Modification of TPN-dependent isocitrate dehydrogenase by the 2', 3'-dialdehyde derivatives of TPNH and TPN

Catalytic reaction of the 2', 3'-dialdehyde analog of TPN (oTPN) with pig heart TPN-dependent isocitrate dehydrogenase in the presence of the substrate manganous isocitrate results in the formation of the dialdehyde derivative of TPNH (oTPNH). In the absence of the substrate, modification by oTPN leads to a progressive inactivation of the enzyme. The dependence of the pseudo-first order rate constants on the reagent concentration indicates the formation of a reversible complex with the enzyme prior to covalent modification (kmax = 5.5 X 10(-2) min-1; K1 = 290 microM). Reaction of [14C]oTPN with the enzyme results in the incorporation of 2 mol of oTPN/mol of peptide chain. No appreciable protection against either inactivation or incorporation by the natural ligands TPN and TPNH was obtained, suggesting different modes of binding of the analog in the presence and absence of the substrate isocitrate. Enzymatically synthesized oTPNH has been isolated and demonstrated to act as an affinity label for a TPNH-binding site of isocitrate dehydrogenase. The inactivation process exhibits saturation kinetics (kmax = 2.67 X 10(-3) min-1; K1 = 33 microM). Protection against activity loss, as well as a decrease in incorporation from 2 to 1 eq of [14C]oTPNH bound/peptide chain was observed in the presence of 1 mM TPNH. From the TPNH concentration dependence of the inactivation rate by oTPNH, a dissociation constant of 3.4 microM is calculated for TPNH, indicating binding of the analog to a specific TPNH-binding site on the enzyme. Although dialdehyde derivatives are frequently assumed to form Schiff bases with proteins, the evidence presented suggests the formation of morpholino derivatives as the products of the covalent reaction of isocitrate dehydrogenase with the dialdehyde derivatives of TPN and TPNH. The new reagent, oTPNH, may serve as an affinity label for other dehydrogenases.     

Modification of glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides with the 2',3'-dialdehyde derivative of NADP+ (oNADP+)

Glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides is irreversibly inactivated by the 2,3'-dialdehyde of NADP+ (oNADP+) in the absence of substrate. The inactivation is first order with respect to NADP+ concentration and follows saturation kinetics, indicating that the enzyme initially forms a reversible complex with the inhibitor followed by covalent modification (KI = 1.8 mM). NADP+ and NAD+ protect the enzyme from inactivation by oNADP+. The pK of inactivation is 8.1. oNADP+ is an effective coenzyme in assays of glucose-6-phosphate dehydrogenase (Km = 200 microM). Kinetic evidence and binding studies with [14C] oNADP+ indicate that one molecule of oNADP+ binds per subunit of glucose-6-phosphate dehydrogenase when the enzyme is completely inactivated. The interaction between oNADP+ and the enzyme does not generate a Schiff's base, or a conjugated Schiff's base, but the data are consistent with the formation of a dihydroxymorpholino derivative.     

Interaction between NAD-dependent isocitrate dehydrogenase, alpha-ketoglutarate dehydrogenase complex, and NADH:ubiquinone oxidoreductase

Interaction between the alpha-ketoglutarate dehydrogenase complex and NAD+-dependent isocitrate dehydrogenase was detected with a variety of techniques including polyethylene glycol precipitation, ultracentrifugation, and centrifugal gel filtration on a Sepharose 6B column. The interaction was specific in that citrate synthase, cytosolic malate dehydrogenase, and NADP-dependent isocitrate dehydrogenase did not interact with alpha-ketoglutarate dehydrogenase complex. The interaction was not inhibited by either 0.1 M KCl or 0.4 M (NH4)2SO4, but was completely prevented by 5% glycerol. A new method for the preparation of NADH: ubiquinone oxidoreductase resulted in an enzyme having a protein subunit composition similar to that of classical complex I preparation. Evidence is given for the existence of ternary complexes containing NADH:ubiquinone oxidoreductase-alpha-ketoglutarate dehydrogenase complex-NAD-dependent isocitrate dehydrogenase and NADH: ubiquinone oxidoreductase-alpha-ketoglutarate dehydrogenase complex-succinate thiokinase. These data suggest that a part of the citric acid cycle may be located in the vicinity of NADH: ubiquinone oxidoreductase. These complexes may facilitate the transport of metabolites among these enzymes without their equilibrating with the whole compartment.     

