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L Dux

Publications and source records attributed to L Dux.

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

Fibre typing using sarcoplasmic reticulum Ca2+-ATPase and myoglobin immunohistochemistry in rat gastrocnemius muscle.

Skeletal muscle fibre types were identified by using immunohistochemical detection of sarcoplasmic reticulum Ca2+-ATPase and myoglobin content in rat gastrocnemius muscle. The strong Ca2+-ATPase-reactive fibres were identical with the fast-twitch population, while the fibres with weak reactivity represented the slow-twitch type. Strong myoglobin immunoreactivity reflected the fast oxidative glycolytic (FOG) and slow oxidative (SO) types. Slight to moderate myoglobin immunostaining was found in the fast glycolytic (FG) fibres. The staining intensity of the different fibre types differed as follows: for Ca2+-ATPase FG greater than FOG greater than SO, and for myoglobin FOG greater than SO greater than FG. The immunoreactivity of Ca2+-ATPase and myoglobin were well preserved after fixation of the muscles in Bouin's solution, or in formol/acetic acid fixative, and paraffin embedding. Detection of the primary antibodies was carried out by using the avidin-biotin-peroxidase complex, and the immunogold-silver-staining methods. The latter was found to be more sensitive and suitable for postembedding ultrastructural demonstration of the Ca2+-pump enzyme on Durcupan-embedded muscles. The method, using 5 nm immunogold conjugate with silver enhancement, offered the advantages of high sensitivity and excellent visualization of the reaction product. The postembedding detection of sarcoplasmic reticulum Ca2+-ATPase also proved to be useful in the retrospective identification of the main fibre classes in human muscle biopsies.

Animals↗
PubMed1989

Structural differences between the Ca2+-ATPase enzymes of sarcoplasmic reticulum membrane from rabbit and carp muscles.

1. Structural features were compared in sarcoplasmic reticulum Ca2+-ATPase enzymes from carp (Cyprinus carpio L.) and rabbit muscles. 2. Both membrane preparations contained the 105,000 mol. wt Ca2+ pump protein in high local density. 3. The tryptic cleavage of the carp enzyme gave different peptide fragments from those observed from rabbit enzyme. 4. Addition of vanadate, Ca2+ or lanthanides did not cause two-dimensional Ca2+-ATPase crystal formation, in contrast to the rabbit enzyme, which forms extensive arrays under these conditions. 5. No differences were found in this respect between microsome preparations derived from warm and cold adapted fishes. 6. A different primary sequence as well as a different disposition of the enzyme in the membrane may stand behind the observed dissimilarities.

Animals↗
PubMed1989

[Characterization of striated muscle fiber types by Ca2+-ATPase and myoglobin immunohistochemistry of the sarcoplasmic reticulum].

By the immunohistochemical demonstration of SR calcium ATPase and myoglobin a fibre classification method was developed. Fast fibres showed intense, while slow fibres weak SR calcium ATPase reactivity. Immunohistochemical reaction of myoglobin characterized the oxidative metabolic state of fibres similar to the succinate dehydrogenase (SDH) reaction. By means of SR calcium ATPase and myoglobin immunohistochemistry fibres were classified as slow oxidative (SO), fast oxidative glycolytic (FOG) and fast glycolytic (Fg) groups. The SR calcium ATPase activity of the different fibres varied in the FG greater than FOG greater than SO order, while myoglobin immunoreactivity in the FOG greater than SO greater than FG order. Both proteins studied preserved their antigenicities in Bouin's fixative or in formol-acetate and paraffin embedding. The light microscopic immunogold-silver method was found suitable also for electron microscopy. The silver intensification of small particle-size (5 nm) gold conjugate results in a reaction with the joint advantages of high sensitivity and optimal visibility. The described immunohistochemical method proved to be suitable for the retrospective differentiation of human biopsy materials.

Calcium-Transporting ATPases↗
PubMed1988

Stabilization and crystallization of Ca2+-ATPase in detergent-solubilized sarcoplasmic reticulum.

