Biomedical subjects

W Hasselbach

Publications and source records attributed to W Hasselbach.

At least 163 records · Page 9Linked to original sources

PubMedDate not supplied

Inhibitors of calmodulin-dependent phosphorylation simultaneously inhibit calcium uptake and calcium-dependent ATPase activity in skeletal muscle sarcoplasmic reticulum and transiently induce calcium release.

Under adequate experimental conditions calmodulin antagonists like compound 48/80 do not dissociate calcium uptake from the calcium-dependent ATP hydrolysis of skeletal muscle sarcoplasmic reticulum membranes but simultaneously inhibit both processes. Apart from the agent's pump inhibiting effect, they interact with the caffeine sensitive calcium channel in the sarcoplasmic reticulum causing a rapid transient calcium release.

Caffeine↗

PubMedDate not supplied

Activation volumes of the calcium dependent para-nitrophenyl phosphate hydrolysis of the sarcoplasmic reticulum calcium transport enzyme.

The effect of pressure on the calcium dependent hydrolysis of para-nitrophenyl phosphate by the calcium transport enzyme of the sarcoplasmic reticulum was studied under different conditions: temperature, solutes, substrate and ion concentrations. The calcium transport enzyme exhibits a large positive activation volume which does neither depend on the enzyme's inhibition by high salt concentrations nor its activation by ethylene glycol. The activation volume further proves to be pressure-independent but exhibits a pronounced negative temperature coefficient. The volume changes connected with the entrance of para-nitrophenyl phosphate, calcium or magnesium ions into the substrate ion complex are quite small, indicating that the transfer of water connected with the binding of these ligands is compensated by volume changes of the protein, accompanying the transition of the enzyme from its activated into its ground state.

Animals↗

PubMedDate not supplied

Invariance of stoichiometry of the sarcoplasmic reticulum calcium pump at physiological calcium concentrations--a reevaluation.

The decline of the transport ratio of the sarcoplasmic calcium pump observed in a recent study (A. Gafni and P. D. Boyer, Proc. Natl. Acad. Sci. USA 82, 89-101 [1985] ) results from the retardation of calcium oxalate precipitation at low calcium/protein ratios. The prevailing high internal calcium level supports a rapid calcium backflux and a compensatory ATP hydrolysis during net calcium uptake which reduces the transport ratio. Yet, the determined calcium backflux does not fully account for the decline of the transport ratio. A supposed modulation of the stoichiometry of the pump by external calcium (0.1 microM) is at variance with results of previous studies showing a constant transport ratio of two in the same calcium concentration range.

Adenosine Triphosphatases↗

PubMedDate not supplied

Thyroxine induced transformation in sarcoplasmic reticulum of rabbit soleus and psoas muscles.

The properties of the sarcoplasmic reticulum membranes isolated from slow-twitch type I soleus and fast-twitch type II psoas muscles of control and thyroxine treated rabbits were comparatively studied. Membrane yield, maximal calcium storing capacity, ATP-supported calcium uptake, calcium-dependent ATPase activity and calcium-dependent phosphoprotein formation were found to be 3-10 fold higher in psoas than in soleus preparations. Membrane yield, calcium-dependent ATPase activity, ATP-supported calcium transport and calcium-dependent phosphoprotein are at least twice enhanced in the membranes from soleus muscles of animals treated for 14-21 days with thyroxine. The corresponding capacities of the membranes from psoas muscles are not further augmented by the same thyroxine treatment. The maximal calcium storing capacity of the psoas membranes is their sole specific property which is significantly increased. The changes in the properties of the soleus muscles' sarcoplasmic reticulum membranes are engendered by an increase from 5 to 30-50% in the number of type II fibres. Since the calcium transporting properties of the sarcoplasmic reticulum membranes from type II fibres qualitatively differ from those of type I fibres, thyroxine does not only affect quantitative but also qualitative parameters of the muscles' sarcoplasmic reticulum membrane system.

Adenosine Triphosphatases↗

PubMedDate not supplied

Interaction of ryanodine with the calcium releasing system of sarcoplasmic reticulum vesicles.

