The role of Ca++ in regulation of oxoglutarate dehydrogenase complex activity from bovine adrenal cortex

Binding of Ca2+ ions by EGTA is established to increase the oxoglutarate dehydrogenase complex K'm for 2-oxoglutarate up to 25 mM in spite of the Mg2+ ions presence in the medium. The maximum reaction rate remains unchanged. Addition, besides EGTA of an equimolar Ca2+ amount to the medium induces a more than 100-fold decrease in K'm. A positive effect of Ca2+ ions is intensified by ADP. Ca2+ counteracts the inhibition of the oxoglutarate dehydrogenase complex activity by NADH. When chelating Ca2+ by EGTA with NADH available, a nonhyperbolic dependence of the reaction rate on the 2-oxoglutarate concentration is observed. When Ca2+ is absent, signs of a positive cooperative interaction of the enzyme with ADP and NADH are observed under conditions when 2-oxoglutarate concentration is much lower than the saturating one.     

Kinetic properties of NADP-specific isocitrate dehydrogenase from bovine adrenals

At low concentrations of Mg2+ or Mn2+ the reaction catalyzed by isocitrate dehydrogenase from bovine adrenal cortex proceeds with a lag period which disappears as a result of the enzyme saturation with Mn2+ or Mg2+. The nu o versus D,L-isocitrate concentration curve is non-hyperbolic, which may be interpreted either by the presence of two active sites with different affinity for the substrate (K'mapp = 2.3 and 63 microM) within the enzyme molecule or by the "negative" cooperativity of these sites. The apparent Km value for NADP lies within the range of 3.6-9 microM. High concentrations of NADP inhibit isocitrate dehydrogenase (Ki = 1.3 mM). NADP.H inhibits the enzyme in a mixed manner with respect to NADP (Ki = 0.32 mM). In the presence of NADP.H the curve nu o dependence on NADP concentration shows a "negative" cooperativity between NADP binding sites. The reverse enzyme-catalyzed reaction of reductive carboxylation of 2-oxoglutarate does not exhibit any significant deviations from the Michaelis-Menten kinetics. The Km value for 2-oxoglutarate is 120 microM, while that for NADP.H is 10 microM.     

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

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.     

Binding studies of a spin-labelled oxidized coenzyme to bovine-liver glutamate dehydrogenase.

NAD+ with a nitroxide piperidine ring linked to the NH2 group of the adenine possesses full coenzymatic activity with glutamate dehydrogenase. Electron spin resonance spectra in the presence of glutamate dehydrogenase show mixtures of free and strongly immobilized spin-label. Binding studies in phosphate buffer demonstrate: (a) weak binary binding to the enzyme with a dissociation constant in the order of 2mM;(b) an indication for negative cooperativity or different sites for binding to enzyme-2-oxoglutarate, with dissociation constants in the order of 20--250muM; (c) similar but much weaker binding to enzyme-2-oxoglutarate-ADP; (d) a strong positive cooperative binding to enzyme-2-oxoglutarate-GTP, dependent on the enzyme concentration. Binding of phosphate to the enzyme with a Kd of about 20 mM or binding of pyrophosphate or tripolyphosphate with a Dd of about 2.5 mM enhances the binding of spin-labelled NAD+ in the presence of 2-oxoglutarate. There is evidence that the binding sites for these phosphates coincide with phosphate binding subsites of GTP.     

Kinetic characterization of the pyruvate and oxoglutarate dehydrogenase complexes from human heart

The Michaelis constant values for the highly purified pyruvate dehydrogenase complex (PDC) from human heart are 25, 13 and 50 microM for pyruvate, CoA and NAD, respectively. Acetyl-CoA produces a competitive inhibition of PDC (Ki = 35 microM) with respect to CoA, whereas NADH produces the same type of inhibition with respect to NAD (Ki = 36 microM). The oxoglutarate dehydrogenase complex (OGDC) from human heart has active sites with two different affinities for 2-oxoglutarate ([S]0.5 of 30 and 120 microM). ADP (1 mM) decreases the [S]0.5 values by a half. The inhibition of OGDC (Ki = 81 microM) by succinyl-CoA is of a competitive type with respect to CoA (Km = 2.5 microM), whereas that of NADH (Ki = 25 microM) is of a mixed type with respect to NAD (Km = 170 microM).     