Conditions were developed for the long-term stabilization of Ca2+-ATPase in detergent-solubilized sarcoplasmic reticulum, purified Ca2+-ATPase, and purified-delipidated Ca2+-ATPase preparations. The standard storage medium contains 0.1 M KCl, 10 mM K-3-(N-morpholino)propanesulfonate, pH 6.0, 3 mM MgCl2, 20 mM CaCl2, 20% glycerol, 3 mM NaN3, 5 mM dithiothreitol, 25 IU/ml Trasylol, 2 micrograms/ml 1,6-di-tert-butyl-p-cresol, 2 mg/ml protein, and 2-4 mg of detergent/mg of protein. Preparations stored under these conditions at 2 degrees C in a nitrogen atmosphere retain significant Ca2+-stimulated ATPase activity for periods of 5-6 months or longer when assayed in the presence of asolectin. The same conditions are also conducive for the formation of three-dimensional microcrystals of Ca2+-ATPase. Of the 49 detergents tested for solubilization, optimal crystallization of Ca2+-ATPase was obtained in sarcoplasmic reticulum solubilized with octaethylene glycol dodecyl ether at a detergent/protein weight ratio of 2, and with Brij 36T, Brij 56, and Brij 96 at a detergent/protein ratio of 4. Similar Ca2+-induced crystals of Ca2+-ATPase were obtained with purified or purified delipidated ATPase preparations at lower detergent/protein ratios. The stabilization of the ATPase activity in the presence of detergents is the combined effect of high Ca2+ (20 mM) and a relatively high glycerol concentration (20%). Ethylene glycol, glucose, sucrose, or myoinositol can substitute for glycerol with preservation of ATPase activity for several weeks in the presence of 20 mM Ca2+.Ca2+-induced association between ATPase molecules may be an essential requirement for preservation of enzymatic activity, both in intact sarcoplasmic reticulum and in solubilized preparations.

Animals↗
PubMed1988

Electron microscope observations on Ca2+-ATPase microcrystals in detergent-solubilized sarcoplasmic reticulum.

Crystalline arrays of Ca2+-ATPase molecules develop in detergent-solubilized sarcoplasmic reticulum during incubation for several weeks at 2 degrees C under nitrogen in a medium of 0.1 M KCl, 10 mM K-3-(N-morpholino)propanesulfonate, pH 6.0, 3 mM MgCl2, 20 mM CaCl2, 20% glycerol, 3 mM NaN3, 5 mM dithiothreitol, 25 IU/ml Trasylol, 2 micrograms/ml 1,6-di-tert-butyl-p-cresol, 2 mg/ml protein, and 2-4 mg of detergent/mg of protein. Electron microscopy of sectioned, negatively stained, freeze-fractured, and frozen-hydrated Ca2+-ATPase crystals indicates that they consist of stacked lamellar arrays of Ca2+-ATPase molecules. Prominent periodicities of ATPase molecules within the lamellae arise from a centered rectangular lattice of dimensions 164 x 55.5 A. The association of lamellae into three-dimensional stacks is assumed to involve interactions between the exposed hydrophilic headgroups of ATPase molecules, that is promoted by glycerol and 20 mM Ca2+. Similar Ca2+-induced crystals were observed with purified or purified and delipidated Ca2+-ATPase preparations at lower detergent/protein ratios. Cross-linking of Ca2+-ATPase crystals with glutaraldehyde protects the structure against conditions such as low Ca2+, high pH, elevated temperature, SH group reagents, high concentration of detergents, and removal of phospholipids by extraction with organic solvents that disrupt unfixed preparations.

Animals↗
PubMed1988

Characterization of membrane calcium pumps by simultaneous immunoblotting and 32P radiography.

Calcium pumps of various plasma membrane, endoplasmic reticulum and sarcoplasmic reticulum preparations were visualized by simultaneous immunoblotting and autoradiography of the 32P-labelled phosphoenzymes. The pump proteins and their fragments produced by a proteolytic pretreatment of the membranes were selectively phosphorylated by [gamma-32P]ATP, separated on an acidic SDS-polyacrylamide gel, blotted onto nitrocellulose and reacted with polyclonal antibodies raised against the purified human erythrocyte and rat skeletal muscle sarcoplasmic reticulum calcium pumps, respectively. The immuno-reaction was detected by peroxidase staining, while the phosphoproteins were shown by autoradiography of the same blot. An antibody against the erythrocyte calcium pump, reacting on the blot with the 140 kDa erythrocyte calcium pump and its 80 kDa proteolytic fragment, did not show a cross-reaction with the calcium pump of similar molecular mass in rat synaptosome membranes or with any of the endoplasmic- or sarcoplasmic-type calcium pumps. An anti-sarcoplasmic reticulum calcium pump antibody cross reacted with several sarcoplasmic and endoplasmic calcium pump proteins and their proteolytic fragments but with none of the plasma membrane pumps. This sensitive double-labelling method can be applied to study structural relationships and molecular alterations in various ion pump proteins.

Animals↗
PubMed1988

Vanadate stimulated NADH oxidation in sarcoplasmic reticulum membrane.

