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W Hasselbach

Publications and source records attributed to W Hasselbach.

At least 19 recordsLinked to original sources
PubMed1998

The Ca(2+)-ATPase of the sarcoplasmic reticulum in skeletal and cardiac muscle. An overview from the very beginning to more recent prospects.

The discovery of the ATP-driven calcium pump in the sarcoplasmic reticulum membranes reaches back to the postwar (World War II) years and would not be possible without the generous support by the American scientific community. It was this community that in pre- and postwar years gave shelter to many European scientists, which in return stimulated scientific development in the United States. These pre- and postwar relations helped to establish the calcium pump as a physiologically relevant mechanism in all kinds of cells. The pump and its counterpart, the calcium release channel, proved to be controlled by various intrinsic mechanisms. Rising hydrogen concentrations as occurring in ischemic muscles switch off pump activity and counteract allosterically caffeine-induced calcium release (CICR). Rising phosphate or the presence of other calcium-precipitating anions, on the other hand, prevents pump inhibition by intraluminal calcium precipitation, which, simultaneously, can increase the quantity of releasable calcium. The inactivation of CICR by removing medium chloride must be considered as a hint of additional mechanisms by which calcium-dependent activity regulation can be modified.

Animals↗
PubMed1998

Cations and anions as modifiers of ryanodine binding to the skeletal muscle calcium release channel.

Rate and equilibrium measurements of ryanodine binding to terminal cysternae fractions of heavy sarcoplasmic reticulum vesicles demonstrate that its activation by high concentrations of monovalent salts is based on neither elevated osmolarity nor ionic strength. The effect of the ions specifically depends on their chemical nature following the Hofmeister ion series for cations (Li+ < NH+4 < K- approximately Cs+ </= Na+) and anions (gluconate- < Cl- < NO3- approximately ClO4- approximately SCN-) respectively, indicating that both are involved in the formation of the salt-protein complex that can react with ryanodine. Activation by rising salt concentrations exhibits saturation kinetics with different dissociation constants (25-11 m) and different degrees of cooperativity (n = 1.5-4.0) for the respective salts. Maximal second order binding rates between 40,000 and 80,000 (m-1 x sec-1) were obtained for chlorides and nitrates of 1a group alkali ions with the exception of lithium supporting only rates of maximally 10,000 (M-1 x sec-1). The nitrogen bases, NH+4 and Tris+, in combination with chloride or nitrate, behave divergently. High maximal binding rates were achieved only with NH4NO3. The dissociation constants for the ryanodine-protein complexes obtained by measurements at equilibrium proved to depend differently on salt concentration, yet, converging to 1-3 nm for the applied salts at saturating concentrations. The salts do not affect dissociation of the ryanodine protein complex proving that the effect of salts on the protein's affinity for ryanodine is determined by their effect on the on-rate of ryanodine binding. ATP and its analogues modify salt action resulting in elevated maximal binding rates and reduction or abolition of binding cooperativity. Linear relations have been obtained by comparing the rates of ryanodine binding at different salt concentrations with the rates or the initial amplitudes (15 sec) of salt induced calcium release from actively loaded heavy vesicles indicating that the various salts promote specifically and concentration dependently channel opening and its reaction with ryanodine.

Adenine Nucleotides↗
PubMed1994

Effect of phospholipid hydrolysis by phospholipase A2 on the kinetics of antagonist binding to cardiac muscarinic receptors.

