Diphosphopyridine Nucleotide Specific Isocitric Dehydrogenase of Mammalian Mitochondria. II. Kinetic Properties of the Enzyme of the Ehrlich Ascites Carcinoma<sup>*</sup>

Stein, Alan; Kirkman, Sandra K.; Stein, Jeanne H.

doi:10.1021/bi00862a030

Cited by 34 publications

(12 citation statements)

References 20 publications

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“…The importance of these findings when considering metabolic regulation is difficult to assess since the function of the enzyme in the cell of eucaryotic organisms which also possess the NAD+-specific enzyme is not known. It is possible that the NADP+-specific enzyme functions to supply NADPH for biosynthetic purposes or to provide NADH via a transhydrogenase reaction (22). In these cases it might be expected that the enzyme would be regulated by the NADPH: NADP+ ratio rather than by the ATP concentration.…”

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confidence: 99%

Nicotinamide Adenine Dinucleotide Phosphate-specific Isocitrate Dehydrogenase from a Higher Plant

Omran

Dennis

1971

Plant Physiol.

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Nicotiniamide adenine dintucleotide plhosphate-specific isocitrate dehydrogenase was extracted from etiolated pea (Pisurn satirum L.) seedlings and was purified 65- (15,(17)(18)(19). The importance of these findings when considering metabolic regulation is difficult to assess since the function of the enzyme in the cell of eucaryotic organisms which also possess the NAD+-specific enzyme is not known. It is possible that the NADP+-specific enzyme functions to supply NADPH for biosynthetic purposes or to provide NADH via a transhydrogenase reaction (22). In these cases it might be expected that the enzyme would be regulated by the NADPH: NADP+ ratio rather than by the ATP concentration.In view of these results it was decided to study the NADP+-specific enzyme from etiolated pea stems and compare its properties with the properties of the NAD+-specific enzyme from the same source which have already been presented (4-7, 9-11). As with the NAD+-specific enzyme (4) there appears to be a considerable difference between the activation of the enzyme by Mn2+ and Mg2+, the latter only acting as cofactor in the forward direction. In the forward reaction the enzyme has a much lower Km for Mn2+ than Mg2+ and the maximal velocity is also considerably higher. In the reverse reaction, only Mn2+ will activate. Unlike the NAD+-specific enzyme the NADP+-specific enzyme is not greatly affected by the mole fraction of reduced pyridine nucleotide when total pyridine nucleotide (NADP+ + NADPH) is kept constant. It is also much more sensitive to Zn2+ inhibition than the NAD+-specific enzyme when Mg2+ but not Mn2+ is the metal activator. Ca + behaves in a similar manner to Zn2+.The enzyme is inhibited by certain nucleosides and nucleotides; part of the inhibition is due to metal complexing. The enzyme is sensitive to p-chloromercuribenzoate but can be protected by isocitrate in the presence of metal ions or by Mn + alone but not by Mg2+ alone. There is also an apparently concerted inhibition by glyoxylate and oxalacetate.

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confidence: 99%

Nicotinamide Adenine Dinucleotide Phosphate-specific Isocitrate Dehydrogenase from a Higher Plant

Omran

Dennis

1971

Plant Physiol.

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“…Nevertheless, the large changes in NADPH and 2-oxoglutarate resulting from additions of NH4Cl and a-methylisocitrate correlate at least qualitatively with regulation of isocitrate oxidation and are consistent with inhibition by these metabolites of purified NAD-specific and NADP-specific isocitrate dehydrogenases [3,5,6,44,451. The matrix isocitrate concentration under the usual conditions of citric acid cycle operation (e.g.…”

Section: E0ect Of Sc-methylisocitrute On Oridution Of Internully Genmentioning

confidence: 59%

“…3), reflecting the rate and equilibrium position of the transhydrogenase reaction [40,41] and the flux of NADP-isocitrate dehydrogenase. The accumulation of NADPH would be expected to decrease the activities of both isocitrate dehydrogenases since it is a potent allosteric inhibitor of the NAD-specific enzyme [3,5,44,45] and NADPH is a product inhibitor of NADP-isocitrate dehydrogenase [6]. To test the relationship between the oxidation-reduction state of pyridine nucleotides and the relative flux through NADP-specific and NAD-specific isocitrate dehydrogenase, a number of substances were added to respiring mitochondria to oxidize matrix NADPH.…”

Section: And I F K T O J Nh4ci and A-methylisocitrate On Isocitrate Oximentioning

confidence: 99%

“…The NADP-isocitrate dehydrogenase activity of tissue homogenates occurs predominantly in the cytosol, and the enzyme in rat liver mitochondria accounts for only 10-12% of the total tissue activity [7,8]. Nevertheless, the maximal capacity of the NADP-specific isocitrate dehydrogenase in liver mitochondria is probably greater than that of the NAD-specific enzyme ; various measurements in disrupted liver mitochondria give values for the maximal activity of NADP-isocitrate dehydrogenase 4-14 times larger than that for the NADlinked enzyme [3,.…”

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confidence: 99%

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Activities of NAD‐Specific and NADP‐Specific Isocitrate Dehydrogenases in Rat‐Liver Mitochondria