Heavy sarcoplasmic reticulum vesicles were reacted with ryanodine in 0.6 M KCl 0.3 M sucrose at pH 6.3 and pH 7.0 at 20 degrees C. The inhibition of caffeine induced calcium release from actively loaded vesicles by ryanodine was applied to monitor time course and attainment of equilibrium of the interaction of ryanodine with its receptors in the vesicular membranes. At ryanodine concentrations rising from 0.1-100 microM, the logarithms of the release amplitudes linearly decline with time. The dependence of the inactivation reaction on the concentration of ryanodine did not saturate in the applicable concentration range. The reaction halflife times are concentration dependent. At pH 7.0, the half times decline from 100 to 10 s when the ryanodine concentration is raised from 0.1 to 1 microM. At pH 6.3 a corresponding decline occurs between 3 microM and 100 microM. The marked dependence of the inactivation reaction on medium pH requires reaction times of one and five hours at pH 7.0 and 6.3, respectively for the attainment of reaction equilibrium at low ryanodine concentrations. The dependence of the amplitude of calcium release on the concentration of added ryanodine has been evaluated as proposed by Gutfreund (Enzymes: Physical Principles, p. 71, Wiley-Interscience, London 1972) for the preparation's affinity for ryanodine and its number of binding sites. At pH 7.0, preparations appear to contain only 0.7 pmol sites per mg protein having an affinity for ryanodine of 0.33 nM-1. The titration curves for caffeine induced calcium release, initial calcium uptake and final calcium level are identical, indicating that the three functions are controlled by the same receptor. Calcium induced calcium release, however, is only partially and differently affected by the occupancy of the high affinity ryanodine binding sites.(ABSTRACT TRUNCATED AT 250 WORDS)

Alkaloids↗

PubMedDate not supplied

Pressure effects on the interactions of the sarcoplasmic reticulum calcium transport enzyme with calcium and dinitrophenyl phosphate.

The effect of hydrostatic pressure on the calcium-dependent hydrolysis of dinitrophenyl phosphate by the sarcoplasmic calcium transport enzyme has been studied. The magnesium dinitrophenyl phosphate complex is the true substrate of the enzyme (K = 7000 M-1) by which it is hydrolyzed at 20 degrees C with a turnover rate of 4 s-1. Activation by calcium ions occurs between 0.1 and 1 microM as observed for ATP hydrolysis. The activation volume of the enzyme saturated with both ligands exhibits pronounced pressure-dependence, rising from 25 ml/mol at atmospheric pressure to 80 ml/mol at 100 MPa. The apparent binding volumes for magnesium dinitrophenyl phosphate and calcium are likewise pressure-dependent. The volume changes connected with the binding of magnesium dinitrophenyl phosphate is quite small approaching zero at 100 MPa. The apparent binding volume for calcium greatly increases with pressure from 35 ml/mol at atmospheric pressure to 150 ml/mol at 70 MPa. A nearly constant binding volume of approximately 40 ml/mol results if the effect of pressure on the respective rate constants that contribute to the apparent binding constant, is taken into account. The pressure-dependence of enzyme activity at subsaturating calcium concentrations yields an activation volume of 250 ml/mol related to the rate of calcium binding indicating the occurrence of a transient large volume expansion of the enzyme complex. The volume changes observed for the calcium-dependent interaction of the enzyme with magnesium dinitrophenyl phosphate well agree with that found for magnesium p-nitrophenyl phosphate (W. Hasselbach and L. Stephan,Z. Naturforsch. 42 c, 641-652 (1987)) indicating that the found volume changes are intrinsic properties of the transport enzyme, independent of the respective energy donor.(ABSTRACT TRUNCATED AT 250 WORDS)

2,4-Dinitrophenol↗

PubMedDate not supplied

Closure of a rapidly exchanging calcium compartment in rat cardiac myocytes by lanthanum.