Activation of phospholipases during prolonged myocardial ischemia could contribute to the functional derangement of myocardial cells by altering the phospholipid environment of a number of membrane bound proteins including receptors. The present study examined the kinetics of muscarinic receptor antagonist [3H]quinuclidinyl benzilate binding ([3H]QNB) to muscarinic receptors of highly purified sarcolemmal membranes under control conditions and after treatment with phospholipase A2 (PLA2; EC 3.1.1.4). Initial binding rates of QNB exhibited saturation kinetics, when plotted against the ligand concentration in control and PLA2 treated sarcolemmal membranes. This kinetic behaviour of QNB-binding is consistent with at least a two step binding mechanism. According to this two step binding hypothesis an unstable intermediate receptor-QNB complex (R*QNB) forms rapidly, and this form undergoes a slow conversion to the high affinity ligand-receptor complex R-QNB. The Michaelis constant Km of R-QNB formation was 1.8 nM, whereas the dissociation constant Kd obtained from equilibrium measurements was 0.062 nM. After 5 min exposure of sarcolemmal membranes to PLA2QNB binding capacity (Bmax) was reduced by 62%, and the affinity of the remaining receptor sites was decreased by 47% (Kd = 0.116 nM). This PLA2-induced increase of Kd was accompanied by a corresponding increase of Km, whereas the rate constants k2 and k-2 of the hypothetical slow conversion step (second reaction step) remained unchanged. These results suggest that binding of QNB to cardiac muscarinic receptors induces a transition in the receptor-ligand configuration, which is necessary for the formation of the final high affinity R-QNB complex. PLA2-induced changes of the lipid environment result in the inability of a part of the receptor population to undergo this transition, thereby inhibiting high affinity QNB-binding.

Animals↗
PubMed1994

Calcium uptake.

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Animals↗
PubMed1992

Expression of myosin heavy and light chains and phosphorylation of the phosphorylatable myosin light chain in the heart ventricle of the European hamster during hibernation and in summer.

We investigated the expression of myosin subunits (myosin heavy chains) as well as light chains and the in vivo phosphorylation of the phosphorylatable myosin light chain in the heart ventricle of the adult male European hamster (Cricetus cricetus L.). Two myosin heavy chain isoenzymes could be detected under native and denaturing electrophoretic conditions having high (alpha-myosin heavy chain) and low (beta-myosin heavy chain) enzymatic activity. Enzymatic activity of alpha- and beta-myosin heavy chain revealed a different temperature dependency. When temperature increased ATPase activity of the alpha-myosin heavy chain isoenzyme increased relatively more than ATPase activity of the beta-myosin heavy chain isoenzyme. Summer animals expressed predominantly the beta-myosin heavy chain (79% of total myosin) while during hibernation the alpha-myosin heavy chain expression increased to 53% of total myosin. Winter-active hamsters kept at 22 degrees C and 12 h day/night rhythm showed the same myosin heavy chain isoenzyme pattern as summer-active animals. Two myosin light chain forms were expressed in the ventricle of all animal groups. The in vivo phosphorylation level of the phosphorylatable myosin light chain decreased from 45% in summer-active hamster to 23% during hibernation.

Animals↗
PubMed1991

Volume changes in high-affinity calcium binding of the sarcoplasmic reticulum calcium-transport enzyme.

The effect which hydrostatic pressure exerts on the hydrolysis of dinitrophenyl phosphate and nitrophenyl phosphate by the sarcoplasmic reticulum calcium-transport enzyme was determined. Activation volumes for substrate hydrolysis at saturating and non-saturating concentrations of calcium were determined and used to evaluate volume increments for initial calcium binding. A reaction scheme in which two unidirectional substrate-driven reactions transfer high-affinity into low-affinity calcium-binding sites was applied to determine binding-volume increments. It has been inferred from the pressure dependence of the volume-generating function, defined as the difference between the reciprocal reaction rates of the saturated and the unsaturated enzyme, that calcium binding proceeds in two steps. The two associated binding constants are endowed with large binding-volume increments of opposite signs (+84 to +207 ml/mol and -3 to -136 ml/mol). Under different experimental conditions, with respect to the temperature, degree of calcium saturation and absence or presence of Me2SO, they add up to the same integral volume increment of 73 +/- 3.5 ml/mol for the entry of two calcium ions into the reaction cycle. In aqueous media, the two binding constants contribute about equally to binding and to the observed binding-volume increment. The presence of Me2SO strongly favours the first binding step. The size of the integral volume increment is in line with that determined for the interaction of calcium with calmodulin [Kupke, D.W. & Dorrier, T.E. (1986) Biochem. Biophys. Res. Commun. 38, 199-204].

