The Experts below are selected from a list of 75 Experts worldwide ranked by ideXlab platform
Aruni Bhatnagar - One of the best experts on this subject based on the ideXlab platform.
-
differential regulation of voltage gated k channels by oxidized and reduced pyridine Nucleotide Coenzymes
American Journal of Physiology-cell Physiology, 2005Co-Authors: Srinivas M Tipparaju, Nina Saxena, Rajiv Kumar, Aruni BhatnagarAbstract:The activity of the voltage-sensitive K+ (Kv) channels varies as a function of the intracellular redox state and metabolism, and several Kv channels act as oxygen sensors. However, the mechanisms u...
-
Differential pyridine Nucleotide coenzyme binding to the β-subunit of the voltage-sensitive K+ channel: a mechanism for redox regulation of excitability?
Chemico-Biological Interactions, 2003Co-Authors: Aruni Bhatnagar, Rajiv Kumar, Srinivas M TipparajuAbstract:Abstract The pore-forming subunits of the voltage-sensitive K+ channel (Kv) associate with ancillary β-subunits that regulate inactivation and voltage-dependence of the channel. The β-subunits are members of the aldo-keto reductase (AKR) superfamily. We have previously demonstrated that recombinant Kvβ2.1 displays tight binding to NADP(H). The protein also binds NAD(H), but with less affinity. To assess the physiological significance of this binding, we examined how pyridine Nucleotides regulate the Kvβ-mediated inactivation of K+ channels. Transient transfection of COS-7 cells with an pIRES-hrGFP vector containing the Kvα1.5 cDNA led to the appearance of the Kv1.5 protein in the membrane fraction and large non-inactivating potassium currents were recorded from the transfected cells. No such currents were observed in cells transfected with the empty vector alone or with Kvβ1.3 (AKR6A3), which was localized to the cytoplasm. In contrast, Kvβ1.3 co-transfected with Kvα1.5 was localized to the membrane, suggesting high affinity binding of the two proteins. Moreover, the K currents recorded from cells transfected with both Kvα1.5 and Kvβ1.3 displayed pronounced inactivation. Inclusion of 1 mM NAD+ in the internal solution of the patch pipette abolished Kvβ-induced inactivation of Kv1.5 currents, but did not affect the non-inactivating currents recorded from cells transfected with Kv1.5 alone, indicating that in the absence of Kvβ, NAD+ does not affect the activity of Kvα. The inactivating currents recorded from cells expressing both Kvα1.5 and Kvβ1.3 were unaffected by the inclusion of 0.1 mM NADPH in the pipette solution. Together, these data suggest that NADPH and NAD+ impart different conformational states to the Kvβ protein and that only the NADPH bound Kvβ imparts inactivation to non-inactivating K+ currents. Thus, differential binding of pyridine Nucleotide Coenzymes to Kvβ could regulate membrane potential and excitability as a function of the cellular redox state. Because NAD+/NADPH ratio is sensitive to oxygen concentration, the differential changes in Kvβ-mediated inactivation of K currents by NAD+ and NADPH could represent an oxygen-sensing mechanism.
-
Differential pyridine Nucleotide coenzyme binding to the beta-subunit of the voltage-sensitive K+ channel: a mechanism for redox regulation of excitability?
