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Rapid purification of pig heart NAD+-isocitrate dehydrogenase. Studies on the regulation of activity by Ca2+, adenine nucleotides, Mg2+ and other metal ions.

ABSTRACT: 1. A new procedure for purifying pig heart NAD+-isocitrate dehydrogenase from mitochondrial extracts has been developed. This relies on the use of f.p.l.c. techniques and exploits the hydrophobic properties of the gel-filtration medium Superose 6 at high ionic strength. A 300-fold purification to apparent homogeneity is achieved within 5 h and with a yield of greater than 20%. 2. The enzyme had an apparent native molecular mass on gel filtration of 320 kDa. In agreement with previous studies [Ramachandran & Colman (1980) J. Biol. Chem. 255, 8859-8864], three subunits (all close to 38 kDa) were separable by isoelectric focusing 3. This preparation was used to investigate the effects of adenine nucleotides, KCl and the required bivalent metal ions, Mg2+ and Mn2+, on the regulation of the enzyme by Ca2+. 4. In the presence of 1.5 mM-ADP, increasing the concentration of Mg2+ from 20 microM to 6.0 mM raised the concentration of Ca2+ required for half-maximal effect (K0.5 value) from 1.2 microM to 232 microM. Similarly, in the presence of 2.5 microM-Mn2+, a K0.5 value for Ca2+ of 3.3 microM was obtained, and this value was increased to 8.9 microM in the presence of 100 microM-Mn2+. In the presence of 1 mM-Mg2+ and 1.5 mM-ADP, the K0.5 value for Ca2+ was raised from 4.7 microM to 10 microM by 75 mM-KCl.

SUBMITTER: Rutter GA

PROVIDER: S-EPMC1133449 | biostudies-other | 1989 Oct

REPOSITORIES: biostudies-other

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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.

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| S-EPMC1149122 | biostudies-other

The binding of Ca2+ ions to pig heart NAD+-isocitrate dehydrogenase and the 2-oxoglutarate dehydrogenase complex.

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| S-EPMC1865649 | biostudies-literature

Metal Ions Sensing by Biodots Prepared from DNA, RNA, and Nucleotides.

Project description:Nucleic acids that exhibit a high affinity toward noble and transition metal ions have attracted growing attention in the fields of metal ion sensing, toxic metal ion removal, and the construction of functional metal nanostructures. In this study, fluorescent nanoparticles (biodots) were synthesized from DNA, RNA, and RNA nucleotides (AMP, GMP, UMP, and CMP) using a hydrothermal (HT) method, in order to study their metal ion sensing characteristics. The fluorescent properties of biodots differ markedly between those prepared from purine and pyrimidine nucleobases. All biodots demonstrate a high sensitivity to the presence of mercury cations (Hg2+), while biodots prepared from DNA, RNA, and guanosine monophosphate (GMP) are also sensitive to Ag+ and Cu2+ ions, but to a lesser extent. The obtained results show that biodots inherit the metal ion recognition properties of nucleobases, while the nucleobase composition of biodot precursors affects metal ion sensitivity and selectivity. A linear response of biodot fluorescence to Hg2+ concentration in solution was observed for AMP and GMP biodots in the range 0-250 μM, which can be used for the analytic detection of mercury ion concentration. A facile paper strip test was also developed that allows visual detection of mercury ions in solutions.

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Comparison of the effects of Ca2+, adenine nucleotides and pH on the kinetic properties of mitochondrial NAD(+)-isocitrate dehydrogenase and oxoglutarate dehydrogenase from the yeast Saccharomyces cerevisiae and rat heart.

Project description:The regulatory properties of NAD(+)-isocitrate dehydrogenase and oxoglutarate dehydrogenase in extracts of yeast and rat heart mitochondria were studied under identical conditions. Yeast NAD(+)-isocitrate dehydrogenase exhibits a low K0.5 for isocitrate and is activated by AMP and ADP, but is insensitive to ATP and Ca2+. In contrast, the rat heart NAD(+)-isocitrate dehydrogenase was insensitive to AMP, but was activated by ADP and by Ca2+ in the presence of ADP or ATP. Both yeast and rat heart oxoglutarate dehydrogenase were stimulated by ADP, but only the heart enzyme was activated by Ca2+. All the enzymes studied were activated by decreases in pH, but to differing extents. The effects of Ca2+, adenine nucleotides and pH were through K0.5 for isocitrate or 2-oxoglutarate. These observations are discussed with reference to the deduced amino acid sequences of the constituent subunits of the enzymes, where they are available.

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The effects of calcium ions and adenine nucleotides on the activity of pig heart 2-oxoglutarate dehydrogenase complex.