2,4-Dinitrophenol↗
PubMed1991

Pressure effects on the binding of vanadate to the sarcoplasmic reticulum calcium-transport enzyme.

The effect which hydrostatic pressure exerts on the binding of vanadate to the calcium-transport enzyme was determined. The recent unavailability of radioactive vanadate prevented direct measurements of vanadate binding. The vanadate-free enzyme fraction was instead monitored by phosphorylating it with ATP according to Medda and Hasselbach [Medda, P. & Hasselbach, W. (1983) Eur. J. Biochem. 137, 7-14]. Vanadate binding is reduced with rising pressure at first markedly and subsequently, above 30 MPa, relatively little. The biphasic pressure-binding relationship was analysed by applying a biexponential fitting procedure to the experimental data. The biphasicity of the pressure-binding relationship indicates that the description of vanadate binding requires at least a two-step reaction sequence. The volume increments which predominate at lower pressure values, range from 200-400 ml.mol-1 depending on the composition of the reaction medium containing 5 microM and 20 microM vanadate and no or 15% (by vol.) Me2SO. The binding volumes deduced for the higher pressure range amount to 20-40 ml.mol-1. Vanadate binding is reduced in the presence of 30 microM calcium, and simultaneously both binding volumes are diminished by 100 ml.mol-1 and 20 ml.mol-1 for the low and high pressure values, respectively, as one can expect for mutual interactions between the two ligands of the transport enzyme.

Adenosine Triphosphate↗
PubMed1991

Activation and binding volumes of the sarcoplasmic reticulum transport enzyme activated by calcium or strontium.

The effect of pressure on the hydrolysis of dinitrophenyl phosphate (DnpP) and p-nitrophenyl phosphate (NpP) by the sarcoplasmatic reticulum transport enzyme in permeabilized and native closed vesicles activated by calcium or strontium, respectively, in aqueous and Me2SO-containing media has been studied. At atmospheric pressure, the enzyme in permeabilized vesicles, saturated with respect to substrates and activating ions, hydrolyzes DnpP ten times faster than NpP; for both substrates, calcium activation exceeds that by strontium only a little (20%). In aqueous media the enzyme displays, under all activating conditions, an almost identical curvilinear relationship between the logarithm of enzyme activity and pressure. The data were analysed on the basis of a simplified reaction scheme, in which two unidirectionally proceeding substrate-driven pressure-dependent reactions (k2, k4) cyclically transfer high-affinity into low-affinity binding sites which are assumed to be in equilibrium with either calcium or strontium. The fitting procedure yielded two sets of positive activation volumes delta V2* = 90-110 ml/mol and delta V4* = 15-25 ml/mol. Substrate specificity, as well as the effect of temperature, are exclusively localized in the pressure-independent rate constants k'2 and k'4. Considerable different pressure/activity relations characterized by a single activation volume of 20 ml/mol were obtained for the strongly suppressed substrate hydrolysis of native closed vesicles. At atmospheric pressure DnpP hydrolysis of open vesicles is inhibited by Me2SO, while NpP hydrolysis is considerably activated, irrespective of its activation by calcium or strontium. In the presence of 22.5% Me2SO, the activation volumes are reduced by 50-70 ml/mol. The rate constants of DnpP and NpP hydrolysis are either augmented or reduced by rising Me2SO concentrations, depending on the corresponding supporting substrate. Me2SO has only a slight effect on the pressure dependence of substrate hydrolysis by native vesicles. The small activation volume observed for the activity of native vesicles could be assigned on account of the simplified reaction scheme of the slow reaction step k4, by which the enzyme is transferred from its low-affinity into its high-affinity binding state. Volume changes connected with the binding of calcium or strontium to the luminal binding site of the enzyme were deduced from the observed activation volume and the computed volume change of the slow reaction step (delta V4*).

Animals↗
PubMed1990

Blockage of a pump-related calcium-efflux pathway in light sarcoplasmic reticulum vesicles by Mops.