Enzymatically isolated myocytes from adult rats were used to measure 45Ca-uptake from salt media of reduced Ca2(+)-content (0.1 mM) with normal (4 mM) or elevated (20 mM) potassium concentrations. Ca2(+)-uptake was interrupted by filtration followed by rapid chasing the filter with salt solutions containing no Ca2+, 2 mM Ca2+ or 2 mM La3+. Rate and extent of 45Ca-uptake of resting cells were found to be 3-fold enhanced when chasing was performed with La3(+)-containing media. In contrast La3+ does not affect Ca2(+)-exchange of depolarized cells, the fluxes of which approximate rates sufficiently high for contraction activation and that are sensitive to Ca2(+)-channel blockers. The effect of La3+ on resting cells suggests the existence of a small rapidly exchanging La3(+)-sensitive Ca2(+)-compartment located adjacent to the plasma membrane. This compartment is though to be either closed or has become La3(+)-insensitive in activated cells. The subsarcolemmal cysternae of the SR, the so-called "peripheral couplings" are most likely the morphological substrate of this compartment.

Animals↗

PubMedDate not supplied

The modulation of the calcium transport by skeletal muscle sarcoplasmic reticulum in the hibernating European hamster.

Calcium transport of skeletal muscle sarcoplasmic reticulum was comparatively studied in hibernating and summer active European hamsters (Cricetus cricetus L.). Crude homogenates from psoas, soleus and mixed skeletal muscles were used. Protein yield was strongly reduced in the muscle homogenates of hibernating hamsters. The calcium concentration in the muscle of hibernating hamsters was increased to a much higher content than in the serum. In the same animals the maximal rate of calcium uptake and the calcium storing capacity of sarcoplasmic reticulum were augmented by 43% and respectively 17%. Kinetic experiments with various concentrations of free calcium revealed in the hibernating animals higher uptake rates and a lower apparent calcium affinity than in the summer active hamsters. Some shift of calcium uptake rate and calcium affinity similar to that of a fast-twitch muscle was also observed in winter active animals kept at 22 degrees C under natural photoperiod. By contrast, the activity of the calcium dependent ATPase was not increased, suggesting a tighter coupling during hibernation between calcium dependent ATP-hydrolysis and calcium transport. No seasonal difference was observed in the calcium release by KCl-caffeine from calcium loaded vesicles of sarcoplasmic reticulum. Proportion and size of fibre types were studied with cold cross sections from psoas and soleus muscles. An average atrophy of about 25% was found during hibernation in both muscles. Cytochemistry revealed, however, a different reduction of cross area between type-I- and type-II-fibres, which reaches values up to 46% in the type-II-fast-fibres of the slow soleus muscle. Electron microscopy did not show any definite change in the distribution and amount of sarcoplasmic reticulum. The results suggest that during hibernation a modulation in the properties of calcium transport ATPase of sarcoplasmic reticulum occurs to better support the calcium transport function at low temperatures, which in turn warrants the restoration of ion homeostasis in the course of the arousal.

Adenosine Triphosphatases↗

PubMedDate not supplied

Modulation by ryanodine of active calcium loading and caffeine induced calcium release of heavy sarcoplasmic reticulum vesicles.

The effect of ATP on the calcium release channel in heavy sarcoplasmic reticulum vesicles modulated by ryanodine has been analyzed by monitoring active calcium uptake and caffeine induced calcium release under near physiological conditions. Native as well as ryanodine reacted vesicles display a complex time course of calcium uptake resulting in nearly complete exhaustion of medium calcium when ATP in combination with an ATP-regenerating system, in contrast to ATP alone, or dinitrophenyl phosphate, were used to support calcium uptake. Applying of dinitrophenyl phosphate as energy yielding substrate, not affecting channel activity, allowed to estimate the fraction of light vesicles devoided of calcium channels contaminating the heavy preparation as the fraction that stores calcium after the preparation has been treated with channel opening, low concentrations of ryanodine (1 microM). Calcium uptake by contaminant light vesicles (25%) cannot account for calcium storage, as well as, abolition of caffeine induced calcium release of ryanodine treated heavy vesicles. Calcium uptake of native and ryanodine treated vesicles is accompanied by the uptake of equivalent amounts of inorganic phosphate arising from ATP hydrolysis indicating that calcium is mainly stored as calcium phosphate. The momentary capability of the preparation to accumulate calcium was measured by activating calcium uptake during the calcium storage period with 0.2 mM 45CaCl2 and 4 mM phosphate at short time intervals. A significant increase of the momentary uptake activity with time was observed being more pronounced for ryanodine treated than for native vesicles indicating that under regenerating conditions, ATP can induce closing of the native and even more effectively of the ryanodine modified calcium release channels.

