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A Margreth

Publications and source records attributed to A Margreth.

At least 55 records · Page 3Linked to original sources
PubMed1988

Isolation of terminal cisternae of frog skeletal muscle. Calcium storage and release properties.

Sarcoplasmic reticulum (SR) terminal cisternae (TC) of frog (Rana esculenta) fast-twitch skeletal muscle have been purified by isopycnic sucrose density gradient centrifugation. Biochemical characteristics and Ca2+ release properties have been investigated and compared to those of the homologous fraction of rabbit skeletal muscle TC. The frog SR fraction obtained at the 38/45% sucrose interface appears to be derived from the terminal cisternae region as judged by: (a) thin section electron microscopy showing vesicles containing electron opaque material and squarelike (feet) projections at the outer surface; (b) protein composition (Ca2+-ATPase, calsequestrin, and high Mr proteins); (c) Ca2+ fluxes properties. The content of calsequestrin was higher in frog TC by 50% and the Ca2+ binding capacity (624 or 45 nmol of Ca2+/mg of TC protein, depending upon experimental conditions) was 3-4 times that of rabbit TC. Species-specific antigenic differences were found between junctional SR proteins of frog and rabbit TC. After active Ca2+ preloading in the presence of pyrophosphate (Palade, P. (1987) J. Biol. Chem. 262, 6135-6141), caffeine and doxorubicin elicited Ca2+ release from either TC fraction but with much faster rates in frog TC than in rabbit TC (14 versus 3 mumol of Ca2+/min/mg of protein). The present results provide new evidence for the existence of marked differences in Ca2+ release properties between TC of amphibian and mammalian fast-twitch muscle. Higher Ca2+ binding capacity and faster release rates in frog TC might compensate for the comparably greater diffusion distance being covered by the released Ca2+ from the Z-line to the actomyosin cross-bridges in the A-I overlap region.

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↗
PubMed1988

Denervation-induced proliferative changes of triads in rabbit skeletal muscle.

Protein compositional and functional differences exist between longitudinal and junctional sarcoplasmic reticulum (SR) in relation to Ca transport and to Ca release. In light of this knowledge, we have reinvestigated the effects of denervation on SR of rabbit gastrocnemius, a predominantly fast muscle. Electron microscopy of 2-weeks denervated muscle showed proliferation of transverse tubules (TT), forming junctional contacts with SR terminal cisternae (TC). At coincident periods, the yield of muscle microsomes was increased, and their fractionation by sucrose-density centrifugation demonstrated a relative increase of heavy vesicles. Thin-section electron microscopy of heavy SR from denervated muscle showed an increased number of vesicles containing calsequestrin (CS) as compared with control muscle. Electrophoretic analysis confirmed the relative decrease of Ca-ATPase protein and the striking increase of CS both in total microsomes and in heavy SR vesicles. Calcium loading and Ca-ATPase activity as well as the density of Ca-ATPase protein were decreased to a similar extent (20-30%) in denervated muscle microsomes. Stimulation of Ca-ATPase activity by Ca-ionophore A23187 showed that the vesicles were tightly sealed. When probed by competitive ELISA with antibody to SR Ca-ATPase from pure fast muscle, the Ca-ATPase of denervated microsomes was found to be highly cross reactive. Cleveland's peptide maps of the Ca-ATPase protein after partial digestion with S. aureus V8 protease also showed no significant change after denervation. Changes in cholesterol content and in the ratio of Mg-ATPase to Ca-ATPase activity of denervated muscle microsomes indicated a 4-fold increase of TT protein, i.e., from about 3% to not more than 12% of total protein, at 2 weeks after denervation. All these changes were totally reversed upon reinnervation of muscle fibers, and the consequent muscle recovery, as obtained by nerve crushing instead of nerve sectioning. From these results, we conclude that denervated adult fast muscle, similarly to immature fast muscle, contains more junctional SR. However, the molecular and catalytic properties of the Ca-ATPase are unaffected by denervation.

Animals↗
PubMed1987

Common structural domains in the sarcoplasmic reticulum Ca-ATPase and the transverse tubule Mg-ATPase.