Chemico-biological interactions, 2003Co-Authors: Aruni Bhatnagar, Rajiv Kumar, Srinivas M TipparajuAbstract:The pore-forming subunits of the voltage-sensitive K(+) channel (K(v)) associate with ancillary beta-subunits that regulate inactivation and voltage-dependence of the channel. The beta-subunits are members of the aldo-keto reductase (AKR) superfamily. We have previously demonstrated that recombinant K(v)beta2.1 displays tight binding to NADP(H). The protein also binds NAD(H), but with less affinity. To assess the physiological significance of this binding, we examined how pyridine Nucleotides regulate the K(v)beta-mediated inactivation of K(+) channels. Transient transfection of COS-7 cells with an pIRES-hrGFP vector containing the Kvalpha1.5 cDNA led to the appearance of the K(v)1.5 protein in the membrane fraction and large non-inactivating potassium currents were recorded from the transfected cells. No such currents were observed in cells transfected with the empty vector alone or with K(v)beta1.3 (AKR6A3), which was localized to the cytoplasm. In contrast, K(v)beta1.3 co-transfected with Kvalpha1.5 was localized to the membrane, suggesting high affinity binding of the two proteins. Moreover, the K currents recorded from cells transfected with both K(v)alpha1.5 and K(v)beta1.3 displayed pronounced inactivation. Inclusion of 1 mM NAD(+) in the internal solution of the patch pipette abolished K(v)beta-induced inactivation of K(v)1.5 currents, but did not affect the non-inactivating currents recorded from cells transfected with K(v)1.5 alone, indicating that in the absence of K(v)beta, NAD(+) does not affect the activity of K(v)alpha. The inactivating currents recorded from cells expressing both K(v)alpha1.5 and K(v)beta1.3 were unaffected by the inclusion of 0.1 mM NADPH in the pipette solution. Together, these data suggest that NADPH and NAD(+) impart different conformational states to the K(v)beta protein and that only the NADPH bound K(v)beta imparts inactivation to non-inactivating K(+) currents. Thus, differential binding of pyridine Nucleotide Coenzymes to K(v)beta could regulate membrane potential and excitability as a function of the cellular redox state. Because NAD(+)/NADPH ratio is sensitive to oxygen concentration, the differential changes in K(v)beta-mediated inactivation of K currents by NAD(+) and NADPH could represent an oxygen-sensing mechanism.
-
binding of pyridine Nucleotide Coenzymes to the β subunit of the voltage sensitive k channel
Journal of Biological Chemistry, 2001Co-Authors: Albert Zacarias, Sanjay Srivastava, Aruni BhatnagarAbstract:Abstract The β-subunit of the voltage-sensitive K+ (Kv) channels belongs to the aldo-keto reductase superfamily, and the crystal structure of Kvβ2 shows NADP bound in its active site. Here we report that Kvβ2 displays a high affinity for NADPH (K d = 0.1 μm) and NADP+(K d = 0.3 μm), as determined by fluorometric titrations of the recombinant protein. The Kvβ2 also bound NAD(H) but with 10-fold lower affinity. The site-directed mutants R264E and N333W did not bind NADPH, whereas, the K d NADPH of Q214R was 10-fold greater than the wild-type protein. TheK d NADPH was unaffected by the R189M, W243Y, W243A, or Y255F mutation. The tetrameric structure of the wild-type protein was retained by the R264E mutant, indicating that NADPH binding is not a prerequisite for multimer formation. A C248S mutation caused a 5-fold decrease inK d NADPH, shifted the pK a of K d NADPH from 6.9 to 7.4, and decreased the ionic strength dependence of NADPH binding. These results indicate that Arg-264 and Asn-333 are critical for coenzyme binding, which is regulated in part by Cys-248. The binding of both NADP(H) and NAD(H) to the protein suggests that several types of Kvβ2-Nucleotide complexes may be formed in vivo.
Srinivas M Tipparaju - One of the best experts on this subject based on the ideXlab platform.
-
differential regulation of voltage gated k channels by oxidized and reduced pyridine Nucleotide Coenzymes
American Journal of Physiology-cell Physiology, 2005Co-Authors: Srinivas M Tipparaju, Nina Saxena, Rajiv Kumar, Aruni BhatnagarAbstract:The activity of the voltage-sensitive K+ (Kv) channels varies as a function of the intracellular redox state and metabolism, and several Kv channels act as oxygen sensors. However, the mechanisms u...
-
Differential pyridine Nucleotide coenzyme binding to the β-subunit of the voltage-sensitive K+ channel: a mechanism for redox regulation of excitability?
