Effects of pH, Ca2+ and lanthanides on conformation of the sarcoplasmic reticulum Ca(2+)-ATPase catalytic site.

The conformational changes at the ATP-catalytic site of the sarcoplasmic reticulum (SR) Ca(2+)-ATPase have been studied by the fluorescence of the fluorescein 5-isothiocyanate (FITC) bound to the adenine subsite. The FITC-SR fluorescence parameters have been examined in the pH range 5.7-8.0 in the presence of EGTA, Ca2+ or Ln3+ (La3+, Pr3+, Nd3+, Tb3+, etc.). A quantitative method to calculate the equilibrium between the protein conformers is proposed on the basis of the fluorometric titration curve analysis. The distance Nd(3+)-FITC was estimated to be about 1 nm at pH 6-7 and 1.7 nm at pH 8 which can be interpreted as an increase of the distance between the nucleotide and phosphorylation domains of Ca(2+)-ATPase in alkaline media. These studies suggest that the ligand-stabilized E1-form of Ca(2+)-ATPase can exist in two conformational states with the closed and opened interdomain cleft in the pH range 5.7-8.0. The pH-dependence of the ratio of these states correlates with that of the E1----E2 equilibrium without ligands. These dependences were approximated by simple Henderson-Hasselbach equations with pK 7.0 +/- 0.1, i.e. the transition between two protein conformations is probably governed by one proton dissociation.

[The effect of the external electric field on Ca2+ transport in the sarcoplasmic reticulum]. / Vliianie vneshnego élektricheskogo polia na transport Ca2+ v sarkoplazmaticheskom retikulume.

The effect of active and passive Ca2+ transport in sarcoplasmic reticulum vesicles on the rate of negative diffusion potential dissipation was investigated by the method of potential-sensitive fluorescent probes. Membrane potentials were generated by K+ gradients in the presence of valinomycin and were measured using dye diS-C3-(5). It was shown that the rate of K+ potential dissipation was accelerated during Ca2+ transport and depended on ATP and Ca2+ concentrations. In its turn the initial rate of ATP hydrolysis was found to be increased in the presence of negative potential. The obtained results support an electrogenic mechanism for the Ca2+ transport by the sarcoplasmic reticulum.

[Permeability of sarcoplasma reticulum membrane for monovalent cations]. / Pronitsaemost' membran sarkoplazmaticheskogo retikuluma dlia odnovalentnykh kationov.

Potassium, sodium and Tris ion permeability of sarcoplasmic reticulum (SR) vesicles was investigated by the method of potential-sensitive probes. Membrane potentials were generated by changing the ionic environment of vesicles and were measured using the probe 3,3'-dipropyl-2,2'-thiodicarbocyanine (diS-C3-(5)). It was shown that 2/3 of the vesicles (Type I) were permeable to K+ and Na+ while the remaining 1/3 (Type II) were not. Both types of vesicles were relatively impermeable to Tris+. The permeability coefficient for Tris+ was estimated to be of the order of 10(-8) cm/sec. The K+ and Na+ permeability coefficients for Type I vesicles were estimated to be greater than 10(-6) sm/sec. A likely physiological function of the K+, Na+-permeable pathways in SR would be to minimize charge and osmotic effects during Ca2+ uptake and release.

[Study of sarcoplasmic reticulum membrane permeability using potential-sensitive fluorescent probes]. / Issledovanie pronitsaemosti membran sarkoplazmaticheskogo retikuluma s pomoshch'iu potentsial-chuvstvitel'nykh fluorestsen tnykh zondov.

A new method is proposed for quantitative studies of SR membrane permeability. It involves measuring to diffusion potentials, arising at salt concentration gradient on the SR membrane due to its anion and cation permeability differences. Visualization of processes of negative chloride potential arising in adding MeCl2 and its further dissipation was realized with diS-C3-(5). A/23187 and X-537A in small concentrations reduce the amplitude and the length of fluorescent signal, while in strong concentrations they fully prevent it. Dissipation kinetics of chloride potential caused by the SR membrane permeability for Me2+ is used for quantitative analysis of this permeability. It is shown that the parameter tau 1/2 corresponds to the moment of time when the inside concentration of Me2+ becomes equal to the geometric average of the initial concentrations of this ion. The relationship between tau 1/2 and the concentrations of the added MeCl2 and the penetration ion (K+) and temperature were studied. It was shown that the membrane permeability gain with increasing MeCl2 concentration is due to accelerating action of the membrane potential. A laser two-wave, two-beam installation was described which allows to study SR membrane permeability with potential-sensitive probe fluorescence and light scattering on the same sample. Correlation between the signals obtained simultaneously with these independent methods was shown.

