E.J. De Haan

Factors affecting the pathway of glutamate oxidation in rat-liver mitochondria

Abstract 1. The pathway of glutamate oxidation in isolated rat-liver mitochondria in the presence of phosphate and phosphate acceptor has been studied. 2. In freshly prepared mitochondria, in experiments extending over 6–40 min at 25°, an average of 90% of the glutamate oxidized was converted to aspartate and 10% to ammonia, independent of the initial concentration of glutamate. Most of the ammonia was formed during the first 10 min. 3. When malonate is present as well as glutamate, the transamination pathway is suppressed and the formation of ammonia is stimulated. 4. Ageing of mitochondria leads to inhibition of the transamination pathway and stimulation of the deamination of glutamate. In aged mitochondria the contribution of the deamination pathway increases as the initial concentration of glutamate is lowered. 5. Uncoupling agents inhibit the transamination pathway of glutamate oxidation. This inhibition is localized in the reactions leading from malate to aspartate. 6. Uncoupling agents stimulate the deamination of glutamate. This is correlated with an increase in the oxidation level of NADP.

Permeability of isolated mitochondria to oxaloacetate

Abstract 1. 1. The transport of oxaloacetate in mitochondria from several sources has been studied. 2. 2. Oxaloacetate is rapidly transported across the membrane of isolated ratliver mitochondria. The V is 80–270 nmoles/min per mg protein at 30 °C. The K m for oxaloacetate is 80–130 μM. 3. 3. Oxaloacetate can exchange either for another dicarboxylate ion or for phosphate. This exchange is inhibited by butylmalonate. It is concluded that oxaloacetate is transported via the dicarboxylate translocator. 4. 4. A rapid rate of transport is found only in tightly coupled mitochondria. The K m for oxaloacetate is independent of the coupling of the mitochondria. 5. 5. A rapid transport of oxaloacetate is found in mitochondria from rat liver and guinea-pig liver, but not in those from rabbit or pigeon liver or from rat heart.

Evidence for a permeability barrier for α-oxoglutarate in rat-liver mitochondria

1. Investigation of the oxidation of glutamate (plus arsenite) in rat-liver mitochondria has provided direct evidence that a permeability barrier for α-oxoglutarate exists in rat-liver mitochondria, and that malonate increases the permeability of the mitochondria for this oxo-acid. 2. During the oxidation of glutamate (plus arsenite), 10–20 nmoles α-oxoglutarate per mg protein accumulate within the mitochondria, and the rate of glutamate oxidation is limited by the rate of efflux of the oxo-acid. When malonate is present in addition to glutamate (plus arsenite) the accumulation of α-oxoglutarate within the mitochondria is strongly decreased and this is accompanied by a 2–3-fold stimulation of the oxidation of glutamate. 3. Malonate stimulates hydrogen transfer from α-oxoglutarate or β-hydroxybutyrate to α-oxoglutarate (plus ammonia), the reduction of intramitochondrial NAD(P)+ by α-oxoglutarate and the oxidation of intramitochondrial NAD(P)H by α-oxoglutarate (plus ammonia). This stimulation is considered to be due to a facilitation of the entry of the added α-oxoglutarate. 4. The effect of malonate on the systems mentioned in the former paragraph can be duplicated by l-malate but not by d-malate. It is suggested that l-malate also affects the permeability of the mitochondria. 5. Half-maximal stimulation of hydrogen transfer from α-oxoglutarate to α-oxoglutarate (plus ammonia), of the reduction of intramitochondrial NAD(P)+ by α-oxoglutarate and of the oxidation of glutamate (plus arsenite) is obtained with about 0.5 mM malonate and maximal stimulation with 5 mM malonate. The corresponding values for l-malate in the first system are 0.5–1.0 mM and 2 mM. 6. The mechanism of action of malonate or l-malate is unknown. It is suggested that a specific carrier for α-oxoglutarate may exist, analogous to those proposed by Chappell and Haarhoff for other anions.

