The Experts below are selected from a list of 192 Experts worldwide ranked by ideXlab platform
Jan Rydstrom - One of the best experts on this subject based on the ideXlab platform.
-
Nicotinamide Nucleotide Transhydrogenase
Encyclopedia of Biological Chemistry, 2013Co-Authors: Jan RydstromAbstract:Membrane-bound Nicotinamide Nucleotide transhydrogenase is located in the bacterial plasma membrane and mitochondrial inner membrane and catalyzes the reversible reduction of NADP + by NADH. The reaction is linked to the electrochemical proton gradient across the membrane, generating NADPH essential for, for example, detoxification of peroxides. In its isolated form, the enzyme is a proton pump where proton translocation is driven by the catalytic reaction, mediated by conformational changes. Extensive investigations of the structure–function relationships, especially of the substrate-binding domains, have established their structures and roles. The membrane domain, in which the proton channel resides, has been structurally predicted, but remains to be determined structurally at the atomic level. Models for the molecular proton translocation mechanism are therefore beginning to emerge.
-
Chapter 25 Insulin Secretion from β‐Cells is Affected by Deletion of Nicotinamide Nucleotide Transhydrogenase
Methods in enzymology, 2009Co-Authors: Kenju Shimomura, Jan Rydstrom, Juris Galvanovskis, Michelle Goldsworthy, Alison Hugill, Stephan Kaizak, Angela Lee, Nicholas A. Meadows, Mohamed M. Quwailid, Lydia TeboulAbstract:Nicotinamide Nucleotide transhydrogenase (NNT) is an inner mitochondrial membrane transmembrane protein involved in regenerating NADPH, coupled with proton translocation across the inner membrane. We have shown that a defect in Nnt function in the mouse, and specifically within the beta-cell, leads to a reduction in insulin secretion. This chapter describes methods for examining Nnt function in the mouse. This includes generating in vivo models with point mutations and expression of Nnt by transgenesis, and making in vitro models, by silencing of gene expression. In addition, techniques are described to measure insulin secretion, calcium and hydrogen peroxide concentrations, membrane potential, and NNT activity. These approaches and techniques can also be applied to other genes of interest.
-
chapter 25 insulin secretion from β cells is affected by deletion of Nicotinamide Nucleotide transhydrogenase
Methods in Enzymology, 2009Co-Authors: Kenju Shimomura, Jan Rydstrom, Juris Galvanovskis, Michelle Goldsworthy, Alison Hugill, Stephan Kaizak, Angela Lee, Nicholas A. Meadows, Mohamed M. Quwailid, Lydia TeboulAbstract:Nicotinamide Nucleotide transhydrogenase (NNT) is an inner mitochondrial membrane transmembrane protein involved in regenerating NADPH, coupled with proton translocation across the inner membrane. We have shown that a defect in Nnt function in the mouse, and specifically within the beta-cell, leads to a reduction in insulin secretion. This chapter describes methods for examining Nnt function in the mouse. This includes generating in vivo models with point mutations and expression of Nnt by transgenesis, and making in vitro models, by silencing of gene expression. In addition, techniques are described to measure insulin secretion, calcium and hydrogen peroxide concentrations, membrane potential, and NNT activity. These approaches and techniques can also be applied to other genes of interest.
-
purification of recombinant membrane proteins tagged with calmodulin binding domains by affinity chromatography on calmodulin agarose example of Nicotinamide Nucleotide transhydrogenase
Nature Protocols, 2007Co-Authors: Nikolay B. Pestov, Jan RydstromAbstract:This protocol describes affinity purification of bacterially expressed, recombinant membrane proteins fused with calmodulin-binding domains. As exemplified by the Escherichia coli Nicotinamide Nucleotide transhydrogenase, this method allows isolation of the protein fusions in a single chromatography step using elution with the calcium chelating agent EDTA and, unlike purification of His-tagged proteins on nickel chelate, it is not sensitive to the presence of strong reducing agents (e.g., DTT). Our protocol involves disruption of host bacteria by sonication, sedimentation of membranes by differential centrifugation, solubilization of membrane proteins and affinity chromatography on calmodulin-agarose. To achieve maximum purity and yield, the use of a combination of non-ionic and anionic detergents is suggested. Purification takes two working days, with an overnight wash of the column to increase the purity of the product.
