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W Hasselbach

Publications and source records attributed to W Hasselbach.

At least 19 recordsLinked to original sources
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↗
PubMed1979

Effects of phospholipase A2 and albumin on the calcium-dependent ATPase and the lipid composition of sarcoplasmic membranes.

1. The calcium-dependent ATPase activity of phospholipase-A2-digested sarcoplasmic vesicles decreases concomitantly with the contents of residual lysophospholipids and fatty acids when increasing albumin concentrations are applied. 2. Delipidated albumin preferentially removes unsaturated fatty acids and lysophosphatidylcholine. A complete removal of the phospholipids by albumin does not occur. 3. The membrane-bound lysophospholipids were analysed with respect to type of phospholipid, plasmalogen content and fatty acid chains by means of thin-layer chromatography and gas chromatography. 4. While the fatty acid composition of the lysophospholipids is independent of the degree of delipidation, the composition of the residual free fatty acids is found to change with the albumin concentration. 5. Reactivation of the Ca2+-ATPase by oleate leads to reasonable activities at room temperature as long as a minimum of about 30 lysophospholipid molec-les per ATPase is left. The course of the residual Ca2+-ATPase activity with the degree of delipidation is related to the presence of unsaturated fatty acids. 6. No specific role of either sphingomyelin or the plasmalogens has been found.

Animals↗
PubMed1979

Comparison between ATP-supported and GTP-supported phosphate turnover of the calcium-transporting sarcoplasmic reticulum membranes.

The study deals with the interrelationship of the phosphate-transferring activities of the calcium-transporting sarcoplasmic reticulum membrane vesicles: the phosphate exchange between nucleoside triphosphate (NTP) and nucleoside diphosphate (NDP) (NTP-NDP exchange), the calcium-dependent NTase, and the phosphorylation of NDP by inorganic phosphate in the presence of NTP (NTP-Pi exchange). Different nucleotides were used as phosphate donors and acceptors. It is demonstrated for the phosphate transfer from ITP to GDP that the NTP-NDP exchange exhibits ping-pong kinetics with Mg-ITP and unliganded GDP as substrates. The apparent affinities of the enzyme for the nucleoside diphosphate and triphosphate species are deduced according to this mechanism. The enzyme's affinity for the nucleoside triphosphates and diphosphates depends on its functional state being considerably lower under conditions of NTP-NDP exchange than during NTP splitting or NTP synthesis. ATP and GTP are split with the same low rates when calcium-activated NTPase is inhibited by high internal calcium concentrations after calcium transport has reached steady state. The rates of the NTP-NDP exchange reactions, however, differ by a factor of about 10 being approximately equal to 3 mumol . mg-1 . min-1 for ATP-ADP and only approximately equal to 0.3 mumol . mg-1 . min-1 (22 degrees C) for GTP-GDP. When the sarcoplasmic reticulum vesicles are made calcium-permeable, the calcium transport ATPase is turned on and the rates of GTP and ATP splitting increase about tenfold. Yet, while the rate of ATP-ADP exchange is little reduced, the rate of GTP-GDP exchange drops by approximately 50%. The persisting exchange activity of calcium-permeable vesicles demonstrates that high internal calcium concentrations are not required for the transfer of the protein-bound phosphoryl group to NDP during NTP-NDP exchange.

Adenosine Diphosphate↗
PubMed1978

The binding of calcium and magnesium to sarcoplasmic reticulum vesicles as studied by manganese electron paramagnetic resonance.

The binding pattern of the biologically relevant ions calcium and magnesium has been investigated via the binding of the ion-analogue manganese. The binding parameters of manganese are obtained conveniently by standard electron paramagnetic resonance techniques, the binding of calcium and magnesium is inferred from competition experiments. The quantitative analysis was carried out with a computer program which was able to treat the competition of three kinds of ions for three classes of independent and one class of cooperative binding sites. It was possible to correlate specific binding classes with biological functions of the sarcoplasmic reticulum membrane. The magnesium and manganese specific binding class of medium affinity is related to the catalytic function of these ions in the ATP-splitting. For the first time a manganese and calcium specific class of cooperative binding sites has been observed and it can be related to the inhibition of the calcium transport at high concentrations of manganese or calcium. From the binding results a model can be developed which allows a full description of the role of the divalent ions and their specific binding sites during calcium transport.

Adenosine Triphosphatases↗
PubMed1977

Phosphorylation from inorganic phosphate and ATP synthesis of sarcoplasmic membranes.

