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O D Lopina - One of the best experts on this subject based on the ideXlab platform.

  • glutathionylation of na k atpase alpha subunit alters Enzyme Conformation and sensitivity to trypsinolysis
    Biochemistry, 2018
    Co-Authors: E A Dergousova, Y M Poluektov, E A Klimanova, I Y Petrushanko, Vladimir A Mitkevich, Alexander A Makarov, O D Lopina
    Abstract:

    We found earlier that Na,K-ATPase is purified from duck salt glands in partially glutathionylated state (up to 13 of the 23 cysteine residues of the Na,K-ATPase catalytic α-subunit can be S-glutathionylated). To determine the effect of glutathionylation on the Enzyme Conformation, we have analyzed the products of trypsinolysis of Na,K-ATPase α-subunit in different Conformations with different extent of glutathionylation. Incubation of the protein in the E1 Conformation with trypsin produced a large fragment with a molecular mass (MM) of 80 kDa with the following formation of smaller fragments with MM 40, 35.5, and 23 kDa. Tryptic digestion of Na,K-ATPase in the E2 Conformation also resulted in the generation of the fragments with MM 40, 35.5, and 23 kDa. Deglutathionylation of Na,K-ATPase α-subunit increases the rate of proteolysis of the Enzyme in both E1 and E2 Conformations. The pattern of tryptic digestion of the α-subunit in E2 Conformation additionally glutathionylated with oxidized glutathione is similar to that of partially deglutathionylated Na,K-ATPase. The pattern of tryptic digestion of the additionally glutathionylated α-subunit in E1 Conformation is similar to that of the native Enzyme. The highest rate of trypsinolysis was observed for the α-subunit in complex with ouabain (E2-OBN Conformation). Additional glutathionylation increased the content of high-molecular-weight fragments among the digestion products, as compared to the native and deglutathionylated Enzymes. The data obtained were confirmed using molecular mod-eling that revealed that number of sites accessible for trypsinolysis is higher in the E2P-OBN Conformation than in the E1-and E2-Conformations and that glutathionylation decreases the number of sites accessible for trypsin. Therefore, glu-tathionylation affects Enzyme Conformation and its sensitivity to trypsinolysis. The mechanisms responsible for the changes in the Na,K-ATPase sensitivity to trypsinolysis depending on the level of Enzyme glutathionylation and increase in the Enzyme sensitivity to proteolysis upon its binding to ouabain, as well as physiological role of these phenomena, are discussed.

  • glutathionylation of na k atpase alpha subunit alters Enzyme Conformation and sensitivity to trypsinolysis
    Biochemistry, 2018
    Co-Authors: E A Dergousova, Y M Poluektov, E A Klimanova, I Y Petrushanko, Vladimir A Mitkevich, Alexander A Makarov, O D Lopina
    Abstract:

    We found earlier that Na,K-ATPase is purified from duck salt glands in partially glutathionylated state (up to 13 of the 23 cysteine residues of the Na,K-ATPase catalytic α-subunit can be S-glutathionylated). To determine the effect of glutathionylation on the Enzyme Conformation, we have analyzed the products of trypsinolysis of Na,K-ATPase α-subunit in different Conformations with different extent of glutathionylation. Incubation of the protein in the E1 Conformation with trypsin produced a large fragment with a molecular mass (MM) of 80 kDa with the following formation of smaller fragments with MM 40, 35.5, and 23 kDa. Tryptic digestion of Na,K-ATPase in the E2 Conformation also resulted in the generation of the fragments with MM 40, 35.5, and 23 kDa. Deglutathionylation of Na,K-ATPase α-subunit increases the rate of proteolysis of the Enzyme in both E1 and E2 Conformations. The pattern of tryptic digestion of the α-subunit in E2 Conformation additionally glutathionylated with oxidized glutathione is similar to that of partially deglutathionylated Na,K-ATPase. The pattern of tryptic digestion of the additionally glutathionylated α-subunit in E1 Conformation is similar to that of the native Enzyme. The highest rate of trypsinolysis was observed for the α-subunit in complex with ouabain (E2-OBN Conformation). Additional glutathionylation increased the content of high-molecular-weight fragments among the digestion products, as compared to the native and deglutathionylated Enzymes. The data obtained were confirmed using molecular mod-eling that revealed that number of sites accessible for trypsinolysis is higher in the E2P-OBN Conformation than in the E1-and E2-Conformations and that glutathionylation decreases the number of sites accessible for trypsin. Therefore, glu-tathionylation affects Enzyme Conformation and its sensitivity to trypsinolysis. The mechanisms responsible for the changes in the Na,K-ATPase sensitivity to trypsinolysis depending on the level of Enzyme glutathionylation and increase in the Enzyme sensitivity to proteolysis upon its binding to ouabain, as well as physiological role of these phenomena, are discussed.