-
A Caenorhabditis elegans mutant lacking functional Nicotinamide Nucleotide transhydrogenase displays increased sensitivity to oxidative stress.
Free radical biology & medicine, 2005Co-Authors: Eva L. Arkblad, Simon Tuck, Nikolay B. Pestov, Ruslan I. Dmitriev, M. B. Kostina, Jörgen Stenvall, Mattias Tranberg, Jan RydstromAbstract:A Caenorhabditis elegans mutant lacking functional Nicotinamide Nucleotide transhydrogenase displays increased sensitivity to oxidative stress.
Youssef Hatefi - One of the best experts on this subject based on the ideXlab platform.
-
The proton channel of the energy-transducing Nicotinamide Nucleotide transhydrogenase of Escherichia coli.
The Journal of biological chemistry, 2002Co-Authors: Mutsuo Yamaguchi, C. David Stout, Youssef HatefiAbstract:Abstract The Nicotinamide Nucleotide transhydrogenases of mitochondria and bacteria are proton pumps that couple direct hydride ion transfer between NAD(H) and NADP(H) bound, respectively, to extramembranous domains I and III to proton translocation by the membrane-intercalated domain II. To delineate the proton channel of the enzyme, 25 conserved and semiconserved prototropic amino acid residues of domain II of the Escherichia coli transhydrogenase were mutated, and the mutant enzymes were assayed for transhydrogenation from NADPH to an NAD analogue and for the coupled outward proton translocation. The results confirmed the previous findings of others and ourselves on the essential roles of three amino acid residues and identified another essential residue. Three of these amino acids, His-91, Ser-139, and Asn-222, occur in three separate membrane-spanning α helices of domain II of the β subunit of the enzyme. Another residue, Asp-213, is probably located in a cytosolic-side loop that connects to the α helix bearing Asn-222. It is proposed that the three helices bearing His-91, Ser-139, and Asn-222 come together, possibly with another highly conserved α helix to form a four-helix bundle proton channel and that Asp-213 serves to conduct protons between the channel and domain III where NADPH binding energy is used via protein conformation change to initiate outward proton translocation.
-
Nicotinamide Nucleotide transhydrogenase: a model for utilization of substrate binding energy for proton translocation.
FASEB journal : official publication of the Federation of American Societies for Experimental Biology, 1996Co-Authors: Youssef Hatefi, Mutsuo YamaguchiAbstract:The energy-transducing Nicotinamide Nucleotide transhydrogenases of mammalian mitochondria and bacteria are structurally related membrane-bound enzymes that catalyze the direct transfer of a hydride ion between NAD(H) and NADP(H) in a reaction that is coupled to transmembrane proton translocation. The protonmotive force alters the affinity of the transhydrogenase for substrates, accelerates the rate of hydride ion transfer from NADH to NADP, and shifts the equilibrium of this reaction toward NADPH formation. Transhydrogenation in the reverse direction from NADPH to NAD is accompanied by outward proton translocation and formation of a protonmotive force. In reverse transhydrogenation, the enzyme utilizes substrate binding energy for proton pumping. Therefore, with regard to the mechanism of energy transduction, the transhydrogenase works according to the same principles as the ATP synthase complex of mitochondria and bacteria, the proton and cation ATPases, and possibly certain redox-linked proton pumps. H...