Reaction of the 2',3'-dialdehyde derivative of NADPH at a nucleotide site of bovine liver glutamate dehydrogenase

The 2',3'-dialdehyde derivative of NADPH (oNADPH) acts as a coenzyme for the reaction catalyzed by bovine liver glutamate dehydrogenase. Incubation of 250 microM oNADPH with enzyme for 300 min at 30 degrees C and pH 8.0 yields covalent incorporation of 1.0 mol of oNADPH/mol of enzyme subunit. The modified enzyme has a functional catalytic site and is activated by ADP, but is no longer inhibited by high NADH concentrations and exhibits decreased sensitivity to GTP inhibition. Using the change in inhibition by 600 microM NADH or 1 microM GTP to monitor the reaction leads to rate constants of 44.0 and 41.5 min-1 M-1, respectively, suggesting that loss of inhibition by the two regulatory compounds results from reaction by oNADPH at a single location. The oNADPH incorporation is proportional to the decreased inhibition by 600 microM NADH or 1 microM GTP, extrapolating to less than 1 mol of oNADPH/mol of subunit when the maximum change in NADH or GTP inhibition has occurred. Modified enzyme is still 93% inhibited at saturating levels of GTP, although its K1 is increased 20-fold to 4.6 microM. The kinetic effects caused by oNADPH are not prevented by alpha-ketoglutarate, ADP, 5 mM NADH, or 200 microM GTP alone, but are prevented by 5 mM NADH with 200 microM GTP. Incorporation of oNADPH into enzyme at 255 min is 0.94 mol/mol of peptide chain in the absence of ligands but only 0.53 mol/mol of peptide chain in the presence of the protectants 5 mM NADH plus 200 microM GTP. These results indicate that oNADPH modifies specifically about 0.4-0.5 sites/enzyme subunit or about 3 sites/enzyme hexamer and that reaction occurs at a GTP-dependent inhibitory NADH site of glutamate dehydrogenase.     

Eubacterial isocitrate dehydrogenase with dual specificity for NAD and NADP from Rhodomicrobium vannielii

Abstract Cell-free extracts of the photosynthetic eubacterium Rhodomicrobium vannielii contained both NADP and NAD-linked isocitrate dehydrogenase activities. Apparent K _m values of 12 μM for NADP, 0.75 mM for NAD, 9.3 μM for isocitrate (NADP utilising) and 8.2 μM for isocitrate (NAD utilising) were determined in such extracts. Four lines of evidence indicated that one enzyme was responsible for the two activities; (i) non-additivity of reaction rates in the presence of both NADP and NAD (ii) the presence of one band which stained for activity with both cofactors on non-denaturing polyacrylamide gels (iii) identical heat inactivation kinetics for both activities (iv) co-elution of both activities after ion-exchange and hydrophobic interaction chromatography. This is the first report of a eubacterial isocitrate dehydrogenase with dual cofactor specificity.     

Effect of 2',3'-dialdehyde NADPH on activation of superoxide-producing NADPH oxidase in a cell-free system of pig neutrophils

We studied the effect of the 2',3'-dialdehyde derivative of NADPH on the activation of superoxide-producing oxidase in a cell-free system of pig neutrophils. The system consisted of a membrane fraction, two cytosolic fractions prepared by gel filtration, and arachidonic acid. Preincubation of one of the cytosolic fractions with the derivatives of NADPH and NADP+ caused the loss of its ability to activate the enzyme. The inactivation was effectively prevented by the addition of NADPH and NADP+. Neither the membrane fraction nor the other cytosolic fraction was affected by the derivatives. The results indicate that the NADPH binding component of the oxidase is present in the cytosolic fraction and may be translocated to the membrane fraction during the activation process in the cell-free system.     