Mops, used as a proton buffer, specifically enhances the accumulation of calcium or strontium by light sarcoplasmic reticulum vesicles driven by ATP or dinitrophenylphosphate as energy-yielding substrates when calcium-precipitating agents are absent. The enhancement of ion uptake by Mops is much greater for strontium than for calcium and is further increased when potassium is replaced by sodium as the dominant monovalent cation. Mops affects neither the activity of the calcium- or strontium-activated transport enzyme nor the active accumulation of calcium in the presence of oxalate, i.e. when the pump runs unidirectionally forward. Passive calcium and strontium efflux rates of approximately 40-50 nmol.mg-1.min-1 are considerably reduced when histidine/glycerophosphate or Tris/maleate are exchanged for Mops. The observed passive efflux rates and their modulation by Mops are too small, in relation to the rate of ion influx, to account for either the relatively small calcium and strontium load in the absence of precipitating agents or for its modulation by Mops. The results imply that the pump itself mediates ion efflux dependent on pump activity and the different degree of saturation of lumenal ion-binding sites by calcium and strontium, as well as their susceptibility to Mops.

Adenosine Triphosphate↗
PubMed1990

Participation of a non-covalent phosphointermediate in ATP hydrolysis by the sarcoplasmic reticulum Ca2(+)-cotransport ATPase.

With increasing SDS/protein ratios, covalent phosphorylation by ATP and Pi is abolished before ATP hydrolysis (Pi production) ceases. We have shown that the SDS-dependent profiles of the decline in covalent phosphorylation by either substrate are virtually identical, reflecting a common mechanism of detergent interaction, while ATP can be hydrolysed via a non-covalent phosphointermediate. Our studies support that the transfer of both terminal Pi from ATP, as well as Pi to its final binding site, is a multistep reaction involving electrostatic interaction with one or more amino acid side chains, including a Lys residue.

Adenosine Triphosphate↗
PubMed1990

Effect of pressure and calcium on the reversible inhibition of the sarcoplasmic-reticulum calcium-transport enzyme and on its tryptic cleavage pattern.

The reversible inhibition of the sarcoplasmic-reticulum calcium-transport enzyme by pressure at room temperature is accompanied by a significant enhancement of the accessibility of the enzyme to tryptic cleavage dependent on the presence of calcium. The calcium-transport enzyme activity was monitored with dinitrophenyl phosphate as substrate. Pressure in the range 0.1-100.0 MPa affects trypsin cleavage of the control substrate N-alpha-benzoyl-L-arginine-4-nitroanilide hydrochloride little in the presence and absence of calcium. In contrast, application of 100.0 MPa to the calcium-transport enzyme at room temperature accelerates subsequent tryptic cleavage at the T2 but not at the T1 cleavage site [C. J. Brandl et al. (1986) Cell 44, 597-607]. Pressure application during tryptic digestion likewise solely affects cleavage at T2 which proceeds slowly in the absence but rapidly in the presence of calcium. At atmospheric pressure in the absence of calcium and at high pressure in the absence and presence of calcium new cleavage sites are exposed giving rise to new subfragments B1-3 in addition to the established peptides A1 and A2. Under pressure and in the presence of calcium, A1 and A2 rapidly disappear indicating the presence of calcium-binding sites in these peptides. In contrast, the B1-3 peptides which are most likely derivates of the B fragment accumulate in the presence and absence of calcium. In contrast to tryptic cleavage at atmospheric pressure, tryptic cleavage of the A as well as the B fragment tends to completion under pressure. In parallel to the disappearance of the A and B fragments calcium-dependent substrate hydrolysis vanishes. Computation of activation volumes for pressure-induced reversible enzyme inhibition and for tryptic cleavage furnished closely related volumes of opposite signs of 20-40 ml/mol and 80-100 ml/mol in the ranges 0.1-40.0 MPa and 40.0-100.0 MPa, respectively. Thus pressure produces reversible changes in the calcium-transport enzyme which activates and modifies tryptic-cleavage patterns at the T2 site of the A segment and at sites in its subfragments in the presence of calcium, i.e. if the enzyme residues in its E1 state. In contrast tryptic cleavage of the B fragment is accelerated by pressure independently of the presence of calcium.