A new glutamate dehydrogenase from Halobacterium halobium with different coenzyme specificity

• 1.1. Halobacterium halobium has two chromatographically distinct forms of glutamate dehydrogenase which differ in their thermolability and other properties. One glutamate dehydrogenase utilizes NAD, the other NADP as a coenzyme.
• 2.2. The NADP-specific glutamate dehydrogenase (EC 1.4.1.4) was purified 65-fold from crude extracts of H. halobium.
• 3.3. The Michaelis constants for 2-oxoglutarate (13.3 mM), ammonium (3.1 mM) and NADPH (0.077 mM) indicate that the enzyme catalyzes in vivo the formation of glutamate from ammonium and 2-oxoglutarate.
• 4.4. The amination of 2-oxoglutarate by NADP-specific glutamate dehydrogenase is optimal at the pH value of 8.0–8.5. The optimal NaCl or KCl concentration for the reaction is 1.6 M.
• 5.5. None of the several metabolites tested for a possible role in the regulation of glutamate dehydrogenase activity appeared to exert an appreciable influence on the enzyme.
• 6.6. NAD- and NADP-dependent glutamate dehydrogenases from H. halobium showed apparent molecular weights of 148,000 and 215,000 respectively.

     

Effects of ATP and adenosine addition on activity of oxoglutarate dehydrogenase and the concentration of cytoplasmic free Ca2+ in rat hepatocytes

Addition of ATP (100 microM) to hepatocytes from starved rats incubated with 5 mM [1-14C]glutamine caused a stimulation of glucose formation; the magnitude of the concomitant increases in 14CO2 production and glutamine consumption indicate that flux from glutamine to glucose was increased. ATP also caused a simultaneous decrease in the cell content of oxoglutarate; together with the increased flux this is consistent with an activation of oxoglutarate dehydrogenase. In corroboration of this, a stimulation by ATP of gluconeogenesis and a decrease in oxoglutarate was also observed with 5 mM proline as substrate. ATP caused an increase in hepatocyte cytoplasmic free Ca2+ concentration, [Ca2+]c, as indicated by the increase in the fluorescence of cytoplasmically trapped quin2, from a resting value of about 0.2 microM to greater than 1 microM. The mechanism of oxoglutarate dehydrogenase activation may be via an increase in mitochondrial Ca2+ content as a consequence of the increase in [Ca2+]c. The effects of 100 microM adenosine were also investigated. An increase in flux from glutamine to glucose was observed together with a decrease in the cell oxoglutarate, thus indicating that adenosine addition to hepatocytes could also activate oxoglutarate dehydrogenase. The activation by adenosine was less than that produced by ATP. Adenosine caused a small apparent increase in [Ca2+]c to 0.3-0.4 microM; it remains to be established if this effect, which is small relative to that of ATP, is sufficient to elicit the activation of oxoglutarate dehydrogenase: alternative mechanisms may exist.     

A kinetic study of the alpha-keto acid dehydrogenase complexes from pig heart mitochondria.

The kinetic mechanisms of the 2-oxoglutarate and pyruvate dehydrogenease complexes from pig heart mitochondria were studied at pH 7.5 and 25 degrees. A three-site ping-pong mechanism for the actin of both complexes was proposed on the basis of the parallel lines obtained when 1/v was plotted against 2-oxoglutarate or pyruvate concentration for various levels of CoA and a level of NAD+ near its Michaelis constant value. Rate equations were derived from the proposed mechanism. Michaelis constants for the reactants of the 2-oxoglutarate dehydrogenase complex reaction are: 2-oxoglutarate, 0.220 mM; CoA, 0.025 mM; NAD+, 0.050 mM. Those of the pyruvate dehydrogenase complex are: pyruvate, 0.015 mM; CoA, 0.021 mM; NAD+, 0.079 mM. Product inhibition studies showed that succinyl-CoA or acetyl-CoA was competitive with respect to CoA, and NADH was competitive with respect to NAD+ in both overall reactions, and that succinyl-CoA or acetyl-CoA and NADH were uncompetitive with respect to 2-oxoglutarate or pyruvate, respectively. However, noncompetitive (rather than uncompetitive) inhibition patterns were observed for succinyl-CoA or acetyl-CoA versus NAD+ and for NADH versus CoA. These results are consistent with the proposed mechanisms.     

