Gerhard W. E. Plaut

Activity of purified NAD-specific isocitrate dehydrogenase at modulator and substrate concentrations approximating conditions in mitochondria

The kinetic parameters of NAD-specific isocitrate dehydrogenase from bovine heart were examined at levels of substrates and effectors approximating the concentrations reported for isolated intact heart mitochondria in different respiratory states. The effect of changing ADP/ATP ratios (with total adenine nucleotides constant at 8 mmol/L) on enzyme activity was measured at constant concentrations of the substrates magnesium D-isocitrate (0.10 mmol/L) and NAD+ (3.0 mmol/L), the positive effector magnesium citrate (1.0 mmol/L) and the negative effector NADPH (1.5 mmol/L) at pH 7.4. Enzyme activity increased with increasing ADP/ATP ratios as a result of activation by rising ADP concentrations and not due to decreasing inhibition by falling levels of ATP. Increasing ADP decreased the inhibition by NADPH, and this effect was enhanced by magnesium citrate and by free Ca2+. In incubation media containing all of the above effectors, the S0.5 for enhancement of activity by free Ca2+ was 10 to 20 mumol/L at ratios of total ADP/total ATP between 2.0 and 0.1. This value is in the range of intramitochondrial concentrations of free Ca2+,1 but it is appreciably larger than S0.5 of Ca2+ (0.6 to 1 mumol/L) for the enhancement of ADP activation, which was determined in the absence of other effectors. When both the NAD+/NADH and the ADP/ATP ratios were decreased, a further decline in activity was found. The effect of the decreasing NAD+/NADH ratio was due to inhibition by NADH (apparent I0.5 = 0.23 +/- 0.03 mmol/L) since NAD+ was saturating over the range examined.

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.

Inhibition and activation of bovine heart NAD-specific isocitrate dehydrogenase by ATP.

In the absence of added calcium, inhibition of NAD-specific isocitrate dehydrogenase by ATP occurred without ADP (I0.5 = 1.8 mM) and with 0.2 mM ADP3- (I0.5 = 1.0 mM) at subsaturating substrate concentrations at pH 7.4. Inhibition by ATP was competitive with NAD+ in the presence and absence of ADP and was not reversed by magnesium citrate. No reversal of ATP inhibition by free Ca2+ was observed in the presence of ADP (0.2 mM). However, when ADP was absent, increasing Ca2+ first caused progressive reversal of ATP inhibition followed by activation by ATP. Without ADP, the S0.5 for calcium activation was 80-140 microM at ATP concentrations between 0.6 and 3.0 mM. The S0.5 for ATP activation, in the absence of ADP, was 1.1 and 2.1 microM when free Ca2+ was held constant at 0.1 and 1.0 mM, respectively. As in activation by ADP, ATP decreased the S0.5 for magnesium isocitrate without affecting V. However, in contrast to ADP, the activation by ATP occurred without lowering the Hill coefficient for the substrate. GDP activated the enzyme at relatively high concentrations of Ca2+ but not without added Ca2+.

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.

Studies of a reactive histidine of diphosphopyridine nucleotide-linked isocitrate dehydrogenase from bovine heart☆

Abstract Diphosphopyridine nucleotide-linked isocitrate dehydrogenase from bovine heart was inactivated at neutral pH by bromoacetate and diethyl pyrocarbonate and by photooxidation in the presence of methylene blue or rose bengal. Inactivation by diethyl pyrocarbonate was reversed by hydroxylamine. Loss of activity by photooxidation at pH 7.07 was accompanied by progressive destruction of histidine with time; loss of 83% of the enzyme activity was accompanied by modification of 1.1 histidyl residues per enzyme subunit. The pH-rate profiles of inactivation by photooxidation and by diethyl pyrocarbonate modification showed an inflection point around pH 6.6, in accord with the p K a for a histidyl residue of a protein. Partial protection against inactivation by photooxidation or diethyl pyrocarbonate was obtained with substrate (manganous isocitrate or magnesium isocitrate) or ADP; the combination of substrate and ADP was more effective than the components singly. As demonstrated by differential enzyme activity assays between pH 6.4 and pH 7.5 with and without 0.67 m m ADP, modification of the reactive histidyl residue of the enzyme caused a preferential loss of the positive modulation of activity by ADP. The latter was particularly apparent when substrate partially protected the enzyme against inactivation by rose bengal-induced photooxidation.