-
Proton-translocating Nicotinamide Nucleotide Transhydrogenase RECONSTITUTION OF THE EXTRAMEMBRANOUS Nucleotide-BINDING DOMAINS
The Journal of biological chemistry, 1995Co-Authors: Mutsuo Yamaguchi, Youssef HatefiAbstract:Abstract The Nicotinamide Nucleotide transhydrogenase of bovine mitochondria is a homodimer of monomer M = 109,065. The monomer is composed of three domains, an NH-terminal 430-residue-long hydrophilic domain I that binds NAD(H), a central 400-residue-long hydrophobic domain II that is largely membrane intercalated and carries the enzyme's proton channel, and a COOH-terminal 200-residue-long hydrophilic domain III that binds NADP(H). Domains I and III protrude into the mitochondrial matrix, where they presumably come together to form the enzyme's catalytic site. The two-subunit transhydrogenase of Escherichia coli and the three-subunit transhydrogenase of Rhodospirillum rubrum have each the same overall tridomain hydropathy profile as the bovine enzyme. Domain I of the R. rubrum enzyme (the α1 subunit) is water soluble and easily removed from the chromatophore membranes. We have isolated domain I of the bovine transhydrogenase after controlled trypsinolysis of the purified enzyme and have expressed in E. coli and purified therefrom domain III of this enzyme. This paper shows that an active bidomain transhydrogenase lacking domain II can be reconstituted by the combination of purified bovine domains I plus III or R. rubrum domain I plus bovine domain III.
-
Chapter 11 The energy-transducing Nicotinamide Nucleotide transhydrogenase
Molecular Mechanisms in Bioenergetics, 1992Co-Authors: Youssef Hatefi, Mutsuo YamaguchiAbstract:Publisher Summary This chapter discusses the energy-transducing Nicotinamide Nucleotide transhydrogenase. The energy-transducing Nicotinamide Nucleotide transhydrogenases of mitochondria and bacteria are membrane-bound enzymes that catalyze the direct and stereospecific transfer of a hydride ion between the 4A position of NAD (H) and the 4B position of NADP (H). In the mitochondria, the enzyme is embedded in the inner membrane, with its Nucleotide binding sites protruding into the matrix, and the transhydrogenation reaction is coupled to transmembrane proton translocation with a H + / H - stoichiometry of unity. Because of the stereospecificity of hydride ion transfer, this type of transhydrogenase is referred to as “AB-transhydrogenase.” The known AB-transhydrogenases are integral membrane proteins, their scalar transhydrogenation reaction is coupled to proton translocation, and they contain separate binding sites for NAD (H) and NADP (H), and have no prosthetic groups. The BB-type transhydrogenases are water-soluble, do not pump protons, are flavoproteins containing FAD, have a single substrate-binding site, and are thought to be concerned with equilibrating the cellular NAD (H) and NADP (H) pools.
-
mitochondrial energy linked Nicotinamide Nucleotide transhydrogenase membrane topography of the bovine enzyme
Journal of Biological Chemistry, 1991Co-Authors: Mutsuo Yamaguchi, Youssef HatefiAbstract:The mitochondrial energy-linked Nicotinamide Nucleotide transhydrogenase is a homodimer of monomer Mr = 109,228. Hydropathy analysis of its cDNA-deduced amino acid sequence (1043 residues) has indicated that the molecule is composed of 3 domains: a 430-residue-long hydrophilic N-terminal domain which binds NAD(H), a 200-residue-long hydrophilic C-terminal domain which binds NADP(H), and a 400-residue-long hydrophobic central domain which appears to be made up mainly of about 14 hydrophobic clusters of approximately 20 residues each. In this study, antibodies were raised to the hydrophilic N- and C-terminal domains cleaved from the isolated transhydrogenase by proteolytic digestion, and to a synthetic, hydrophilic pentadecapeptide, which corresponded to position 540-554 within the central hydrophobic domain. Immunochemical experiments with mitoplasts (mitochondria denuded of outer membrane) and submitochondrial particles (inside-out inner membrane vesicles) as sources of antigens showed that essentially the entire N- and C-terminal hydrophilic domains of the transhydrogenase, as well as epitopes from the central pentadecapeptide, protrude from the inner membrane into the mitochondrial matrix, where the N- and C-terminal domains would be expected to come together to form the enzyme's catalytic site. Treatment of mitoplasts with several proteolytic enzymes indicated that large protease-sensitive masses of the transhydrogenase are not exposed on the cytosolic side of the inner membrane, which agreed with the exception that the central highly hydrophobic domain of the molecule should be largely membrane-intercalated. Trypsin, alpha-chymotrypsin, and papain had little or no effect on the mitoplast-embedded transhydrogenase. Proteinase K, subtilisin (Nagarse), thermolysin, and pronase E each split the mitoplast-embedded enzyme into two fragments only, a fragment of approximately 70 kDa containing the N-terminal hydrophilic domain, and one of approximately 40 kDa bearing the C-terminal hydrophilic domain. The cleavage site of proteinase K was determined to be A690 -A691, which is located in a small hydrophilic segment within the central hydrophobic domain. This protease-sensitive loop appears to be exposed on the cytosolic side of the inner membrane. The proteinase K-nicked enzyme containing two peptides of 71 and 39 kDa was isolated from mitoplasts and shown to have high transhydrogenase activity.