  • binding of ouabain and marinobufagenin leads to different structural changes in na k atpase and depends on the Enzyme Conformation
    FEBS Letters, 2015
    Co-Authors: E A Klimanova, I Y Petrushanko, Vladimir A Mitkevich, Alexander A Makarov, Anastasia A Anashkina, Sergey N Orlov, O D Lopina
    Abstract:

    Ion pump, Na,K-ATPase specifically binds cardiotonic steroids (CTS), which leads to inhibition of the Enzyme activity and activation of signaling network in the cell. We have studied interaction of Na,K-ATPase with CTS of two different types – marinobufagenin and ouabain. We have shown that both CTS inhibit activity of Na,K-ATPase with the same Ki values, but binding of ouabain is sensitive to the Conformation of Na,K-ATPase while binding of marinobufagenin is not. Furthermore, binding of ouabain and marinobufagenin results in different structural changes in Na,K-ATPase. Our data allow to explain the diversity of effects on the receptor function of Na,K-ATPase caused by different types of CTS.

Christine D Keating - One of the best experts on this subject based on the ideXlab platform.

  • interactions of macromolecular crowding agents and cosolutes with small molecule substrates effect on horseradish peroxidase activity with two different substrates
    Journal of Physical Chemistry B, 2014
    Co-Authors: William M Aumiller, Bradley W Davis, Emmanuel Hatzakis, Christine D Keating
    Abstract:

    The importance of solution composition on enzymatic reactions is increasingly appreciated, particularly with respect to macromolecular cosolutes. Macromolecular crowding and its effect on enzymatic reactions has been studied for several Enzymes and is often understood in terms of changes to Enzyme Conformation. Comparatively little attention has been paid to the chemical properties of small-molecule substrates for Enzyme reactions in crowded solution. In this article, we studied the reaction of horseradish peroxidase (HRP) with two small-molecule substrates that differ in their hydrophobicity. Crowding agents and cosolutes had quite different effects on HRP activity when the substrate used was 3,3′,5,5′-tetramethylbenzidine (TMB, which is hydrophobic) as compared to o-phenylenediamine (OPD, which is more hydrophilic). Reaction rates with TMB were much more sensitive to the presence of crowding agents and cosolutes than OPD, suggesting that the small-molecule substrates may themselves be interacting with c...

  • interactions of macromolecular crowding agents and cosolutes with small molecule substrates effect on horseradish peroxidase activity with two different substrates
    The Journal of Physical Chemistry, 2014
    Co-Authors: William M Aumiller, Bradley W Davis, Emmanuel Hatzakis, Christine D Keating
    Abstract:

    The importance of solution composition on enzymatic reactions is increasingly appreciated, particularly with respect to macromolecular cosolutes. Macromolecular crowding and its effect on enzymatic reactions has been studied for several Enzymes and is often understood in terms of changes to Enzyme Conformation. Comparatively little attention has been paid to the chemical properties of small-molecule substrates for Enzyme reactions in crowded solution. In this article, we studied the reaction of horseradish peroxidase (HRP) with two small-molecule substrates that differ in their hydrophobicity. Crowding agents and cosolutes had quite different effects on HRP activity when the substrate used was 3,3′,5,5′-tetramethylbenzidine (TMB, which is hydrophobic) as compared to o-phenylenediamine (OPD, which is more hydrophilic). Reaction rates with TMB were much more sensitive to the presence of crowding agents and cosolutes than OPD, suggesting that the small-molecule substrates may themselves be interacting with crowders and cosolutes. At high polyethylene glycol (PEG) concentrations (25–30 wt/wt %), no reaction was observed for TMB. Even at lower concentrations, Michaelis constants (KM) for HRP with the more hydrophobic substrate increased in the presence of crowding agents and cosolutes, particularly with PEG. Diffusion of TMB and OPD in the PEG and dextran reaction media was evaluated using pulsed field gradient nuclear magnetic resonance (PFG-NMR). The diffusivity of the TMB decreased 3.9× in 10% PEG 8k compared to that in buffer and decreased only 1.7× for OPD. Together, these data suggest that weak attractive interactions between small-molecule substrates and crowders or cosolutes can reduce substrate chemical activity and consequently decrease Enzyme activity and that these effects vary with the identity of the molecules involved. Because many Enzymes can act on multiple substrates, it is important to consider substrate chemistry in understanding enzymatic reactions in complex media such as biological fluids.

E A Klimanova - One of the best experts on this subject based on the ideXlab platform.

  • glutathionylation of na k atpase alpha subunit alters Enzyme Conformation and sensitivity to trypsinolysis
    Biochemistry, 2018
    Co-Authors: E A Dergousova, Y M Poluektov, E A Klimanova, I Y Petrushanko, Vladimir A Mitkevich, Alexander A Makarov, O D Lopina
    Abstract:

    We found earlier that Na,K-ATPase is purified from duck salt glands in partially glutathionylated state (up to 13 of the 23 cysteine residues of the Na,K-ATPase catalytic α-subunit can be S-glutathionylated). To determine the effect of glutathionylation on the Enzyme Conformation, we have analyzed the products of trypsinolysis of Na,K-ATPase α-subunit in different Conformations with different extent of glutathionylation. Incubation of the protein in the E1 Conformation with trypsin produced a large fragment with a molecular mass (MM) of 80 kDa with the following formation of smaller fragments with MM 40, 35.5, and 23 kDa. Tryptic digestion of Na,K-ATPase in the E2 Conformation also resulted in the generation of the fragments with MM 40, 35.5, and 23 kDa. Deglutathionylation of Na,K-ATPase α-subunit increases the rate of proteolysis of the Enzyme in both E1 and E2 Conformations. The pattern of tryptic digestion of the α-subunit in E2 Conformation additionally glutathionylated with oxidized glutathione is similar to that of partially deglutathionylated Na,K-ATPase. The pattern of tryptic digestion of the additionally glutathionylated α-subunit in E1 Conformation is similar to that of the native Enzyme. The highest rate of trypsinolysis was observed for the α-subunit in complex with ouabain (E2-OBN Conformation). Additional glutathionylation increased the content of high-molecular-weight fragments among the digestion products, as compared to the native and deglutathionylated Enzymes. The data obtained were confirmed using molecular mod-eling that revealed that number of sites accessible for trypsinolysis is higher in the E2P-OBN Conformation than in the E1-and E2-Conformations and that glutathionylation decreases the number of sites accessible for trypsin. Therefore, glu-tathionylation affects Enzyme Conformation and its sensitivity to trypsinolysis. The mechanisms responsible for the changes in the Na,K-ATPase sensitivity to trypsinolysis depending on the level of Enzyme glutathionylation and increase in the Enzyme sensitivity to proteolysis upon its binding to ouabain, as well as physiological role of these phenomena, are discussed.