Inactivation of beef heart mitochondrial F1-ATPase by the 2',3'-dialdehyde derivatives of adenine nucleotides

Beef heart mitochondrial F1-ATPase was inactivated by the 2',3'-dialdehyde derivatives of ATP, ADP and AMP (oATP, oADP, oAMP). In the absence of Mg2+, inactivation resulted from the binding of 1 mol nucleotide analog per active unit of F1. The most efficient analog was oADP, followed by oAMP and oATP. Complete inactivation was correlated with the binding of about 11 mol [14C]oADP/mol F1. After correction for non-specific labeling, the number of specifically bound [14C]oADP was 2-3 mol per mol F1. By SDS-polyacrylamide gel electrophoresis, [14C]oADP was found to bind covalently mainly to the alpha and beta subunits. In the presence of Mg2+, oATP behaved as a substrate and was slowly hydrolyzed.     

Inhibition of adenylate cyclase by the 2',3'-dialdehyde of adenosine triphosphate

The periodate-oxidized analog of ATP, 2',3'-dialATP, competitively inhibited bovine brain and rat liver adenylate cyclase. The apparent Ki for inhibition of brain adenylate cyclase by 2',3'-dialATP was 196 microM in the presence of Mg2+ and 37 microM in the presence of Mn2+. The Ki values for inhibition of rat liver adenylate cyclase by 2',3'-dialATP were 48 and 30 microM in the presence of Mg2+; and Mn2+, respectively. Adenylate cyclase activity was irreversibly inactivated by 2'3'-dialATP in the presence of NaCNBH3 and the kinetics for loss in enzyme activity were pseudo-first order. Both ATP and Tris protected adenylate cyclase from irreversible inhibition by 2',3'-dialATP and NaCNBH3. It is proposed that 2',3'-dialATP forms a Schiff's base with an amino group at the active site of the enzyme and that Na-CNBH3 reduction of this Schiff's base causes irreversible modification of the catalytic subunit. The Km for 2',3'-dialATP inactivation, the maximal rate constant of inactivation, and protection of the enzyme by ATP were not affected by the presence or absence of free Mg2+. These data indicate that a divalent cation is not required for binding of 2',3'-dialATP to the active site of adenylate cyclase.     

Regulation of replicative senescence by NADP+ -dependent isocitrate dehydrogenase

The free radical hypothesis of aging postulates that senescence is due to an accumulation of cellular oxidative damage, caused largely by reactive oxygen species that are produced as by-products of normal metabolic processes. Recently, we demonstrated that the control of cytosolic and mitochondrial redox balance and the cellular defense against oxidative damage is one of the primary functions of cytosolic (IDPc) and mitochondrial NADP+ -dependent isocitrate dehydrogenase (IDPm) by supplying NADPH for antioxidant systems. In this paper, we demonstrate that modulation of IDPc or IDPm activity in IMR-90 cells regulates cellular redox status and replicative senescence. When we examined the regulatory role of IDPc and IDPm against the aging process with IMR-90 cells transfected with cDNA for IDPc or IDPm in sense and antisense orientations, a clear inverse relationship was observed between the amount of IDPc or IDPm expressed in target cells and their susceptibility to senescence, which was reflected by changes in replicative potential, cell cycle, senescence-associated beta-galactosidase activity, expression of p21 and p53, and morphology of cells. Furthermore, lipid peroxidation, oxidative DNA damage, and intracellular peroxide generation were higher and cellular redox status shifted to a prooxidant condition in the cell lines expressing the lower level of IDPc or IDPm. The results suggest that IDPc and IDPm play an important regulatory role in cellular defense against oxidative stress and in the senescence of IMR-90 cells.     