The incorporation of inorganic phosphate in the fragmented sarcoplasmic membranes induced by the removal of calcium ions bound to high affinity binding sites at the cytoplasmic surface of the membranes gives rise to the formation of two species of phosphoenzyme. The properties of the phosphoproteins formed depend on the absence or the presence of a gradient of calcium ions across the membranes. The phosphoenzymes differ by the affinity of the protein for phosphate, the enthalpy of formation, the kinetics of phosphate incorporation, and by the sensitivity to ionophores and ADP. In the absence of a calcium gradient less than 0.5 nmol phosphoenzyme per mg protein are formed in media containing less than 5 mM phosphate at pH7 and 10 degrees C. Under the same conditions approximately 2 nmol of phosphoenzyme per mg protein are formed with an initial rate of 0.5 nmol mg-1-s-1 when a calcium gradient exists. When the gradient is abolished by the addition of the ionophore X537A, the level of phosphoprotein drops to the same value as observed in the absence of a gradient. On addition of ADP at concentrations increasing from 0.3 to 10 muM continuous ATP formation is activated to its maximum rate, and simultaneously, the level of phosphoprotein declines. These concentrations of ADP scarcely affect phosphoprotein formed in the absence of a gradient, the phosphoryl residue of which is displaced when the concentration of ADP exceeds 10 micrometer without the formation of an equivalent amount of ATP. Minimum mechanisms for the formation of gradient-independent and gradient-dependent phosphoprotein are discussed.

Adenosine Diphosphate↗
PubMed1977

The sarcoplasmic calcium pump - a most efficient ion translocating system.

In contrast to the sodium-potassium transporting plasma membranes, the sarcoplasmic membranes (SR) are highly specialized structures into which only two major intrinsic proteins, a calcium transporting protein and a calcium binding protein are embedded. The calcium transporting protein is a highly asymmetric molecule. It binds two calcium ions with a very high affinity at its external, and two calcium ions with low affinity at the internal section of the molecule. ATP is bound with high afffinity to an external binding site, inducing a conformational change. When the vesicular membranes are exposed to solutions containing Ca++, Mg++ and ATP, ATP is hydrolyzed and simultaneously calcium ions are translocated from the external medium into the vesicular space. When calcium ions are translocated in the opposite direction, ATP is synthesized. The calcium-ATP ratio for ATP cleavage as well as for ATP synthesis is 2. Thus, the SR membranes can transform reversibly chemical into osmotical energy. Inward and outward movements of calcium ions are relatively slow processes connected with the appearance and disappearance of different phosphorylated intermediates. One phosphorylated intermediate is formed by phosphoryltransfer from ATP when calcium ions are present in the medium. In contrast, when calcium ions are absent from the external medium, two different intermediates can be formed by the incorporation of inorganic phosphate. Only when calcium ions present in the internal space of the vesicles are released, the incorporation of inorganic phosphate gives rise to an intermediate who phosphoryl group can be transferred to ADP.

Biological Transport, Active↗
PubMed1977

Stimulatory and inhibitory effects of dimethyl sulfoxide and ethylene glycol on ATPase activity and calcium transport of sarcoplasmic membranes.

1. The effect of dimethyl sulfoxide (Me2SO) and ethylene glycol on two different preparations of the sarcoplasmic reticulum, i.e. native membranes and membranes whose phospholipids were hydrolyzed by phospholipase A, were investigated using ATP and p-nitrophenylphosphate as substrates. 2. Me2SO and ethylene glycol inhibit both calcium-dependent ATP hydrolysis and ATP-supported calcium transport by native vesicles. 3. In contrast, calcium-dependent p-nitrophenylphosphatase activity as well as p-nitrophenyl-phosphate-supported calcium transport are activated by both agents at concentrations lower than 30% (v/v). 4. Me2SO strongly stimulates p-nitrophenylphosphate activity of vesicles treated with phospholipase A, but has relatively little effect on p-nitrophenylphosphatase activity of native vesicles. 5. Up to a concentration of approximately 40% Me2SO (v/v) the inhibiting effect on the calcium-dependent ATPase is fully reversible, but only partially reversible on calcium transport. 6. In the concentration range where Me2SO inhibits ATP hydrolysis and calcium transport, it does not affect ATP binding to the membranes nor calcium-dependent formation of phospho-protein. 7. The rate of dephosphorylation as well as the rate of Pi exchange between ATP and ADP are markedly reduced by the presence of 30% Me2SO (v/v). 8. While Me2SO inhibits passive calcium efflux, ethylene glycol produces a considerable activation. 9. ADP-dependent calcium efflux and ATP synthesis are activated by 15% Me2SO (v/v). Ethylene glycol reduces both activities. 10. The results suggest that the respective substrate-enzyme complexes are differently affected by the agents, resulting either in inhibition or stimulation

4-Nitrophenylphosphatase↗
PubMed1977

Dependence of ionophore- and caffeine-induced calcium release from sarcoplasmic reticulum vesicles on external and internal calcium ion concentrations.