Animals↗
PubMed1990

On the problem of season and cold dependence of calcium transport by skeletal muscle sarcoplasmic reticulum.

Calcium transport of skeletal muscle sarcoplasmic reticulum from golden hamsters was studied in January and in June on animals kept at 22 degrees C under natural photoperiod and in January after cold-acclimation at +/- 2 degrees C in the dark for 55 days. Crude homogenates from psoas and soleus muscles and from mixed skeletal muscles were used. No differences were observed in the calcium storing capacity of sarcoplasmic reticulum among the three groups of animals. Kinetic studies on the dependence of the calcium uptake rate on the concentration of free calcium revealed a significant increase of the uptake rates and a decrease of the calcium affinity in the control animals sacrificed in winter as compared to those killed in June. Cold-acclimation in winter leads to a further small reduction of the calcium affinity. This shift of calcium uptake rate and affinity in the sense of that of a fast-twitch muscle may be related to the functional demands of the cold season and cold-acclimation respectively.

Acclimatization↗
PubMed1989

The discrepancy between ryanodine binding and its effects on the calcium releasing system of the sarcoplasmic reticulum.

Heavy sarcoplasmic reticulum vesicles isolated from rabbit skeletal muscle were reacted in high ionic strength solutions with ryanodine. The effect of this reaction on ATP - and dinitrophenyl phosphate supported calcium uptake and caffeine induced calcium release were studied. At pH 7.0 calcium uptake and caffeine induced calcium release are simultaneous affected by the occupation of 0.5 pmol ryanodine binding sites/mg protein, having an affinity of 0.33 nM-1.

Alkaloids↗
PubMed1988

Inactivation of detergent-solubilized sarcoplasmic reticulum ATPase.

Inactivation of sarcoplasmic ATPase in the solubilized state was studied in the absence and presence of Ca2+, Mg2+ and glycerol. The effects of the detergents octa(ethyleneglycol) mono-n-dodecyl ether (C12E8), 1-O-tetradecylpropanediol-(1,3)-3-phosphorylcholine and myristoylglycerophosphocholine were compared. All three detergents caused a rapid decline of the dinitrophenyl phosphatase activity of the unprotected enzyme. The stabilizing effect of Ca2+ ions was kinetically analysed. It was found that the stability of the solubilized enzyme depends on the Ca2+ concentration in a manner which is best explained by assuming rapid inactivation of Ca2+-free enzyme accompanied by slow inactivation of a calcium-enzyme complex (E1Ca). The apparent affinity constants obtained are in the order of 10(6)M-1, suggesting that high-affinity Ca2+ binding must be involved. No indications of a contribution were found, either of low-affinity Ca2+-binding sites of the conformational state E2 or of the high-affinity calcium complex E1Ca2. If Ca2+ was replaced by Mg2+, which exerts a weaker protection, the apparent affinity constants for Mg2+ are in the range of 1 mM-1. The stoichiometry of the effect of Mg2+ depends on the detergent.

4-Nitrophenylphosphatase↗
PubMed1987

Activation and inhibition of the calcium gate of sarcoplasmic reticulum by high-affinity ryanodine binding.

The occupancy of high-affinity ryanodine-binding sites of isolated heavy sarcoplasmic reticulum vesicles occurring in concentrated salt solutions affects ATP-dependent calcium accumulation and caffeine-induced calcium release. The initial suppression of calcium uptake is followed by a marked uptake activation resulting in a reduction of the final calcium level in the medium. Simultaneously, caffeine-induced calcium release is blocked. The dependence of inhibition of calcium uptake and caffeine-induced calcium release observed in assay media containing physiological concentrations of magnesium and ATP on the concentration of ryanodine corresponds to the drug's effectiveness in living muscles.

Adenosine Triphosphate↗
PubMed1987

Electrical pump currents generated by the Ca2+-ATPase of sarcoplasmic reticulum vesicles adsorbed on black lipid membranes.