Inactivation of NADP(+)-dependent isocitrate dehydrogenase by nitric oxide

Recently, we demonstrated that the control of cytosolic and mitochondrial redox balance and oxidative damage is one of the primary functions of NADP(+)-dependent isocitrate dehydrogenase (ICDH) through to supply NADPH for antioxidant systems. NO donors such as S-nitrosothiols, diethylamine NONOate, spermine NONOate, and 3-morpholinosydnomine N-ethylcarbamide (SIN-1)/superoxide dismutase inactivated ICDH in a dose- and time-dependent manner. The inhibition of ICDH by S-nitrosothiol was partially reversed by thiol, such as dithiothreitol or 2-mercaptoethanol. Loss of enzyme activity was associated with the depletion of the cysteine-reactive 5,5'-dithiobis-(2-nitrobenzoate) and the loss of fluorescent probe N,N'-dimethyl-N(iodoacetyl)-N'-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) ethyleneamine accessible thiol groups. Using electrospray ionization mass spectrometry with tryptic digestion of protein, we found that nitric oxide forms S-nitrosothiol adducts on Cys305 and Cys387. These results indicate that S-nitrosylation of cysteine residues on ICDH is a mechanism involving the inactivation of ICDH by NO. The structural alterations of modified enzyme were indicated by the changes in protease susceptibility and intrinsic tryptophan fluorescence. When U937 cells were incubated with 200 microM SNAP for 1 h, a significant decrease in both cytosolic and mitochondrial ICDH activities were observed. Furthermore, stimulation with lipopolysaccharide significantly decreased intracellular ICDH activity in RAW 264.7 cells, and this effect was blocked by NO synthase inhibitor N(omega)-methyl-L-arginine. This result indicates that ICDH was also inactivated by endogenous NO. The NO-mediated damage to ICDH may result in the perturbation of cellular antioxidant defense mechanisms and subsequently lead to a pro-oxidant condition.     

Purification and characterization of NAD-dependent isocitrate dehydrogenase from Dictyostelium discoideum

Isocitrate dehydrogenase (threo-d_s-isocitrate: NAD oxidoreductase (decar☐ylating) EC 1.1.1.41) from Dictyostelium dicoideum was purified 161-fold. The purified enzyme was NAD specific and required Mn²⁺ for activity. Isocitrate consumption and 2-oxoglutarate and NADH production were stoichiometric; no NADH oxidase or glutamate dehydrogenase activities were detected. The pH optimum range for activity was pH 7.5–8.5. Reductive car☐ylation of 2-oxoglutarate with NADH could not be demonstrated. Lineweaver - Burk plots of data from initial velocity studies were linear. There was no evidence of allosteric control by reported effectors (AMP, ADP, citrate) of isocitrate dehydrogenase activity. The reaction was inhibited by NADH. The inhibition by NADH was competitive when either isocitrate or NAD was the variable substrate. 2-Oxoglutarate was not inhibitory at concentrations below 4 mm. The Michaelis constant (K_m) and dissociation constant (K_ib) for isocitrate were 0.16 mm; and K_m and dissociation constant (K_ia) for NAD were 0.34 mm. The inhibition constant for NADH was 0.02 mm. The data are consistent with a rapid equilibrium random bi-bi reaction mechanism (Cleland nomenclature). The NAD-linked isocitrate dehydrogenase activity was also demonstrated in crude extracts of isolated mitochondria.     

Affinity modification of creatine kinase from rabbit skeletal muscle by 2',3'-dialdehyde derivatives of ADP and ATP

Periodate-oxidized ADP and ATP (oADP and oATP) are substrates and affinity reagents for creatine kinase from rabbit skeletal muscle. oADP and oATP modified a lysine epsilon-amino group in the nucleotide-binding site of the enzyme. Complete inactivation is observed upon binding 2 moles oADP per 1 mole of the enzyme dimer. Modification with oADP is described by a liner dependence of the log of enzyme activity on time, testifying to a pseudo-first-order of the reaction. The reaction rate constant (ki = 8.10(3) min-1) and dissociation constant for the reversible enzyme-oADP complex (Kd = 62 microM) were determined. ADP protected the enzyme from inactivation and covalent binding of the analog, whereas oADP covalently bound to the enzyme was phosphorylated by phosphocreatine. The data obtained allow to suggest that the epsilon-amino group of a lysine residue of the active site is located in close proximity to ribose of ATP and ADP forming a complex with the enzyme. This group seems essential for correct orientation of the nucleotide polyphosphate chain in the enzyme active center, but take no immediate part in the transphosphorylation process.     