  • glutathionylation of na k atpase alpha subunit alters Enzyme Conformation and sensitivity to trypsinolysis
    Biochemistry, 2018
    Co-Authors: E A Dergousova, Y M Poluektov, E A Klimanova, I Y Petrushanko, Vladimir A Mitkevich, Alexander A Makarov, O D Lopina
    Abstract:

    We found earlier that Na,K-ATPase is purified from duck salt glands in partially glutathionylated state (up to 13 of the 23 cysteine residues of the Na,K-ATPase catalytic α-subunit can be S-glutathionylated). To determine the effect of glutathionylation on the Enzyme Conformation, we have analyzed the products of trypsinolysis of Na,K-ATPase α-subunit in different Conformations with different extent of glutathionylation. Incubation of the protein in the E1 Conformation with trypsin produced a large fragment with a molecular mass (MM) of 80 kDa with the following formation of smaller fragments with MM 40, 35.5, and 23 kDa. Tryptic digestion of Na,K-ATPase in the E2 Conformation also resulted in the generation of the fragments with MM 40, 35.5, and 23 kDa. Deglutathionylation of Na,K-ATPase α-subunit increases the rate of proteolysis of the Enzyme in both E1 and E2 Conformations. The pattern of tryptic digestion of the α-subunit in E2 Conformation additionally glutathionylated with oxidized glutathione is similar to that of partially deglutathionylated Na,K-ATPase. The pattern of tryptic digestion of the additionally glutathionylated α-subunit in E1 Conformation is similar to that of the native Enzyme. The highest rate of trypsinolysis was observed for the α-subunit in complex with ouabain (E2-OBN Conformation). Additional glutathionylation increased the content of high-molecular-weight fragments among the digestion products, as compared to the native and deglutathionylated Enzymes. The data obtained were confirmed using molecular mod-eling that revealed that number of sites accessible for trypsinolysis is higher in the E2P-OBN Conformation than in the E1-and E2-Conformations and that glutathionylation decreases the number of sites accessible for trypsin. Therefore, glu-tathionylation affects Enzyme Conformation and its sensitivity to trypsinolysis. The mechanisms responsible for the changes in the Na,K-ATPase sensitivity to trypsinolysis depending on the level of Enzyme glutathionylation and increase in the Enzyme sensitivity to proteolysis upon its binding to ouabain, as well as physiological role of these phenomena, are discussed.

  • binding of ouabain and marinobufagenin leads to different structural changes in na k atpase and depends on the Enzyme Conformation
    FEBS Letters, 2015
    Co-Authors: E A Klimanova, I Y Petrushanko, Vladimir A Mitkevich, Alexander A Makarov, Anastasia A Anashkina, Sergey N Orlov, O D Lopina
    Abstract:

    Ion pump, Na,K-ATPase specifically binds cardiotonic steroids (CTS), which leads to inhibition of the Enzyme activity and activation of signaling network in the cell. We have studied interaction of Na,K-ATPase with CTS of two different types – marinobufagenin and ouabain. We have shown that both CTS inhibit activity of Na,K-ATPase with the same Ki values, but binding of ouabain is sensitive to the Conformation of Na,K-ATPase while binding of marinobufagenin is not. Furthermore, binding of ouabain and marinobufagenin results in different structural changes in Na,K-ATPase. Our data allow to explain the diversity of effects on the receptor function of Na,K-ATPase caused by different types of CTS.

William M Aumiller - One of the best experts on this subject based on the ideXlab platform.