Chemico-Biological Interactions, 2003Co-Authors: Aruni Bhatnagar, Rajiv Kumar, Srinivas M TipparajuAbstract:Abstract The pore-forming subunits of the voltage-sensitive K+ channel (Kv) associate with ancillary β-subunits that regulate inactivation and voltage-dependence of the channel. The β-subunits are members of the aldo-keto reductase (AKR) superfamily. We have previously demonstrated that recombinant Kvβ2.1 displays tight binding to NADP(H). The protein also binds NAD(H), but with less affinity. To assess the physiological significance of this binding, we examined how pyridine Nucleotides regulate the Kvβ-mediated inactivation of K+ channels. Transient transfection of COS-7 cells with an pIRES-hrGFP vector containing the Kvα1.5 cDNA led to the appearance of the Kv1.5 protein in the membrane fraction and large non-inactivating potassium currents were recorded from the transfected cells. No such currents were observed in cells transfected with the empty vector alone or with Kvβ1.3 (AKR6A3), which was localized to the cytoplasm. In contrast, Kvβ1.3 co-transfected with Kvα1.5 was localized to the membrane, suggesting high affinity binding of the two proteins. Moreover, the K currents recorded from cells transfected with both Kvα1.5 and Kvβ1.3 displayed pronounced inactivation. Inclusion of 1 mM NAD+ in the internal solution of the patch pipette abolished Kvβ-induced inactivation of Kv1.5 currents, but did not affect the non-inactivating currents recorded from cells transfected with Kv1.5 alone, indicating that in the absence of Kvβ, NAD+ does not affect the activity of Kvα. The inactivating currents recorded from cells expressing both Kvα1.5 and Kvβ1.3 were unaffected by the inclusion of 0.1 mM NADPH in the pipette solution. Together, these data suggest that NADPH and NAD+ impart different conformational states to the Kvβ protein and that only the NADPH bound Kvβ imparts inactivation to non-inactivating K+ currents. Thus, differential binding of pyridine Nucleotide Coenzymes to Kvβ could regulate membrane potential and excitability as a function of the cellular redox state. Because NAD+/NADPH ratio is sensitive to oxygen concentration, the differential changes in Kvβ-mediated inactivation of K currents by NAD+ and NADPH could represent an oxygen-sensing mechanism.
-
Differential pyridine Nucleotide coenzyme binding to the beta-subunit of the voltage-sensitive K+ channel: a mechanism for redox regulation of excitability?
Chemico-biological interactions, 2003Co-Authors: Aruni Bhatnagar, Rajiv Kumar, Srinivas M TipparajuAbstract:The pore-forming subunits of the voltage-sensitive K(+) channel (K(v)) associate with ancillary beta-subunits that regulate inactivation and voltage-dependence of the channel. The beta-subunits are members of the aldo-keto reductase (AKR) superfamily. We have previously demonstrated that recombinant K(v)beta2.1 displays tight binding to NADP(H). The protein also binds NAD(H), but with less affinity. To assess the physiological significance of this binding, we examined how pyridine Nucleotides regulate the K(v)beta-mediated inactivation of K(+) channels. Transient transfection of COS-7 cells with an pIRES-hrGFP vector containing the Kvalpha1.5 cDNA led to the appearance of the K(v)1.5 protein in the membrane fraction and large non-inactivating potassium currents were recorded from the transfected cells. No such currents were observed in cells transfected with the empty vector alone or with K(v)beta1.3 (AKR6A3), which was localized to the cytoplasm. In contrast, K(v)beta1.3 co-transfected with Kvalpha1.5 was localized to the membrane, suggesting high affinity binding of the two proteins. Moreover, the K currents recorded from cells transfected with both K(v)alpha1.5 and K(v)beta1.3 displayed pronounced inactivation. Inclusion of 1 mM NAD(+) in the internal solution of the patch pipette abolished K(v)beta-induced inactivation of K(v)1.5 currents, but did not affect the non-inactivating currents recorded from cells transfected with K(v)1.5 alone, indicating that in the absence of K(v)beta, NAD(+) does not affect the activity of K(v)alpha. The inactivating currents recorded from cells expressing both K(v)alpha1.5 and K(v)beta1.3 were unaffected by the inclusion of 0.1 mM NADPH in the pipette solution. Together, these data suggest that NADPH and NAD(+) impart different conformational states to the K(v)beta protein and that only the NADPH bound K(v)beta imparts inactivation to non-inactivating K(+) currents. Thus, differential binding of pyridine Nucleotide Coenzymes to K(v)beta could regulate membrane potential and excitability as a function of the cellular redox state. Because NAD(+)/NADPH ratio is sensitive to oxygen concentration, the differential changes in K(v)beta-mediated inactivation of K currents by NAD(+) and NADPH could represent an oxygen-sensing mechanism.
Michael W W Adams - One of the best experts on this subject based on the ideXlab platform.