Transverse tubule (TT) membranes isolated from chicken skeletal muscle possess a very active magnesium-stimulated ATPase (Mg-ATPase) activity. The Mg-ATPase has been tentatively identified as a 102-kD concanavalin A (Con A)-binding glycoprotein comprising 80% of the integral membrane protein (Okamoto, V.R., 1985, Arch. Biochem. Biophys., 237:43-54). To firmly identify the Mg-ATPase as the 102-kD TT component and to characterize the structural relationship between this protein and the closely related sarcoplasmic reticulum (SR) Ca-ATPase, polyclonal antibodies were raised against the purified SR Ca-ATPase and the TT 102-kD glycoprotein, and the immunological relationship between the two ATPases was studied by means of Western immunoblots and enzyme-linked immunosorbent assays (ELISA). Anti-chicken and anti-rabbit SR Ca-ATPase antibodies were not able to distinguish between the TT 102-kD glycoprotein and the SR Ca-ATPase. The SR Ca-ATPase and the putative 102-kD TT Mg-ATPase also possess common structural elements, as indicated by amino acid compositional and peptide mapping analyses. The two 102-kD proteins exhibit similar amino acid compositions, especially with regard to the population of charged amino acid residues. Furthermore, one-dimensional peptide maps of the two proteins, and immunoblots thereof, show striking similarities indicating that the two proteins share many common epitopes and peptide domains. Polyclonal antibodies raised against the purified TT 102-kD glycoprotein were localized by indirect immunofluorescence exclusively in the TT-rich I bands of the muscle cell. The antibodies substantially inhibit the Mg-ATPase activity of isolated TT vesicles, and Con A pretreatment could prevent antibody inhibition of TT Mg-ATPase activity. Further, the binding of antibodies to intact TT vesicles could be reduced by prior treatment with Con A. We conclude that the TT 102-kD glycoprotein is the TT Mg-ATPase and that a high degree of structural homology exists between this protein and the SR Ca-ATPase.

Animals↗
PubMed1986

Direct photoaffinity labeling of junctional sarcoplasmic reticulum with [14C]doxorubicin.

Doxorubicin, an anticancer drug, induces Ca2+ release from the terminal cisternae (TC) of skeletal muscle (Zorzato, F., Salviati, G., Facchinetti, T., and Volpe, P. (1985) J. Biol. Chem. 260, 7349-7355). Long wave ultraviolet irradiation of a TC fraction with morphologically intact feet structures (Saito, A., Seiler, S., Chu, A., and Fleischer, S. (1984) J. Cell Biol. 99, 875-885) in the presence of [14C]doxorubicin, led to covalent photolabeling of two proteins that exhibited apparent Mr values of 350,000 and 170,000. Such proteins were found to be absent in a fraction of longitudinal sarcoplasmic reticulum but enriched in junctional face membranes obtained by Triton X-100 treatment of the TC fraction. Three additional proteins with Mr values of 80,000, 60,000, and 30,000 were also faintly labeled in the junctional face membrane fraction. On a molar basis the highest level of incorporation was found in the 170,000-Da protein, probably a Ca2+-binding protein (Campbell, K. P., MacLennan, D. H., and Jorgensen, A. O. (1983) J. Biol. Chem. 258, 11267-11273). A lower level of labeling was observed in the 350,000-Da protein, tentatively identified as a component of the feet structures (Cadwell, J. J. S., and Caswell, A. H. (1982) J. Cell Biol. 93, 543-550). Photolabeling of junctional TC proteins did not occur if a 10-50-fold excess cold doxorubicin was included in the assay medium, indicating that it was displaceable and specific, and if ultraviolet irradiation was omitted. Photolabeling was inhibited by caffeine or ruthenium red, i.e. by an activator and an inhibitor of Ca2+ release from TC, respectively. Furthermore, photolabeling was prevented by [ethylenebis(oxyethylenenitrilo)]tetraacetic acid suggesting that doxorubicin binding is Ca2+-dependent. Doxorubicin-binding proteins are constituents of the junctional sarcoplasmic reticulum and might be involved in modulating Ca2+ release from TC.

Affinity Labels↗
PubMed1986

Characteristics of skeletal muscle calsequestrin: comparison of mammalian, amphibian and avian muscles.

