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A M Rubtsov

Publications and source records attributed to A M Rubtsov.

At least 19 recordsLinked to original sources
PubMed2001

Phosphorylation of the alpha-subunit of Na,K-ATPase from duck salt glands by cAMP-dependent protein kinase inhibits the enzyme activity.

Although it was shown earlier that phosphorylation of Na,K-ATPase by cAMP-dependent protein kinase (PKA) occurs in intact cells, the purified enzyme in vitro is phosphorylated by PKA only after treatment by detergent. This is accompanied by an unfortunate side effect of the detergent that results in complete loss of Na,K-ATPase activity. To reveal the effect of Na,K-ATPase phosphorylation by PKA on the enzyme activity in vitro, the effects of different detergents and ligands on the stoichiometry of the phosphorylation and activity of Na,K-ATPase from duck salt glands (alpha1beta1-isoenzyme) were comparatively studied. Chaps was shown to cause the least inhibition of the enzyme. In the presence of 0.4% Chaps at 1 : 10 protein/detergent ratio in medium containing 100 mM KCl and 0.3 mM ATP, PKA phosphorylates serine residue(s) of the Na,K-ATPase with stoichiometry 0.6 mol Pi/mol of alpha-subunit. Phosphorylation of Na,K-ATPase by PKA in the presence of the detergent inhibits the Na,K-ATPase. A correlation was found between the inclusion of P(i) into the alpha-subunit and the loss of activity of the Na,K-ATPase.

Adenosine Triphosphate↗
PubMed2001

Characteristics of sarcoplasmic reticulum membrane preparations isolated from skeletal muscles of active and hibernating ground squirrel Spermophilus undulatus.

The total Ca-ATPase activity in the sarcoplasmic reticulum (SR) membrane fraction isolated from skeletal muscles of winter hibernating ground squirrel Spermophilus undulatus is approximately 2.2-fold lower than in preparations obtained from summer active animals. This is connected in part with approximately 10% decrease of the content of Ca-ATPase protein in SR membranes. However, the enzyme specific activity calculated with correction for its content in SR preparations is still approximately 2-fold lower in hibernating animals. Analysis of the protein composition of SR membranes has shown that in addition to the decrease in Ca-ATPase content in hibernating animals, the amount of SR Ca-release channel (ryanodine receptor) is decreased approximately 2-fold, content of Ca-binding proteins calsequestrin, sarcalumenin, and histidine-rich Ca-binding protein is decreased approximately 3-4-fold, and the amount of proteins with molecular masses 55, 30, and 22 kD is significantly increased. Using the cross-linking agent cupric-phenanthroline, it was shown that in SR membranes of hibernating ground squirrels Ca-ATPase is present in a more aggregated state. The affinity of SR membranes to the hydrophilic fluorescent probe ANS is higher and the degree of excimerization of the hydrophobic probe pyrene is lower (especially for annular lipids) in preparations from hibernating than from summer active animals. The latter indicates an increase in the microviscosity of the lipid environment of Ca-ATPase during hibernation. We suggest that protein aggregation as well as the changes in protein composition and/or in properties of lipid bilayer SR membranes can result in the decrease of enzyme activity during hibernation.

Anilino Naphthalenesulfonates↗
PubMed2001

Molecular mechanisms of regulation of the activity of sarcoplasmic reticulum Ca-release channels (ryanodine receptors), muscle fatigue, and Severin's phenomenon.

In this short review of the literature and our own data the characteristics of structural organization of sarcoplasmic reticulum Ca-release channels (ryanodine receptors) in different types of muscles, the participation of other sarcoplasmic reticulum proteins in excitation-contraction coupling and Ca-release channel operation, and the regulation of the channel activity by endogenous low molecular weight compounds are analyzed. Special attention is given to changes that occur in muscle cells during exhausting work and to the role of sarcoplasmic reticulum Ca-release channels in the loss of muscle contractile activity during the development of fatigue. It is concluded that the protection of muscle fibers against fatigue in the presence of the histidine-containing dipeptide carnosine, called in the literature "Severin's phenomenon", is primarily connected with modulation of sarcoplasmic reticulum Ca-release channel activity by carnosine.