Adenosine Triphosphate↗

PubMedDate not supplied

Modulation by monovalent anions of calcium and caffeine induced calcium release from heavy sarcoplasmic reticulum vesicles.

Both calcium and caffeine induced calcium release from actively loaded heavy sarcoplasmic reticulum vesicles were studied to analyze the dependence of both activities on the composition of the release medium with respect to monovalent anions. Calcium is unable to induce net calcium release while caffeine remains effective as releasing agent when the experimental media contain neither chloride nor nitrate ions. Caffeine induced calcium release is not suppressed by chelating residual medium calcium (approximately 0.5-1 microM) with 2 mM EGTA added 15 s prior to 10 mM caffeine. Calcium release from vesicles loaded in media containing 0.2 M gluconate as monovalent anion is induced when the medium is supplemented with chloride or nitrate. The release amplitude increases linearly when K-gluconate is replaced by KCl. At constant ionic strength the release amplitude becomes maximal at a chloride concentration of 0.2 M. The chloride effect completely disappears when 2 mM EGTA are added simultaneously. When chloride is replaced by nitrate, as releasing agent, maximal release is achieved already by addition of 0.1 M K-nitrate. The releasing effect of nitrate can only partially be suppressed by EGTA. The different effectiveness of gluconate, chloride and nitrate as calcium release supporting ions corresponds to their activating effect on the binding of ryanodine to the calcium release channel in the vesicular membranes.

Animals↗

PubMedDate not supplied

How many ryanodine binding sites are involved in caffeine induced calcium release from sarcoplasmic reticulum terminal cysternae vesicles?

The inhibition by ryanodine of caffeine induced calcium release from actively loaded heavy sarcoplasmic vesicles has been studied in order to analyse the relation between the occupancy of the vesicular calcium release channels by ryanodine and channel function. Ryanodine binding was monitored with [3H]ryanodine under ionic conditions favouring the establishment of binding equilibrium. Binding follows 1:1 stoichiometry yielding dissociations constants between 7-12 nM and 12-15 pmol ryanodine/mg vesicular protein as maximum number of ryanodine binding sites. When ryanodine labeling was monitored by measuring the decline of the amplitude of caffeine induced calcium release 50% inhibition occurred at a free ryanodine concentration of 1 nM. At this concentration less than 10% of the available ryanodine binding sites are occupied. Caffeine induced calcium release is completely abolished when 3 pmol ryanodine/mg have reacted. A corresponding divergence between ryanodine binding and its effect on caffeine induced calcium release was observed when the initial rate of ryanodine binding was measured either by labeling the vesicles with [3H]ryanodine or by following the decline with time of caffeine induced calcium release. Caffeine induced calcium release declines four times faster than the fraction of unoccupied ryanodine binding sites, k = 4.3 x 10(4) M-1 s-1 versus 1.2 x 10(4) M-1 s-1. The observed interrelation between the occupation of ryanodine binding sites and its effect on caffeine induced calcium release indicates that the caffeine sensitive calcium channel functions as an assembly of at least 4 ryanodine binding sites whereby the occupation of one site suffices to abolish calcium release. The stoichiometric composition appears to be not fixed but might change according to the size of the fraction of ryanodine receptors exhibiting caffeine sensitivity. The reported data were evaluated according to the algorithm derived by H. Asai and M. F. Morales, J. Biol. Chem. 4, 830-838 (1965) for the activity of a macromolecule and the extent of an inhibiting reaction.

Animals↗

PubMedDate not supplied

Micromorphometric evaluation of changes in symmetry of sarcoplasmic reticulum membranes induced by vanadate.

