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W Hasselbach

Publications and source records attributed to W Hasselbach.

At least 37 records · Page 2Linked to original sources
PubMed1987

Phosphorylation of the sarcoplasmic calcium-activated adenosinetriphosphatase as studied by 31P nuclear magnetic resonance.

A reinvestigation of a study of Fossel et al. [Fossel, E. T., Post, R. L., O'Hara, D.S., & Smith, T. W. (1981) Biochemistry 20, 7215-7219] in which the 31P nuclear magnetic resonance (NMR) signal of the phosphointermediate of the sarcoplasmic (Ca2+, Mg2+)-ATPase has been identified shows that the signal they describe most probably originates from free Mg . ATP but not from the phosphoenzyme itself. It was possible to detect the 31P NMR signal of the phosphoenzyme in peptic fragments of sarcoplasmic ATPase phosphorylated either by ATP or by inorganic phosphate. The two products exhibit the same spectral characteristics in 31P NMR, implying that most probably both reaction pathways yield the same chemical product. Chemical shifts at low pH (-6.5 ppm) and high pH (-1.4 ppm) of the phosphoryl group are indicative of a beta-phosphoaspartyl moiety, thus confirming independently the results from chemical analysis. The relatively low pK value of 4.3 of the phosphoryl group suggests an interaction with a positively charged group of the enzyme.

Animals↗
PubMed1987

Palmitate uptake in calcium tolerant, adult rat myocardial single cells--evidence for an albumin mediated transport across sarcolemma.

The dependence of (1-14C)-palmitate uptake in adult rat heart single cells on albumin was investigated. The apparent initial rate of palmitate uptake vs total palmitate concentration exhibited saturation kinetics, provided the concentration ratio of palmitate vs albumin was kept constant. However, if total palmitate was increased at constant albumin concentrations, the dependence of the initial rate on palmitate concentration was linear. Within the concentration range of total palmitate investigated in this study, the concentration of free palmitate remained almost constant. These results favour the hypothesis, that palmitate uptake in rat myocardium may be mediated by an albumin dependent sarcolemmal transport system.

Albumins↗
PubMed1987

Pressure effects on the interactions of the sarcoplasmic reticulum calcium transport enzyme with calcium and para-nitrophenyl phosphate.

The effect of hydrostatic pressure on calcium dependent p-nitrophenyl phosphate hydrolysis of the sarcoplasmic reticulum calcium transport enzyme has been investigated at different degree of enzyme saturation by calcium and Mg-p-nitrophenyl phosphate to distinguish between activation and binding volumes. The enzyme saturated by both ligands displays a significant dependence of the activation volume on pressure, rising from 20 ml/mol at atmospheric pressure (0.1 MPa) to 80 ml/mol at 100 MPa. At subsaturating concentration of Mg-p-nitrophenyl phosphate an activation volume of 35 ml/mol prevails between 0.1 and 40 MPa. At subsaturating concentration of calcium the activation volume approximates 80 ml/mol in the same pressure range. The binding volume for both substrates is likewise pressure dependent falling from 20 ml/mol to 0 ml/mol for Mg-p-nitrophenyl phosphate and rising from 67 ml/mol to 155 ml/mol for calcium. The pressure dependence of activation and binding volumes is analysed on account of a simplified reaction scheme yielding activation volumes and rate constants for individual reaction steps.

Animals↗
PubMed1986

The dependence on internal pH of Ca2+-fluxes across sarcoplasmic reticulum vesicular membranes.

The interdependence of the competition between Ca2+ and hydrogen ions for the internally located low-affinity Ca2+ binding sites of sarcoplasmic reticulum vesicles and the pH-dependent splitting rate of phosphoenzyme was investigated. Sarcoplasmic reticulum vesicles were preincubated at a selected pH and passive Ca2+ loading, active Ca2+ uptake at the same pH as well as active Ca2+ uptake at a distinct pH (pH-jump method) were observed. In addition, Cai-Cao exchange in the absence and presence of ADP and ATP-ADP exchange were measured. The overall ATP splitting rate was assayed with leaky vesicles in the presence of varied Ca2+ concentration and four different pH. All experiments were carried out at Ca2+ concentrations sufficient to saturate the externally located activating high-affinity binding sites at all pH and in the absence of affecting concentrations of monovalent cations. Active Ca2+ transport (particularly evident applying the pH-jump method) is facilitated at low intravesicular pH, reflecting the favoured Ca2+ release to the intravesicular space, in contrast to the reverse pH-dependence of passive Ca2+ accumulation and the initial rate of Cai-Cao exchange, both favoured by elevated internal Ca2+ binding capacity. The rates of ATP splitting, the continuing slow rate of Cai-Cao exchange, and the ATP-ADP exchange are optimal at an intermediate proton concentration, reflecting the influence of protons on partial reaction steps occurring later in the reaction cycle and the accelerated exchange of Ca2+ at the internal low-affinity sites as well as the establishment of a new pseudo equilibrium between the possible reaction intermediates. The pool of rapidly exchangeable Ca2+ is enlarged whereas the rate of slow exchange is unaltered or diminished (pH 7.8) by ADP.