Helen Freeman - One of the best experts on this subject based on the ideXlab platform.
-
Nicotinamide Nucleotide transhydrogenase a link between insulin secretion glucose metabolism and oxidative stress
Biochemical Society Transactions, 2006Co-Authors: Helen Freeman, Kenju Shimomura, Frances M. AshcroftAbstract:This paper reviews recent studies on the role of Nnt (Nicotinamide Nucleotide transhydrogenase) in insulin secretion and detoxification of ROS (reactive oxygen species). Glucose-stimulated insulin release from pancreatic β-cells is mediated by increased metabolism. This elevates intracellular [ATP], thereby closing K ATP channels (ATP-sensitive potassium channels) and producing membrane depolarization, activation of voltage-gated Ca 2+ channels, Ca 2+ influx and, consequently, insulin secretion. The C57BL/6J mouse displays glucose intolerance and reduced insulin secretion, which results from a naturally occurring deletion in the Nnt gene. Transgenic expression of the wild-type Nnt gene in C57BL/6J mice rescues the phenotype. Knockdown of Nnt in the insulin-secreting cell line MIN6 with small interfering RNA dramatically reduced Ca 2+ influx and insulin secretion. Similarly, mice carrying ENU ( N -ethyl- N -nitrosourea)-induced loss-of-function mutations in Nnt were glucose intolerant and secreted less insulin during a glucose tolerance test. Islets isolated from these mice showed impaired insulin secretion in response to glucose, but not to the K ATP channel blocker tolbutamide. This is explained by the fact that glucose failed to elevate ATP in Nnt mutant islets. Nnt is a nuclear-encoded mitochondrial protein involved in detoxification of ROS. β-Cells isolated from Nnt mutant mice showed increased ROS production on glucose stimulation. We hypothesize that Nnt mutations enhance glucose-dependent ROS production and thereby impair β-cell mitochondrial metabolism, possibly via activation of uncoupling proteins. This reduces ATP production and lowers K ATP channel activity. Consequently, glucose-dependent electrical activity and insulin secretion are impaired.
-
deletion of Nicotinamide Nucleotide transhydrogenase a new quantitive trait locus accounting for glucose intolerance in c57bl 6j mice
Diabetes, 2006Co-Authors: Helen Freeman, Frances M. Ashcroft, Alison Hugill, Neil Dear, Roger D. CoxAbstract:The C57BL/6J mouse displays glucose intolerance and reduced insulin secretion. The genetic locus underlying this phenotype was mapped to Nicotinamide Nucleotide transhydrogenase (Nnt) on mouse chromosome 13, a nuclear-encoded mitochondrial protein involved in beta-cell mitochondrial metabolism. C57BL/6J mice have a naturally occurring in-frame five-exon deletion in Nnt that removes exons 7-11. This results in a complete absence of Nnt protein in these mice. We show that transgenic expression of the entire Nnt gene in C57BL/6J mice rescues their impaired insulin secretion and glucose-intolerant phenotype. This study provides direct evidence that Nnt deficiency results in defective insulin secretion and inappropriate glucose homeostasis in male C57BL/6J mice.
