level of the cardiac isoform. No evidence was obtained for developmentally regulated alternative splicing of the calsequestrin transcript in mature slow or fast-twitch muscle.

Animals↗
PubMed1989

Molecular cloning and expression of cDNA encoding a lumenal calcium binding glycoprotein from sarcoplasmic reticulum.

Antibody screening was used to isolate a cDNA encoding the 160-kDa glycoprotein of rabbit skeletal muscle sarcoplasmic reticulum. The cDNA is identical to that encoding the 53-kDa glycoprotein except that it contains an in-frame insertion of 1308 nucleotides near its 5' end, apparently resulting from alternative splicing. The protein encoded by the cDNA would contain a 19-residue NH2-terminal signal sequence and a 453-residue COOH-terminal sequence identical to the 53-kDa glycoprotein. It would also contain a 436-amino acid insert between these sequences. This insert would be highly acidic, suggesting that it might bind Ca2+. The purified 160-kDa glycoprotein and the glycoprotein expressed in COS-1 cells transfected with cDNA encoding the 160-kDa glycoprotein were shown to bind 45Ca2+ in a gel overlay assay. The protein was shown to be located in the lumen of the sarcoplasmic reticulum and to be associated through Ca2+ with the membrane. We propose that this lumenal Ca2+ binding glycoprotein of the sarcoplasmic reticulum be designated "sarcalumenin."

Amino Acid Sequence↗
PubMed1989

Parvalbumin in mouse muscle in vivo and in vitro.

Parvalbumin is a cytosolic calcium-binding protein found in adult fast-twitch mammalian muscle. Using an antibody to paravalbumin, we have shown that its distribution in adult mouse muscles is associated with certain fibre types. It is absent from slow-twitch type 1 fibres, is absent or at low levels in fast-twitch type 2A fibres, but is present at moderate or high levels in fast-twitch type 2B fibres. When adult mouse muscle is cultured with embryonic mouse spinal cord, the regenerated fibres become innervated, express the adult fast isoform of myosin heavy chain and appear histochemically as fast-twitch fibres. We therefore investigated whether these apparently mature fibres also contained parvalbumin. Parvalbumin was not found in any fibres of twenty mature cultures, suggesting that neurotrophic activity in the absence of specific adult nerve activity patterns was insufficient to cause the expression of parvalbumin in the cultures.

Adenosine Triphosphatases↗
PubMed1988

Postnatal development of Ca2+-sequestration by the sarcoplasmic reticulum of fast and slow muscles in normal and dystrophic mice.

Ca2+-uptake activities of the sarcoplasmic reticulum (SR) were determined with a Ca2+-sensitive electrode in homogenates from fast- and slow-twitch muscles from both normal and dystrophic mice (C57BL/6J strain) of different ages. Immunochemical quantification of tissue Ca2+-ATPase content allowed determination of the specific Ca2+-transport activity of the enzyme. In 3-week-old mice of the dystrophic strain specific Ca2+ transport was already significantly lower than in the normal strain. It progressively decreased with maturation and reached only 40-50% and 30-50% of the normal values in fast- and slow-twitch muscles of adult dystrophic animals, respectively. Tissue contents of calsequestrin were reduced in both types of muscle leading to an increased Ca2+-ATPase to calsequestrin protein ratio. Equal amounts of the Ca2+-ATPase protein (detected by Coomassie blue staining of polyacrylamide gels) were present in SR vesicles isolated by Ca2+-oxalate loading from adult normal and dystrophic fast-twitch muscles. However, the specific ATP-hydrolysing activity of the enzyme was approximately 50% lower in dystrophic than in normal SR. The reduced ATP-hydrolysing activity was correlated with decreased Ca2+-transport activity, phosphoprotein formation and fluorescein isothiocyanate labeling as determined in total microsomal and heavy SR fractions. Although the Ca2+ and ATP affinities of the enzyme were unaltered, its ATPase activity was reduced at all levels of ATP in the dystrophic SR. Taken together, these findings point to a markedly impaired function of the SR and an increase in the population of inactive SR Ca2+-ATPase molecules in murine muscular dystrophy.

Adenosine Triphosphate↗
PubMed1988

Distribution of sarcoplasmic reticulum Ca-ATPase and of calsequestrin at the polar regions of rat, rabbit and cat intrafusal fibers.