Animals↗
PubMed2001

Ca-ATPase activity and protein composition of sarcoplasmic reticulum membranes isolated from skeletal muscles of typical hibernator, the ground squirrel Spermophilus undulatus.

Ca-ATPase activity in sarcoplasmic reticulum (SR) membranes isolated from skeletal muscles of the typical hibernator, the ground squirrel Spermophilus undulatus, is about 2-fold lower than that in SR membranes of rats and rabbits and is further decreased 2-fold during hibernation. The use of carbocyanine anionic dye Stains-All has revealed that Ca-binding proteins of SR membranes, histidine-rich Ca-binding protein and sarcalumenin, in ground squirrel, rat, and rabbit SR have different electrophoretic mobility corresponding to apparent molecular masses 165, 155, and 170 kDa and 130, 145, and 160 kDa, respectively; the electrophoretic mobility of calsequestrin (63 kDa) is the same in all preparations. The content of these Ca-binding proteins in SR membranes of the ground squirrels is decreased 3-4 fold and the content of 55, 30, and 22 kDa proteins is significantly increased during hibernation.

Animals↗
PubMed2000

Ankyrins.

This review is focused on ankyrin which is a protein linker between the integral membrane proteins and spectrin-based cytoskeleton. Structure and distribution of different ankyrin isoforms that are products of alternative-spliced genes are described. Interaction of ankyrins with various membranes is considered. Special attention is paid to ankyrin participation in signal transduction and in assembly of integral membrane proteins in specialized membrane domains.

Animals↗
PubMed2000

Ankyrin: structure, properties, and functions.

Recent data on characteristics of the structure, functions, and main properties of ankyrins (proteins that are linkers between the spectrin-based cytoskeleton and integral membrane proteins) are summarized. The interactions of ankyrins with band-3 protein, P-type ATPases, ion channels, receptors, and protein kinase C are considered. The structure of ankyrin repeats that are often contained in other proteins (which are not classified with the ankyrin family) and ensure protein-protein interactions as well as interactions between proteins and nucleic acids is described in details. The mechanisms of regulation of the ability of ankyrins to interact with other proteins (alternative splicing and post-translational modification, including phosphorylation) are also considered.

Animals↗
PubMed1999

Phosphorylation of H,K-ATPase alpha-subunit in microsomes from rabbit gastric mucosa by cAMP-dependent protein kinase.

A 100-kDa protein that is a main component of the microsomal fraction from rabbit gastric mucosa is phosphorylated by cAMP-dependent protein kinase (PKA) in the presence of 0.2% Triton X-100. Microsomes from rabbit gastric mucosa possess activity of H,K-ATPase but not activity of Na,K-ATPase. Incubation of microsomes with 5 microM fluorescein 5'-isothiocyanate (FITC) results in both an inhibition of H,K-ATPase and labeling of a protein with an electrophoretic mobility corresponding to the mobility of the protein phosphorylated by PKA. The data suggest that the alpha-subunit of H,K-ATPase can be a potential target for PKA phosphorylation.

Animals↗
PubMed1999

Characteristics of the interaction of melittin with sarcoplasmic reticulum membranes.

Addition of an amphipathic bee venom peptide, melittin, to sarcoplasmic reticulum (SR) vesicles isolated from rabbit skeletal muscles resulted in a fast (<1 min) blue shift in the fluorescence maximum of the melittin--SR membrane complex. Over the following 45 min the position of the fluorescence maximum did not change, but the fluorescence intensity of the melittin--SR membrane complex decreased by approximately 35% with rate constant 0.14 min-1. Melittin rapidly quenched the isotropic signal in the EPR spectrum of spin-labeled stearic acid added to SR membranes. Further changes in the spectral parameters of the spin probe bound to SR membranes in the presence of melittin indicated an increase of the viscosity of the probe microenvironment (empiric parameter T/eta was decreased by approximately 35% with rate constant 0.11 min-1). The surface potential of SR membranes measured using a pH-sensitive dye, neutral red, decreased after melittin addition from -60 to -30 mV. It was demonstrated with the use of a cross-linking agent, cupric o-phenanthroline, that melittin induced slow aggregation of Ca-ATPase protein in SR membranes; the content of enzyme in the monomeric form decreased with rate constant 0.14 min-1. It is concluded that melittin binds rapidly to SR membranes, inducing slow changes in Ca-ATPase conformation and oligomeric state as well as structural transitions in the lipid bilayer of SR membranes.