-
Deletion of Nicotinamide Nucleotide Transhydrogenase: A New Quantitive Trait Locus Accounting for Glucose Intolerance in C57BL/6J Mice
Diabetes, 2006Co-Authors: Helen Freeman, Frances M. Ashcroft, Alison Hugill, Neil Dear, Roger D. CoxAbstract:The C57BL/6J mouse displays glucose intolerance and reduced insulin secretion. The genetic locus underlying this phenotype was mapped to Nicotinamide Nucleotide transhydrogenase (Nnt) on mouse chromosome 13, a nuclear-encoded mitochondrial protein involved in beta-cell mitochondrial metabolism. C57BL/6J mice have a naturally occurring in-frame five-exon deletion in Nnt that removes exons 7-11. This results in a complete absence of Nnt protein in these mice. We show that transgenic expression of the entire Nnt gene in C57BL/6J mice rescues their impaired insulin secretion and glucose-intolerant phenotype. This study provides direct evidence that Nnt deficiency results in defective insulin secretion and inappropriate glucose homeostasis in male C57BL/6J mice.
-
Nicotinamide Nucleotide transhydrogenase: a key role in insulin secretion.
Cell metabolism, 2006Co-Authors: Helen Freeman, Kenju Shimomura, Emma Horner, Roger D. Cox, Frances M. AshcroftAbstract:The C57BL/6J mouse displays glucose intolerance and reduced insulin secretion. QTL mapping identified Nicotinamide Nucleotide Transhydrogenase (Nnt), a nuclear-encoded mitochondrial protein thought to be involved in free radical detoxification, as a candidate gene. To investigate its functional role, we used siRNA to knock down Nnt in insulin-secreting MIN6 cells. This produced a dramatic reduction in insulin secretion and the rise in [Ca2+]i evoked by glucose, but not tolbutamide. We identified two ENU-induced point mutations in Nnt (N68K, G745D). Nnt mutant mice were glucose intolerant and secreted less insulin during a glucose tolerance test. Isolated islets showed impaired insulin secretion in response to glucose, but not to tolbutamide, and glucose failed to enhance ATP levels. Glucose utilization and production of reactive oxygen species were increased in Nnt beta cells. We hypothesize that Nnt mutations/deletion uncouple beta cell mitochondrial metabolism leading to less ATP production, enhanced KATP channel activity, and consequently impaired insulin secretion.
Roger D. Cox - One of the best experts on this subject based on the ideXlab platform.
-
Nicotinamide Nucleotide transhydrogenase (NNT) acts as a novel modulator of macrophage inflammatory responses
FASEB journal : official publication of the Federation of American Societies for Experimental Biology, 2012Co-Authors: Vera M. Ripoll, Nicholas A. Meadows, Mathieu Bangert, Angela W. Lee, Aras Kadioglu, Roger D. CoxAbstract:Nicotinamide Nucleotide transhydrogenase (NNT) is a mitochondrial redox-driven proton pump that couples the production of NADPH to the mitochondrial metabolic rate. In this study, we demonstrated for the first time that NNT has a significant effect in the modulation of the immune response and host defense against pathogens. We found that NNT mRNA is enriched in immune system-related tissues and regulated during macrophage activation. Overexpression of NNT in a macrophage cell-line resulted in decreased levels of reactive oxygen species (ROS) and nitric oxide upon induction of the macrophage inflammatory responses. These cells failed to fully activate MAPK signaling pathways, resulting in defective secretion of proinflammatory cytokines in response to LPS, and were inefficient in clearance of intracellular bacteria. We have shown that C57BL/6J mice, which have a deletion in the Nnt gene, exhibited greater resistance to acute pulmonary infection with Streptococcus pneumoniae. Macrophages from these mice generated more ROS and established a stronger inflammatory response to this pathogen. Our results demonstrate a novel role for NNT as a regulator of macrophage-mediated inflammatory responses.