Sarcoplasmic reticulum (SR) Ca2+-pumping ATPase (Ca-ATPase) and calsequestrin (CaS) were visualized by indirect immunofluorescence at the polar regions of adult rat, rabbit and cat intrafusal fibers. The immunohistochemical reaction products were regarded as histochemical markers of the SR and as valid indicators of the distribution of the two Ca2+-sequestering proteins. Static nuclear bag2 fibers displayed lower levels of both Ca-ATPase and CaS than the other two intrafusal fiber types. Nuclear chain fibers presented the highest Ca-ATPase levels and, together with dynamic nuclear bag1 fibers, they also exhibited relatively high amounts of CaS. The level of Ca-ATPase was lower in bag 1 fibers than in nuclear chain fibers, but not as low as in bag2 fibers. The comparatively high levels of Ca-ATPase and CaS seen in nuclear chain fibers coincided with their reported faster contractile speeds compared to nuclear bag fibers.

Animals↗
PubMed1988

Relationship between parvalbumin content and the speed of relaxation in chronically stimulated rabbit fast-twitch muscle.

The time courses of changes in parvalbumin (PA) content, isometric twitch tension, and half-relaxation time (1/2 RT) were studied in rabbit tibialis anterior muscle following chronic 10 Hz nerve stimulation of 1-21 days. Up to 5 days stimulation had no effect on PA content, but it induced a slight (10-15%) increase in the 1/2 RT. This change occurred together with the previously observed 50% decrease in Ca2+-uptake by the SR (Leberer et al. 1987). While prolonged stimulation produced no further decrease in the Ca2+-uptake by the SR, PA content declined after 5 days of stimulation. The reduction in PA content was accompanied by a progressive lengthening of the 1/2 RT. However, the increase in 1/2 RT was particularly pronounced after PA had fallen below 50% of its normal value. A 90% reduction in PA coincided with a 60% increase in the 1/2 RT. By this time the staircase phenomenon, normally observed in fast-twitch muscle, was completely abolished. Although the changes in PA content and 1/2 RT were not linearly related, these results suggest that PA plays an important role in the relaxation process of mammalian fast-twitch muscle.

Animals↗
PubMed1988

Fibre types, calcium-sequestering proteins and metabolic enzymes in denervated and chronically stimulated muscles of the rat.

1. Fibre types, Ca2+-sequestering proteins (parvalbumin, sarcoplasmic reticulum Ca2+-ATPase), enzyme activities of energy metabolism, and lactate dehydrogenase isozymes were studied in extensor digitorum longus (EDL) and soleus muscles of adult rats after denervation and direct stimulation for 45-61 days. The stimulation resembled the firing patterns of normal motor units (Hennig & Lømo, 1985) in either soleus (20 Hz, high amount) or EDL (150 Hz, low and high amount). 2. Low- and high-amount 150 Hz stimulation maintained essentially normal properties in the denervated EDL and induced many EDL-like properties in the denervated soleus, e.g. pronounced increases in Ca2+-ATPase and parvalbumin contents, a rise in the glycolytic enzyme activities, as well as a reversal of the lactate dehydrogenase H/M subunit ratio. The 150 Hz high-amount stimulation was not as efficient as the 150 Hz low amount in maintaining the fast properties of denervated EDL with the exception of the enzymes of aerobic oxidative metabolism. High-amount 20 Hz stimulation maintained essentially normal properties in the soleus but induced only moderate slow muscle characteristics in the EDL. 3. The maintenance of essentially normal properties by 'native' and the changes induced by 'foreign' stimulus patterns in the absence of the nerve indicate that evoked muscle activity plays a major role in controlling the phenotypic expression of muscle properties. However, the different responses of the denervated fast- and slow-twitch muscles to identical stimulus patterns further suggest that rat EDL and soleus muscles contain intrinsically different muscle fibres.

Animals↗
PubMed1987

Reversible inhibition of sarcoplasmic reticulum Ca-ATPase by altered neuromuscular activity in rabbit fast-twitch muscle.

A 50% decrease in both the initial rate and the total capacity of Ca2+ uptake by the sarcoplasmic reticulum (SR) occurred 2 days after the onset of chronic (10 Hz) nerve stimulation in rabbit fast-twitch muscle. Prolonged stimulation (up to 28 days) did not lead to further decreases. This reduction, which was detected in muscle homogenates using a Ca2+-sensitive electrode, was reversible after 6 days cessation of stimulation and was not accompanied by changes in the immunochemically (ELISA) determined tissue level or isozyme characteristics of the SR Ca2+-ATPase protein. However, as measured in isolated SR, it correlated with a reduced specific activity of the Ca2+-ATPase. Kinetic analyses demonstrated that affinities of the SR Ca2+-ATPase towards Ca2+ and ATP were unaltered. Positive cooperativity for Ca2+ binding (h = 1.5) was maintained. However, a 50% decrease in Ca2+-dependent phosphoprotein formation indicated the presence of inactive forms of Ca2+-ATPase in stimulated muscle. The reduced phosphorylation of the enzyme was accompanied by an approximately 50% lowered binding of fluorescein isothiocyanate, a competitor at the ATP-binding site. In view of the unaltered affinity for ATP, this finding suggests that active Ca2+-ATPase molecules coexist in stimulated muscle with inactive enzyme molecules, the latter displaying altered properties at the nucleotide-binding site.