Vanadate stimulated NADH oxidation was detected in sarcoplasmic reticulum membrane preparations. The reaction showed enzymatic character, with half maximal activating concentration of 1.2 mM vanadate and maximal NADH oxidation 50 nmol/mg protein/minute. Acidic pH, micromolar free Ca2+ concentration and decavanadate addition increased the rate of NADH oxidation. The described enzyme activity is similar to the ones observed in erythrocyte liver and cardiac plasma membranes. The vanadate stimulated NADH oxidation in sarcoplasmic reticulum preparations does not seem to originate from other contaminating membrane elements. The presence of this enzyme activity in the sarcoplasmic reticulum should be taken into consideration when planning experiments with vanadate, especially when measuring ATPase activity through NADH oxidation with coupled enzymatic assay.

Animals↗
PubMed1987

Crystallization of Ca2+-ATPase in detergent-solubilized sarcoplasmic reticulum.

Microcrystalline arrays of Ca2+-transporting ATPase (EC 3.6.1.38) develop in detergent-solubilized sarcoplasmic reticulum upon exposure to 10-20 mM CaCl2 at pH 6.0 for several weeks at 2 degrees C, in a crystallization medium that preserves the ATPase activity for several months. Of 48 detergents tested, optimal crystallization was obtained with Brij 36T, Brij 56, and Brij 96 at a detergent:protein weight ratio of 4:1 and with octaethylene glycol dodecyl ether at a ratio of 2:1. Similar Ca2+-induced crystalline arrays were obtained with the purified or delipidated Ca2+-ATPase of sarcoplasmic reticulum but at lower detergent:protein ratios. The crystals are stabilized by fixation with glutaraldehyde and persist even after the removal of phospholipids by treatment with phospholipases A or C and by extraction with organic solvents. The crystals obtained so far can be used only for electron microscopy, but ongoing experiments suggest that under similar conditions large ordered arrays may develop that are suitable for x-ray diffraction analysis.

Calcium-Transporting ATPases↗
PubMed1987

Effect of chemical modification on the crystallization of Ca2+-ATPase in sarcoplasmic reticulum.

The influence of chemical modification on the morphology of crystalline ATPase aggregates was analyzed in sarcoplasmic reticulum (SR) vesicles. The Ca2+-ATPase forms monomer-type (P1) type crystals in the E1 and dimer-type (P2) crystals in the E2 conformation. The P1 type crystals are induced by Ca2+ or lanthanides; P2 type crystals are observed in Ca2+-free media in the presence of vanadate or inorganic phosphate. P1- and P2-type Ca2+-ATPase crystals do not coexist in significant amounts in native sarcoplasmic reticulum membrane. The crystallization of Ca2+-ATPase in the E2 conformation is inhibited by guanidino-group reagents (2,3-butanedione and phenylglyoxal), SH-group reagents, phospholipases C or A2, and detergents, together with inhibition of ATPase activity. Amino-group reagents (fluorescein 5'-isothiocyanate, pyridoxal phosphate and fluorescamine) inhibit ATPase activity but do not interfere with the crystallization of Ca2+-ATPase induced by vanadate. In fluorescamine-treated sarcoplasmic reticulum the vanadate-induced crystals contain significant P1-type regions in addition to the dominant P2 form.

Animals↗
PubMed1987

Electron microscopic analysis of two-dimensional crystals of the Ca2+-transport ATPase--a freeze-fracture study.

Two distinct forms of Ca2+-ATPase crystals have been analysed in sarcoplasmic reticulum (SR) membranes. The E1-type crystals, induced by Ca2+ or lanthanide ions, consist of single chains of ATPase monomers, and the E2-type crystals, induced by vanadate ions, consist of dimer chains. Using improved freeze-fracture techniques we have obtained high-resolution images of complementary surface replicas of SR membranes containing these crystal forms. In E1 crystals, the concave fracture (P) faces display obliquely oriented rows of intramembrane particles (IMPs) spaced at congruent to 6-7 nm along both crystal axes, while the convex fracture (E) faces show corresponding rows of pits. In E2 crystals, regular arrays of oblique parallel ridges with spacing of congruent to 10.5-11 nm appear on the P-faces and complementary grooves or furrows on the E-faces. In many instances the ridges break up into elongated particles repeating every 5.5 nm. When the direction of the shadow is almost parallel to the axis of the ridges, these 9.5 nm particles can be resolved into two domains, which represent intramembranous contacts between the two monomers of the two adjacent dimer chains. Complementary grooves on the E-faces can also be resolved into rows of pits complementary to the particles of the ridges on the P-faces. In the control SR membranes, randomly dispersed IMPs and corresponding pits are observed on the P- and E-faces, respectively. The data suggest that transport of Ca2+ involves significant structural changes of the enzyme molecule, reflected in the ATPase-ATPase interactions both on the cytoplasmic surface and in the lipid bilayer.

Animals↗
PubMed1987

Effects of systemic kainic acid administration on regional Na+, K+-ATPase activity in rat brain.

