Stimulus-dependent control of inositol 1,4,5-trisphosphate-induced Ca(2+) oscillation frequency by the endoplasmic reticulum Ca(2+)-ATPase.

In many cell types, receptor stimulation evokes cytosolic calcium oscillations with a frequency that depends on agonist dose. Previous studies demonstrated controversial effects of changing the activity of the endoplasmic reticulum Ca(2+)-ATPase upon the frequency of oscillations. By numerical simulations, we found that the model of De Young and Keizer (J. Keizer and G.W. De Young, 1994, J. Theor. Biol. 166: 431-442), unlike other models, can explain the observed discrepancies, assuming that the different experiments were performed at different stimulus levels. According to model predictions, partial inhibition of internal calcium pumps is expected to increase frequency at low stimulus strength and should have an opposite effect at strong stimuli. Similar results were obtained using an analytical estimation of oscillation period, based on calcium-dependent channel activation and inactivation. In experiments on HeLa cells, 4 nM thapsigargin increased the frequency of calcium oscillations induced by 1 and 2.5 microM histamine but had no effect on supramaximally stimulated cells. In HEp-2 cells, 2 nM thapsigargin slowed down the rapid, ATP-induced oscillations. Our results suggest that in the investigated cell types, the De Young-Keizer model based on inositol 1,4,5-trisphosphate-dependent calcium-induced calcium release can properly describe intracellular calcium oscillations.

Protein flexibility as revealed by fluorescence resonance energy transfer: an extension of the method for systems with multiple labels.

The temperature profile of the normalized fluorescence resonance energy transfer efficiency is capable of monitoring the relative change of flexibility and/or conformational state of macromolecules [Biochemistry 23 (1984) 3403]. The method described earlier for one donor-one acceptor systems is extended to multiple fluorophore systems when the energy transfer occurs between either one donor-m acceptors, or n donors-one acceptor or n donors-m acceptors (where n and m are integer values). It is shown that the normalized energy transfer efficiency obtained for systems containing multiple labels is a linear combination of the normalized transfer efficiency assigned to individual donor-acceptor pairs of the system, thus its temperature profile is capable of monitoring the change of intramolecular flexibility and/or conformational state.

Effect of Ca2+-Mg2+ exchange on the flexibility and/or conformation of the small domain in monomeric actin.

A fluorescence resonance energy transfer (FRET) parameter, f' (defined as the average transfer efficiency, (E), normalized by the actual fluorescence intensity of the donor in the presence of acceptor, F(DA)), was previously shown to be capable of monitoring both changes in local flexibility of the protein matrix and major conformational transitions. The temperature profile of this parameter was used to detect the change of the protein flexibility in the small domain of the actin monomer (G-actin) upon the replacement of Ca2+ by Mg2+. The Cys-374 residue of the actin monomer was labeled with N-iodoacetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine (IAEDANS) to introduce a fluorescence donor and the Lys-61 residue with fluorescein-5-isothiocyanate (FITC) to serve as an acceptor. The f' increases with increasing temperature over the whole temperature range for Mg-G-actin. This parameter increases similarly in the case of Ca-G-actin up to 26 degrees C, whereas an opposite tendency appears above this temperature. These data indicate that there is a conformational change in Ca-G-actin above 26 degrees C that was not detected in the case of Mg-G-actin. In the temperature range between 6 degrees C and 26 degrees C the slope of the temperature profile of f' is the same for Ca-G-actin and Mg-G-actin, suggesting that the flexibility of the protein matrix between the two labels is identical in the two forms of actin.

Alteration of the intramolecular dynamics of glycogen phosphorylase b by allosteric ligands.

Phosphorylase b (E.C. 2.4.1.1), prepared from rabbit skeletal muscle, was used to study whether the binding of allosteric ligands modifies the intramolecular dynamics of the protein matrix. Protein dynamics were monitored through the fluorescence and phosphorescence parameters of the 12 tryptophan (Trp) residues (one monomer) of the enzyme. The phosphorescence lifetime was measured at room temperature both in the absence and the presence of ligands. The addition of an allosteric inhibitor (ATP) decreased the lifetime, while the presence of activator (AMP) and/or substrate (G-1-P) had no detectable effect. The lifetime data allow us to conclude that the environment of the buried tryptophans becomes more flexible upon the binding of ATP, while the other ligands did not induce such change. The ATP-induced perturbation was also examined by the quenching of Trp fluorescence by acrylamide. The quenching parameters did not show any change, suggesting that the effect of ATP is localized to the vicinity of the phosphorescent Trp residues.

Effect of haematoporphyrin-induced photosensitization on lipid membranes.