Control of glutamate dehydrogenase activity during glutamate oxidation in isolated rat-liver mitochondria

Abstract 1. The kinetics of the reaction of glutamate dehydrogenase with the intramitochondrial nicotinamide nucleotides has been followed in isolated rat-liver mitochondria preincubated with phosphate and phosphate acceptor in order largely to oxidize intramitochondrial nicotinamide nucleotides. In the presence of rotenone and arsenite, the oxidation of glutamate to α-oxoglutarate is accompanied by extensive reduction of NADP + and very little reduction of NAD + , and ceases when NADP is maximally reduced, even though the level of NAD + is still quite high. 2. In mitochondria preincubated in the presence of ATP, oligomycin or rotenone in order largely to reduce intramitochondrial nicotinamide nucleotides, NADPH was oxidized much more rapidly than NADH on the addition of ammonia (α-oxoglutarate was already present). 3. On addition of glutamate to mitochondria preincubated as in 1, a small amount of ammonia is formed and some α-oxoglutarate accumulates in the first few minutes of incubation, during which NADP + is rapidly reduced and NAD + slowly reduced. Aspartate is not formed in the first few seconds. α-Oxoglutarate formation, on the other hand, is greater during the first few seconds than in the subsequent 2 min. 4. When malonate is added together with glutamate, there is a marked deamination of glutamate and only a partial reduction of NADP + . 5. 2-Methyl-1,4-naphthoquinone stimulates the deamination of glutamate markedly both in the absence and presence of malonate, and brings about an oxidation of NADPH. 6. It is concluded that the most important factor controlling the activity of glutamate dehydrogenase during glutamate oxidation in isolated rat-liver mitochondria is the oxidoreduction state of NADP.

The nicotinamide nucleotide specificity of glutamate dehydrogenase in intact RAT-liver mitochondria

Abstract 1. The kinetics of oxidation of intramitochondrial reduced nicotinamide nucleotides by α-oxoglutarate plus ammonia in intact rat-liver mitochondria have been reinvestigated. It is demonstrated that the preferential oxidation of NADPH observed on addition of ammonia to mitochondria, preincubated under energized conditions in the presence of α-oxoglutarate, is due to a transhydrogenation catalysed by glutamate dehydrogenase rather than to an energy-dependent modification of the nicotinamide nucleotide specificity of the enzyme in intact mitochondria. 2. When mitochondria are preincubated at 25 °C under energized conditions in the presence of respiratory inhibitors with the substrates of glutamate dehydrogenase, an oxidation of NADPH, but not of NADH, is brought about by decreasing the reaction temperature. Both the rate of NADPH oxidation and the final steady-state mass-action ratio of nicotinamide nucleotides are dependent on the concentration of ammonia and on the final reaction temperature. A similar effect is observed when rhein is added to the reaction medium at 25 °C in order to inhibit the energy-linked transhydrogenase reaction. 3. In the presence of the substrates of glutamate dehydrogenase, intact ratliver mitochondria catalyse an ATPase reaction due to the simultaneous activity of the energy-linked transhydrogenase and the non-energy-linked transhydrogenation catalysed by glutamate dehydrogenase. 4. These findings are discussed in relation to the nicotinamide nucleotide specificity of glutamate dehydrogenase and to a possible compartmentation of nicotinamide nucleotides in intact rat-liver mitochondria.

Anaerobic chemical changes and mechanical output during isometric tetani of rat skeletal muscle in situ

The time course was examined of the energy-rich phosphate usage and exerted isometric tetanic force in electrically stimulated rat quadriceps muscle. The maximal rate of energy-rich phosphate usage was calculated from the changes in the intramuscular concentrations of phosphocreatine, lactate, ATP and inosine monophosphate (IMP) and was somewhat higher than those calculated on the basis of exercise in vivo. The IMP concentration increased directly from the onset of the contraction until after about 11 s it remained constant. The increase in the IMP concentration coincided with a decrease in the ATP concentration. The relationship between mechanical output and energy usage was examined in two wasy (i) by calculating the ratio time integral of the force (FTI) and the total energy-rich usage (Ptot) and (ii) by calculating the ratio Force (Ft) to the energy flux (dPtot/dt) at a certain timet.Whereas the ratio FTI/Ptot showed a hyperbolic relationship, the ratioFt/(dPtot/dt) showed a parabolic relationship From the latter finding and from the results described in the literature it is concluded that the ratio mechanical output/energy-rich phosphate usage depends on the conditions under which exercise is carried out. Recovery under aerobic conditions from a maximal tetanic isometric contraction sustained for 15 s was slow compared to results of experiments in vivo.