Sarcoplasmic reticulum vesicles adsorbed on a black lipid membrane generate an electrical current after a fast increment of the concentration of ATP. This demonstrates directly that the sarcoplasmic Ca2+-ATPase from skeletal muscle acts as an electrogenic ion pump. The increment of the concentration of ATP is achieved by the photolysis of caged ATP (P3-1-(2-nitro)phenylethyl adenosine 5'-triphosphate) a protected analogue of ATP (Kaplan, J.H. et al. (1978) Biochemistry 17, 1929-1935), which is split into ATP and 2-nitroso acetophenone. The release of ATP leads to a transient current flow across the lipid membrane indicating that the vesicles are capacitatively coupled to the underlying lipid membrane. In addition to this transient signal, a stationary current flow is obtained in the presence of ionophores which increase the conductance of the bilayer system and prevent the accumulation of Ca2+ in the lumen of the vesicles. The direction of the transient and the stationary current is in accordance with the concept that Ca2+ is pumped into the lumen of the vesicles. The transient current depends on the concentration of ATP, Ca2+ and Mg2+ as would be the case for a current generated by the sarcoplasmic Ca2+-ATPase. Its amplitude is half-maximal at 10 microM ATP and 1 microM Ca2+. At Ca2+ concentrations above 0.1 mM the amplitude of the current signal declines again. The Mg2+ concentration dependence of the current amplitude at a constant ATP concentration indicates that the MgATP complex is the substrate for the activation of the current. The pump current is inhibited by vanadate and ADP. No current signal is observed if caged ATP is replaced by caged ADP. However, the release of ADP from caged ADP generates a pump current in the presence of an ATP generating system such as creatine phosphate and creatine kinase.

Adenosine Diphosphate↗
PubMed1987

The effect of monovalent and divalent cations on the ATP-dependent Ca2+-binding and phosphorylation during the reaction cycle of the sarcoplasmic reticulum Ca2+-transport ATPase.

The coupling of Ca2+ movements and phosphate fluxes as well as the time-dependent occurrence of sequential reaction intermediates in the forward mode of the Ca,Mg-dependent ATPase reaction have been investigated using leaky vesicles (A23187) in the presence of varying Ca2+, Mg2+, and K+ concentrations. The employed ATP concentration of 2 microM does not allow more than one reaction cycle to occur. The respective fractions of ADP-sensitive and ADP-insensitive phosphoenzyme have been determined. The chosen experimental conditions (0-1 degree C, pH 6.0, absence of solubilizers) allow a prolonged time of observation and exclude interfering alterations of coupling and binding parameters, respectively. It is shown that under the experimental conditions K+ interacts with at least four different reaction steps (phosphoenzyme formation, E1P----E2P transition, E2P hydrolysis, and E2----E1 transformation). Mg2+ represents the sole ionic co-factor for the formation of the substrate MgATP if it is present in high concentrations (5 mM). Additional Ca2+ is bound to the substrate as well as to unspecific sites otherwise occupied by Mg2+ if Mg2+ is reduced to 0.1 mM. In this case the E1P----E2P transition rate (including Ca2+ translocation and Ca2+ release from low-affinity sites) is little diminished. If, in the absence of K+, both Mg2+ and Ca2+ are deficient E2P hydrolysis is vastly retarded. We find Ca2+ release to occur time-coincidently with E1P formation and not concomitantly with the comparably slow appearance of E2P; the molar amount of Ca2+ released, however, rather agreed with that of E2P formed. This suggests that under the prevailing conditions of a high proton concentration, phosphoenzyme states containing occluded Ca2+ or Ca2+ bound to low-affinity sites are transitional and not detectable. Preliminary findings on this subject have been published by us and colleagues from this laboratory [Hasselbach, W., Agostini, B., Medda, P., Migala, A. & Waas, W. (1985) in The sarcoplasmic reticulum calcium pump: Early and recent developments critically overviewed (Fleischer, S. & Tonomura, Y., eds) pp. 19-49, Academic Press, Orlando].

Adenosine Triphosphate↗