Activation of oxoglutarate dehydrogenase in the kidney in response to acute acidosis.

1. Activation by H+ and by Ca2+ of 2-oxoglutarate dehydrogenase extracted from mitochondria of normal or acidotic rat kidney is described. This effect, first shown for the enzyme from heart by McCormack & Denton [Biochem. J. (1979) 180, 533--544], is of a regulatory importance in kidney, in which organ, in contrast with heart, increased flux occurs during acute acidosis. 2. In renal-cortical tubules, 2-oxoglutarate concentration fell within 1 min of decreasing the pH and rose again 1--3 min after increasing the pH of the medium. The extent of the decrease in 2-oxoglutarate was directly related to the decrease in pH. A similar fall in the oxoglutarate concentration in the whole perfused kidney was noted within 5 min of inducing acidosis. 3. In tubules, the rates of gluconeogenesis and ammoniagenesis from 1 mM-glutamine were increased by 64 and 33% respectively on decreasing pH to 7.0, the increase in rates being proportional to the fall in pH between 7.4 and 7.0. 4. The increased rates of renal ammoniagenesis and gluconeogenesis seen in acute acidosis in vitro can be accounted for by the increased activity of 2-oxoglutarate dehydrogenase and the tissue concentrations of 2-oxoglutarate when calculated from the Km determined at normal and acidotic pH. 5. The decrease in 2-oxoglutarate concentration seen in acute acidosis implies a fall in intramitochondrial pH in kidney, and is the result of two phenomena, accelerated disposal via 2-oxoglutarate dehydrogenase and maintenance of near equilibrium of glutamate dehydrogenase.     

Induction, isolation, and some properties of the NADPH-dependent glutamate dehydrogenase from the nonheterocystous cyanobacterium Phormidium laminosum.

The level of the NADPH-dependent glutamate dehydrogenase activity (EC 1.4.1.4) from nitrate-grown cells of the thermophilic non-N2-fixing cyanobacterium Phormidium laminosum OH-1-p.Cl1 could be significantly enhanced by the presence of ammonium or nitrite, as well as by L-methionine-DL-sulfoximine and other sources of organic nitrogen (L-Glu, L-Gln, and methylamine). The enzyme was purified more than 4,400-fold by ultracentrifugation, ion-exchange chromatography, and affinity chromatography, and at 30 degrees C it showed a specific activity of 32.9 mumol of NADPH oxidized per min per mg of protein. The purified enzyme showed no aminotransferase activity and catalyzed the amination of 2-oxoglutarate preferentially to the reverse catabolic reaction. The enzyme was very specific for its substrates 2-oxoglutarate (Km = 1.25 mM) and NADPH (Km = 64 microM), for which hyperbolic kinetics were obtained. However, negative cooperativity (Hill coefficient h = 0.89) and [S]0.5 of 18.2 mM were observed for ammonium. The mechanism of the aminating reaction was of a random type with independent sites. The purified enzyme showed its maximal activity at 60 degrees C (Ea = 5.1 kcal/mol [21.3 kJ/mol]) and optimal pH values of 8.0 and 7.5 when assayed in Tris hydrochloride and potassium phosphate buffers, respectively. The native molecular mass of the enzyme was about 280 kilodaltons. The possible physiological role of the enzyme in ammonia assimilation is discussed.     