Adenosine Triphosphate↗
PubMed1986

Neural control of gene expression in skeletal muscle. Calcium-sequestering proteins in developing and chronically stimulated rabbit skeletal muscles.

Tissue contents of the sarcoplasmic-reticulum Ca2+-ATPase (Ca2+ +Mg2+-dependent ATPase), of calsequestrin and of parvalbumin were immunochemically quantified in homogenates of fast- and slow-twitch muscles of embryonic, maturing and adult rabbits. Unlike parvalbumin, Ca2+-ATPase and calsequestrin were expressed in embryonic muscles. Presumptive fast-twitch muscles displayed higher contents of these two proteins than did presumptive slow-twitch muscles. Calsequestrin steeply increased before birth and reached adult values in the two muscle types 4 days after birth. The main increase in Ca2+-ATPase occurred during the first 2 weeks after birth. Denervation of postnatal fast- and slow-twitch muscles decreased calsequestrin to amounts typical of embryonic muscle and suppressed further increases of Ca2+-ATPase. Denervation caused slight decreases in Ca2+-ATPase in adult fast-twitch, but not in slow-twitch, muscles, whereas calsequestrin was greatly decreased in both. Chronic low-frequency stimulation induced a rapid decrease in parvalbumin in fast-twitch muscle, which was preceded by a drastic decrease in the amount of its polyadenylated RNA translatable in vitro. Tissue amounts of Ca2+-ATPase and calsequestrin were essentially unaltered up to periods of 52 days stimulation. These results indicate that in fast- and slow-twitch muscles different basal amounts of Ca2+-ATPase and calsequestrin are expressed independent of innervation, but that neuromuscular activity has a modulatory effect. Conversely, the expression of parvalbumin is greatly enhanced by phasic, and drastically decreased by tonic, motor-neuron activity.

Animals↗
PubMed1986

Neural control of gene expression in skeletal muscle. Effects of chronic stimulation on lactate dehydrogenase isoenzymes and citrate synthase.

The aim of this study was to investigate the effects of neural activity on the expression of fibre-type-specific patterns of metabolic enzymes at the levels of transcription and translation. For this purpose, changes in tissue amounts of citrate synthase (CS) and the H- and M-subunits of lactate dehydrogenase (LDH) were followed in fast-twitch rabbit muscles during low-frequency (10 Hz, 12 h/day) nerve stimulation. These stimulation-induced alterations were correlated with changes in tissue amounts of the total poly(A)+ (polyadenylated) RNA, poly(A)+ RNAs specifically translatable in vitro, yield of total ribosomes and distributions of monosomes and polysomes. The tissue contents of poly(A)+ RNAs translatable in vitro coding for CS and H- and M-LDH were quantified by immunoprecipitation of their translation products. Increases in total ribosome yields occurred after 4 days' stimulation, reaching a maximum between 14 and 21 days. Stimulation for only 1-2 days greatly increased the amount of monosomes. An increase in polysomes occurred before that in total ribosomes, suggesting that monosomes were integrated into polysomes. Total poly(A)+ RNA significantly increased in muscles stimulated for more than 6 days. A maximum increase of 2.5-fold was attained after 14-21 days. Chronic stimulation progressively induced the appearance of LDH isoenzymes containing the H-subunit, with a predominance of LDH-3. This shift corresponded to a slow decay of the M-subunit and a 2-fold steep increase in the H-subunit. These changes correlated with those of the respective poly(A)+ RNAs translatable in vitro, thus indicating that the re-arrangement of the LDH isoenzyme pattern is mainly due to qualitatively and quantitatively altered transcription. The increase in CS was biphasic and consisted of a moderate rise during the first 4 days of stimulation and a steep rise thereafter. The latter coincided with a steep increase in poly(A)+ RNA translatable in vitro coding for CS. In view of the early increase in translational capacity, it was concluded that the initial rise in CS resulted from selective post-transcriptional control and enhanced translation in vivo of existing mRNA, whereas its steep increase was due to enhanced transcription. These results indicate that the neurally regulated expression of phenotype-specific properties in muscle includes control of both transcription and translation.