Inhibition of tonoplast ATPase by 2',3'-dialdehyde derivative of ATP

The 2′,3′-dialdehyde derivative of ATP (dial-ATP) has been shown to be an affinity label for the ATP binding site of the H⁺-ATPase from tonoplast of etiolated mung bean seedlings (Vigna radiata L.). The dial-ATP caused marked inactivation of enzymatic activities of both membrane-bound and soluble ATPase and its associated proton translocation. The inactivation was reversible, but could be stabilized by NaBH₄. The sodium dodecyl sulfatepolyacrylamide gel electrophoresis pattern revealed that the dial-ATP binding site was in the large (A) subunit of ATPase. The inhibition could be substantially protected by its physiological substrate ATP, pyrophosphate, and nucleotides in the decreasing order: ATP > pyrophosphate > ADP = AMP > GTP > CTP = UTP. A Lineweaver-Burk plot showed that the mode of inhibition was competitive with respect to ATP. Loss of ATPase activity followed pseudo-first order kinetics with a K_i of 4.1 millimolar, a minimum inactivation half-time of 20 seconds, and a pseudo-first order rate constant of 0.035 s⁻¹. The double logarithmic plot of apparent rate constant versus dial-ATP concentration gave a slope of 0.927, indicating that inactivation results from reaction of at least one lysine residue at the catalytic site of the large subunit. Labeling studies with [³H]dial-ATP indicate that the incorporation of approximately 1 mole of dial-ATP per mole ATPase is sufficient to completely inhibit the ATPase. A working model of nonequivalent subunits for enzymatic mechanism of vacuolar ATPase is suggested.     

Higher plant NADP+-dependent isocitrate dehydrogenases,ammonium assimilation and NADPH production

The assimilation of ammonium into glutamate is mainly achieved by the GS/GOGAT pathway and requires carbon skeletons in the form of 2-oxoglutarate. To date, the exact enzymatic origin of this organic acid for plant ammonium assimilation is unknown. NADP⁺-dependent isocitrate dehydrogenases can carry out this function and the recent efforts concentrated on evaluating the involvement of different isoforms, distinguished by their subcellular localisation, are analysed. Furthermore, a possible role for these enzymes in the production of NADPH for redox-regulated cell metabolism, such as the recycling of glutathione required in response to oxidative stress will be discussed.     

Regulation of NAD+-linked isocitrate dehydrogenase and 2-oxoglutarate dehydrogenase by Ca2+ ions within toluene-permeabilized rat heart mitochondria. Interactions with regulation by adenine nucleotides and NADH/NAD+ ratios.

1. Toluene-permeabilized rat heart mitochondria have been used to study the regulation of NAD+-linked isocitrate dehydrogenase and 2-oxoglutarate dehydrogenase by Ca2+, adenine and nicotinamide nucleotides, and to compare the properties of the enzymes in situ, with those in mitochondrial extracts. 2. Although K0.5 values (concn. giving half-maximal effect) for Ca2+ of 2-oxoglutarate dehydrogenase were around 1 microM under all conditions, corresponding values for NAD+-linked isocitrate dehydrogenase were in the range 5-43 microM. 3. For both enzymes, K0.5 values for Ca2+ observed in the presence of ATP were 3-10-fold higher than those in the presence of ADP, with values increasing over the ADP/ATP range 0.0-1.0. 4. 2-Oxoglutarate dehydrogenase was less sensitive to inhibition by NADH when assayed in permeabilized mitochondria than in mitochondrial extracts. Similarly, the Km of NAD+-linked isocitrate dehydrogenase for threo-Ds-isocitrate was lower in permeabilized mitochondria than in extracts under all the conditions investigated. 5. It is concluded that in the intact heart Ca2+ activation of NAD+-linked isocitrate dehydrogenase may not necessarily occur in parallel with that of the other mitochondrial Ca2+-sensitive enzymes, 2-oxoglutarate dehydrogenase and the pyruvate dehydrogenase system.     