Glutamate Dehydrogenase Reaction as a Source of Glutamic Acid in Synaptosomes

The role of the glutamate dehydrogenase reaction as a pathway of glutamate synthesis was studied by incubating synaptosomes with 5 mM 15NH4Cl and then utilizing gas chromatography-mass spectrometry to measure isotopic enrichment in glutamate and aspartate. The rate of formation of [15N]glutamate and [15N]aspartate from 5 mM 15NH4Cl was approximately 0.2 nmol/min/mg of protein, a value much less than flux through glutaminase (4.8 nmol/min/mg of protein) but greater than flux through glutamine synthetase (0.045 nmol/min/mg of protein). Addition of 1 mM 2-oxoglutarate to the medium did not affect the rate of [15N]glutamate formation. O2 consumption and lactate formation were increased in the presence of 5 mM NH3, whereas the intrasynaptosomal concentrations of glutamate and aspartate were unaffected. Treatment of synaptosomes with veratridine stimulated reductive amination of 2-oxoglutarate during the early time points. The production of ([15N]glutamate + [15N]aspartate) was enhanced about twofold in the presence of 5 mM beta-(+/-)-2-aminobicyclo [2.2.1]heptane-2-carboxylic acid, a known effector of glutamate dehydrogenase. Supplementation of the incubation medium with a mixture of unlabelled amino acids at concentrations similar to those present in the extracellular fluid of the brain had little effect on the intrasynaptosomal [glutamate] and [aspartate]. However, the enrichment in these amino acids was consistently greater in the presence of supplementary amino acids, which appeared to stimulate modestly the reductive amination of 2-oxoglutarate. It is concluded: (a) compared with the phosphate-dependent glutaminase reaction, reductive amination is a relatively minor pathway of synaptosomal glutamate synthesis in both the basal state and during depolarization; (b) NH3 toxicity, at least in synaptosomes, is not referable to energy failure caused by a depletion of 2-oxoglutarate in the glutamate dehydrogenase reaction; and (c) transamination is not a major mechanism of glutamate nitrogen production in nerve endings.     

Purification and properties of three NAD(P)+ isozymes of L-glutamate dehydrogenase of Chlamydomonas reinhardtii.

Three isozymes of glutamate dehydrogenase (GDH) of Chlamydomonas reinhardtii, induced under different trophic and stress conditions, have been purified about 800-1000-fold to electrophoretic homogeneity. They are hexamers of Mr 266,000-269,000 as deduced from gel filtration and sedimentation coefficient data. GDH1 consisted of six identical subunits of 44 kDa each, whereas both GDH2 and GDH3 consisted of six similar-sized monomers (4 of 44 kDa and 2 of 46 kDa). Optimum pH for the three activities with each pyridine nucleotide was identical (8.5 with NADH; 7.7 with NADPH; and 9.0 with NAD+). The isozymes exhibited similar high optimum temperature values (60-62 degrees C) and isoelectric points (7.9-8.1). Activity was enhanced in vitro by Ca2+ ions and strongly inhibited by pyridoxal 5'-phosphate, KCN, o-phenanthroline and EDTA, and to a lesser extent by pHMB and methylacetimidate. In the aminating reaction the three isozymes were inhibited in a concentration-dependent process by both NADH and NADPH, with apparent Km values for NH4+ ranging from 13-53 mM; 0.36-1.85 mM for 2-oxoglutarate and 0.07-0.78 mM for NADH and NADPH. In the deaminating reaction apparent Km values ranged from 0.64-3.52 mM for L-glutamate and 0.20-0.32 for NAD+. In addition, the three isozymes exhibited a non-hyperbolic kinetics for NAD+ with negative cooperativity (n = 0.8).     

NADP+-specific 2-oxoglutarate dehydrogenase in denitrifying Pseudomonas species

Several denitrifying Pseudomonas strains contained an NADP⁺-specific 2-oxoglutarate dehydrogenase, in contrast to an NAD⁺-specific pyruvate dehydrogenase, if the cells were grown anaerobically with aromatic compounds. With non-aromatic substrates or after aerobic growth the coenzyme specificity of 2-oxoglutarate dehydrogenase changed to NAD⁺-specificity. The reaction stoichiometry and the apparent K _m-values of the enriched enzymes were determined: pyruvate 0.5 mM, coenzyme A 0.05 mM, NAD⁺ 0.25 mM; 2-oxoglutarate 0.6 mM, coenzyme A 0.05 mM, NADP⁺ 0.03 mM. Isocitrate dehydrogenase was NADP⁺-specific. The findings suggest that these strains contained at least two lipoamide dehydrogenases, one NAD⁺-specific, the other NADP⁺-specific.     