Animals↗
PubMed1986

Immunochemical quantification of sarcoplasmic reticulum Ca-ATPase, of calsequestrin and of parvalbumin in rabbit skeletal muscles of defined fiber composition.

Antibodies directed against purified Ca-ATPase from sarcoplasmic reticulum, calsequestrin and parvalbumin from rabbit fast-twitch muscle were raised in sheep. The specificity of the antibodies was shown by immunoblot analysis and by enzyme-linked immunoadsorbent assays (ELISAs). IgG against the sarcoplasmic reticulum Ca-ATPase inhibited the catalytic activities of Ca-ATPase from fast-twitch (psoas, tibialis anterior) and slow-twitch (soleus) muscles to the same degree. In non-equilibrium competitive ELISAs the anti(Ca-ATPase) IgG displayed a slightly higher affinity for the Ca-ATPase from fast-twitch muscle than for that from slow-twitch muscle. This suggests a fiber-type-specific polymorphism of the sarcoplasmic reticulum Ca-ATPase. Quantification of Ca-ATPase, calsequestrin and parvalbumin in various rabbit skeletal muscles of histochemically determined fiber composition was achieved by sandwich ELISA. Ca-ATPase was found to be 6-7 times higher in fast than in slow-twitch muscles. A slightly higher concentration was found in fast-twitch muscles with a higher percentage of IIb fibers when compared with fast-twitch muscles with a higher percentage of IIa fibers. Thus Ca-ATPase is distributed as follows, IIb greater than or equal to IIa much greater than I. Calsequestrin was uniformly distributed in fast-twitch muscles independently of their IIa/IIb fiber ratio and displayed 50% lower concentrations in slow than in fast-twitch muscles (IIb = IIa greater than I). Parvalbumin contents were 200-300-fold higher in fast than in slow-twitch muscles. Significantly lower parvalbumin concentrations were found in fast-twitch muscles with a higher percentage of IIa fibers than in fast-twitch muscles with a higher percentage of IIb fibers (IIb greater than IIa much greater than I).

Animals↗
PubMed1986

Neural regulation of parvalbumin expression in mammalian skeletal muscle.

Parvalbumin was purified from rabbit fast skeletal muscle and used to raise antibodies in sheep. Subsequently, a sensitive 'sandwich' enzyme-linked immunoadsorbent assay permitted quantification of parvalbumin in homogenates of embryonic, maturing, innervated, denervated and chronically stimulated skeletal muscles of the rabbit. High concentrations of parvalbumin were detected in various adult fast-twitch muscles of the rabbit (700-1200 micrograms/g of muscle), whereas slow-twitch muscles contained negligible concentrations (3-5 micrograms/g of muscle). Parvalbumin was not detectable in embryonic-rabbit muscles (21, 25, 28 days of gestation), either presumptive fast- or slow-twitch. However, parvalbumin concentrations did increase during postnatal development in presumptive fast-twitch muscles. Thus the onset of parvalbumin synthesis appears to be correlated with the neonatal-to-adult transition of motor-neuron activity [Navarrete & Vrbová (1983) Dev. Brain Res. 8, 11-19]. The increase of parvalbumin in maturing, presumptive fast-twitch muscle was suppressed by denervation. In the adult rabbit, denervation of the tibialis anterior muscle caused a reduction of parvalbumin to a level normally found in slow-twitch muscles. In contrast, the already low levels of parvalbumin in maturing and adult slow-twitch soleus muscle were unaffected by denervation. Chronic low-frequency stimulation of adult fast-twitch muscle resulted in a rapid reduction of parvalbumin to a level normally found in slow-twitch muscle. These data support the hypothesis that the expression of parvalbumin is under positive control of fast-type motor-neuron activity.

Animals↗
PubMed1986

Distribution of sarcoplasmic reticulum Ca-ATPase and of calsequestrin in rabbit and rat skeletal muscle fibers.

