Time-resolved phosphorescence anisotropy as a tool to study the conformation and oligomeric structure of ion-transport ATPases.
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Animals↗
Biomedical subjects
A M Rubtsov
Publications and source records attributed to A M Rubtsov.
[Study of the rotational mobility of E1- and E2- conformers of Ca-ATPase in sarcoplasmic reticulum membranes using a time-resolved phosphorescence anisotropy method].
Membrane preparations of sarcoplasmic reticulum Ca-ATPase from rabbit skeletal muscles were covalently labelled with eosin-5'-isothiocyanate at Lys 515 residue in a putative active site and with 4-(iodoacetamido)-eosin (presumably, at Cys 670 and Cys 674 residues). These preparations were used to measure the laser flash induced phosphorescence anisotropy in a microsecond time scale. An analysis of effects of diethyl ether, glycerol, the nonionic detergent C12E9 and Ca-ATPase ligands stabilizing the enzyme in the E2 or E1 conformeric state, on the anisotropy parameters showed that Ca-ATPase of sarcoplasmic reticulum membranes is present in both monomeric and oligomeric states. The enzyme transition from the E1 to the E2 conformation is due to the increase in the content of oligomeric complexes and their average size. These data support the hypothesis that the oligomeric state of Ca-ATPase can be changed during enzyme reaction cycle.
Animals↗
[Rotational mobility of membrane-bound Na,K-ATPase].
The rotational mobility of E1 and E2 conformers of duck salt gland Na,K-ATPase labelled with eosine-5'-isothiocyanate (EITC) was studied using a time-resolved phosphorescence anisotropy approach. For each conformer, two types of the rotational mobility were found. The rotational correlation time of the faster component equal to about 15 microseconds at 20 degrees for the both conformers, was ascribed to the rotation of the (alpha beta) protomer with an apparent radius 2.4 nm. The slower component (100-500 microseconds depending on experimental conditions) was suggested to reflect the presence in the bilayer of associates between Na,K-ATPase molecules or those with other protein constituents of the membrane bilayer. A rise in temperature tends to decrease the fast component with a subsequent increase in the slow component of the experimental curve, apparently due to oligomerisation of the protomers into oligomers. The size of the oligomers depends on pH and temperature and under favourable conditions may come up to octamers.
Animals↗
[Mechanism of temperature uncoupling of the sarcoplasmic reticulum calcium pump: change in the functional properties and structural state of the enzyme molecule].
The effect of short-term thermal treatment causing the uncoupling of the hydrolytic and transport functions of skeletal muscle sarcoplasmic reticulum Ca-ATRase on the conformational state of the enzyme and the physico-chemical characteristics of the membrane lipid bilayer has been studied. It has been found that enhanced passive permeability of the membrane for Ca2+ is the main cause for the decreased efficiency of the Ca-pump function. Thermal treatment induced no changes in the conformation of the hydrophilic domain of the Ca-ATRase; however, the accessibility of the transmembrane hydrophobic domain for some solutes turned out to be restricted, the microviscosity of the lipid bilayer decreased and the protein-lipid and protein-protein interactions within the membrane disturbed after the heat exposure.
Animals↗
[The effect of carnosine on Ca-channels in rabbit skeletal muscle sarcoplasmic reticulum].
Carnosine (beta-alanyl-L-histidine), which is present in millimolar concentrations in skeletal muscles, induces Ca2+ release from the heavy fraction of rabbit skeletal muscle sarcoplasmic reticulum by activation ruthenium red-sensitive Ca-release channels. The effect of carnosine is dose-dependent, which indicates the presence of saturable carnosine-binding sites in the Ca-release channel molecule. The half-maximal Ca2+ release is observed in the presence of 8.7 mM carnosine. At the same time, carnosine addition to the medium increases the affinity of sarcoplasmic reticulum Ca-channels for the Ca-release activators, caffeine and adenine nucleotides. It is concluded that carnosine is an endogenous regulator of skeletal muscle sarcoplasmic reticulum Ca-channels which modulates the affinity of these channels for different ligands.
Adenine Nucleotides↗
[Direct measurement of the interaction of carnosine and its analogs with free radicals].
An ESR study of interactions of carnosine and its derivatives with free radicals has been carried out. In model systems the spin trap OH. radical adduct generation has been shown to decrease significantly in the presence of carnosine in a pronounced concentration-dependent manner. A comparative study of effects of some other histidine-containing dipeptides on this process has revealed a similarity in anserine, homocarnosine, and acetylcarnosine actions.
Animals↗
A biochemical approach to essential hypertension.
Using multivariate statistical analysis, an attempt has been made to select hypertensive and normotensive sub-groups of subjects on the basis of certain parameters of their blood serum, such as the inhibition of purified Na,K-ATPase by serum and the content of two proteins with molecular masses of 12 and 15 kDa. An analysis of 20 human beings (10 hypertensive and 10 normotensive individuals) revealed that the best division into sub-groups is achieved only through the use of a combination of these three parameters.