Animals↗
PubMed1999

Comparative characteristics of sarcoplasmic reticulum preparations from skeletal muscles of the ground squirrel Spermophilus undulatus, rats, and rabbits.

A comparison of sarcoplasmic reticulum (SR) preparations from skeletal muscles of ground squirrels Spermophilus undulatus, rats, and rabbits established that on the basis of protein yield and phospholipid/protein ratio these preparations are practically the same. Nevertheless, the specific activity of Ca-ATPase, the main protein component of SR membranes, in SR preparations of the ground squirrel skeletal muscles is only about half of the activity in SR preparations of rats and rabbits. Significant differences in protein composition of the preparations were detected: ground squirrel SR differed by an unusually high content of a 205 kD protein (probably myosin) and a number of low-molecular-weight SR protein components, and the SR preparations of rabbits are characterized by a high content of the Ca-binding proteins calsequestrin and sarcalumenin. Use of the anionic carbocyanine dye Stains-All established that all preparations contained only three proteins which are stained dark blue by this dye: calsequestrin, sarcalumenin, and a histidine-rich Ca-binding protein. The electrophoretic mobility of calsequestrin was identical in all preparations (molecular mass 63 kD), whereas sarcalumenin and histidine-rich Ca-binding protein are probably present in different isoforms with molecular masses of 130, 145, and 160 and 165, 155, and 170 kD, respectively, in SR preparations of ground squirrels, rats, and rabbits. Analysis of the fluorescence parameters of the fluorescent probes 8-anilino-1-naphthalene sulfonic acid and pyrene bound to SR membranes showed that the properties of the lipid bilayer in the SR membranes of the preparations differed considerably. It is suggested that the differences in protein composition and/or structural state of the ground squirrel SR membrane lipid bilayer could be the reason for the low Ca-ATPase activity in these preparations.

Animals↗
PubMed1997

Melittin-induced inhibition and aggregation of Ca-ATPase in skeletal muscle sarcoplasmic reticulum: a comparative study.

Incubation of melittin with sarcoplasmic reticulum membranes at pH 7. 0 and different melittin:Ca-ATPase molar ratios results in the progressive loss of enzyme activity. At high melittin:Ca-ATPase molar ratios (10:1 and 30:1), enzyme inhibition may be described by a biexponential curve. At pH 7.0, the values of the pseudo-first-order rate constants are 1.0 and 0.1 min-1 for the fast and slow phases of inhibition, respectively, at a melittin:Ca-ATPase molar ratio of 30:1. At pH 6.0 and a melittin:Ca-ATPase molar ratio of 30:1, melittin does not inhibit Ca-ATPase. Melittin-induced aggregation of Ca-ATPase molecules was studied using cupric phenanthroline as a chemical cross-linking agent. At a melittin:Ca-ATPase molar ratio of 5:1, aggregation of Ca-ATPase protein was not observed; however, the loss of enzyme activity was about 30% after 30 min. At melittin:Ca-ATPase molar ratios of 10:1 and 30:1, significant aggregation of Ca-ATPase protein takes place. The rate of Ca-ATPase aggregation is much lower than the rate of enzyme inhibition. At melittin:Ca-ATPase molar ratios of 10:1 and 30:1, the rate of Ca-ATPase protein aggregation is close to that for the slow phase of enzyme inhibition. At pH 6.0 and a melittin:Ca-ATPase molar ratio of 30:1, significant aggregation of Ca-ATPase occurs. It is concluded that melittin induces both Ca-ATPase inhibition and aggregation. These two processes may occur simultaneously, but under some conditions either inhibition or aggregation takes place independently of each other. Therefore, the aggregation of Ca-ATPase induced by melittin is not necessary for enzyme inhibition.