2-Oxoglutarate transport: A protential mechanism for regulating glutamate and tricarboxylic acid cycle intermediates in neurons

2-Oxoglutarate (-ketoglutarate) is transported into synaptosomal and synaptoneurosomal preparations by a Na⁺-dependent, high-affinity process that exhibits complex kinetics, and is differentially modulated by glutamate, glutamine, aspartate, malate, and a soluble, heat-labile substance of high molecular weight present in rat brain extracts. Glutamate and aspartate generally inhibit 2-oxoglutarate uptake, but under certain conditions may increase uptake. Glutamine generally increases 2-oxoglutarate uptake, but under certain conditions may inhibit uptake. One interpretation of our results is that 2-oxoglutarate uptake is mediated primarily by a transporter that exhibits negative cooperativity and possesses three regulatory sites that differentially modulate substrate affinity, V_max, and negative cooperativity. Glutamate, aspartate, malate, and 2-oxoglutarate itself may interact with a site that reduces substrate affinity; whereas glutamine, and possibly glutamate and aspartate, appear to interact with another site that increases V_max. A putative regulatory protein appears to abolish negative cooperativity and increases substrate affinity in the absence of glutamine. Based on the evidence that glutamatergic and GABAergic neurons depend on astrocytes to supply precursors to replenish their neurotransmitter and tricarboxylic acid cycle pools, the uptake of 2-oxoglutarate, presumably into synaptic terminals, may reflect a role for this metabolite in replenishing the transmitter and tricarboxylic acid pools, and a role for the transporter as a site at which these pools are regulated.Abbreviations used AAT aspartate aminotransferase - glu glutamate - gln glutamine - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - LDS low-density synaptosomes - OAA oxaloacetate - 2-OG 2-oxoglutarate (-ketoglutarate) - PC pyruvate carboxylase - PDH pyruvate dehydrogenase - TCA tricarboxylic acid Special issue dedicated to Dr. Claude Baxter.     

2-Oxoglutarate transport system in Staphylococcus aureus

2-[(14)C]oxoglutarate uptake in resting cells of Staphylococcus aureus 17810S occurs via two kinetically different systems: (1) a secondary, electrogenic 2-oxoglutarate:H(+) symporter (K(m)=0.105 mM), energized by an electrochemical proton potential (Delta mu H(+)) that is generated by the oxidation of endogenous amino acids and sensitive to ionophores, and (2) a Delta mu H(+)-independent facilitated diffusion system (K(m)=1.31 mM). The 2-oxoglutarate transport system of S. aureus 17810S can be classified as a new member of the MHS (metabolite:H(+) symporter) family. This transporter takes up various dicarboxylic acids in the order of affinity: succinate = malate > fumarate > 2-oxoglutarate > glutamate. Energy conservation with 2-oxoglutarate was studied in starved cells of strain 17810S. Initial transport of 2-oxoglutarate in these cells is energized by Delta mu H(+) generated via hydrolysis of residual ATP. Subsequent oxidation of the accumulated 2-oxoglutarate generates Delta mu H(+) for further, autoenergized transport of this 2-oxoacid and also for Delta mu H(+)-linked resynthesis of ATP. In the cadmium-sensitive S. aureus 17810S, Cd(2+) accumulation strongly inhibits energy conservation with 2-oxoglutarate at the level of Delta mu H(+) generation, without direct blocking of the 2-oxoglutarate transport system or ATP synthase complex. In the cadmium-resistant S. aureus 17810R, Cd(2+) does not affect energy conservation due to its extrusion by the Cd(2+) efflux system (Cd(2+)-ATPase of P-type), which prevents Cd(2+) accumulation.     

