Search PubMed⌕ Search

Biomedical subjects

W Stürmer

Publications and source records attributed to W Stürmer.

16 recordsLinked to original sources
PubMed1994

Binding of sodium ions and cardiotonic steroids to native and selectively trypsinized Na,K pump, detected by charge movements.

A fluorescent dye, RH421, has been used to characterize charge movements associated with cation and cardiotonic steroid binding to Na,K-ATPase and to a specifically trypsinized preparation, so-called "19-kDa membranes." A fluorescence decrease induced by Na+ is attributed to electrogenic binding of one Na+ ion from the cytoplasm. The apparent affinity for Na+ is the same in both preparations. (ATP + Na + Mg) or (P(i) + Mg)-induced fluorescence signals observed with native enzyme are not observed in 19-kDa membranes, consistent with loss of ATP binding and phosphorylation. Cardiotonic steroids (CS) bind to native enzyme and 19-kDa membranes as judged by RH421 signals, fluorescence of anthroyl ouabain, and inhibition of Rb+ occlusion. Binding affinities to both preparations are in the micromolar range, and binding is prevented by the presence of Na+ or K+. The kinetics of glycone binding and dissociation are identical in both preparations, but aglycones bind and dissociate about 6-fold faster to 19-kDa membranes. Binding of Na+ and cardiotonic steroids is inactivated upon heating or extensive Pronase digestion of 19-kDa membranes. This suggests that cation and CS binding depend on the structural integrity of a complex of the proteolytic fragments, and that sites for both cations or CS consist of ligating groups located on more than one fragments of 19-kDa membranes.

Animals↗
PubMed1994

Favourable glycaemic effects of a new balanced liquid diet for enteral nutrition - Results of a short-term study in 30 type II diabetic patients.

A new fibre enriched (15 g/l soy polysaccharides) balanced liquid diet was developed containing 53% (of energy) as carbohydrate, 32% fat and 15% protein. 71% of carbohydrates in this formula consist of modified starch, 29% of fructose. The glycaemic response to this diabetes adapted diet (DD) was compared with an isoenergetic standard diet (SD) containing maltodextrins and sucrose and a typical continental breakfast (CB). 30 type II diabetics orally ingested isoenergetic amounts of these test diets in randomised order. Each diet contained 48 g carbohydrates. The 4 h postprandial (p.p.) observation period was characterised by a markedly improved response of blood glucose following ingestion of DD. This effect was seen as significantly reduced p.p. peaks (p < 0.00001 vs SD; p = 0.00001 vs CB) as well as diminished areas under the curve. (p < 0.00001 vs SD; p = 0.00001 vs CB). Similar results were obtained by evaluation of the corresponding insulin levels. The differences between the test diets were greater in insulin treated patients than in diabetics on sulfonylureas or a dietary regimen only. We conclude that this new balanced liquid diet can be helpful in maintaining a good glycaemic control in diabetics receiving artificial enteral nutrition and may be administered in a similar way to normal foods without changes in the patients antidiabetic therapy.

Journal Article↗
PubMed1994

Partial reactions of the Na,K-ATPase: determination of rate constants.

Experiments were designed to characterize several partial reactions of the Na,K-ATPase and to demonstrate that a model can be defined that reproduces most of the transport features of the pump with a single set of kientic parameters. We used the fluorescence label 5-iodoacetamidofluorescein, which is thought to be sensitive to conformational changes, and the styryl dye RH 421, which can be applied to detect ion-binding and -release reactions. In addition transient electric currents were measured, which are associated mainly with the E1-->E2 conformational transition. Numerical simulations were performed on the basis of a reaction model, that has been developed from the Post-Albers cycle. Analysis of the experimental data allows the determination of several rate constants of the pump cycle. Our conclusions may be summarized as follows: (a) binding of one Na+ ion at the cytoplasmic face is electrogenic. This Na+ ion is specifically bound to a neutral binding site with an affinity of 8 mM in the presence of 10 mM Mg2+. In the absence of divalent cations, the intrinsic binding affinity was found to be 0.7 mM. (b) The analysis of fluorescence experiments with the cardiotonic steroid strophanthidin indicates that the 5-iodoacetamidofluorescein label monitors the conformational transition (Na3)E1-P-->P-E2(Na2), which is accompanied by the release of one Na+ ion. 5-IAF does not respond to the release of the subsequent two Na+ ions, which can be monitored by the RH 421 dye. These experiments indicate further that the conformational transition E1P-->P-E2 is the rate limiting process of the Na+ translocation. The corresponding rate constant was determined to be 22 s-1 at 20 degrees C. From competition experiments with cardiotonic steroids, we estimated that the remaining 2 Na+ ions are released subsequently with a rate constant of at least 5,000 s-1 from their negatively charged binding sites. (c) Comparing the fluorescence experiments with electric current transients, which were performed at various Na concentrations in the absence and presence of strophanthidin, we found that the transition (Na3).E1-P-->P-E2.(Na2) is the major charge translocating step in the reaction sequence Na3.E1-->(Na3).E1-P-->P-E2.(Na2)-->P-E2. The subsequent release of 2 Na+ ions contributed less than 25% to the total electric current transient. (d) The well known antagonism between cardiotonic steroids and K+ binding can be explained by a kinetic model. A quantitative description has been obtained under the assumption that these inhibitors bind only to the states P-E2(Na2) and P-E2(K2).(ABSTRACT TRUNCATED AT 400 WORDS)