Inactivation of NADP+-dependent isocitrate dehydrogenase by lipid peroxidation products

Membrane lipid peroxidation processes yield products that may react with proteins to cause oxidative modification. Recently, we demonstrated that the control of cytosolic and mitochondrial redox balance and oxidative damage is one of the primary functions of NADP₊-dependent isocitrate dehydrogenase (ICDH) through to supply NADPH for antioxidant systems. When exposed to lipid peroxidation products, such as malondialdehyde (MDA), 4-hydroxynonenal (HNE) and lipid hydroperoxide, ICDH was susceptible to oxidative damage, which was indicated by the loss of activity and the formation of carbonyl groups. The structural alterations of modified enzymes were indicated by the change in thermal stability, intrinsic tryptophan fluorescence and binding of the hydrophobic probe 8-anilino 1-napthalene sulfonic acid. Upon exposure to 2,2'-azobis(2-amidinopropane) hydrochloride (AAPH), which induces lipid peroxidation in membrane, a significant decrease in both cytosolic and mitochondrial ICDH activities were observed in U937 cells. Using immunoprecipitation and immunoblotting, we were able to isolate and positively identify HNE adduct in mitochondrial ICDH from AAPH-treated U937 cells. The lipid peroxidation-mediated damage to ICDH may result in the perturbation of the cellular antioxidant defense mechanisms and subsequently lead to a pro-oxidant condition.     

Inactivation of NADP(+)-dependent isocitrate dehydrogenase by reactive oxygen species

Recently, we demonstrated that the control of cytosolic and mitochondrial redox balance and the cellular defense against oxidative damage is one of the primary functions of NADP(+)-dependent isocitrate dehydrogenase (ICDH) through supply of NADPH for antioxidant systems. When exposed to various reactive oxygen species such as hydrogen peroxide, singlet oxygen generated by photoactivated dye, superoxide anion, and hydroxyl radical produced by metal-catalyzed Fenton reactions, ICDH was susceptible to oxidative modification and damage, which was indicated by the loss of activity, fragmentation of the peptide as well as by the formation of carbonyl groups. Oxidative damage to ICDH was inhibited by antioxidant enzymes, free radical scavengers, and spin-trapping agents. The structural alterations of modified enzymes were indicated by the increase in thermal instability and binding of the hydrophobic probe 8-anilino-1-naphthalene sulfonic acid (ANSA). The reactive oxygen species-mediated damage to ICDH may result in the perturbation of cellular antioxidant defense mechanisms and subsequently lead to a pro-oxidant condition.     

Inactivation of mitochondrial NADP+-dependent isocitrate dehydrogenase by hypochlorous acid

Myeoloperoxidase catalyses the formation of hypochlorous acid (HOCl) via reaction of H(2)O(2) with Cl(-) ion. Although HOCl is known to play a major role in the human immune system by killing bacteria and other invading pathogens, excessive generation of this oxidant is known to cause damage to tissue. Recently, it was demonstrated that the control of mitochondrial redox balance and oxidative damage is one of the primary functions of mitochondrial NADP(+)-dependent isocitrate dehydrogenase (IDPm) to supply NADPH for antioxidant systems. This study investigated whether the IDPm would be a vulnerable target of HOCl as a purified enzyme and in intact cells. Loss of enzyme activity was observed and the inactivation of IDPm was reversed by thiols. Transfection of HeLa cells with an IDPm small interfering RNA (siRNA) markedly enhanced HOCl-induced oxidative damage to cells. The HOCl-mediated damage to IDPm may result in the perturbation of the cellular antioxidant defense mechanisms and subsequently lead to a pro-oxidant condition.