Calsequestrin was identified in the isolated sarcoplasmic reticulum from skeletal muscle of three mammalian species (man, rat and rabbit) and from frog and chicken muscle, using electrophoretic and immunoblot techniques. It was further characterized in sarcoplasmic reticulum protein mixtures and at several stages of purification, following extraction with EDTA. We found extensive similarities in apparent molecular weight values, Stains All staining properties and in Cleveland's peptide maps, between mammalian calsequestrins, and no detectable difference within a species between fast and slow muscle. Human calsequestrin, with an apparent molecular weight of 60,000 when measured at alkaline pH and of 41,000 when measured at neutral pH, appears to be the smallest in size. Frog calsequestrin, although weakly cross-reactive with rabbit calsequestrin and having a relatively higher apparent molecular weight at alkaline pH (72,000), shares several significant properties with mammalian calsequestrins. It bound calcium with a high capacity (1300 nmol per mg protein), it contained about 32% acidic amino acid residues and focused at closely similar pI values. We observed the formation of a complex with Stains All absorbing maximally at 535 nm, rather than at 600 nm, and an even more marked shift in apparent molecular weight at neutral pH. We found distinct differences in the case of chicken calsequestrin, in addition to those previously reported. It is a highly acidic, calcium-precipitable protein, but its amino acid composition is contradistinguished by a higher ratio of glutamate to aspartate and its rate of electrophoretic mobility is minimally affected by changes in pH. It stained deep bluish with Stains All after gel electrophoresis and yielded a protein-dye complex in aqueous solution, absorbing maximally at 560 nm, and finally, it bound fluorescent Concanavalin A.

Animals↗
PubMed1985

Ca2+ channel agonist BAY-k 8644 does not elicit Ca2+ release from skeletal muscle sarcoplasmic reticulum.

BAY-k 8644, a nifedipine analogue, promotes Ca2+ influx into excitable cells via plasma membrane voltage-sensitive Ca2+ channels. We report here that sarcoplasmic reticulum (SR) Ca2+ release channels are insensitive to BAY-k 8644, as studied in highly purified isolated fractions and in chemically skinned fibers of rabbit skeletal muscle. This result suggests that a subcellular heterogeneity exists among Ca2+ channels, at least with respect to drug-receptor sites. In the course of this study, however we found that BAY-k 8644 reversibly inhibits the SR Ca2+ pump, i.e., it decreases Ca2+ influx into the SR lumen, although at concentrations (IC50 = 3-5 X 10(-5) M) much higher than those effective on voltage-sensitive Ca2+ channels.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1985

Tubular aggregates: sarcoplasmic reticulum origin, calcium storage ability, and functional implications.

Muscle biopsy specimens from three patients with an autosomal dominant myopathy and tubular aggregates in both type 1 and type 2 fibers were investigated for immunofluorescent staining with antibodies to sarcoplasmic reticulum (SR) Ca-pump protein and calsequestrin and for Ca2+ loading ability. The results show that type 1 and type 2 fibers are differentially reactive to anti-Ca-pump protein IgG and similarly reactive with affinity-purified antibody to calsequestrin, which is in agreement with earlier observations in rat skeletal muscle. Tubular aggregates, which are shown to be highly reactive with either kind of antibody, appear to be sites of calcium accumulation for oxalate-facilitated adenosine triphosphate (ATP) dependent Ca uptake by chemically skinned fibers and thereby increase markedly the Ca loading capacity of the affected fibers.

Blotting, Western↗
PubMed1985

Slow myosin heavy chain isozyme in nemaline myopathy.

Muscle biopsies from two sporadic cases of congenital nemaline myopathy were examined for myosin heavy chain composition. Electrophoresis of congenital nemaline myopathy (CNM) muscle myosin in SDS-5% polyacrylamide gels gave rise to a single heavy chain band, with a migration rate and antigenic properties identical to that of the adult slow form, as demonstrated by Western blot techniques and by using specific antibody. Immunofluorescent studies indicate that CNM muscle fibers, including the most severely atrophic fibers, are homogeneous with respect to myosin heavy chain composition.

Adult↗
PubMed1984

Myosin subunit composition in human developing muscle.

Previous pyrophosphate-gel studies have reported the existence of embryonic neonatal myosin isoenzymes in human developing muscle. The present investigation was undertaken to characterize their subunit composition more precisely. Two immature muscle myosins are contrasted with adult myosin: neonatal myosin and foetal myosin. The neonatal form of myosin is weakly cross-reactive with rabbit slow myosin and contains only fast-type light chains (LC), LC1F and LC2F. The associated heavy chains consist of a single electrophoretic component that reacts exclusively with antibodies against human foetal myosin and has a mobility and peptide pattern distinct from that of adult fast and slow heavy chains. Foetal myosin is distinguished by the presence of low amounts of a heavy chain immunologically cross-reactive with the adult slow form and of two additional light-chain components: a LC2S light chain and a foetal-specific light chain (LCemb.). The foetal-specific light chain, as shown by one-dimensional-peptide-map analysis, is structurally unrelated to both LC1S and LC1F light chains of human adult myosin. We conclude from these results that the ontogenesis of human muscle myosin shares certain common features with that observed in other species, except for the persistence until birth of a foetal form of heavy chain (HCemb.).