Muscle fibers in rabbit extensor digitorum longus (EDL), tibialis anterior (TA) and soleus, and rat soleus, were examined immunohistochemically for two proteins of the sarcoplasmic reticulum. Ca-ATPase and calsequestrin (CaS). Fibers were typed with the histochemical reaction for actomyosin ATPase. In the rabbit EDL and TA, type I fibers clearly reacted less for Ca-ATPase and CaS than type II fibers, but the difference was less with CaS than with Ca-ATPase. Although the differences were relatively small, IIB fibers consistently presented greater amounts of Ca-ATPase than IIA fibers. No type II subgroups could be recognized after incubation with anti-CaS. These findings confirm results from previous immunochemical measurements on whole muscles containing different proportions of IIA and IIB fibers (Leberer and Pette 1986). Type IIA and IIC in the rabbit and rat soleus reacted stronger for Ca-ATPase and for CaS than type I fibers. Small differences in Ca-ATPase, but not in CaS, were recognized within the type I fiber population. Therefore, type I fibers in the rabbit and rat soleus are not a homogeneous population.

Animals↗
PubMed1986

Actin-severing activity copurifies with phosphofructokinase.

Microinjection of muscle 6-phosphofructokinase (PFK; EC 2.7.1.11) into tissue culture cells led to a reversible disintegration of microfilament bundles (stress fibers). The mode of disruption as well as of recovery of stress fibers was very similar to that found previously in experiments performed with the actin-severing protein brevin, an extracellular variant of gelsolin. PFK, like brevin, was also capable of disrupting stress fibers in detergent-extracted cells and in ethanol-fixed cells, in a Ca2+-dependent manner. When compared with heart muscle gelsolin, PFK comigrated with the 85- to 90-kDa band. Antibodies against PFK crossreacted with gelsolin from the same species. These results point to a tight association between polypeptides with similar biochemical and immunological parameters present in both preparations. They suggest hitherto unexpected cellular control mechanisms for both microfilament functions and glycolysis.

Actin Cytoskeleton↗
PubMed1984

Lactate dehydrogenase isozymes in type I, IIA and IIB fibres of rabbit skeletal muscles.

Lactate dehydrogenase (LDH) isozyme patterns were analyzed by polyacrylamide (PAA) slab gel electrophoresis in extracts prepared from various rabbit skeletal muscles of defined fibre composition and by PAA microelectrophoresis of microdissected, histochemically typed single muscle fibres. The results obtained by electrophoresis of whole muscle extracts generally agreed with the data obtained from single fibre electrophoresis, i.e. the LDH isozyme pattern corresponded to that of the predominant fibre type. Type I fibres from soleus and semitendinosus muscles were characterized by a unique pattern of all 5 LDH isozymes with a predominance of LDH-1, 2 and 3. The major fraction (80%) of the type II fibres from extensor digitorum longus and tibialis anterior muscles contained only LDH-5 (M4). About 20% of the type II fibres contained in addition to LDH-5 small amounts of LDH-4 and LDH-3. The fraction of fibres containing LDH-5, LDH-4, and LDH-3 was similar (ca. 20%) in the histochemically defined IIA and IIB subpopulations. In view of the fact that the major fractions of rabbit IIB fibres display low and of IIA fibres high aerobic oxidative capacities ( Reichmann and Pette 1982), these data indicate that the expression of the H-subunit of LDH is not correlated with the aerobic-oxidative capacity of the fibre. It also appears not to be correlated with the presence of different myosin isoforms in IIA and IIB fibres.

Animals↗
PubMed1984

Effects of different patterns of long-term stimulation on blood flow, fuel uptake and enzyme activities in rabbit fast skeletal muscles.

Long-term electrical stimulation (14-28 days) of rabbit fast muscles (tibialis anterior, TA and extensor digitorum longus, EDL) using intermittent high frequency (3 trains per min of 5 s duration at 40 Hz, for 8 h per day) produced changes in enzyme activities similar to those found with continuous stimulation at a frequency occurring in nerves to slow muscles (10 Hz). The activity of citrate synthetase, 3-hydroxyacyl-CoA dehydrogenase and succinate dehydrogenase increased two to 3-fold within 28 days. There was a 4-fold increase in hexokinase whereas phosphofructokinase, pyruvate kinase, lactate dehydrogenase and fructose-1,6-diphosphatase decreased to about 60% of the activity levels in the contralateral unstimulated muscles. Blood flow and oxygen consumption at rest were not changed even after 28 days of stimulation, but were increased during contractions in muscles stimulated at either frequency, the level being twice as high as in control muscles. Glucose uptake was similar to that in control muscles both at rest and during contractions and the output of lactate was similar to that found in control muscles in muscles stimulated at 40 Hz. Muscles stimulated at 10 Hz had smaller lactate output. Thus intermittent stimulation at high frequency (40 Hz) and continuous low frequency (10 Hz) produced similar changes in aerobic metabolism and fuel uptake provided that the total number of stimuli was comparable and that the stimulation was carried out for sufficiently long period.

Animals↗