Effects of ischaemia on metabolite concentrations in rat liver

1. Changes in the concentrations of ammonia, glutamine, glutamate, 2-oxoglutarate, 3-hydroxybutyrate, acetoacetate, alanine, aspartate, malate, lactate, pyruvate, NAD(+), NADH and adenine nucleotides were measured in freeze-clamped rat liver during ischaemia. 2. Although the concentrations of most of the metabolites changed rapidly during ischaemia the ratios [glutamate]/[2-oxoglutarate][NH(4) (+)] and [3-hydroxybutyrate]/[acetoacetate] changed equally and the value of the expression [3-hydroxybutyrate][2-oxoglutarate][NH(4) (+)]/[acetoacetate][glutamate] remained approximately constant, indicating that the 3-hydroxybutyrate dehydrogenase and glutamate dehydrogenase systems were at near-equilibrium with the mitochondrial NAD(+) couple. 3. The value of the expression [alanine][oxoglutarate]/[pyruvate][glutamate] was about 0.7 in vivo and remained fairly constant during the ischaemic period of 5min, although the concentrations of alanine and oxoglutarate changed substantially. No explanation can be offered why the value of the ratio differed from that of the equilibrium constant of the alanine aminotransferase reaction, which is 1.48. 4. Injection of l-cycloserine 60min before the rats were killed increased the concentration of alanine in the liver fourfold and decreased the concentration of the other metabolites measured, except that of pyruvate. During ischaemia the concentration of alanine did not change but that of aspartate almost doubled. 5. After treatment with l-cycloserine the value in vivo of the expression [alanine][oxoglutarate]/[pyruvate][glutamate] rose from 0.7 to 2.4. During ischaemia the value returned to 0.8. 6. The effects of l-cycloserine are consistent with the assumption that it specifically inhibits alanine aminotransferase. 7. Most of the alanine formed during ischaemia is probably derived from pyruvate and from ammonia released by the deamination of adenine nucleotides and glutamine. The alanine is presumably formed by the combined action of glutamate dehydrogenase and alanine aminotransferase. 8. The rate of anaerobic glycolysis, calculated from the increase in the lactate concentration, was 1.3mumol/min per g fresh wt. 9. Although the concentrations of the adenine nucleotides changed rapidly during ischaemia, the ratio [ATP][AMP]/[ADP](2) remained constant at 0.54, indicating that adenylate kinase established near-equilibrium under these conditions.     

Effects of micromolar concentrations of free calcium ions on the reduction of heart mitochondrial NAD(P) by 2-oxoglutarate.

1. The reduction of mitochondrial NAD(P) by 2-oxoglutarate was monitored as a measure of 2-oxoglutarate dehydrogenase activity in its intramitochondrial locale. In the absence of ADP, steady-state reduction of NAD(P) by 0.5 mM-2-oxoglutarate in the presence of 0.5 mM-L-malate was markedly increased by extramitochondrial Ca2+, with 50% activation at pCa 6.58, when the Na+ concentration was 10 mM, the Pi concentration ws 5 mM and the added Mg2+ concentration was 1 mM. Omission of Pi resulted in 50% activation at pCa 6.77; omission of Mg2+ resulted in 50% activation at pCA greater than or equal to 7.3. 2. The activation of 2-oxoglutarate dehydrogenase could be reversed on addition of an excess of EGTA. The rate of inactivation was dependent on the concentration of Na+, with K0.5 2.5 mM, which is consistent with the rate of withdrawal of Ca2+ from the mitochondria being the limiting factor. 3. The steady-state reduction of cytochrome c by 2-oxoglutarate (0.5 mM) also showed a marked dependence on pCa in the absence of ADP; in the presence of an excess of ADP, no such effect of Ca2+ was detectable. 4. Mitochondria from the hearts of senescent rats showed an undiminished rate of dehydrogenase activation by Ca2+ but a rate of inactivation by excess EGTA that was diminished by 40%. Direct studies of Ca2+ egress with Arsenazo III confirmed a decrement in rate with old age. 5. Studies of 2-oxoglutarate dehydrogenase activity as a function of the mitochondrial context of Ca2+, as measured by atomic-absorption spectrophotometry, showed half-maximal activation at a mitochondrial content of 1.0 nmol of Ca2+/mg of protein, and saturation at 3 nmol/mg. 6. These findings support the model advanced by Denton, Richards & Chin [(1978) Biochem. J. 176, 899-906], of a control of the tricarboxylate cycle by intramitochondrial Ca2+, and demonstrate the range of mitochondrial Ca2+ content over which this may occur. In addition, they raise the possibility of a disturbance of this control mechanism in old age.     