Adenosine Triphosphate↗
PubMed1993

Fluorescence spectroscopic studies on equilibrium dipole-relaxational dynamics of Na,K-ATPase.

Intramolecular dynamics in Na,K-ATPase molecules have been studied by ultraviolet fluorescence spectroscopic methods: determination of temperature-dependent shifts in steady-state spectra, site-selective red-edge effects and their temperature dependence, and time-resolved emission decay as a function of excitation and emission wavelengths. The combination of these methods allows the characterization of the dipolar-relaxational mobility in the environment of the tryptophan residues. Our results show that the mean dipolar-relaxational time is of the order of one nanosecond at room temperature. This is much faster than what is usually observed in globular proteins. The fast dynamics of the protein dipoles are rapid enough so that the dipoles are in dielectric equilibrium during the slower ion transfer processes; this may have important functional consequences.

Buffers↗
PubMed1992

Fluorescence study on cardiac glycoside binding to the Na,K-pump. Ouabain binding is associated with movement of electrical charge.

Recently we have presented evidence that the fluorescence probe RH 421 can be used to detect binding and release of ions at the extracellular face of the pump since these processes are associated with translocation of electrical charge. Applying this method to experiments with cardiac glycosides we found that: (1) ouabain induced fluorescence changes of the electrochromic dye, RH 421, were caused by the change of charges bound to the enzyme; (2) independent of the sodium concentration, the final fluorescence amplitude indicated that approximately 2 Na+ ions were bound to the pump; (3) the sodium release to the extracellular side involved two distinct electrogenic steps; (4) the kinetics of inhibition depended on the Na(+)-concentration. Experiments with hydrophobic ions indicated that the kinetics of ouabain binding to the Na-ATPase is voltage dependent; and (5) the applied technique is a convenient tool to characterize binding of cardiac glycosides to the Na,K-pump.

Animals↗
PubMed1991

[Lipid status and basal steroid hormone level following 16 weeks of lovastatin therapy in primary hypercholesterolemia].

We examined the effect of a 16 week therapy with the HMG CoA reductase inhibitor lovastatin in 29 patients (mean age 43 years) with primary hypercholesterolemia. All patients had cholesterol levels above 250 mg/dl (mean 348 +/- 96 mg/dl) inspite of a lipid lowering diet and a therapy with conventional lipid lowering drugs during a three month screening period. After 4 weeks on placebo 20 mg lovastatin was given orally for 4 weeks. If total cholesterol exceeded 200 mg/dl the dose of lovastatin was increased monthly by 20 mg up to the maximal dose of 80 mg/day. After 16 weeks lipid values changed compared with the placebo period: total-cholesterol -25%, triglycerides -8.6%, LDL-cholesterol -31%, APO B -25%, HDL-cholesterol +5.8%, APO AI +0.8%, total-cholesterol/HDL-cholesterol -25%. There was a significant improvement of lipid parameters after lovastatin therapy compared with conventional lipid lowering drugs at the end of the screening period. Lovastatin was well tolerated. A small and reversible rise of transaminases and/or creatinine kinase was observed in 6 patients. Basal levels of ACTH in the morning increased significantly during lovastatin therapy within the normal range. This observation was more frequent in females (10/12) than in males (10/17).

Adrenocorticotropic Hormone↗
PubMed1991

Charge translocation by the Na,K-pump: I. Kinetics of local field changes studied by time-resolved fluorescence measurements.

Membrane fragments containing a high density of Na,K-ATPase can be noncovalently labeled with amphiphilic styryl dyes (e.g., RH 421). Phosphorylation of the Na,K-ATPase by ATP in the presence of Na+ and in the absence of K+ leads to a large increase of the fluorescence of RH 421 (up to 100%). In this paper evidence is presented that the styryl dye mainly responds to changes of the electric field strength in the membrane, resulting from charge movements during the pumping cycle: (i) The spectral characteristic of the ATP-induced dye response essentially agrees with the predictions for an electrochromic shift of the absorption peak. (ii) Adsorption of lipophilic anions to Na,K-ATPase membranes leads to an increase, adsorption of lipophilic cations to the decrease of dye fluorescence. These ions are known to bind to the hydrophobic interior of the membrane and to change the electric field strength in the boundary layer close to the interface. (iii) The fluorescence change that is normally observed upon phosphorylation by ATP is abolished at high concentrations of lipophilic ions. Lipophilic ions are thought to redistribute between the adsorption sites and water and to neutralize in this way the change of field strength caused by ion translocation in the pump protein. (iv) Changes of the fluorescence of RH 421 correlate with known electrogenic transitions in the pumping cycle, whereas transitions that are known to be electrically silent do not lead to fluorescence changes. The information obtained from experiments with amphiphilic styryl dyes is complementary to the results of electrophysiological investigations in which pump currents are measured as a function of transmembrane voltage. In particular, electrochromic dyes can be used for studying electrogenic processes in microsomal membrane preparations which are not amenable to electrophysiological techniques.