The effects of the ionophore, X537A, and caffeine on ATP-dependent calcium transport by fragmented sarcoplasmic reticulum were studied in the absence (calcium storage) or presence (calcium uptake) of calcium-precipitating anions. The ionophore caused rapid calcium release after calcium storage, the final level of calcium storage being the same whether a given concentration of X537A was added prior to initiation of the reaction or after calcium storage had reached a steady state. Although 10 to 12 muM X537A caused approximately 90% inhibition of oxalate-supported calcium uptake when added prior to the start of the reaction, this ionophore concentration caused only a small calcium release when added after a calcium oxalate precipitate had formed within the vesicles, and only slight inhibition of calcium uptake velocity when added during the calcium uptake reaction. When low initial calcium loads limited calcium uptake to 0.4 mumol of calcium/mg of protein, subsequent calcium additions in the absence of the ionophore led to renewed calcium uptake. Uptake of the subsequent calcium additions was not significantly inhibited by 10 to 12 muM X537A. These phenomena are most readily understood in terms of constraints imposed by fixed Cai (calcium ion concentration inside the vesicles) on the pump-leak situation in sarcoplasmic reticulum vesicles containing a large amount of an insoluble calcium precipitate, where most of the calcium is within the vesicles and Cai is maintained at a relatively low level. These constraints restrict calcium loss after calcium permeability is increased because calcium release can end when the calcium pump is stimulated by the increased Cao (calcium concentration outside the vesicles) so as to compensate for the increased efflux rate. In contrast, an increased permeability in vesicles that have stored calcium in the absence of a calcium-precipitating ion causes a much larger portion of the internal calcium store to be released. Under these conditions calcium storage capacity is low so that release of stored calcium is less able to raise Cao to levels where the calcium pump can compensate for the increased efflux rate. The constraints imposed by anion-supported calcium uptake explain the finding that more calcium is released by X537A or caffeine when these agents are added at higher levels of Cao, and that more calcium leaves the vesicles in response to a given increase in calcium permeability at higher Cai. Although such calcium release is amplified by increased Cao, the amplification is attributable to the constraints described above and does not represent a "calcium-triggered calcium release."

Animals↗
PubMed1977

Dependence of calcium permeability of sarcoplasmic reticulum vesicles on external and internal calcium ion concentrations.

The ability of sarcoplasmic reticulum vesicles to retain calcium following ATP-supported calcium uptake in the presence of the calcium-precipitating anions oxalate and phosphate depends on Cao (calcium ion concentration outside the vesicles) and Cai (calcium ion concentration within the vesicles). Calcium efflux rates at any level of Cai are accelerated when Cao is increased. Higher Cao at the time that calcium uptake reactions reach steady state is associated with a spontaneous calcium release that reflects this effect of increased Cao. Increasing Cai at any level of Cao causes little or no acceleration of calcium efflux rate so that calcium permeability coefficients, estimated by dividing calcium efflux rates by Cai, the "driving force", are inversely proportional to Cai. Calcium permability coefficients thus correlate, as a first approximation, with the ratio Cai/Cao, decreasing 1000-fold as this ratio increases over a 3000-fold range (Cao = 0.1 to 3.3 muM, Cai =4 to 750 muM). Oscillations in both the calcium content of the vesicles and Cao are seen as calcium uptake reactions approach steady state, suggesting that calcium permeability undergoes time-dependent variations. Sudden reduction of Cao to levels that markedly inhibit calcium influx via the calcium pump unmasks a calcium efflux that decreases slowly over 60 to 90 s. The maximal calcium permeability observed in the present study would allow the calcium efflux rate from the sarcoplasmic reticulum at a Cai of 100 muM to be approximately 10(-10) mol/cm2/s, which is about 1 order of magnitude less than that estimated for the sarcoplasmic reticulum of activated skeletal muscle in vivo. The release of most of the stored calcium in some experiments indicates that the observed permeability changes can occur over a large portion of the surface of the sarcoplasmic reticulum.

Animals↗
PubMed1977

Effect of 1,25-dihydroxycholecalciferol on impaired calcium transport by the sarcoplasmic reticulum in experimental uremia.

In the fragmented sarcoplasmic reticulum from skeletal muscle of rabbits with experimental uremia, defective calcium ion transport is found. An impairment of all parameters is observed (initial rate of uptake, storing capacity with and without oxalate, and concentrating ability). In vivo administration of 1,25-dihydroxycholecalciferol (1,25-(OH)2-vitamin D3)(2 X 27 ng X kg of body wt-1 X day-1 and 6 X 27 ng X kg-1 X day-1, respectively) improved the kinetic parameters. The low dose improved storing capacity, and the higher dose, in addition to the storing capacity, also corrected concentrating ability and the initial rate of uptake. It is concluded that active calcium transport in the sarcoplasmic reticulum is impaired by uremia and that this defect is responsive to the administration of 1,25-(OH)2-vitamin D3.