FLUORESCENCE OF THE COENZYME ANALOG NICOTINAMIDE FORMYCIN DINUCLEOTIDE

Abstract— Emission spectra of unbound reduced nicotinamide formycin dinucleotide (NFDH) revealed the presence of two major conformations of the coenzyme in solution: when examined at excitation wavelengths at or below 307 nm, emission spectra contained peaks at 343 and 447 nm; when excited above 307 nm, an additional maximum appeared at 355 nm and the peak of the dihydronicotinamide emission band shifted from 447 to 440 nm. Both conformers are probably detected at the longer wavelengths since the emission peak at 343 nm was retained. Identical changes occurred in the emission spectra of NFD+, however, the dihydronicotinamide emission between 440 and 447 nm was absent. Several mechanisms which may account for the presence of these conformers have been considered. The choice has been narrowed to conformations with ring‐ring interactions of the formycin and nicotinamide moieties resulting from (a) formycin tautomerization or (b) heterogeneity of glycosidic bond angles in the structures. The efficiency of intramolecular energy transfer from the formycin to the dihydronicotinamide moiety for free NFDH in aqueous solution was 84% and declined slightly (to 77%) when measured in 1,2‐propanediol. NFD+ has coenzyme activity for NAD‐specific isocitrate dehydrogenase (Plaut et al., 1979). The emission spectrum of enzyme bound NFDH was altered markedly in the presence of manganese isocitrate; emission intensity at 343 and 355 nm decreased while the emission from the dihydronicotinamide ring at 433 nm increased, when NFDH was excited at 310 nm. This shift in emission intensity was indicative of an increase in energy transfer within the NFDH molecule, caused by a change in coenzyme conformation upon binding to the enzyme‐substrate complex.

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...

p-Nitrobenzyl p-Toluenesulfonyl-L-Arginine: a Chromogenic Substrate for Thrombin, Plasmin and Trypsin

p-Nitrobenzyl p-toluenesulfonyl-L-arginine is hydrolyzed by thrombin, plasmin, and trypsin to p-nitrobenzyl alcohol and tosyl-L-arginine. The absorption of p-nitrobenzyl alcohol formed is measured at 271 nm (AmM 8.89). With 0.10 mM of the ester in 0.1 M Tris-HCl at pH 8.4 and 30 degrees C, the hydrolysis catalyzed by thrombin, plasmin, and trypsin is linearly proportional to time up to consumption of 60% of the substrate. Km is 14 micron and Vmax is 0.037 mumol/min/NIH unit for bovine thrombin, Km is 78 micron and Vmax is 0.31 mumol/min/CTA unit/ml for human plasmin, and Km is 12 micron and Vmax is 138 mumol/min/mg protein/ml for bovine trypsin. Samples of bovine and human thrombin ranging in specific clotting activity from 59 to 2,133 NIH units/mg protein showed esterase activities ranging from 0.15 to 0.4 mumol p-nitrobenzyl alcohol formed/10 min/NIH unit. Useful ranges for assay of enzymes were (per milliliter): 0.05-0.2 NIH units (thrombin), 0.005-0.02 CTA units (plasmin), and 0.01-0.04 microgram (trypsin).

The ultraviolet fluorescence spectra of DPN-linked isociatrate dehydrogenase from bovine heart

Abstract The emission maximum of DPN-linked isocitrate dehydrogenase from bovine heart shifted from 316 nm to 324 nm as the excitation wavelength was varied from 265 nm to 300 nm. This shift was accompanied by a nonproportional change in fluorescence intensity. Comparisons of the emission spectra of model compounds in aqueous buffer at pH 7.07 and n-butanol showed that lowered solvent polarity led to a blue shift of the peak of free tryptophan without significant change of fluorescence intensity, whereas the fluorescence intensity of tyrosine amide increased markedly without change in emission maximum. The emission peak of mixtures of tryptophan and tyrosine amide shifted to shorter wavelengths as the proportion of tyrosine amide increased. The results suggest a major contribution of tyrosine to the overall fluorescence of the dehydrogenase. DPNH caused quenching and a blue shift of the protein fluorescence maximum when excited between 270 nm and 290 nm, indicating that the two tryptophan residues per subunit of enzyme are located in different microenvironments of the protein and that DPNH may interact preferentially with the residue emitting at the longer wavelength.