Adult↗
PubMed1982

Polymorphism of myosin light chains. An electrophoretic and immunological study of rabbit skeletal-muscle myosins.

Antibodies specific for rabbit fast-twitch-muscle myosin LCIF light chain were purified by affinity chromatography and characterized by both non-competitive and competitive enzyme-linked immunosorbent assay (ELISA) and a gel-electrophoresis-derived assay (GEDELISA). The antibodies did not cross-react with myosin heavy chains, and were weakly cross-reactive with the LC2F [5,5'-dithio-(2-nitrobenzoic acid)-dissociated] light chain and with all classes of dissociated light chains (LC1Sa, LC1Sb and LC2S), as well as with the whole myosin, from hind-limb slow-twitch muscle. The immunoreactivity of myosins with a truly mixed light-chain pattern (e.g. vastus lateralis and gastrocnemius) correlated with percentage content of fast-twitch-muscle-type light chains. A more extensive immunoreactivity was observed with diaphragm and masseter myosins, which were also characterized, respectively, by a relative or absolute deficiency of LC1Sa light chain. Furthermore, it was found that the LC1Sb light chain of masseter myosin is antigenically different from its slow-twitch-muscle myosin analogue, and is immunologically related to the LC1F light chain. Rabbit masseter muscle from its metabolic and physiological properties and the content, activity and immunological properties of sarcoplasmic-reticulum adenosine triphosphatase, is classified as a red, predominantly fast-twitch, muscle. Therefore our results suggest that the two antigenically different iso-forms of LC1Sb light chain are associated with the myosins of fast-twitch red and slow-twitch red fibres respectively.

Animals↗
PubMed1982

Biochemical heterogeneity of skeletal-muscle microsomal membranes. Membrane origin, membrane specificity and fibre types.

1. Microsomes were isolated from rabbit fast-twitch and slow-twitch muscle and were separated into heavy and light fractions by centrifugation in a linear (0.3-2m) sucrose density gradient. The membrane origin of microsomal vesicles was investigated by studying biochemical markers of the sarcoplasmic-reticulum membranes and of surface and T-tubular membranes, as well as their freeze-fracture properties. 2. Polyacrylamide-gel electrophoresis showed differences in the Ca(2+)-dependent ATPase/calsequestrin ratio between heavy and light fractions, which were apparently consistent with their respective origin from cisternal and longitudinal sarcoplasmic reticulum, as well as unrelated differences, such as peptides specific to slow-muscle microsomes (mol.wts. 76000, 60000, 56000 and 45000). 3. Freeze-fracture electron microscopy of muscle microsomes demonstrated that vesicles truly derived from the sarcoplasmic reticulum, with an average density of 9nm particles on the concave face of about 3000/mum(2) for both fast and slow muscle, were admixed with vesicles with particle densities below 1000/mum(2). 4. As determined in the light fractions, the sarcoplasmic-reticulum vesicles accounted for 84% and 57% of the total number of microsomal vesicles, for fast and slow muscle respectively. These values agreed closely with the percentage values of Ca(2+)-dependent ATPase protein obtained by gel densitometry. 5. The T-tubular origin of vesicles with a smooth concave fracture face in slow-muscle microsomes is supported by their relative high content in total phospholipid and cholesterol, compared with the microsomes of fast muscle, and by other correlative data, such as the presence of (Na(+)+K(+))-dependent ATPase activity and of low amounts of Na(+)-dependent membrane phosphorylation. 6. Among intrinsic sarcoplasmic-reticulum membrane proteins, a proteolipid of mol.wt. 12000 is shown to be identical in the microsomes of both fast and slow muscle and the Ca(2+)-dependent ATPase to be antigenically and catalytically different, though electrophoretically homogeneous. 7. Basal Mg(2+)-activated ATPase activity was found to be high in light microsomes from slow muscle, but its identification with an enzyme different from the Ca(2+)-dependent ATPase is still not conclusive. 8. Enzyme proteins that are suggested to be specific to slow-muscle longitudinal sarcoplasmic reticulum are the flavoprotein NADH:cytochrome b(5) reductase (mol.wt. 32000), cytochrome b(5) (mol.wt. 17000) and the stearoyl-CoA desaturase, though essentially by criteria of plausibility.

Animals↗
PubMed1982

Transitions in membrane composition during postnatal development of rabbit fast muscle.