Adolescent↗
Pathways of calcium release from heavy sarcoplasmic reticulum vesicles isolated from rabbit skeletal muscle.
The active uptake and efflux of Ca2+ from suspensions of vesicles from heavy rabbit muscle sarcoplasmic reticulum have been examined using the antipyrylazo III dye method in the presence of various nucleotide triphosphate substrates to support active Ca2+ accumulation. On addition of ATP, Ca2+ is rapidly accumulated and maintained at high internal concentrations until the substrate for pump protein is exhausted. Ca2+-induced Ca2+ release which is inhibited by ruthenium red can be demonstrated. The kinetics of Ca2+ release via these channels is different from the Ca2+ efflux observed after substrate exhaustion. This rate was found to be dependent on the type of nucleotide triphosphate, decreasing in the order ATP greater than GTP greater than CTP greater than ITP UTP. It is suggested that different conformations of the Ca2+ pump protein induced by the different substrates may result in the creation of pathways for the facilitated diffusion of Ca2+.
Adenosine Monophosphate↗
Caffeine interaction with the Ca-release channels of heavy sarcoplasmic reticulum. Evidence that 170 kD Ca-binding protein is a caffeine receptor of the Ca-channels.
The study of Ca2+- and caffeine-induced Ca2+ release from heavy sarcoplasmic reticulum vesicles under the different conditions suggests that Ca2+ and caffeine can interact with the common receptor of the Ca-release channels. The reticulum membranes were solubilized using nonionic detergent polyoxyethylene 9-lauryl ether, and affinity chromatography on reactive red 120-agarose was carried out. The 170 kD Ca-binding protein which is eluted by caffeine is the most probable candidate for the caffeine receptor of the Ca-channels.
Animals↗
Interaction of different nucleotides with Ca-release channels from heavy sarcoplasmic reticulum.
Calcium accumulation and release from the heavy fraction of sarcoplasmic reticulum vesicles have been studied in the presence of different nucleotides with the use of the Ca-sensitive dye antipyrylazo III for monitoring of the free Ca2+ concentration. The calcium- and caffeine-induced Ca2+ release is observed only with ATP but not with any of nonadenine nucleotides used as substrates for the Ca-pump. Adenine nucleotides provide for a rapid ruthenium red sensitive Ca2+ release from the vesicles, when nonadenine nucleotides are used as energy sources for Ca2+ uptake. A comparison of the nucleotides interaction with Ca-channels and Ca-ATPase supports the hypothesis that Ca-ATPase is involved in the operation of Ca-channels.
Animals↗
Effect of temperature on Ca-ATPase from sarcoplasmic reticulum membranes: ESR studies.
Using spin-labeled fatty acid derivatives and maleimide, the effect of temperature on the structural state of various parts of the lipid bilayer of sarcoplasmic reticulum (SR) membranes and the segmental motion of the Ca-ATPase molecule were investigated. The mobility of the spin probes localized in the hydrophobic zone and the outer part of the SR membrane was shown to increase with a rise in temperature from 4 to 44 degrees C, the temperature of 20 degrees C being critical for these changes. In the presence of ATP, critical changes in the spin probe mobility occur at lower temperatures, while in the presence of ATP and Ca2+ they are observed at 20 degrees C for a spin probe localized in the outer part of the SR membrane. The mobility of a spin probe localized in the hydrophobic part of the membrane increases linearly with a rise in temperature. In the absence of ligands, the segmental motion of Ca-ATPase changes linearly within a temperature range of 10-30 degrees C. However, when ATP alone or ATP and Ca2+ are simultaneously added to the incubation mixture, the protein mobility undergoes critical changes at 20 degrees C. The Arrhenius plots for ATPase activity and Ca2+ uptake rate in SR membrane preparations also have a break at 20 degrees C. It is assumed that changes in the structural state of membrane lipids produce conformational changes in the Ca-ATPase molecule; the enzyme seems to be unsensitive to the structural state of the membrane lipid matrix in the absence of the ligands.
Animals↗
[Effects of Ca2+-ATPase substrates on the kinetic properties of SH-groups from the sarcoplasmic reticulum].
The effects of ATP, GTP, ITP, UTP, CTP, AcP, pNPP and ADP on the kinetic properties of SH-groups of Ca2+-ATPase from sarcoplasmic reticulum were studied. Among the substrates used only ATP and ADP changed the reactivity of SH-groups of the enzyme; the effects of Ca2+ and Mg2+ in this process were investigated. Inactivation of Ca2+-ATPase by NBD-chloride was performed. In the presence of high concentrations of ATP and ADP the enzyme inactivation was correlated with modification of fast SH-groups. Two different values of apparent Kd found for the interaction of ATP and ADP with the enzyme suggest that SR preparations contain two types of substrate binding sites. The data obtained are discussed within the framework of a scheme according to which Ca2+-ATPase functions in SR membranes as an oligomeric complex.