In vitro cultured mouse myeloma (Sp-2/0-Ag14) cells and phosphatidylcholine liposomes were used to study the membrane effects of photosensitization with an He-Ne laser activated haematoporphyrin (HP). Lipophilic HP molecules, intercalated between the membrane lipid molecules, caused morphological changes of cell membranes on light activation. Steady state and time-resolved fluorescence spectroscopic studies of membrane-bound HP molecules provide information about the change in membrane lipid dynamics (fluidity). Increased HP fluorescence anisotropy was found after laser irradiation in the case of cell membrane. This finding can be related either to the increased rotation correlation time of the rotating fluorophore (HP) (decreased membrane fluidity) or to the decrease in the angular range of molecular rotation, which corresponds to an increased lipid order after photosensitization. Changes in the ratio of saturated:unsaturated fatty acid content of membrane lipids or other chemical events such as cross-linking of membrane components during the photosensitization process can also account for the observed effects.

The local dynamics of the active site region of glycogen phosphorylase B.

The parameters characterizing the quenching of fluorescence emitted by the coenzyme (pyridoxal-5-phosphate) of phosphorylase b (EC 2.4.1.1) by anions are good indicators of conformational/dynamic changes at the active center. Reinvestigation of this quenching process resulted in a non-linear Stern-Volmer plot. This non-linearity is described by a simple kinetic model which assumes two parallel processes, one represented by bound and the second by free quencher molecules. Analysis of separate parts of the non-linear Stern-Volmer plot results in the values of rate constants for the bound and free quencher molecules as well as the value of dissociation constant of the anions.

Fluorescence quenching in membrane phase.

Membrane-related events can be investigated when the fluorescence of an intramembrane fluorophore is quenched by molecules that are dissolved in lipid phase. In this case the bimolecular quenching constant characterises the relative transport rate of the fluorophore and quencher molecules in the membrane interior and thereby it is related to the dynamics or structure of the membrane. Unlike classic quenching experiments, the crucial point in such studies is that the concentration of the quencher in the lipid phase differs from that in the bulk. As a consequence, it is usually described by different models, or regarded as the total concentration added. Here a simple fluorometric study is presented for distinguishing between the solvation mechanisms (partition or binding) of quencher molecules in membrane phase.

Protein dynamics and fluorescence quenching.

There are both theoretical and experimental data which strongly suggest that the intramolecular dynamics of the protein matrix play an important role in protein functions. The interrelationship between theory and experiments is rather weak mainly because of the lack of relevant experimental methods and (model-dependent) misinterpretation of experimental data. We give a short account of a few fluorescence-quenching techniques that can provide adequate information concerning protein dynamics provided that the experimental data sets are correctly processed.

The effect of transmembrane potential on the dynamic behavior of cell membranes.

The relationship between transmembrane potential and lipid dynamics in the cytoplasmic membrane of mouse thymus cells has been investigated. Changes of transmembrane potential was followed by measuring the fluorescence emission of the anionic dye, bis-(1,3-dibutylbarbiturate)trimethine oxonol (diBa-C4-(3)). Assessment of lipid fluidity was carried out applying three fluorescent lipid probes, 1-[4-(trimethylammonium)phenyl]-6-phenyl-1,3,5-hexatriene (TMA-DPH), 12-(9-anthroyloxy)stearic acid (12-AS) and 1,6-diphenyl-1,3,5-hexatriene (DPH) used to monitor different structural regions of the bilayer. The fluorescence anisotropy of these probes was measured as a function of temperature at two values of transmembrane potential. In the case of DPH it proved to depend on the membrane potential in the higher temperature range (above 28 degrees C), while no such a dependence could be observed for DPH below this temperature range and for TMA-DPH and 12-AS in between 20 and 37 degrees C. These data suggest that changes in transmembrane potential are accompanied with some local alteration in membrane lipid dynamics and/or structure.

The use of a new fluorescent viscosity indicator to study the relationship between transmembrane potential and membrane dynamics of lymphocytes.

A new fluorescent dye, polymethine derivative 4501 U, was applied to label the cytoplasmic membrane of mouse lymphocytes to investigate whether the membrane lipid dynamics is related to the transmembrane potential. The dye was found not to alter the cell viability. The dye is localized close to the external surface of the membrane, and its spectroscopic characteristics make it possible to obtain information on membrane fluidity using the intensity ratio of its two emission peaks. In contrast to recent data indicating that transmembrane potential alters the membrane lipid dynamics (as revealed by anisotropy studies with diphenyl hexatriene) 4501 U does not show such a relationship. The investigations presented support the hypothesis that the change in the transmembrane potential affects the dynamics/structure of the membrane region only at the interface between the two lipid layers.