Early postnatal changes (4-5 days to 15 days after birth) in the biochemical composition of microsomes were investigated in rabbit skeletal muscles destined to become fast-twitch muscles. During this period, a steady decrease in the microsomal content of cholesterol and of ouabain-sensitive Na + /K + -ATPase activity, as well as a decrease in protein electrophoretic components in the 80 000-70 000 molecular weight range, were observed. These changes are probably due to a diminishing yield of microsomal membranes derived from T-tubules, as the age of the animals increases, and are indicated from a knowledge of the mixed composition of muscle microsomes and previous biochemical data on isolated T-tubules. The content of cytochrome b5, which was found to be high in muscle microsomes of newborn animals, decreased strikingly as the amount of membrane-bound Ca2 + -ATPase protein increased, with a crossing-over point at about 7-10 days after birth. These changes, possibly corresponding to a transition from precursor sarcoplasmic reticulum (SR) to mature SR, were found to be temporally correlated with changes in [3H] alpha-tocopherol binding ability of the microsomes and in the mitochondrial content of glycerol phosphate dehydrogenase. At the same critical periods, coincident with the onset of motile activity, the immunological cross-reactivity of the Ca2 + -ATPase protein of microsomal vesicles, with antibody specific for the Ca2 + -ATPase of adult fast SR, was found to increase markedly, as tested by competitive enzyme-linked immunosorbent assay (ELISA). The immunological data are consistent with data in the literature demonstrating an increase in the concentration of Ca2 + -ATPase molecules in the SR membranes during ontogenic development. Both these data and catalytic data, however, suggest that the Ca2 + -ATPase protein is present in the same form in the SR of immature and of adult fast muscle and, in an antigenically different form, in slow muscle SR.

Animals↗
PubMed1982

Fast to slow change of myosin in nemaline myopathy: electrophoretic and immunologic evidence.

Muscle biopsies from two familial and one sporadic case with congenital nemaline myopathy and seven healthy family members were examined for myosin composition. Myosin was characterized with respect to light chain (LC) composition by one-dimensional and two-dimensional electrophoresis, and by immunologic methods (enzyme-linked immunosorbent assay [ELISA]), using specific antibody for rabbit fast myosin LCl (LC1F). Type I fiber predominance was associated with the substitution of a hybrid, predominantly "slow" to a virtually pure "slow" myosin LC pattern for the "mixed" pattern found with myosin of normal muscle. Muscle myosin from the relatives had apparently normal light chain composition.

Adult↗
PubMed1981

Molecular and antigenic properties of cytochrome b5 from slow-muscle sarcoplasmic reticulum.

NADH--cytochrome b5 reductase and cytochrome b5 associated with slow-muscle sarcoplasmic reticulum and liver microsomal fraction were identified with discrete protein bands of molecular weights 33000 and 16700 by polyacrylamide-gel electrophoresis. Purified detergent-extracted cytochrome b5 from muscle sarcoplasmic reticulum is indistinguishable from liver microsomal cytochrome b5 with respect to spectral properties, pI values and immunological reactivity with antibody to the liver cytochrome b5. Reaction of the antibody with membrane-bound cytochrome b5 inhibits the sarcoplasmic-reticulum NADH--cytochrome c reductase activity.

Animals↗
PubMed1981

Polymorphism of sarcoplasmic-reticulum adenosine triphosphatase of rabbit skeletal muscle.

Antibody was raised in chickens against purified sarcoplasmic-reticulum Ca2+-activated ATPase (Ca2+-ATPase). The immunological relationship between the Ca2+-ATPase of fast-muscle and slow-muscle sarcoplasmic reticulum was investigated by a one-step and a two-step competitive enzyme-linked immunosorbent assay (ELISA). The results show marked antigenic differences between the membrane-bound Ca2+-ATPase of the sarcoplasmic-reticulum vesicles from fast muscle and slow muscle, beside differences in the membrane content of ATPase protein.

Animals↗
PubMed1981

Characterization of human muscle myosins with respect to the light chains.

Isolated myosins from human predominantly fast and slow muscles, human neonatal and foetal muscle were examined for light chain composition by one- and two-dimensional electrophoresis. The LC1F, LC2F and LC3F light chains were identical with their counterparts from rabbit fast myosin. Human LC1S was identified by correlative criteria as a single component having a molecular weight slightly lower than, but an electric charge similar to, that of rabbit LC1Sb. Consequently, human LC1S appears to be much less heterogeneous relative to LC1F than is the case with other mammalian species. A high immunological cross-reactivity was likewise observed, with antibody specific to rabbit LC1F, between the isolated myosins from several human mixed muscles and rabbit fast myosin, though reactivity was highest with foetal myosin (having a pure-fast-light-chain pattern).

Animals↗