-
deletion of Nicotinamide Nucleotide transhydrogenase a new quantitive trait locus accounting for glucose intolerance in c57bl 6j mice
Diabetes, 2006Co-Authors: Helen Freeman, Frances M. Ashcroft, Alison Hugill, Neil Dear, Roger D. CoxAbstract:The C57BL/6J mouse displays glucose intolerance and reduced insulin secretion. The genetic locus underlying this phenotype was mapped to Nicotinamide Nucleotide transhydrogenase (Nnt) on mouse chromosome 13, a nuclear-encoded mitochondrial protein involved in beta-cell mitochondrial metabolism. C57BL/6J mice have a naturally occurring in-frame five-exon deletion in Nnt that removes exons 7-11. This results in a complete absence of Nnt protein in these mice. We show that transgenic expression of the entire Nnt gene in C57BL/6J mice rescues their impaired insulin secretion and glucose-intolerant phenotype. This study provides direct evidence that Nnt deficiency results in defective insulin secretion and inappropriate glucose homeostasis in male C57BL/6J mice.
-
Deletion of Nicotinamide Nucleotide Transhydrogenase: A New Quantitive Trait Locus Accounting for Glucose Intolerance in C57BL/6J Mice
Diabetes, 2006Co-Authors: Helen Freeman, Frances M. Ashcroft, Alison Hugill, Neil Dear, Roger D. CoxAbstract:The C57BL/6J mouse displays glucose intolerance and reduced insulin secretion. The genetic locus underlying this phenotype was mapped to Nicotinamide Nucleotide transhydrogenase (Nnt) on mouse chromosome 13, a nuclear-encoded mitochondrial protein involved in beta-cell mitochondrial metabolism. C57BL/6J mice have a naturally occurring in-frame five-exon deletion in Nnt that removes exons 7-11. This results in a complete absence of Nnt protein in these mice. We show that transgenic expression of the entire Nnt gene in C57BL/6J mice rescues their impaired insulin secretion and glucose-intolerant phenotype. This study provides direct evidence that Nnt deficiency results in defective insulin secretion and inappropriate glucose homeostasis in male C57BL/6J mice.
-
Nicotinamide Nucleotide transhydrogenase: a key role in insulin secretion.
Cell metabolism, 2006Co-Authors: Helen Freeman, Kenju Shimomura, Emma Horner, Roger D. Cox, Frances M. AshcroftAbstract:The C57BL/6J mouse displays glucose intolerance and reduced insulin secretion. QTL mapping identified Nicotinamide Nucleotide Transhydrogenase (Nnt), a nuclear-encoded mitochondrial protein thought to be involved in free radical detoxification, as a candidate gene. To investigate its functional role, we used siRNA to knock down Nnt in insulin-secreting MIN6 cells. This produced a dramatic reduction in insulin secretion and the rise in [Ca2+]i evoked by glucose, but not tolbutamide. We identified two ENU-induced point mutations in Nnt (N68K, G745D). Nnt mutant mice were glucose intolerant and secreted less insulin during a glucose tolerance test. Isolated islets showed impaired insulin secretion in response to glucose, but not to tolbutamide, and glucose failed to enhance ATP levels. Glucose utilization and production of reactive oxygen species were increased in Nnt beta cells. We hypothesize that Nnt mutations/deletion uncouple beta cell mitochondrial metabolism leading to less ATP production, enhanced KATP channel activity, and consequently impaired insulin secretion.
Mutsuo Yamaguchi - One of the best experts on this subject based on the ideXlab platform.
-
The proton channel of the energy-transducing Nicotinamide Nucleotide transhydrogenase of Escherichia coli.