-
[4] Ferredoxin:NADP oxidoreductase from Pyrococcus furiosus
Methods in Enzymology, 2004Co-Authors: Kesen Ma, Michael W W AdamsAbstract:Publisher Summary Ferredoxin:NADP + oxidoreductase (FNOR) is a flavoenzyme that catalyzes electron transfer between the redox protein, ferredoxin, and the pyridine Nucleotide Coenzymes, NADP(H) and/or NAD(H). Enzymes of this type have been characterized from many organisms, including from both the bacterial and eukaryotic domains. However, only one such enzyme has been purified from the hypertherophilic archaea, that from Pyrococcus furiosus. The P. Furiosus enzyme not only functions as a very efficient FNOR, but also catalyzes a variety of reactions, including the reduction of polysulfide using NADPH as electron donor. This chapter describes the assay methods, purification procedure, and properties of P. furiosus FNOR.
-
characterization of pyridine Nucleotide Coenzymes in the hyperthermophilic archaeon pyrococcus furiosus
Extremophiles, 2001Co-Authors: Marc F J M Verhagen, Michael W W AdamsAbstract:Pyridine-type Nucleotides were identified in cell-free extracts of the hyperthermophilic archaeon Pyrococcus furiosus by their ability to replace authentic nicotinamide adenine diNucleotide (phosphate) [NAD(P)] in assays using pure P. furiosus enzymes. The Nucleotides were purified using a combination of ion-exchange and reverse-phase chromatography. They were identified as NAD and NADP by analyses using liquid chromatography–mass spectrometry and high performance liquid chromatography. Their intracellular concentrations were measured in P. furiosus grown using maltose and peptides as the carbon sources. The concentrations decreased during the lag phase but remained constant during the exponential phase at approximately 0.17 and 0.13 mM, respectively. The amount of NAD was significantly lower (more than four-fold lower) than that in mesophilic bacteria, although the NADP concentration was comparable. The internal concentrations of NADH and NADPH in P. furiosus were determined to be 0.14 mM and 0.04 mM, respectively. The overall cellular concentration of NAD(P)(H) in P. furiosus (0.48 mM) is about half the value in the mesophiles. The NAD(H)/NADP(H) ratio in P. furiosus is consistent with the preferred use of NADP by several catabolic enzymes that have been purified from this organism. The mechanisms by which hyperthermophiles stabilize these thermally labile nicotinamide Nucleotides are not known.
William Curtis - One of the best experts on this subject based on the ideXlab platform.
-
the great controlling Nucleotide Coenzymes
Iubmb Life, 2019Co-Authors: Richard L Veech, Michael Todd King, Robert Pawlosky, Yoshihiro Kashiwaya, Patrick C Bradshaw, William CurtisAbstract:: Nucleotide Coenzymes dot the map of metabolic pathways providing energy to drive the reactions of the pathway and play an important role in regulating and controlling energy metabolism through their shared potential energy, which is widely unobserved due to the paradox that the energy in the coenzyme pools cannot be determined from the concentration of the coenzyme couples. The potential energy of the Nucleotide couples in the mitochondria or the cytoplasm is expressed in the enzyme reactions in which they take part. The energy in these couples, [NAD+]/[NADH], [NADP+]/[NADPH], [acetyl CoA]/[CoA], and [ATP]/[ADP]x[Pi], regulates energy metabolism. The energy contained in the couples can be altered by suppling energy equivalents in the form of ketones, such as, D-β-hydroxybutyrate to overcome insulin resistance, to restore antioxidants capacity, to form potential treatments for Alzheimer's and Parkinson's diseases, to enhance life span, and to increase physiological performance. © 2019 IUBMB Life, 71(5):565-579, 2019.
Rajiv Kumar - One of the best experts on this subject based on the ideXlab platform.
-
differential regulation of voltage gated k channels by oxidized and reduced pyridine Nucleotide Coenzymes
American Journal of Physiology-cell Physiology, 2005Co-Authors: Srinivas M Tipparaju, Nina Saxena, Rajiv Kumar, Aruni BhatnagarAbstract:The activity of the voltage-sensitive K+ (Kv) channels varies as a function of the intracellular redox state and metabolism, and several Kv channels act as oxygen sensors. However, the mechanisms u...
-
Differential pyridine Nucleotide coenzyme binding to the β-subunit of the voltage-sensitive K+ channel: a mechanism for redox regulation of excitability?