The effect of intensive interval training on the anaerobic power of the rat quadriceps muscle

The effect of intensive interval training on the maximal anaerobic power of the rat quadriceps muscle was investigated. The anaerobic energy production was estimated from the changes in the concentrations of phosphocreatine, adenine nucleotides, inosine monophosphate and lactate in freeze-clamped muscle tissue after electrical stimulation for 2-30 s. The results showed that the maximal running speed of rats tested increased by 24%, the maximal force exerted increased by 13%, and the succinate dehydrogenase activity by 48%, while the adenylate kinase activity was the same before and after training. No difference could be observed between the maximal anaerobic power of the quadriceps muscles of trained and sedentary animals. It seems that trained muscles may be able to work with a higher degree of economy than untrained muscles.

Control of nicotinamide nucleotide-linked oxidoreductions in rat-liver mitochondria

Abstract 1. 1. With α-oxoglutarate as the hydrogen donor and the substrate-linked phosphorylation step as the source of energy, hydrogen transfer to α-oxoglutarate (plus ammonia) in rat-liver mitochondria was inhibited by dicoumarol or oligomycin, and concomitantly, intramitochondrial NAD became more reduced. Hydrogen transfer to α-oxoglutarate (plus CO2) was also inhibited by the uncoupler, but not that to acetoacetate or oxaloacetate. 2. 2. Hydrogen transfer from β-hydroxybutyrate to α-oxoglutarate (plus ammonia) was stimulated by ATP. The stimulation was oligomycin-sensitive. 3. 3. Hydrogen transfer from glutamate to acetoacetate or oxaloacetate was inhibited by ATP. This inhibition was oligomycin-sensitive. 4. 4. With isocitrate as hydrogen donor, the reduction of acetoacetate was slightly stimulated by ATP. Reduction to α-oxoglutarate (plus ammonia) was not affected by ATP or by dicoumarol. 5. 5. The reduction of acetoacetate or oxaloacetate with glutamate as hydrogen donor was inhibited by ATP, and concomitantly, NADP became more reduced. The effect of ATP was oligomycin-sensitive. 6. 6. The effect of the mitochondrial energy level on the reductive amination of α-oxoglutarate in rat-liver mitochondria with α-oxoglutarate as the hydrogen donor was studied in detail. Following a preincubation in order largely to oxidize the intramitochondrial nicotinamide nucleotides, the overall reaction was separated into two steps: the reduction of intramitochondrial NAD(P)+ by α-oxoglutarate, and the oxidation of the NAD(P)H formed by α-oxoglutarate (plus ammonia). 7. 7. When dicoumarol plus oligomycin was present during the preincubation, the subsequent oxidation of intramitochondrial NAD(P)H by α-oxoglutarate (plus ammonia) was strongly inhibited. The inhibition could be prevented by removal of the dicoumarol after the preincubation and allowing α-oxoglutarate to be oxidized via the respiratory chain. 8. 8. Preincubation with dicoumarol plus oligomycin led to inhibition of the accumulation of α-oxoglutarate by the mitochondria. However, a kinetic analysis showed that diminished substrate penetration was not the cause of the inhibition in the uncoupled mitochondria of the oxidation of intramitochondrial NAD(P)H by α-oxoglutarate (plus ammonia). 9. 9. The results are discussed in relation to the NADP specificity of glutamate dehydrogenase in intact rat-liver mitochondria, the role of the energy-linked transhydrogenase, and a possible energy requirement for the oxidation of intramitochondrial nicotinamide nucleotides (previously reduced by NAD-linked substrates) by α-oxoglutarate (plus ammonia).

Glutamate oxidation in rat-liver homogenate

Abstract 1. In rat-liver homogenate ammonia can be produced from adenine nucleotides by deamination of AMP when no oxidizable substrate is added. Glutamate prevents ammonia formation by lowering the AMP level and by removal of ammonia as glutamine. 2. The time course of glutamate oxidation in rat-liver homogenates is biphasic. In the first 10 min of the reaction aspartate production and deamination of glutamate are equal. In the 10–30-min period ammonia formation from glutamate declines, while the aspartate formation is correspondingly stimulated. 3. Ammonia formed by deamination of glutamate cannot be used for the synthesis of citrulline under the conditions used, due to its efficient removal as glutamine. Aspartate formed via the transamination pathway can be used as nitrogen donor for arginine synthesis from citrulline. This causes a stimulation of the transamination pathway. 4. The factors influencing the pathway of glutamate oxidation in rat-liver homogenate are discussed in relation to the metabolism of nitrogen in vivo .