The Journal of biological chemistry, 2002Co-Authors: Mutsuo Yamaguchi, C. David Stout, Youssef HatefiAbstract:Abstract The Nicotinamide Nucleotide transhydrogenases of mitochondria and bacteria are proton pumps that couple direct hydride ion transfer between NAD(H) and NADP(H) bound, respectively, to extramembranous domains I and III to proton translocation by the membrane-intercalated domain II. To delineate the proton channel of the enzyme, 25 conserved and semiconserved prototropic amino acid residues of domain II of the Escherichia coli transhydrogenase were mutated, and the mutant enzymes were assayed for transhydrogenation from NADPH to an NAD analogue and for the coupled outward proton translocation. The results confirmed the previous findings of others and ourselves on the essential roles of three amino acid residues and identified another essential residue. Three of these amino acids, His-91, Ser-139, and Asn-222, occur in three separate membrane-spanning α helices of domain II of the β subunit of the enzyme. Another residue, Asp-213, is probably located in a cytosolic-side loop that connects to the α helix bearing Asn-222. It is proposed that the three helices bearing His-91, Ser-139, and Asn-222 come together, possibly with another highly conserved α helix to form a four-helix bundle proton channel and that Asp-213 serves to conduct protons between the channel and domain III where NADPH binding energy is used via protein conformation change to initiate outward proton translocation.
-
Nicotinamide Nucleotide transhydrogenase: a model for utilization of substrate binding energy for proton translocation.
FASEB journal : official publication of the Federation of American Societies for Experimental Biology, 1996Co-Authors: Youssef Hatefi, Mutsuo YamaguchiAbstract:The energy-transducing Nicotinamide Nucleotide transhydrogenases of mammalian mitochondria and bacteria are structurally related membrane-bound enzymes that catalyze the direct transfer of a hydride ion between NAD(H) and NADP(H) in a reaction that is coupled to transmembrane proton translocation. The protonmotive force alters the affinity of the transhydrogenase for substrates, accelerates the rate of hydride ion transfer from NADH to NADP, and shifts the equilibrium of this reaction toward NADPH formation. Transhydrogenation in the reverse direction from NADPH to NAD is accompanied by outward proton translocation and formation of a protonmotive force. In reverse transhydrogenation, the enzyme utilizes substrate binding energy for proton pumping. Therefore, with regard to the mechanism of energy transduction, the transhydrogenase works according to the same principles as the ATP synthase complex of mitochondria and bacteria, the proton and cation ATPases, and possibly certain redox-linked proton pumps. H...
-
Proton-translocating Nicotinamide Nucleotide Transhydrogenase RECONSTITUTION OF THE EXTRAMEMBRANOUS Nucleotide-BINDING DOMAINS
The Journal of biological chemistry, 1995Co-Authors: Mutsuo Yamaguchi, Youssef HatefiAbstract:Abstract The Nicotinamide Nucleotide transhydrogenase of bovine mitochondria is a homodimer of monomer M = 109,065. The monomer is composed of three domains, an NH-terminal 430-residue-long hydrophilic domain I that binds NAD(H), a central 400-residue-long hydrophobic domain II that is largely membrane intercalated and carries the enzyme's proton channel, and a COOH-terminal 200-residue-long hydrophilic domain III that binds NADP(H). Domains I and III protrude into the mitochondrial matrix, where they presumably come together to form the enzyme's catalytic site. The two-subunit transhydrogenase of Escherichia coli and the three-subunit transhydrogenase of Rhodospirillum rubrum have each the same overall tridomain hydropathy profile as the bovine enzyme. Domain I of the R. rubrum enzyme (the α1 subunit) is water soluble and easily removed from the chromatophore membranes. We have isolated domain I of the bovine transhydrogenase after controlled trypsinolysis of the purified enzyme and have expressed in E. coli and purified therefrom domain III of this enzyme. This paper shows that an active bidomain transhydrogenase lacking domain II can be reconstituted by the combination of purified bovine domains I plus III or R. rubrum domain I plus bovine domain III.