  • interactions of macromolecular crowding agents and cosolutes with small molecule substrates effect on horseradish peroxidase activity with two different substrates
    Journal of Physical Chemistry B, 2014
    Co-Authors: William M Aumiller, Bradley W Davis, Emmanuel Hatzakis, Christine D Keating
    Abstract:

    The importance of solution composition on enzymatic reactions is increasingly appreciated, particularly with respect to macromolecular cosolutes. Macromolecular crowding and its effect on enzymatic reactions has been studied for several Enzymes and is often understood in terms of changes to Enzyme Conformation. Comparatively little attention has been paid to the chemical properties of small-molecule substrates for Enzyme reactions in crowded solution. In this article, we studied the reaction of horseradish peroxidase (HRP) with two small-molecule substrates that differ in their hydrophobicity. Crowding agents and cosolutes had quite different effects on HRP activity when the substrate used was 3,3′,5,5′-tetramethylbenzidine (TMB, which is hydrophobic) as compared to o-phenylenediamine (OPD, which is more hydrophilic). Reaction rates with TMB were much more sensitive to the presence of crowding agents and cosolutes than OPD, suggesting that the small-molecule substrates may themselves be interacting with c...

  • interactions of macromolecular crowding agents and cosolutes with small molecule substrates effect on horseradish peroxidase activity with two different substrates
    The Journal of Physical Chemistry, 2014
    Co-Authors: William M Aumiller, Bradley W Davis, Emmanuel Hatzakis, Christine D Keating
    Abstract:

    The importance of solution composition on enzymatic reactions is increasingly appreciated, particularly with respect to macromolecular cosolutes. Macromolecular crowding and its effect on enzymatic reactions has been studied for several Enzymes and is often understood in terms of changes to Enzyme Conformation. Comparatively little attention has been paid to the chemical properties of small-molecule substrates for Enzyme reactions in crowded solution. In this article, we studied the reaction of horseradish peroxidase (HRP) with two small-molecule substrates that differ in their hydrophobicity. Crowding agents and cosolutes had quite different effects on HRP activity when the substrate used was 3,3′,5,5′-tetramethylbenzidine (TMB, which is hydrophobic) as compared to o-phenylenediamine (OPD, which is more hydrophilic). Reaction rates with TMB were much more sensitive to the presence of crowding agents and cosolutes than OPD, suggesting that the small-molecule substrates may themselves be interacting with crowders and cosolutes. At high polyethylene glycol (PEG) concentrations (25–30 wt/wt %), no reaction was observed for TMB. Even at lower concentrations, Michaelis constants (KM) for HRP with the more hydrophobic substrate increased in the presence of crowding agents and cosolutes, particularly with PEG. Diffusion of TMB and OPD in the PEG and dextran reaction media was evaluated using pulsed field gradient nuclear magnetic resonance (PFG-NMR). The diffusivity of the TMB decreased 3.9× in 10% PEG 8k compared to that in buffer and decreased only 1.7× for OPD. Together, these data suggest that weak attractive interactions between small-molecule substrates and crowders or cosolutes can reduce substrate chemical activity and consequently decrease Enzyme activity and that these effects vary with the identity of the molecules involved. Because many Enzymes can act on multiple substrates, it is important to consider substrate chemistry in understanding enzymatic reactions in complex media such as biological fluids.

E A Dergousova - One of the best experts on this subject based on the ideXlab platform.

  • glutathionylation of na k atpase alpha subunit alters Enzyme Conformation and sensitivity to trypsinolysis
    Biochemistry, 2018
    Co-Authors: E A Dergousova, Y M Poluektov, E A Klimanova, I Y Petrushanko, Vladimir A Mitkevich, Alexander A Makarov, O D Lopina
    Abstract:

    We found earlier that Na,K-ATPase is purified from duck salt glands in partially glutathionylated state (up to 13 of the 23 cysteine residues of the Na,K-ATPase catalytic α-subunit can be S-glutathionylated). To determine the effect of glutathionylation on the Enzyme Conformation, we have analyzed the products of trypsinolysis of Na,K-ATPase α-subunit in different Conformations with different extent of glutathionylation. Incubation of the protein in the E1 Conformation with trypsin produced a large fragment with a molecular mass (MM) of 80 kDa with the following formation of smaller fragments with MM 40, 35.5, and 23 kDa. Tryptic digestion of Na,K-ATPase in the E2 Conformation also resulted in the generation of the fragments with MM 40, 35.5, and 23 kDa. Deglutathionylation of Na,K-ATPase α-subunit increases the rate of proteolysis of the Enzyme in both E1 and E2 Conformations. The pattern of tryptic digestion of the α-subunit in E2 Conformation additionally glutathionylated with oxidized glutathione is similar to that of partially deglutathionylated Na,K-ATPase. The pattern of tryptic digestion of the additionally glutathionylated α-subunit in E1 Conformation is similar to that of the native Enzyme. The highest rate of trypsinolysis was observed for the α-subunit in complex with ouabain (E2-OBN Conformation). Additional glutathionylation increased the content of high-molecular-weight fragments among the digestion products, as compared to the native and deglutathionylated Enzymes. The data obtained were confirmed using molecular mod-eling that revealed that number of sites accessible for trypsinolysis is higher in the E2P-OBN Conformation than in the E1-and E2-Conformations and that glutathionylation decreases the number of sites accessible for trypsin. Therefore, glu-tathionylation affects Enzyme Conformation and its sensitivity to trypsinolysis. The mechanisms responsible for the changes in the Na,K-ATPase sensitivity to trypsinolysis depending on the level of Enzyme glutathionylation and increase in the Enzyme sensitivity to proteolysis upon its binding to ouabain, as well as physiological role of these phenomena, are discussed.

  • glutathionylation of na k atpase alpha subunit alters Enzyme Conformation and sensitivity to trypsinolysis
    Biochemistry, 2018
    Co-Authors: E A Dergousova, Y M Poluektov, E A Klimanova, I Y Petrushanko, Vladimir A Mitkevich, Alexander A Makarov, O D Lopina
    Abstract:

    We found earlier that Na,K-ATPase is purified from duck salt glands in partially glutathionylated state (up to 13 of the 23 cysteine residues of the Na,K-ATPase catalytic α-subunit can be S-glutathionylated). To determine the effect of glutathionylation on the Enzyme Conformation, we have analyzed the products of trypsinolysis of Na,K-ATPase α-subunit in different Conformations with different extent of glutathionylation. Incubation of the protein in the E1 Conformation with trypsin produced a large fragment with a molecular mass (MM) of 80 kDa with the following formation of smaller fragments with MM 40, 35.5, and 23 kDa. Tryptic digestion of Na,K-ATPase in the E2 Conformation also resulted in the generation of the fragments with MM 40, 35.5, and 23 kDa. Deglutathionylation of Na,K-ATPase α-subunit increases the rate of proteolysis of the Enzyme in both E1 and E2 Conformations. The pattern of tryptic digestion of the α-subunit in E2 Conformation additionally glutathionylated with oxidized glutathione is similar to that of partially deglutathionylated Na,K-ATPase. The pattern of tryptic digestion of the additionally glutathionylated α-subunit in E1 Conformation is similar to that of the native Enzyme. The highest rate of trypsinolysis was observed for the α-subunit in complex with ouabain (E2-OBN Conformation). Additional glutathionylation increased the content of high-molecular-weight fragments among the digestion products, as compared to the native and deglutathionylated Enzymes. The data obtained were confirmed using molecular mod-eling that revealed that number of sites accessible for trypsinolysis is higher in the E2P-OBN Conformation than in the E1-and E2-Conformations and that glutathionylation decreases the number of sites accessible for trypsin. Therefore, glu-tathionylation affects Enzyme Conformation and its sensitivity to trypsinolysis. The mechanisms responsible for the changes in the Na,K-ATPase sensitivity to trypsinolysis depending on the level of Enzyme glutathionylation and increase in the Enzyme sensitivity to proteolysis upon its binding to ouabain, as well as physiological role of these phenomena, are discussed.