Changes in the activity of Na+,K+-ATPase and in the water, Na+, and K+ levels in the parietal cortex, hippocampus, and thalamus were investigated in rats 1, 3, 6, and 24 h following systemic kainic acid injection. An increase in Na+,K+-ATPase activity was observed in all three regions 3 h after the treatment, with a subsequent decrease in enzyme activity. The elevation in Na+,K+-ATPase activity was accompanied by an increase in the Na+ content and a decrease in the K+ content. These changes are presumed to occur because of repeated discharges and excessive prolonged depolarization in response to kainic acid. The decreases in Na+,K+-ATPase activity 6 and 24 h following kainic acid treatment coincide with neuropathological damage and edema formation, mainly in the hippocampus and thalamus.

Animals↗
PubMed1987

Effects of disuse on the function of fragmented sarcoplasmic reticulum of rabbit M. gastrocnemius.

A preparation method has been described to obtain a relatively pure and functionally intact fragmented sarcoplasmic reticulum (SR) vesicles fraction from normal and atrophied muscles. Purified SR preparations from rabbit gastrocnemius muscle atrophied by disuse showed similar protein composition (gel electrophoresis; Laemmli 1970) and similar vanadate induced crystallization (Dux and Martonosi 1983) properties of Ca2+-ATPase as those of control preparations. In the early period of atrophy (1-2 weeks) both the Ca2+-ATPase activity and Ca2+ uptake showed a 2-3-fold increase (from 3.42 +/- 0.24 to 7.34 +/- 0.25 mumol Pi X mg-1 prot X min-1 and from 1.26 +/- 0.10 to 3.36 +/- 0.22 mumol/l Ca2+ X min-1 X mg-1 prot. respectively).

Animals↗
PubMed1986

Three-dimensional reconstruction of negatively stained crystals of the Ca2+-ATPase from muscle sarcoplasmic reticulum.

The structure of the Ca2+ transport ATPase from rabbit skeletal muscle sarcoplasmic reticulum has been determined to 25 A resolution by three-dimensional image reconstruction of crystalline membrane tubules induced through exposure to Na3VO4 and preserved for electron microscopy in negative stain. The crystalline arrays have projection symmetry p2 and consist of chains of Ca2+-ATPase dimers arranged in a right-handed helix. The density map shows protein features that project from the membrane surface into the cytoplasm. The luminal side of the membrane tubules is featureless, presumably because very little of the Ca2+-ATPase molecule projects into the luminal space. The cytoplasmic region of the Ca2+-ATPase molecule is pear-shaped, with a lobe oriented nearly parallel to the axis of the dimer ribbons, about 16 A above the surface of the membrane bilayer. The structure seen in the maps has a volume of 71,000 A3, corresponding to a molecular weight of 57,000. The two Ca2+-ATPase profiles that constitute a dimer are connected by a stain-excluding bridge that is oriented parallel with the axis of the tubule at a height of about 42 A above the surface of the bilayer.

Animals↗
PubMed1986

Affi-gel blue treatment simplifies the protein composition of sarcoplasmic reticulum vesicles.

Sarcoplasmic reticulum vesicles isolated by conventional techniques usually contain, in addition to the recognized sarcoplasmic reticulum components, several other proteins (phosphorylase, myosin, glyceraldehyde-3-phosphate dehydrogenase, etc.) in variable amounts; these proteins complicate the interpretation of chemical modification data. Incubation of sarcoplasmic reticulum vesicles with Affi-Gel blue particles for 1-4 h at 2 degrees C, followed by sedimentation of the Affi-Gel in a clinical centrifuge, simplifies the protein composition by selective adsorption of the accessory proteins, and improves the consistency of the preparations. The Affi-Gel blue treatment is recommended as part of the standard procedure for the isolation of sarcoplasmic reticulum vesicles.

Animals↗
PubMed1985

Conformational responses of the tryptic cleavage products of the Ca2+-ATPase of sarcoplasmic reticulum.

Trypsin cleaves the Ca2+-ATPase of sarcoplasmic reticulum into two major fragments (A and B), followed by subsequent cleavage into smaller peptides. Although the ATP-dependent Ca2+ transport is still observed after cleavage of the ATPase into the A and B fragments, the Ca2+ transport energized by acetyl phosphate is strongly inhibited. Covalent labeling of the Ca2+-ATPase by fluorescein 5'-isothiocyanate inhibited both the ATP and acetyl phosphate-dependent Ca2+ transport. Vanadate protected the A and B fragments from further hydrolysis and preserved the ability of the cleaved Ca2+-ATPase to form crystals and to show the characteristic conformational changes in response to Ca2+ and EGTA that are observed with the intact enzyme. The protective effect of vanadate may be useful for the isolation of the A and B fragments in functional form.

Animals↗