Adenosine Triphosphate↗
PubMed1991

Charge translocation by the Na,K-pump: II. Ion binding and release at the extracellular face.

In the first part of the paper, evidence has been presented that electrochromic styryl dyes, such as RH 421, incorporate into Na,K-ATPase membranes isolated from mammalian kidney and respond to changes of local electric field strength. In this second part of the paper, fluorescence studies with RH-421-labeled membranes are described, which were carried out to obtain information on the nature of charge-translocating reaction steps in the pumping cycle. Experiments with normal and chymotrypsin-modified membranes show that phosphorylation by ATP and occlusion of Na+ are electroneutral steps, and that release of Na+ from the occluded state to the extracellular side is associated with translocation of charge. Fluorescence signals observed in the presence of K+ indicate that binding and occlusion of K+ at the extracellular face of the pump is another major electrogenic reaction step. The finding that the fluorescence signals are insensitive to changes of ionic strength leads to the conclusion that the binding pocket accommodating Na+ or K+ is buried in the membrane dielectric. This corresponds to the notion that the binding sites are connected with the extracellular medium by a narrow access channel ("ion well"). This notion is further supported by experiments with lipophilic ions, such as tetraphenylphosphonium (TPP+) or tetraphenylborate (TPB-), which are known to bind to lipid bilayers and to change the electrostatic potential inside the membrane. Addition of TPP+ leads to a decrease of binding affinity for Na+ and K+, which is thought to result from the TPP(+)-induced change of electric field strength in the access channel.

Adenosine Triphosphate↗
PubMed1989

Conformational transitions and change translocation by the Na,K pump: comparison of optical and electrical transients elicited by ATP-concentration jumps.

The electrogenic properties of the Na,K-ATPase were studied by correlating transient electrical events in the pump molecule with conformational transitions elicited by an ATP-concentration jump. Flat membrane fragments containing a high density (approximately 8000 microm(-2)) of oriented Na,K-ATPase molecules were bound to a planar lipid bilayer acting as a capacitive electrode. ATP was released in the medium from a photolabile inactive ATP derivative ("caged" ATP) by a 40-microsec light flash. Electrical signals resulting from transient charge movements in the protein under single-turnover conditions were recorded in the external measuring circuit. In parallel experiments carried out under virtually identical conditions, the fluorescence of membrane fragments containing Na,K-ATPase with covalently-bound 5-iodoacetamido-fluorescein (5-IAF) was monitored after the ATP-concentration jump. When the medium contained Na+, but no K+, the fluorescence of the 5-IAF-labeled protein decreases monotonously after release of ATP. In the experiments with membrane fragments bound to a planar bilayer, a transient pump current was observed which exhibited virtually the same time behavior as the fluorescence decay. This indicates that optical and electrical transients are governed by the same rate-limiting reaction step. Experiments with chymotrypsin-modified Na,K-ATPase suggest that both the fluorescence change as well as the charge movement are associated with the deocclusion of Na+ and release to the extracellular side. In experiments with Na+-free K+ media, a large inverse fluorescence change is observed after the ATP-concentration jump, but no charge translocation can be detected. This indicates that deocclusion of K+ is an electrically silent process.

Animals↗
PubMed1989

Enteral nutrition in diabetes mellitus.

In the diabetic insulin deficiency gives rise to a series of special metabolic features which must be taken into consideration in artificial nutrition. Quantitative and temporal harmonization of nutrient intake and endogenous insulin reserve or exogenous insulin administration is more important than the ratio of nutrients. In critical situations, parenteral administration of insulin is to be recommended for specific modulation. If absorption of insulin from the subcutaneous tissue and of nutrients from the intestine is certain, the enteral route is feasible. Continuous nutrient provision via a pump is to be recommended, since blood glucose peaks can be avoided in this way. However, since the comparison of liquid diets with conventional food showed similar postprandial blood glucose profiles, we also regard a bolus application and a conventional subcutaneous insulin treatment regime possible in diabetes mellitus. With newly developed fiber-enriched diets, more favourable effects on metabolism may be achieved in the future.

Blood Glucose↗