Tadashi Aogaichi

Inhibition of d(-)-3-hydroxybutyrate dehydrogenase by malonate analoges

Abstract (1) d (-)-3-Hydroxybutyrate dehydrogenase activity from guinea pig, rat, and bovine heart and from guinea pig liver is inhibited by malonate and tartronate, and more potently by the analogs methylmalonate, bromomalonate, chloromalonate, and mesoxalate. Little or no inhibitory effect was found for aminomalonate, ethylmalonate, dimethylmalonate, succinate, glutarate, oxaloacetate, malate, propionate, pyruvate, d - and l -lactate, n -butyrate, isobutyrate, and cyclopropanecarboxylate. (2) In initial velocity kinetics at pH 8.1 with a soluble enzyme preparation from bovine heart, the inhibition by the active malonate derivatives is competitive with respect to 3-hydroxybutyrate and uncompetitive with respect to acetoacetate, NAD + or NADH. With d -3-hydroxybutyrate as the variable reactant ( K m app = 0.26 mM) the inhibition constant of methylmalonate ( K is ) was 0.09 m m . (3) The rate of utilization of d -3-hydroxybutyrate (78 μ m ) by coupled rat heart mitochondria in the presence of ADP was inhibited 50% by 150 μ m methylmalonate. (4) With coupled guinea pig liver mitochondria oxidizing n -octanoate in the absence of added ADP, methylmalonate (1–3 m m ) depressed 3-hydroxybutyrate formation substantially more than total ketone production. However, the intramitochondrial NADH (or NADPH) levels were unchanged by the addition of methylmalonate, indicating that the changes in ratios of accumulated 3-hydroxybutyrate and acetoacetate were caused by direct inhibition of 3-hydroxybutyrate dehydrogenase. Methylmalonate had the same effect on 3-hydroxybutyrate/acetoacetate ratios and ketone body formation with pyruvate or acetate as the source of acetyl groups. Similar results were obtained with malonate (10 m m ) although the inhibition of total ketone formation from octanoate was more severe.

The effects of calcium and lanthanide ions on the activity of bovine heart nicotinamide adenine dinucleotide-specific isocitrate dehydrogenase

Abstract (i) The activity of purified NAD-specific isocitrate dehydrogenase from bovine heart was stimulated by free Ca 2+ in the presence of ADP and subsaturating levels of magnesium isocitrate, but not in absence of ADP. However, Ca 2+ was not absolutely required for ADP activation. This was particularly apparent when free Mg 2+ was kept low (0.0024–0.020 m m ) and the substrate magnesium dl -isocitrate ranged from 0.07–0.25 m m . When kinetic constants were determined at pH 7.4 under these conditions and in the absence of ethylene glycol bis(β-aminoethyl ether) N,N′ -tetraacetate, Ca 2+ had little or no effect on K m (app) for ADP; the stimulation of rate by Ca 2+ was mainly due to increased V (app). With subsaturating ADP, there was an interdependence in the interaction of the enzyme with substrate and Ca 2+ . Thus, with ADP constant (0.30 m m ) the values of K m (app) for magnesium dl -isocitrate declined from 0.35 m m at zero Ca 2+ to 0.19 m m with saturating Ca 2+ without affecting V ; K m (app) for free Ca 2+ declined with increasing magnesium isocitrate to a limiting K m of 0.3 μ m . (ii) Ethylene glycol bis(β-aminoethyl ether)- N,N′ -tetraacetate, frequently used as a calcium buffer, inhibited enzyme activity with and without ADP. (iii) The enzyme was not inhibited by the calmodulin inhibitors trifluoperazine and chlorpromazine. Inhibition by lanthanide ions of the isocitrate dehydrogenase was competitive with magnesium isocitrate and not with respect to Ca 2+ . The values of K is (1.8 to 3.1 μ m ) for La 3+ , Yb 3+ , Gd 3+ , Eu 3+ , Tb 3+ , and Er 3+ were about two orders of magnitude smaller than K m for magnesium dl -isocitrate.

β-Sulfur substituted α-ketoglutarates as inhibitors and alternate substrates for isocitrate dehydrogenases and certain other enzymes☆

Abstract The RS-isomers of β-mercapto-α-ketoglutarate, β-methylmercapto-α-ketoglutarate and β-methylmercapto-α-hydroxyglutarate have been synthesized. β-Mercapto-α-ketoglutarate was a potent inhibitor, competitive with isocitrate and noncompetitive with NADP+, of the mitochondrial NADP-specific isozyme from pig heart ( K i = 5 nM ; K m ( DL-isocitrate ) K i (RS-β- mercapto-α-ketoglutarate ) = 650) and pig liver, the cytosolic isozyme from pig liver (I0·5 = 23 n m ), and the NADP-linked enzymes from yeast (Ki = 58 nM) and Escherichia coli (Ki = 58 nM) at pH 7.4 and with Mg2+ as activator. β-Mercapto-α-ketoglutarate was also an effective inhibitor of NADP-isocitrate-dehydrogenase activity in intact liver mitochondria. β-Mercapto-α-ketoglutarate was a much less potent inhibitor for heart NAD-isocitrate dehydrogenase (Ki = 520 nM) than for the NADP-specific enzyme. β-Methylmercapto-α-ketoglutarate (I0·5 = 10 μ m ) was a much less effective inhibitor than the β-mercapto derivative for heart NADP-isocitrate dehydrogenase. The β-sulfur substituted α-ketoglutarates were substrates for the oxidation of NADPH by heart NADP-isocitrate dehydrogenase without requiring CO2. β-Methylmercapto-α-hydroxyglutarate, the expected product of reduction of β-methylmercapto-α-ketoglutarate, did not cause reduction of NADP+ but it was an inhibitor competitive with isocitrate for NADP-isocitrate dehydrogenase. The β-sulfur substituted α-ketoglutarate derivatives were alternate substrates for α-ketoglutarate dehydrogenase and the cytosolic and mitochondrial isozymes of heart aspartate aminotransferase but had no effect on glutamate dehydrogenase or alanine aminotransferase.

Apparent Stability Constants of Magnesium and Calcium Complexes of Tricarboxylates

Abstract Arsenazo I was used as a metallochromic indicator for the spectrophotometric determination at 560 nm to 570 nm of apparent stability constants of magnesium and calcium complexes of tricarb...