Smith

Plaut

1979

European Journal of Biochemistry

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The contributions of NAD-specific and NADP-specific isocitrate dehydrogenases to isocitrate oxidation in isolated intact rat liver mitochondria were examined using DL-threo-a-methylisocitrdte (3-hydroxy-l,2,3-butanetricarboxylate) to specifically inhibit flux through NADP-specific isocitrate dehydrogenase.Under a range of conditions tested with respiring mitochondria, the rate of isocitrate oxidation was decreased by about 20-40 ' %; by inhibition of NADP-isocitrate dehydrogenase, and matrix NADP became more oxidized. (a) For mitochondria incubated with externally added DL-isocitrate and citrate, the rate of isocitrate oxidation obtained by extrapolation to infinite a-methylisocitrate concentration was approximately 70% of the uninhibited rate in both state 3 and state 4. (b) With pyruvate plus malate added as substrates of citric acid cycle oxidation and isocitrate generated intramitochondrially, a concentration of a-methylisocitrate (400 pM) sufficient for 99.99 x, inhibition of NADP-isocitrate dehydrogenase inhibited isocitrate oxidation in states 4 and 3 by 21 i 6 ' %; and 19 k 11 (mean i -SEM), respectively. (c) With externally added isocitrate and citrate, the addition of NH4Cl increased isocitrate oxidation by 3 -4-fold, decreased NADPH levels by 30 -40 % and 2-oxoglutarate accumulation by about 40 %. The further addition of 600 pM a-methylisocitrate decreased the NH4C1-stimulated isocitrate oxidation by about 40 and decreased NADPH to about 30% of the level prevailing in the absence of NH4Cl; nevertheless, the rate of isocitrate oxidation was still twice as large in the presence of NH4Cl and a-methylisocitrate as in their absence.Experiments were also performed with intact mitochondria incubated with respiratory inhibitors to determine additional factors which might affect the flux through the two isocitrate dehydrogenases. (a) In the coupled reduction of acetoacetate by isocitrate, where the rate of reoxidation of reduced pyridine nucleotides is limited by NAD-specific 3-hydroxybutyrate dehydrogenase, 85 -100% of the rate of 3-hydroxybutyrate formation was retained in the presence of 400-900 pM a-methylisocitrate. (b) In a system where the rate of isocitrate oxidation is limited by the rate of NADPH reoxidation by glutathione reductase, the rate of glutathione reduction extrapolated to infinite a-methylisocitrate concentration was from 20-40% of the uninhibited rate. (c) In the coupled synthesis of glutamate from isocitrate and NH4C1, where the reoxidation of NADPH and NADH can occur via glutamate dehydrogenase, the rate of glutamate production extrapolated to infinite a-methylisocitrate concentration was about 60 % of the uninhibited rate.

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“…8, 9), thereby suggesting a regulatory function. Conversely, ATP is primarily an inhibitor (9,10). The NADP9-specific enzyme, on the other hand, is located primarily in the soluble fraction (1 1).…”

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Properties of an NADP⁺-specific isocitric dehydrogenase from Thiobacillus novellus

Am¹

1970

Can. J. Biochem.

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C~R L E S , A. M. Properties of an NADP+-specific isocitric dehydrogenase from Thiobacillus novellas. Can. J. Biochem. 48,95-103 (1970).Isocitric dehydrogenase from Thiobacillus nooellus has been purified 1Zfold and some of its properties were studied. The enzyme is NADP+-specific and shows an absolute requirement for a divalent cation. Mn2+ was the most effective of those tested but was replaceable to varying degrees by any of several other divalent cations. The order of effectiveness at a concentration of 3.The optimal pH was about 8.0 with both potassium phosphate and Tris-HC1 buffers. Arsenate and phosphate were capable of increasing enzyme activity in the presence of low concentrations of buffer. Added phosphate resulted in maximal activity at a concentration of 40 mM. Double reciprocal plots of velocity against Pi concentration gave a Kp sf 0.38 mM. The reaction was not reversible under any of the conditions of the investigation, nor was actibity enhanced by AMP. In fact, depending upon the concentration used, most of the nucleotides tested were inhibitory. The order of efFectiveness was mononuclestide a dinucleotide < trinucleotide. Of interest is the observation that the enzyme was inhibited by low concentrations of oxalacetate and a concerted effect was demonstrated by glyoxylate and oxalacetate.

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Diphosphopyridine Nucleotide Specific Isocitric Dehydrogenase of Mammalian Mitochondria. II. Kinetic Properties of the Enzyme of the Ehrlich Ascites Carcinoma^*

Cited by 34 publications

References 20 publications

Nicotinamide Adenine Dinucleotide Phosphate-specific Isocitrate Dehydrogenase from a Higher Plant

Nicotinamide Adenine Dinucleotide Phosphate-specific Isocitrate Dehydrogenase from a Higher Plant

Activities of NAD‐Specific and NADP‐Specific Isocitrate Dehydrogenases in Rat‐Liver Mitochondria

Properties of an NADP⁺-specific isocitric dehydrogenase from Thiobacillus novellus

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Diphosphopyridine Nucleotide Specific Isocitric Dehydrogenase of Mammalian Mitochondria. II. Kinetic Properties of the Enzyme of the Ehrlich Ascites Carcinoma*

Cited by 34 publications

References 20 publications

Nicotinamide Adenine Dinucleotide Phosphate-specific Isocitrate Dehydrogenase from a Higher Plant

Nicotinamide Adenine Dinucleotide Phosphate-specific Isocitrate Dehydrogenase from a Higher Plant

Activities of NAD‐Specific and NADP‐Specific Isocitrate Dehydrogenases in Rat‐Liver Mitochondria

Properties of an NADP+-specific isocitric dehydrogenase from Thiobacillus novellus

Contact Info

Product

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Diphosphopyridine Nucleotide Specific Isocitric Dehydrogenase of Mammalian Mitochondria. II. Kinetic Properties of the Enzyme of the Ehrlich Ascites Carcinoma^*

Properties of an NADP⁺-specific isocitric dehydrogenase from Thiobacillus novellus