Kinetic characterization of the rotenone-insensitive internal NADH: ubiquinone oxidoreductase of mitochondria from Saccharomyces cerevisiae

Saccharomyces cerevisiae mitochondria contain an NADH:Q6 oxidoreductase (internal NADH dehydrogenase) encoded by NDI1 gene in chromosome XIII. This enzyme catalyzes the transfer of electrons from NADH to ubiquinone without the translocation of protons across the membrane. From a structural point of view, the mature enzyme has a single subunit of 53 kDa with FAD as the only prosthetic group. Due to the fact that S. cerevisiae cells lack complex I, the expression of this protein is essential for cell growth under respiratory conditions. The results reported in this work show that the internal NADH dehydrogenase follows a ping-pong mechanism, with a Km for NADH of 9.4 microM and a Km for oxidized 2,6-dichorophenolindophenol (DCPIP) of 6.2 microM. NAD+, one of the products of the reaction, did not inhibit the enzyme while the other product, reduced DCPIP, inhibited the enzyme with a Ki of 11.5 microM. Two dead-end inhibitors, AMP and flavone, were used to further characterize the kinetic mechanism of the enzyme. AMP was a linear competitive inhibitor of NADH (Ki = 5.5 mM) and a linear uncompetitive inhibitor of oxidized DCPIP (Ki = 11.5 mM), in agreement with the ping-pong mechanism. On the other hand, flavone was a partial inhibitor displaying a hyperbolic uncompetitive inhibition regarding NADH, and a hyperbolic noncompetitive inhibition with respect to oxidized DCPIP. The apparent intercept inhibition constant (Kii = 5.4 microM) and the slope inhibition constant (Kis = 7.1 microM) were obtained by non linear regression analysis. The results indicate that the ternary complex F-DCPIPox-flavone catalyzes the reduction of DCPIP, although with lower efficiency. The effect of pH on Vmax was studied. The Vmax profile shows two groups with pKa values of 5.3 and 7.2 involved in the catalytic process.     

Purification and characterization of the 2-oxoglutarate dehydrogenase complex from Dictyostelium discoideum

The 2-oxoglutarate dehydrogenase complex was isolated from the cellular slime mould, Dictyostelium discoideum, and purified 113-fold. The enzyme exhibited Michaelis-Menten kinetics and the Km values for 2-oxoglutarate, CoA, and NAD were 1.0 mM, 0.002 mM, and 0.07 mM, respectively. The Ki value for succinyl-CoA was determined to be 0.004 mM and the Ki for NADH was 0.018 mM. AMP had positive effects whereas ATP had negative effects on the enzyme activity. The kinetic constants determined in this study and the reaction mechanism suggested can now be incorporated into a transition model of the tricarboxylic acid cycle during differentiation of D. discoideum.     

Rate enhancement of cytokinin oxidase/dehydrogenase using 2,6-dichloroindophenol as an electron acceptor

Cytokinin oxidase/dehydrogenase degrades cytokinins by dehydrogenating the N6-C1 bond of cytokinins. The resulting imine is then hydrolyzed. For example, isopentenyl-adenine is cleaved into 3-methyl-2-butenal (isopentenyl-aldehyde) and adenine . The reducing equivalents from dehydrogenation are transferred to an unknown sink, in vivo. It has been hypothesized that the enzyme requires oxygen , possibly resulting in the formation of hydrogen peroxide. 2,6-dichloroindophenol (DCPIP) can function as an acceptor of reducing equivalents for in vitro cytokinin oxidase/dehydrogenase reactions. For the predominant cytokinin oxidase/dehydrogenase in maize, ZmCKX1, the addition of DCPIP to in vitro reactions increases the reaction rate to nearly 4000-fold faster than the oxygen-dependent rate. Further, the change in absorbance of DCPIP at 600 nm, as it is reduced, forms the basis for an assay suitable for following biochemical purification of cytokinin oxidase/dehydrogenases , detailed kinetic studies , and rapid measurement of cytokinin oxidase/dehydrogenase activity in large numbers of samples.