Project description:1. The effects of Ca2+ (mainly by using EGTA buffers), pH, ATP and ADP on the activity of the 2-oxoglutarate dehydrogenase complex from pig heart were explored. 2. Ca2+ (about 30 micrometer) resulted in a decrease in the apparent Km for 2-oxoglutarate from 2.1 to 0.16 mM (at pH 7) without altering the maximal velocity. At 0.1 mM-oxoglutarate there was a 4--5-fold activation by Ca2+, with an apparent Km for Ca2+ of 1.2 micrometer. A similar activation was also observed with Sr2+ (Km 15.1 micrometer), but not wised markedly from pH 7.4 TO 6.6. The effects of Ca2+ remained evident over this pH range. 4. In the presence of Mg2+, ATP resulted in a marked increase in the apparent Km for oxoglutarate, whereas ADP greatly decreased thisp arameter. The concentrations of adenine nucleotide required for half-maximal effects were about 10 micrometer in each case. 5. The effects of the adenine nucleotides and Ca2+ on the apparent Km for oxoglutarate appeared to be essentially independent of each other, reversible, and demonstrable in the presence of end product inhibition by NADH and obtained. 6. Effects similar to those described above were also observed on the activity of 2-oxoglutarate dehydrogenase from rat heart and brown adipose tissue. 7. We discuss the mechanisms controlling this enzyme's activity and compare these regulatory features with those of NAD-isocitrate dehydrogenase and the pyruvate dehydrogenase system, which are also sensitive to Ca2+ and adenine nucleotides.

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NAD-specific isocitrate dehydrogenase from bovine heart. Interaction with Ca2+ chelators.

Project description:The activity of NAD-specific isocitrate dehydrogenase was inhibited by EDTA, EGTA and other nitrogen-containing polycarboxylate Ca2+ chelators in the absence and in the presence of ADP by a mechanism that could not be attributed solely to the removal of free Ca2+. Carboxymethyltartronate (2-oxapropane-1,1,3-tricarboxylate), an oxygen ether polycarboxylate chelator, did not inhibit when ADP was absent. The activation by ADP, a positive effector of the enzyme, decreased with increasing concentration of carboxymethyltartronate, paralleling the removal of free Ca2+ by this chelator. The following were found when free Ca2+ was decreased to negligible concentrations (5-50 nM) with carboxymethyltartronate. (1) Free Ca2+ enhanced, but was not absolutely required for, activation by ADP. (2) Activation of enzyme activity by magnesium citrate neither required nor was increased by Ca2+ when ADP was absent. However, the potentiation of citrate activation by ADP was facilitated by free Ca2+. (3) The reversal of NADPH inhibition of enzyme activity by ADP did not absolutely require Ca2+, but it was enhanced by free Ca2+. (4) The inhibition of enzyme activity by NADH was not reversed by ADP either with or without Ca2+.

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Roles of metal ions in the selective inhibition of oncogenic variants of isocitrate dehydrogenase 1.

Project description:Cancer linked isocitrate dehydrogenase (IDH) 1 variants, notably R132H IDH1, manifest a 'gain-of-function' to reduce 2-oxoglutarate to 2-hydroxyglutarate. High-throughput screens have enabled clinically useful R132H IDH1 inhibitors, mostly allosteric binders at the dimer interface. We report investigations on roles of divalent metal ions in IDH substrate and inhibitor binding that rationalise this observation. Mg2+/Mn2+ ions enhance substrate binding to wt IDH1 and R132H IDH1, but with the former manifesting lower Mg2+/Mn2+ KMs. The isocitrate-Mg2+ complex is the preferred wt IDH1 substrate; with R132H IDH1, separate and weaker binding of 2-oxoglutarate and Mg2+ is preferred. Binding of R132H IDH1 inhibitors at the dimer interface weakens binding of active site Mg2+ complexes; their potency is affected by the Mg2+ concentration. Inhibitor selectivity for R132H IDH1 over wt IDH1 substantially arises from different stabilities of wt and R132H IDH1 substrate-Mg2+ complexes. The results reveal the importance of substrate-metal ion complexes in wt and R132H IDH1 catalysis and the basis for selective R132H IDH1 inhibition. Further studies on roles of metal ion complexes in TCA cycle and related metabolism, including from an evolutionary perspective, are of interest.

| S-EPMC8560763 | biostudies-literature

The effects of Mg2+ and adenine nucleotides on the sensitivity of the heart mitochondrial Na+-Ca2+ carrier to extramitochondrial Ca2+. A study using arsenazo III-loaded mitochondria.

Project description:The technique of reversible Ca2+-induced permeabilization [Al Nasser & Crompton (1986) Biochem. J. 239, 19-29, 31-40] has been applied to the preparation of heart mitochondria loaded with the Ca2+ indicator arsenazo III (2 nmol of arsenazo III/mg of mitochondrial protein). The loaded mitochondria ('mitosomes') were used to study the control of the Na+-Ca2+ carrier by extramitochondrial Ca2+ mediated by putative regulatory sites. The Vmax. of the Na+-Ca2+ carrier and the degree of regulatory-site-mediated inhibition were similar to normal heart mitochondria. Ca2+ occupation of the sites in mitosomes yields partial inhibition, which is half-maximal with 0.8 microM external free Ca2+. The inhibition consists of a small decrease in Vmax. and a relatively large increase in apparent Km for internal Ca2+. Mg2+ also appears to interact with the sites, but this is largely abolished by ATP and ADP (but not AMP) under conditions in which the free [Mg2+] is maintained constant. The results indicate that the regulatory sites are effective in controlling the Na+-Ca2+ carrier at physiological concentrations of adenine nucleotides, Mg2+, intra- and extra-mitochondrial free Ca2+.

| S-EPMC1148028 | biostudies-other

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