Animals↗
PubMed1976

Comparison between strontium and calcium uptake by the fragmented sarcoplasmic reticulum.

The ATP-supported uptake of strontium by the fragmented sarcoplasmic reticulum is monophasic and proceeds more rapidly than the fast uptake of calcium. Strontium uptake is not activated by Pi. The accumulation of strontium is nearly proportional to the external strontium concentration even in the millimolar range. Internal and external strontium quickly equilibrate. One mole of strontium is stored for every mole of ATP split by the Sr2+-activated ATPase. In the absence of oxalate most of the strontium is taken up with a transport ratio of one. On the opposite, the transport ratio of calcium decreases immediately, especially when ADP is not instantaneously phosphorylated to ATP. In this case, energy conversion is uncoupled more effectively by the simultaneous action of ADP and free internal calcium, resulting in the interruption of the fast uptake. After depletion of ATP most of the stored strontium is released and the remaining fraction appears to be not exchangeable. Strontium activates the slow uptake of calcium, but reduces the amplitude of the fast uptake. The calcium induced release of strontium, and vice versa, is partial and transient. The strontium activated ATPase does not transport calcium at low ionic calcium concentrations.

Adenosine Triphosphatases↗
PubMed1976

The biphasic active transport of calcium by the fragmented sarcoplasmic reticulum as revealed by the flow dialysis method.

The calcium accumulated by the fast uptake has an apparent association constant of 0.8 X 10(6) M-1 and a maximum of 80 nmol/mg protein. The fast uptake and the initial rate of the slow uptake show a similar dependence on the calcium concentration when the latter ranges from 5 to 50 muM. The fast uptake is a linear and the slow uptake rate an exponential function of the reticulum concentration. Both uptakes of calcium display a fast and nearly total isotopic equilibration between intra- and extravesicular calcium. After depletion of ATP the calcium accumulated during the slow uptake is released, while that sequestered during the fast uptake is retained by the vesicles, though it remains rapidly exchangeable. After depletion of ITPor acetylphosphate, or addition of EGTA, the release is more substantial, but is almost complete only after addition of ionophore X537A or deoxycholate. The presence of oxalate strongly reduces the rates of these releases. It is concluded that in the steady state observed after the depletion of ATP, a Ca2+ gradient exists through the sarcoplasmic membrane, and the sarcoplasmic pump works at equilibrium. The fast uptake is an active transport and not an active binding. The slow uptake represents an extension of the calcium capacity of the vesicles due to the phosphate liberated by the sarcoplasmic ATPase.

Adenosine Triphosphate↗
PubMed1976

Characterization of cardiac sarcoplasmic reticulum ATP-ADP phosphate exchange and phosphorylation of the calcium transport adenosine triphosphatase.

1. The terminal phosphate of (gamma-32P)ATP is rapidly incorporated into cardiac sarcoplasmic reticulum membranes (0.7--1.3 mumol/g protein) in the presence of calcium and magnesium. Cardiac sarcoplasmic reticulum membranes catalize an ATP-ADP phosphate exchange in the presence of calcium and magnesium. 2. Half-maximum activation of the phosphoprotein formation and ATP-ADP phosphate exchange is reached at an ionized calcium concentration of about 0.3 muM. The Hill coefficients are 1.3. 3. Transphosphorylation and ATP-ADP phosphate exchange require magnesium and are maximally activated at magnesium concentrations close to or equal to the ATP concentration. 4. The phosphoprotein level is reduced to about 45% at an ADP/ATP ratio of 0.1. The rate of calcium-dependent ATP splitting declines, whilst the rate of the calcium-dependent ATP-ADP phosphate exchange increases when the ADP/ATP ratio is varied from 0.1 to 1. The sum of both, the rate of ATP splitting and the rate of ADP-ATP phosphate exchange remains constant. 5. Phosphoprotein formation and ATP-ADP phosphate exchange are not affected by azide, dinitrophenol, dicyclohexyl carbodiimide and oubain, whilst both activities are reduced by blockade of -SH groups localized on the outside of the sarcoplasmic reticulum membrane. 6. The isolated phosphoprotein is acid stable. The trichloroacetic acid denatured 32P-labelled membrane complex is dephosphorylated by hydroxylamine, which might indicate that the phosphorylated protein is an acyl-phosphate. 7. Polyacrylamide gel elctrophoresis (performed with phenol/acetic acid/water) of phosphorylated sarcoplasmic reticulum fractions demonstrates that the 32P-incorporation occurs into a protein of about 100000 molecular weight. 8. It is suggested that the phosphoprotein represents a phosphorylated intermediate of the calcium-dependent ATPase which formation occurs as an early step in the reaction sequence of calcium translocation by cardiac sarcoplasmic reticulum similar as in skeletal muscle.

Adenosine Diphosphate↗