Adenosine Diphosphate↗
PubMed1985

Preparation of a highly concentrated, completely monomeric, active sarcoplasmic reticulum Ca2+-ATPase.

Sarcoplasmic reticulum vesicles from fast skeletal muscle were partially delipidated with sodium cholate at high ionic strength and sedimented in a discontinuous sucrose gradient. Phospholipid content was reduced from 0.777 mumol/mg protein to 0.242 mumol/mg protein. As judged from gel electrophoresis and high pressure liquid gel chromatography, accessory proteins were removed during centrifugation and the Ca2+-ATPase was obtained in an almost pure form. Addition of myristoylglycerophosphocholine (1 mg/mg protein) reactivates ATPase and dinitrophenylphosphatase activity to the same degree obtained with native vesicles. Using the analytical ultracentrifuge it could be demonstrated that the reactivated Ca2+-ATPase was present exclusively in a monomeric state. These results were obtained at high and low ionic strength and up to a protein concentration of 10 mg/ml. Therefore this preparation should be very useful to investigate differences between oligomeric and monomeric Ca2+-ATPase.

Animals↗
PubMed1985

Presence of a 1,25-dihydroxy-vitamin D3 receptor in chick skeletal muscle myoblasts.

The presence of a specific receptor for 1,25-dihydroxy-vitamin D3 was investigated in myoblasts released from chick embryo skeletal muscle by trypsin and collagenase treatment. Density gradient analysis of the cytosol obtained from these muscle cell preparations showed that 1,25-dihydroxy-vitamin D3 binds specifically to a 3.7 S macromolecule. Scatchard analysis yielded an equilibrium dissociation constant of 2.46 x 10(-10) M and a Nmax of 74 fmol/mg of cytosol protein. The data is in agreement with previous evidence which indicates that the action of the vitamin D metabolite on muscle Ca uptake is mediated by de novo protein and RNA synthesis, and supports the concept that muscle is a target organ for 1,25-dihydroxy-vitamin D3.

Animals↗
PubMed1985

Tryptophan fluorescence of sarcoplasmic reticulum ATPase. A fluorescence quench study.

The calcium-dependent change in the tryptophan fluorescence intensity of the sarcoplasmic reticulum Ca2+- and Mg2+-ATPase was investigated using different quenching reagents. It is demonstrated that only those compounds which are bound to the enzyme (i.e., 1-(9,10-dibromomyristoyl)-sn-2-glycerophosphorylcholine and 1-(9,10-dibromostearoyl)-sn-glycero-3-phosphorylcholine) are able to decrease the amplitude of the fluorescence decrement observed after removal of calcium ions. From the position of the bromine atom within the lysophosphatidylcholines, it is concluded that the tryptophan residues involved are located in the hydrophobic part of the ATPase molecule and are in contact with the hydrocarbon chains of the phospholipids.

Adenosine Triphosphatases↗
PubMed1985

Lipid requirement of the vanadate effect on the binding of calcium and ATP to the calcium transport ATPase of the sarcoplasmic reticulum.

Lipid deprivation of the sarcoplasmic reticulum calcium-transport ATPase neither affects the enzyme's affinity for ATP nor that of calcium. In contrast, vanadate binding is almost completely abolished. Lipid substitution by oleic acid which at a ratio of 0.3 mg/mg protein completely reactivates the calcium-dependent ATP hydrolysis restores vanadate binding. Concomitantly the mutual interactions between vanadate and calcium or ATP and ADP, respectively are restored. The vanadate-induced disappearance of the enzyme's ATP binding sites as well as its high-affinity binding sites for calcium follow the same time course. Conversely, the displacement of vanadate by calcium proceeds in parallel with the recovery of ADP binding. In lipid-restituted preparations as well as in native membranes vanadate induces the disappearance of external high-affinity and simultaneously the appearance of internal low-affinity calcium binding sites.

Adenosine Triphosphate↗
PubMed1985

Kinetics of 13N-ammonia uptake in myocardial single cells indicating potential limitations in its applicability as a marker of myocardial blood flow.