[Transmembrane potential formation upon ATP hydrolysis in sarcoplasmic reticulum]. / Obrazovanie transmembrannogo potentsiala pri gidrolize ATF v sarkoplazmaticheskom retikulume.

Fluorescent cyanine (diS-C3-(5), diS-C2-(5), diO-C3-(5)) and oxonol (diBA-C4-(5)) potential-dependent dyes appeared to be extremely effective in detecting and studying the potential formed on the fragmented sarcoplasmic reticulum membrane under Ca2+ transport When [Ca2+] less than 5 X 10(-7) M ATP hydrolysis leads to formation of transmembrane potential (positive inside vesicules) caused by the Ca-independent ATPase activity. The potential is formed by a monovalent ion, presumably by H+, and possibly by Mg2+ ions. Ca-dependent ATPase activation by Ca2+ makes the potential to drop sharply and successive Ca2+ transport proceeds at low potential value. When Ca2+ has been accumulated by vesicules the Ca-independent ATPase restores positive potential. The potentials generated by both Ca-independent (10--30 mv) and Ca-dependent (-20 divided by -40 mv) ATPases have been estimated on the basis of the Nernst's equation with the help of positive and negative diffusion potentials formed by MgCl2 and CaCl2 gradients. The Ca2+ transport is shown not to be due to transmembrane electrophoresis but Ca-dependent ATPase action. The results suggest quite clearly that Ca-dependent ATPase operates as electrogenic Ca2+/H+, Mg2+-exchanger. The functional role of Ca-independent ATPase is, possibly, in compensation of charge effects when Ca2+ ions are passing through the membranes. The model illustrating the electrogenicity of Ca-independent and Ca-dependent ATPases action during Ca2+ transport in SR membranes has been proposed.

[The role of histidine residues in conformational changes in the sarcoplasmic reticulum Ca-ATPase active site]. / Rol' gistidinovykh ostatkov v konformatsionnykh izmeneniiakh v aktivnom tsentre Ca-ATFazy sarkoplazmaticheskogo retikuluma.

Conformational pH-induced changes of Mg-ATP binding site of the sarcoplasmic reticulum Ca-ATPase (SR-ATPase) were investigated by fluorescence energy transfer between covalently bound fluorescent label (fluorescein-5-isothiocyanate, FITC) and lanthanide ion (Nd3+). These changes were approximated by simple Henderson-Hasselbach equation with the apparent pK 7.0 +/- 0.1 which is similar that of a histidyl residue [3]. In this work it was used the double chemical modification of SR-ATPase to research the role of histidyl residues in this conformational transition. Diethyl pyrocarbonate was used to modify the histidyl residues of the SR-ATPase. The influence of histidyl modification on the functional parameters (the rates of ATP and p-nitrophenyl phosphate hydrolysis, the Ca transport and the level of Ca2+ accumulation) was monitored by the fluorescent probes (Quin-2, chlortetracycline) using fluorescent, spectrophotometric and pH-metric measurements. In the result of these experiments it was found the appropriate conditions to carry out the second modification. The DEPC-SR-ATPase was labeled by FITC. The pH-dependent conformational changes in the active site of FITC-DEPC-SR-ATPase were studied by the method of the fluorescence energy transfer between FITC and Nd3+ in the region of pH 6-8. The histidyl modification of FITC-DEPC-SR-ATPase resulted in the significant shift of the curve of fluorescence energy transfer efficiency (the apparent pK > 7.5). These results suggest that the conformational transition in the active site of SR-ATPase was controlled by the histidyl residues.

[Mechanism of the fluorescent response of carbocyanine probe diS-C3-(5) to membrane potential change]. / O mekhanizme fluorestsentnogo otveta karbotsianinovogo zonda diS-C3-(5) na izmenenie transmembrannogo potentsiala.

A method for estimation of constants of carbocyanine probe diS-C3-(5) partition between water and membrane phases was developed based on the analysis of fluorometric titration curves. The partition constants (K) of this dye were calculated (the ratio of dye mole fraction in membrane and water phases) for sarcoplasmic reticulum (SR) vesicles in sucrose K = (1.15 +/- 0.04) X 10(7) and salt K = (6.12 +/- 0.07) X 10(6) media and for the asolectine liposomes in salt medium K = (3.4 +/- 0.2) X 10(7). The probe partition between water and lipid phases was calculated for different membrane concentrations. Critical aggregation concentrations of probe in lipid phase were estimated to be approximately 8 mol. probe per 1000 mol. lipid for asolectine liposomes and approximately 9 mol. per 1000 mol. lipid for SR vesicles (both in salt medium), approximately 6 mol. per 1000 mol. lipid for SR vesicles in sucrose medium. On the basis of own and literature data a mechanism of voltage-sensitive probe response related to probe aggregation in membranes was suggested.