Chemico-Biological Interactions, 2003Co-Authors: Aruni Bhatnagar, Rajiv Kumar, Srinivas M TipparajuAbstract:Abstract The pore-forming subunits of the voltage-sensitive K+ channel (Kv) associate with ancillary β-subunits that regulate inactivation and voltage-dependence of the channel. The β-subunits are members of the aldo-keto reductase (AKR) superfamily. We have previously demonstrated that recombinant Kvβ2.1 displays tight binding to NADP(H). The protein also binds NAD(H), but with less affinity. To assess the physiological significance of this binding, we examined how pyridine Nucleotides regulate the Kvβ-mediated inactivation of K+ channels. Transient transfection of COS-7 cells with an pIRES-hrGFP vector containing the Kvα1.5 cDNA led to the appearance of the Kv1.5 protein in the membrane fraction and large non-inactivating potassium currents were recorded from the transfected cells. No such currents were observed in cells transfected with the empty vector alone or with Kvβ1.3 (AKR6A3), which was localized to the cytoplasm. In contrast, Kvβ1.3 co-transfected with Kvα1.5 was localized to the membrane, suggesting high affinity binding of the two proteins. Moreover, the K currents recorded from cells transfected with both Kvα1.5 and Kvβ1.3 displayed pronounced inactivation. Inclusion of 1 mM NAD+ in the internal solution of the patch pipette abolished Kvβ-induced inactivation of Kv1.5 currents, but did not affect the non-inactivating currents recorded from cells transfected with Kv1.5 alone, indicating that in the absence of Kvβ, NAD+ does not affect the activity of Kvα. The inactivating currents recorded from cells expressing both Kvα1.5 and Kvβ1.3 were unaffected by the inclusion of 0.1 mM NADPH in the pipette solution. Together, these data suggest that NADPH and NAD+ impart different conformational states to the Kvβ protein and that only the NADPH bound Kvβ imparts inactivation to non-inactivating K+ currents. Thus, differential binding of pyridine Nucleotide Coenzymes to Kvβ could regulate membrane potential and excitability as a function of the cellular redox state. Because NAD+/NADPH ratio is sensitive to oxygen concentration, the differential changes in Kvβ-mediated inactivation of K currents by NAD+ and NADPH could represent an oxygen-sensing mechanism.
-
Differential pyridine Nucleotide coenzyme binding to the beta-subunit of the voltage-sensitive K+ channel: a mechanism for redox regulation of excitability?
Chemico-biological interactions, 2003Co-Authors: Aruni Bhatnagar, Rajiv Kumar, Srinivas M TipparajuAbstract:The pore-forming subunits of the voltage-sensitive K(+) channel (K(v)) associate with ancillary beta-subunits that regulate inactivation and voltage-dependence of the channel. The beta-subunits are members of the aldo-keto reductase (AKR) superfamily. We have previously demonstrated that recombinant K(v)beta2.1 displays tight binding to NADP(H). The protein also binds NAD(H), but with less affinity. To assess the physiological significance of this binding, we examined how pyridine Nucleotides regulate the K(v)beta-mediated inactivation of K(+) channels. Transient transfection of COS-7 cells with an pIRES-hrGFP vector containing the Kvalpha1.5 cDNA led to the appearance of the K(v)1.5 protein in the membrane fraction and large non-inactivating potassium currents were recorded from the transfected cells. No such currents were observed in cells transfected with the empty vector alone or with K(v)beta1.3 (AKR6A3), which was localized to the cytoplasm. In contrast, K(v)beta1.3 co-transfected with Kvalpha1.5 was localized to the membrane, suggesting high affinity binding of the two proteins. Moreover, the K currents recorded from cells transfected with both K(v)alpha1.5 and K(v)beta1.3 displayed pronounced inactivation. Inclusion of 1 mM NAD(+) in the internal solution of the patch pipette abolished K(v)beta-induced inactivation of K(v)1.5 currents, but did not affect the non-inactivating currents recorded from cells transfected with K(v)1.5 alone, indicating that in the absence of K(v)beta, NAD(+) does not affect the activity of K(v)alpha. The inactivating currents recorded from cells expressing both K(v)alpha1.5 and K(v)beta1.3 were unaffected by the inclusion of 0.1 mM NADPH in the pipette solution. Together, these data suggest that NADPH and NAD(+) impart different conformational states to the K(v)beta protein and that only the NADPH bound K(v)beta imparts inactivation to non-inactivating K(+) currents. Thus, differential binding of pyridine Nucleotide Coenzymes to K(v)beta could regulate membrane potential and excitability as a function of the cellular redox state. Because NAD(+)/NADPH ratio is sensitive to oxygen concentration, the differential changes in K(v)beta-mediated inactivation of K currents by NAD(+) and NADPH could represent an oxygen-sensing mechanism.