Animals↗
[Investigation of sarcoplasmic reticulum SH-groups].
The amount and the reaction capacity of the thiol groups in the sarcoplasmic reticulum containing up to 86% of Ca-ATPase were determined using 7-chloro-4-nitrobenzo-2-hydroxo-1,3-diazole (NBD-chloride). The total amount of SH-groups interacting with NBD-chloride is about 9 moles/10(5) g of protein as determined in the excess of NBD-chloride (750 micrometers). With respect to their sensitivity to NBD-chloride the SH-groups may be divided into two classes: slow and fast ones (5,3 and 3,5 moles/10(5) g of protein, respectively). The modification constants for the fast and slow SH-groups are 0,16 and 0,015min-1. ATP (30 micrometers) decreases the number of fast groups by 1 mole/10(5) g of protein. At higher concentrations of ATP (1--3 mM) the amount of fast SH-groups is decreased by 3 moles/10(5) g of protein, their modification rate constant being decreased 2-fold. ATP at concentration of 1 mM, decreases the rate constant for the Ca-ATPase inactivation by NBD-chloride from 0.68 down to 0,073 min-1, which coincides with the modification rate constant for fast SH-groups (0,071 min-1) under the same conditions. Ca2+ at concentration of 10(-4) M increases the amount of fast thiol groups by 1 mole/10(5) g of protein, the rate constant of their modification by NBD-chloride being increased 2-fold. A half-maximal effect was observed in the presence of 5.10(-7) M Ca2+ . Mg2+ did not affect the total amount of fast thiol groups; however, it decreased their modification rate constant.
4-Chloro-7-nitrobenzofurazan↗
[Calcium release from vesicles of heavy sarcoplasmic reticulum of rabbit skeletal muscles].
The release of Ca2+ from vesicles of heavy sarcoplasmic reticulum after its accumulation due to hydrolysis of ATP, GTP, CTP, UTP or ITP has been studied using Antipyrylazo III, a metal-chromic Ca-indicator. All the studied substrates of the Ca-pump provide Ca2+ accumulation inside the heavy sarcoplasmic reticulum vesicles, the spontaneous Ca2+ outflux rate being different for different nucleoside triphosphates. It is only ATP that provides Ca-(caffeine)-induced Ca2+ release, however AMP, ADP, beta, gamma-methylene-ATP induce Ca2+ ejection in the presence of nonadenylic nucleotides. The ruthenium red (10(-7M) inhibits the induced ejection of Ca2+ from vesicles of the heavy sarcoplasmic reticulum, but does not prevent the spontaneous release of Ca2+ in the same concentrations. A conclusion is drawn that besides Ca-channels sensitive to Ca2+ and caffeine in the presence of ATP (or to AMP, ADP, beta, gamma-methylene-ATP in the presence of nonadenylic nucleotides) and possessing high sensitivity to the ruthenium red there is another pathway for Ca2+ in the heavy reticulum membranes along which its spontaneous release occurs after the substrate exhaustion. It is supposed that this release is provided by the presence of the Ca-ATPase protein.
Adenosine Monophosphate↗
[The effect of verdazyl radicals on Ca2+ metabolism in preparations of heavy sarcoplasmic reticulum].
Three verdazyl radicals were studied for their effect on calcium accumulation and outflux (passive and Ca- or caffeine-induced) and conformational state of the Ca-ATPase. All three compounds differently affected the ATP-dependent Ca-accumulation. Their effect on the Ca-release from the sarcoplasmic reticulum vesicles could not be explained by their influence on the Ca-accumulation system. The Ca2+ amount liberated by the calcium or caffeine addition was equal in both cases but was modified differently by the used verdazyl compounds. The data obtained suggest that Ca-induced and caffeine-induced calcium release is realized by different mechanisms.
Animals↗
[Interaction of ATP with sarcoplasmic reticulum Ca2+-ATPase; effect on the conformational state of the enzyme].
It was studied how temperature influences the NBD-Cl inactivation of sarcoplasmic reticulum Ca2+-ATPase and the protective effect of ATP under conditions preventing ATP hydrolysis. Two types of ATP-binding sites with Kd equal to 30 and 220 microM at 37 degrees C were found. ADP interacts with these sites with the (K'd = 20 and 200 microM). The temperature decrease from 25 degrees to 5 degrees C induces the abrupt increase in the Kd for the low affinity site. The possible reasons for heterogeneity of ATP-binding sites are discussed. The conclusion is made that interaction of monomers in oligomeric complex of Ca2+-ATPase induces heterogeneity of ATP-binding sites.
4-Chloro-7-nitrobenzofurazan↗