Electron micrographs of light sarcoplasmic vesicles fixed with glutaraldehyde and osmium tetroxide followed by contrasting with uranyl acetate and lead citrate have been evaluated by registering their membrane profiles with a microdensitometer. The asymmetric arrangement of the two layers of the vesicular membrane could be ascertained by demonstrating a ratio of 1.5 for the thickness of the outer versus the inner membrane layer which is in general agreement with the proposed protein structure of the calcium transport enzyme. Treatment of the vesicles with low concentrations of vanadate (0.1 mM) results in a significant lowering of the symmetry ratio by 20% by reducing mainly the thickness of the outer membrane leaflet. Removal of the membrane lipids by treating the vesicles with phospholipase A2 and bovine serum albumin diminishes the membrane surface by 50% resulting in a significant increase of both the membrane thickness and the asymmetry ratio by 30 and 12% respectively. The vanadate induced reduction of membrane asymmetry is accentuated after delipidation indicating that the membrane lipids are not essential for the asymmetric appearance of the native membrane. The stability of the spherical form of the vesicles to delipidation implies that the transport molecules are conically shaped allowing strong mutual interactions. At a measured height of the molecule of 80 A in the membrane, the vanadate induced change in symmetry would be brought about by compensatory changes of less than 3 A of the outer (35 A) and the inner (25 A) diameter of the cone.

Animals↗

PubMedDate not supplied

The effect of calcium and phosphate on the biphasic calcium uptake by the sarcoplasmic reticulum.

The amplitude of the fast uptake and the initial rate of the slow uptake increase with increasing free calcium concentrations, up to 30 muM. In that range, both processes are correlated to each other. At higher concentrations, the slow uptake is more inhibited than the fast uptake. The fast uptake shows a maximum amplitude which remains unchanged in the presence of phosphate. The slow uptake leads to a nearly complete depletion of the external calcium, and its rate is proportional to the phosphate concentration, even at physiological range. The sarcoplasmic ATPase liberates inorganic phosphate and the slow uptake is an autocatalytic process.

Adenosine Triphosphatases↗

PubMedDate not supplied

Fusion of isolated sarcoplasmic reticulum membranes.

Fragmented sarcoplasmic reticulum (FSR) vesicles from rabbit muscle were suspended in 1.5-5% glycerol solutions and were pelleted onto aluminum foil disks in a modified centrifuge tube. Examination of these pellets in the electron microscope after drying for 2--2.5, 4--5.5, and 21 hours revealed a progression of changes. First, distances between individual, round vesicles decreases. Next, somewhat flattened vesicles establish limited areas of contact with adjacent vesicles. Finally, vesicle fusion occurs and extended areas of double bilayers are formed. A water loss-time interaction appears to be needed for the fusion process. A Hg-phenyl azoferritin compound was used as a marker to identify intra- and extra-vesicular space in the fused samples. Quantitative measurements of birefringence during imbibition of pellet slices in a graded series (eta-1.41-1.43) is reached. The plateau seen in this part of the curve is again followed by steadily increasing birefringence at higher glycerol concentrations. This interruption in the birefringence curve is presumably due to a matching of the refractive indices of the glycerol solution and a lipid component in the membranes.

Animals↗

PubMedDate not supplied

Calcium transport and release by the sarcoplasmic reticulum.

The slow rate of calcium transport by cardiac sarcoplasmic reticulum vesicles, compared to those prepared from skeletal muscle, is due mainly to a lesser density of transport sites and a lower Ca2+ affinity. Because the turnover rate of the cardiac sarcoplasmic reticulum calcium pump is similar to that of comparable skeletal muscle preparations, the cardiac sarcoplasmic reticulum seems able to effect the slower relaxation of the heart. Calcium permeability of skeletal sarcoplasmic reticulum vesicles is increased when the Ca2+ concentration outside the vesicles is increased, or when that inside the vesicle is decreased.

Adenosine Triphosphatases↗

PubMedDate not supplied

Effect of non-solubilizing SDS concentrations on high affinity Ca2+ binding and steady state phosphorylation by inorganic phosphate of the sarcoplasmic reticulum ATPase.