Animals↗
PubMed1997

Histidine-containing dipeptides as endogenous regulators of the activity of sarcoplasmic reticulum Ca-release channels.

It is shown that histidine-containing dipeptide carnosine (beta-alanyl-L-histidine), which is present in skeletal muscles in millimolar concentrations, decreases the rate of Ca2+ accumulation by the heavy fraction of sarcoplasmic reticulum from rabbit skeletal muscles. This effect results from the ability of carnosine to induce a rapid Ca2+ release from the heavy sarcoplasmic reticulum vesicles via activation of the ruthenium red-sensitive Ca-channels. The effect of carnosine is dose-dependent that indicates the presence of saturable site(s) for carnosine in the molecules of Ca-channels. The C0.5 value carnosine (the concentration that induces the half-maximal Ca2+ release) is 8.7 mM. The 1 N-methylated derivative of carnosine, i.e., anserine, also induces a rapid Ca2+ release with the half-maximal effect at 2.7 mM. Conversely, neither histidine nor beta-alanine (both separately and in the mixture) cause Ca2+ release. In addition, carnosine increases the sensitivity of Ca-channels to their well-known activators (caffeine, AMP, and Ca2+) and decreases inhibitory effect of low concentrations of Mg2+. It is concluded that carnosine as a component of skeletal muscles can be an endogenous regulator of the sarcoplasmic reticulum Ca-channel activity.

Adenosine Monophosphate↗
PubMed1997

Mechanism of inhibition of E1-E2 ATPases by melittin.

The inhibition of Na,K-ATPase from duck salt gland and Ca-ATPase from rabbit skeletal muscle sarcoplasmic reticulum by melittin, a 26-residue peptide from bee venom, was studied. Melittin irreversibly inhibits both enzymes. At melittin/ATPase molar ratio (30-50):1, the time dependence of the inhibition is described by the sum of two exponential curves. At pH 7.0, the fast phase of the inhibition provides for about 50% of total loss of activity with pseudo-first order rate constants of 1.52 +/- 0.17 and 1.20 +/- 0.21 min-1 for Na,K- and Ca-ATPase, respectively. The corresponding pseudo-first order rate constants for the slow phase were 0.12 +/- 0.02 and 0.09 +/- 0.02 min-1. The inhibition of both enzymes by melittin depends upon pH; the inhibition increases when the pH is increased from 6.0 to 8.5. The enhancement of the inhibition concomitant to increase in pH is mainly due to an increase in the rate constant of the fast phase. ATP protects both enzymes from the inhibition by melittin; however, the character of protection is different for Ca-versus Na,K-ATPase. The protection of Ca-ATPase activity by ATP is due to an increase in melittin-insensitive activity. The protective effect of ATP on Na,K-ATPase is due to a decrease in the rate constant of fast phase as well as an increase in melittin-insensitive activity. The data suggest that the inhibition of Ca- and Na,K-ATPases by melittin results from the interaction of the peptide with two different sites. One of the sites may be located on the catalytic subunit of the enzymes, the other can be related to the lipid bilayer of the membrane.

Animals↗
PubMed1997

Ca-release channels (ryanodine receptors) of sarcoplasmic reticulum: structure and properties. A review.

Recent data on characteristics of the structure, function, and regulation of sarcoplasmic reticulum Ca-release channels (ryanodine receptors) from various types of muscles are summarized. The history of identification and purification of Ca-release channels, the properties of isoforms expressed in various tissues, their electric characteristics, and regulation by endogenous and exogenous compounds are described. The phosphorylation of Ca-release channels by protein kinases and their interaction with various intracellular regulatory proteins are discussed. The proposed mechanisms of excitation-contraction coupling and the role of the Ca-release channels in this process are analyzed.

Animals↗
PubMed1997

H,K-ATPase and acid secretion control in gastric mucosa.