-
Chapter 11 The energy-transducing Nicotinamide Nucleotide transhydrogenase
Molecular Mechanisms in Bioenergetics, 1992Co-Authors: Youssef Hatefi, Mutsuo YamaguchiAbstract:Publisher Summary This chapter discusses the energy-transducing Nicotinamide Nucleotide transhydrogenase. The energy-transducing Nicotinamide Nucleotide transhydrogenases of mitochondria and bacteria are membrane-bound enzymes that catalyze the direct and stereospecific transfer of a hydride ion between the 4A position of NAD (H) and the 4B position of NADP (H). In the mitochondria, the enzyme is embedded in the inner membrane, with its Nucleotide binding sites protruding into the matrix, and the transhydrogenation reaction is coupled to transmembrane proton translocation with a H + / H - stoichiometry of unity. Because of the stereospecificity of hydride ion transfer, this type of transhydrogenase is referred to as “AB-transhydrogenase.” The known AB-transhydrogenases are integral membrane proteins, their scalar transhydrogenation reaction is coupled to proton translocation, and they contain separate binding sites for NAD (H) and NADP (H), and have no prosthetic groups. The BB-type transhydrogenases are water-soluble, do not pump protons, are flavoproteins containing FAD, have a single substrate-binding site, and are thought to be concerned with equilibrating the cellular NAD (H) and NADP (H) pools.
-
mitochondrial energy linked Nicotinamide Nucleotide transhydrogenase membrane topography of the bovine enzyme
Journal of Biological Chemistry, 1991Co-Authors: Mutsuo Yamaguchi, Youssef HatefiAbstract:The mitochondrial energy-linked Nicotinamide Nucleotide transhydrogenase is a homodimer of monomer Mr = 109,228. Hydropathy analysis of its cDNA-deduced amino acid sequence (1043 residues) has indicated that the molecule is composed of 3 domains: a 430-residue-long hydrophilic N-terminal domain which binds NAD(H), a 200-residue-long hydrophilic C-terminal domain which binds NADP(H), and a 400-residue-long hydrophobic central domain which appears to be made up mainly of about 14 hydrophobic clusters of approximately 20 residues each. In this study, antibodies were raised to the hydrophilic N- and C-terminal domains cleaved from the isolated transhydrogenase by proteolytic digestion, and to a synthetic, hydrophilic pentadecapeptide, which corresponded to position 540-554 within the central hydrophobic domain. Immunochemical experiments with mitoplasts (mitochondria denuded of outer membrane) and submitochondrial particles (inside-out inner membrane vesicles) as sources of antigens showed that essentially the entire N- and C-terminal hydrophilic domains of the transhydrogenase, as well as epitopes from the central pentadecapeptide, protrude from the inner membrane into the mitochondrial matrix, where the N- and C-terminal domains would be expected to come together to form the enzyme's catalytic site. Treatment of mitoplasts with several proteolytic enzymes indicated that large protease-sensitive masses of the transhydrogenase are not exposed on the cytosolic side of the inner membrane, which agreed with the exception that the central highly hydrophobic domain of the molecule should be largely membrane-intercalated. Trypsin, alpha-chymotrypsin, and papain had little or no effect on the mitoplast-embedded transhydrogenase. Proteinase K, subtilisin (Nagarse), thermolysin, and pronase E each split the mitoplast-embedded enzyme into two fragments only, a fragment of approximately 70 kDa containing the N-terminal hydrophilic domain, and one of approximately 40 kDa bearing the C-terminal hydrophilic domain. The cleavage site of proteinase K was determined to be A690 -A691, which is located in a small hydrophilic segment within the central hydrophobic domain. This protease-sensitive loop appears to be exposed on the cytosolic side of the inner membrane. The proteinase K-nicked enzyme containing two peptides of 71 and 39 kDa was isolated from mitoplasts and shown to have high transhydrogenase activity.