In this investigation low, non-solubilizing concentrations of the strong anionic detergent SDS were used to perturbate the interaction of Ca2+ and Pi with their respective binding domains on the sarcoplasmic reticulum Ca-transport ATPase. Rising SDS concentrations produce a two-step decline of Ca2+-dependent ATP hydrolysis. At pH 6.15, SDS differently affects high affinity Ca2+ binding and phosphorylation by inorganic phosphate and releases the "mutual exclusion" of these two ligand binding steps. The degree of uncoupling is considerably more pronounced in the presence of 20% Me2SO. The reduction of Ca2+ binding by SDS is demonstrated to be a result of decreased affinity of one of the two specific high affinity binding sites and of perturbation of their cooperative interaction. Higher SDS partially restores the original high Ca2+ affinity but not the cooperativity of binding. Phosphorylation exhibits a higher SDS sensitivity than Ca2+ binding: Increasing SDS competitively inhibits and then completely abolishes phosphoenzyme formation. Thus, SDS binds to the phosphorylation domain, evidently involving the Lys352 residue of the ATPase molecule; this is accompanied by a more unspecific concentration-dependent SDS effect, probably mediated by hydrophobic force, which, finally, suppresses phosphorylation. Me2SO does neither qualitatively affect the SDS-dependent chemical properties of the vesicular material nor the SDS-dependent perturbation of the investigated reaction steps.

Animals↗

PubMedDate not supplied

Reaction of fluorescein isothiocyanate with thiol and amino groups of sarcoplasmic ATPase.

Several model compounds containing thiol and/or amino groups (mercaptoethanol, glutathione, cysteine, ethanolamine, glycine) were studied with respect to their reactivity towards fluorescein isothiocyanate (followed spectrophotometrically at 504 and 412 nm), stability of product and long-wave absorption maximum of the fluorescein residue attached. Thiol groups reacted by far more readily than amino groups. A specific effect was observed with cysteine, indicating an intramolecular transfer of the fluorescein residue from SH to NH2. With sarcoplasmic vesicles both types of reactions were observed. The ratio of products, which can be distinguished by their different stabilities and absorption spectra, depended on the absence or presence of detergents. While with native vesicles the NH2 reaction predominated, with vesicles solubilized with sodium dodecylsulfate, octaethyleneglycol mono-n-dodecyl ether or 1-0-tetradecyl-propanediol-(1,3)-3-phosphorylcholine the SH reaction became prevailing. Already 0.35 mg sodium dodecylsulfate per mg protein were sufficient to give rise to dithiourethane formation exclusively. Excess fluorescein isothiocyanate reacted with several thiol groups of dodecylsulfate-solubilized vesicles. In the presence of ATP binding of fluorescein isothiocyanate to native vesicles was significantly reduced. Total blockage of the vesicular SH groups with N-ethyl-maleimide led to preparations that reacted with fluorescein isothiocyanate much more slowly, compared to native vesicles. Octaethyleneglycol mono-n-dodecyl ether or 1-0-tetradecyl-propanediol-(1,3)-3-phosphorylcholine in the assay accelerated the thioureide formation from N-ethylmaleimide modified vesicles, whereas sodium dodecylsulfate prevented it almost completely. Our results support the suggestion that one or several thiol groups in vicinity of the highly reactive lysyl residue might play a role in the fast specific reaction, which is only observed with intact native vesicles.

Adenosine Triphosphatases↗

PubMedDate not supplied

Formation and decay of the vanadate complex of the sarcoplasmic reticulum calcium transport protein.

The calcium free sarcoplasmic reticulum calcium transport ATPase incorporates in the presence of magnesium ions approx. 8 nmol monovanadate per mg protein, indicating the formation of a complex containing one vanadate residue per enzyme molecule. On ligand-removal or dilution, the saturated enzyme complex displays biphasic decay kinetics, while the unsaturated complex slowly dissociates monophasically. -Ligand competition by raising the concentrations of unlabeled vanadate results in a progressive decrease of the dissociation rate of the unsaturated enzyme. The complicated dissociation kinetics indicate a sequential mode of interaction between two ligand binding sites. The one to one stoichiometry of the complex suggests that the two sites are located at adjacent ATPase molecules. -It appears unlikely that the decay of the enzyme, vanadate complex is retarded by the formation of a stable quaternary complex between the enzyme, magnesium, mono- and polyvanadate.

Adenosine Triphosphatases↗