H,K-ATPase from gastric mucosa is responsible for HCI secretion in the gastric lumen and is a member of the P-type ATPase family. The structure of enzyme subunits, their functions and topology, the mechanism of ATP hydrolysis and transport function of the enzyme, its specific inhibitors, and the success of their pharmacological application are reviewed. The methods for isolation of membrane fractions with H,K-ATPase activity and attempts for solubilization and purification of the enzyme are described. Data demonstrating the presence of H,K-ATPase in other tissues are considered. Information about other enzyme systems of parietal cells involved in transepithelial transport of HCl (the Cl- and K-channels of the apical membrane, the HCO3-/Cl- anion exchanger and Na+/H+ cation exchanger of the basolateral membrane) is presented. Mechanisms of activation of acid secretion by parietal cells via gastrin, acetylcholine, and histamine receptors and the role of cytoskeletal proteins in activation are reviewed.

Biological Transport↗
PubMed1995

[Effect of ligands on rotational mobility of Na,K-ATPase].

Phosphorescence anisotropy of eosin-5'-isothiocyanate labelled Na,K-ATPase purified from duck salt glands has been studied. The initial anisotropy value is 0.235 +/- 0.015 (room temperature) and does not depend on the enzyme conformation (sodium or potassium). The experimental curve is fitted into a two-exponential curve with residual term, the fast component corresponds to the rotational mobility of the functional unit of Na,K-ATPase (promoter), while the slow one--to that of larger associates. In the presence of ligands modifying the conformational state of Na,K-ATPase (sodium, potassium, ATP) the rotational mobility of the fast component does not change in contrast with the slow one. A comparison of the enzyme rotational mobility in the presence of ligands simulating different steps of hydrolytic cycle suggests that interprotomer interactions are changed in the course of hydrolytic cycle: the fraction of larger associates increases at the step of the enzyme interactions with potassium ions, whereas their mobility in the bilayer enhances sharply after interaction with ATP. In the presence of the 2% non-ionic detergent, C12E9, the initial anisotropy value decreases down to 0.1; the residual term disappears thereby, while the curve is still two-exponential. However, the difference in the rotational mobility of sodium and potassium conformers diminishes. At the same time, the ratios between protomers and oligomers in the presence of sodium and potassium become approximated. This indicates that in the presence of the detergent high molecular weight associates are solubilized, the mobility of the both protomers and oligomers of Na,K-ATPase increases, while the difference between the mobilities of sodium and potassium conformers is disappeared.

Animals↗
PubMed1994

Thermal uncoupling of the Ca(2+)-transporting ATPase in sarcoplasmic reticulum. Changes in surface properties of light vesicles.

It is known that the light fraction of rabbit skeletal muscle sarcoplasmic reticulum vesicles can release Ca2+ from the intravesicular space, although the Ca(2+)-conductive channels are present only in the heavy fraction of sarcoplasmic reticulum vesicles. To study the possible pathways of the Ca2+ leakage from light vesicles we have used a short-term treatment for 4.5 min at 45 degrees C which quickly decreases the efficiency of Ca(2+)-transporting ATPase operation without any visible effects on the hydrolytic activity of the Ca(2+)-ATPase in the membranes. The treatment of the vesicles decreased the negative membrane surface potential created by the Ca(2+)-ATPase. Comparative titration of control and heat-treated preparations of light sarcoplasmic reticulum vesicles by K+, Na+, Mg2+, and Ca2+ revealed clear differences in their surface properties. The short-term heating resulted in release of Ca2+ from the vesicles previously loaded with 45Ca2+, which indicates an increase in passive membrane permeability to Ca2+. Study of Ca(2+)-ATPase protein arrangement in the membrane indicated that the heat treatment induced protein oligomerization and some of the Ca(2+)-ATPase molecules acquired intermolecular and intramolecular covalent bonds. From these data, we have concluded that the changes in the surface and structure properties of the vesicle membranes after the short-term heat treatment were the result of clustering of the Ca(2+)-ATPase molecules. This protein rearrangement may create channels for calcium leakage from light sarcoplasmic reticulum vesicles.

Animals↗
PubMed1994

[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↗