To study kinetics and principles of cellular uptake of 13N-ammonia, a marker of coronary perfusion in myocardial scintigraphy, heart muscle cells of adult rats were isolated by perfusion with collagenase and hyaluronidase. Net uptake of 13N, measured by flow dialysis, reached equilibrium within 20 sec in the presence of sodium bicarbonate and carbon dioxide (pH 7.4, 37 degrees C). Total extraction, 80 sec after the reaction start, was 786 +/- 159 mumol/ml cell volume. Cells destroyed by calcium overload were unable to extract 13N-ammonia. Omission of bicarbonate and carbon dioxide reduced total extraction to 36% of control. 13N-Ammonia uptake could also be reduced by 50 muM 4,4' diisothiocyanostilbene 2,2' disulfonic acid, by 100 micrograms/ml 1-methionine sulfoximine, and by preincubation with 5 muM free oleic acid. These results indicate that in addition to metabolic trapping by glutamine synthetase, the extraction of 13N-ammonia by myocardial cells is influenced by cell membrane integrity, intracellular-extracellular pH gradient, and possibly an anion exchange system for bicarbonate. For this reason, the uptake of 13N-ammonia may not always provide a valid measurement of myocardial perfusion.

Ammonia↗
PubMed1984

State of aggregation of detergent-solubilized sarcoplasmic reticulum adenosine triphosphatase investigated by high-performance liquid chromatography.

The state of aggregation of purified sarcoplasmic reticulum adenosine triphosphatase (ATPase) was investigated by high-performance liquid chromatography (LKB TSK-G 4000 SW column) in the presence of various detergents: sodium dodecylsulphate, dodecyl octaethylene glycol monoether (C12E8), sodium deoxycholate, Triton X-100 and myristoylglycerophosphocholine. When the protein (5 mg ml-1) was solubilized with detergent (2 mg per mg protein) and the column was equilibrated with 1 mg ml-1 of the respective detergent, a molecular weight for the monomeric ATPase protein ranging from 100,000 to 200,000 was obtained. In addition to the monomeric form, significant amounts (more than 20%) of aggregated ATPase protein were observed when C12E8 or deoxycholate was used. These results are in agreement with the observation of a great tendency for self-aggregation of the ATPase protein in conventional gel filtration chromatography and ultracentrifugation experiments. The dimeric form of the ATPase protein was detected only when deoxycholate and, probably, when C12E8 was used.

Adenosine Triphosphatases↗
PubMed1984

Separation of the tryptic fragments of sarcoplasmic reticulum ATPase with high performance liquid chromatography. Identification of the calcium binding site.

The four tryptic fragments of the Ca2+- and Mg2+-dependent ATPase from sarcoplasmic reticulum were separated on a TSK-G 4000 SW preparative high performance liquid chromatography column in the presence of sodium dodecyl sulfate. Using atomic absorption spectroscopy, the analysis of the protein peaks demonstrated that the calcium binding sites with high affinities are located in the smallest fragment (A2; 25 kDa).

Adenosine Triphosphatases↗
PubMed1984

Caffeine-induced calcium release from isolated sarcoplasmic reticulum of rabbit skeletal muscle.

The essential conditions for the Ca2+ releasing action of caffeine from isolated sarcoplasmic reticulum (SR) of rabbits were evaluated by an investigation into the effects of Ca2+, Mg2+, MgATP2-, and ATP concentration, ionic strength, and degree of loading. The heavy fraction (4,500 X g) of the reticulum was used. Except for the study on degree of loading, 0.2 mg protein X ml-1 SR was loaded actively with 0.02 mM 45CaCl2, resulting in greater than 90 nmol X mg protein -1 at steady state, and then the effects of various parameters with or without (control) caffeine were tested. It was found that (1) caffeine induces a transient, dose-dependent release of Ca2+, (2) the absolute amount of Ca2+ released by caffeine increases with the Ca2+ load of the SR, (3) increasing the ionic strength (mu) from 0.09 to 0.3 lowers the threshold concentration of caffeine, (4) the SR is refractory to a repeated challenge by a caffeine concentration causing maximal effect, (5) caffeine-induced Ca2+ release increases with increasing (a) external Ca2+ concentrations up to 5 microM total Ca2+ (or 3 microM free Ca2+) and (b) free ATP concentrations up to 0.45 mM, and (6) caffeine-induced Ca2+ release is not affected by changes of either the Mg2+ or the MgATP2- concentration.

Adenosine Triphosphate↗
PubMed1983

The vanadate complex of the calcium-transport ATPase of the sarcoplasmic reticulum, its formation and dissociation.

Vanadate binding to different sarcoplasmic reticulum membrane preparations was determined by measuring bound vanadate colorimetrically and by phosphorylating the vanadate-free enzyme fraction with [gamma-32P] ATP. Colorimetry allowed the study of the dependence of equilibrium vanadate binding on ionized magnesium and the displacing effect of ionized calcium at vanadate concentrations greater than 0.1 mM only. At saturating magnesium concentration the enzyme binds 6-8 nmol vanadate/mg protein and half-maximum saturation is reached at 40 microM. Vanadate is displaced from the enzyme when its high-affinity calcium-binding sites are saturated and conversely calcium is solely displaced from its high-affinity binding sites by vanadate. The phosphorylation procedure allowed the measurement of equilibrium binding as well as the kinetics of vanadate binding and release at vanadate concentrations below 0.1 mM. Half-times of 30s and 3s were observed for vanadate release induced by 0.1 mM and 1 mM calcium respectively. Millimolar concentrations of ATP are required for vanadate displacement. Under equilibrium conditions the enzyme displays an affinity for vanadate of 1.6 X 10(6) M-1. The dependence on the concentration of vanadate of the rate of vanadate binding yielded an affinity of only 1 X 10(4) M-1. Closed vesicles bind vanadate much more slowly than calcium-permeable preparations. The initial rate of calcium-induced vanadate dissociation is accelerated considerably when the vesicles are made calcium permeable. The rate of vanadate dissociation from calcium-permeable vesicles reaches half-maximum values at 1-2 mM calcium indicating that the internal low-affinity calcium-binding sites must first be occupied in order to release bound vanadate. The results suggest that vanadate binding leads to a transition of the external high to internal low-affinity calcium-binding sites.

Animals↗
PubMed1983

The influence of sodium trichloroacetate on the tryptophan fluorescence of sarcoplasmic reticulum ATPase.

The chaotropic anion trichloroacetate quenches the tryptophan fluorescence of the sarcoplasmic reticulum calcium transport ATPase. Half-maximum quenching was observed at 50 mM trichloroacetate. In contrast to native preparations, in trichloroacetate-treated sarcoplasmic reticulum vesicles a decrease of the tryptophan fluorescence is observed on addition of millimolar concentrations of calcium. It is concluded that trichloroacetate renders the tryptophan fluorescence of the ATPase sensitive to the occupancy of its low-affinity sites.

Animals↗
PubMed1983

Inactivation of the calcium-transport ATPase in the sarcoplasmic reticulum by the combined effect of lasolocid and Triton X-100.

The calcium-transport ATPase of the sarcoplasmic reticulum membranes is irreversibly inactivated by the combined action of Lasolocid and Triton X-100 at concentrations which separately do not interfere with the enzyme's activity. In the presence of Lasolocid the enzyme is most susceptible to inactivation when the Triton X-100 concentration just exceeds its critical micellar concentration, approximately, 0.2 mg X ml-1. Lasolocid becomes effective at a concentration of 10 microM and produces rapid inactivation at 100 microM. Phosphoprotein formation is less affected than phosphate liberation. The influence of the ATPase protein on the fluorescence intensity of Lasolocid passes a distinct maximum at the most effective Triton X-100 concentration of 0.2 mg X ml-1.

Adenosine Triphosphatases↗
PubMed1983

Excimer formation of ATPase from sarcoplasmic reticulum labeled with N-(3-pyrene)maleinimide.

Sarcoplasmic reticulum ATPase from fast skeletal muscle was labeled in native vesicles with N-(3-pyrene)maleinimide. At labeling ratios larger than 1 mol pyrenemaleinimide/2.5 mol ATPase significant amounts of excimers are detected. Excimer concentration decreases at low, non-solubilizing amounts of detergents (0.2 mg X mg protein-1) and completely disappears after solubilization of the membranes. These results exclude that excimers are formed due to 'double-labeling' of one ATPase molecule. It is concluded that the ATPase exists as an oligomer within the membrane of native vesicles.

Calcium-Transporting ATPases↗
PubMed1983

Effect of 1,25-dihydroxycholecalciferol on sarcoplasmic reticulum calcium transport in strontium-fed chicks.

Feeding of chicks with strontium, an inhibitor of 1,25-dihydroxycholecalciferol synthesis in kidney, during 7 days, significantly depressed the initial rate of calcium uptake and calcium storing capacity of sarcoplasmic reticulum membranes from skeletal muscle. Oral administration of 1,25(OH)2D3 to strontium-fed animals returned calcium transport values to normal. The changes observed could not be related to differences in the relative proportions of transport ATPase and calcium binding proteins. The results are consistent with a role of 1,25(OH)2D3 in muscle function.

Animals↗