The oxidation of glutamate by rat-liver mitochondria

1. In rat-liver mitochondria both the dehydrogenase and transaminase routes participate in glutamate oxidation. However, the rate of ammonia production by the dehydrogenase pathway progressively decreases with the time of incubation. 2. Glutamate deamination is stimulated by blocking the transaminase pathway with arsenite or malonate. On the other hand, this process is completely suppressed by succinate, malate, pyruvate and oxaloacetate. Succinate and pyruvate inhibit, whereas malate and oxaloacetate stimulate, aspartate formation. 3. Glutamate deamination increases with increasing concentrations of 2,4-dinitrophenol from 0·05 to 0·2mm, and then becomes inhibited, together with the rate of oxygen consumption. Aspartate formation is progressively inhibited with increasing 2,4-dinitrophenol concentration from 0·05 to 0·8mm. In the presence of 0·20mm-2,4-dinitrophenol the rate of ammonia production is higher than in the presence of phosphate acceptors and decreases much slower and linearly with the time of incubation. 4. The addition of NAD⁺ enhances glutamate deamination without affecting oxygen uptake.     

Phosphate transport in rat-liver mitochondria

1. The kinetics of the efflux of P_i and malate as well as the relationship between P_i transport and intra- and extramitochondrial pH changes were studied in rat-liver mitochondria in the presence of rotenone and oligomycin at different pH's.

2. At high pH a fast efflux of P_i from the mitochondria occurs in the first few seconds, followed by a slow re-entry of P_i into the mitochondria. Under the same conditions the exit of malate shows a time lag of 2–4 sec. The exit of malate coincides with the re-entry of P_i.

3. In the presence of butylmalonate the exit of endogenous P_i is coupled with a concomitant alkalinization of the mitochondrial matrix space, as calculated from the distribution of 5,5-[¹⁴C]dimethyloxazolidine-2,4-dione.

4. The stoicheiometry of the P_i-hydroxyl exchange was found to be 1:1.

5. The kinetics of P_i transport are consistent with previous observations that there is a direct exchange between OH⁻ and P_i, but not between OH⁻ and malate. The equilibrium distribution of H₂PO₄⁻ and OH⁻ deviates from the Donnan distribution. This may be explained by assuming a pH-dependent binding of P_i in the mitochondria.     

Nigericin-induced transient changes in rat-liver mitochondria

Addition of nigericin to mitochondria oxidizing succinate in a choline- and Tris-supplemented, low-KCI medium leads to a transient matrix acidification, followed by a return of pH_in to values very close to pH_out. The initial inhibition of stimulated respiration is gradually relieved as pH_in returns to higher values. Matrix realkalinization depends on the operation of the H⁺ pumps and on the electrogenic influx of cations and efflux of anions. The process leads to replacement of much of the matrix K⁺ by other cations. Throughout the acidification/realkalinization cycle $\Delta {\overline{\mu }}_H$ variations, if any, are small, even though there are profound changes in the relative contributions of its two components, Δψ and ΔpH.     

Compartmentation of acetyl-coA in rat-liver mitochondria.

The ratio of the specific radioactivities of 3-hydroxybutyrate: citrate was determined in rat liver mitochondria which were incubated in the presence of [1-14C]palmitate, pyruvate, bicarbonate, ATP, phosphate and malonate. Without compartmentation this ratio would maximally be 2, however, under our conditions values of 2.5-3.7 were observed. In further experiments with mitochondria, the sensitivity of pyruvate carboxylase for acetyl-CoA produced from various precursors was tested. It was found that acetyl-CoA produced from L-acetylcarnitine or by oxidation from either pyruvate, octanoate or palmitylcarnitine but not from leucine led to a stimulation of pyruvate carboxylation. These results demonstrate a compartmentation of acetyl-CoA in liver mitochondria. The further finding that different mitochondrial fractions showed varying ratios of specific radioactivities of 3-hydroxybutyrate:citrate indicates that the observed compartmentation may be explained by the existence of different types of mitochondria with varying enzyme patterns and acetyl-CoA pools.     

Transport of glutamine and glutamate in kidney mitochondria in relation to glutamine deamidation

1. In the absence of added ADP glutamine is transformed by pig kidney mitochondria to ammonium glutamate, which appears in the external medium. This reaction is stimulated only slightly by the addition of ADP, but under these conditions about 20% of the glutamate is oxidized to aspartate. 2. Externally added glutamate is oxidized to aspartate, and at about the same rate as glutamine. 3. The net rates of glutamine and glutamate influx into the intramitochondrial compartment are very slow. 4. The phosphate-dependent glutaminase activity of intact mitochondria is stimulated by the provision of energy. 5. The provision of energy also decreases the concentration of glutamate and increases the concentration of glutamine in the intramitochondrial compartment. These energy-linked changes in the glutamine and glutamate concentrations are of equal magnitude. 6. It is suggested that transport of glutamine and glutamate across the inner membrane of kidney mitochondria occurs by an obligatory exchange between the two metabolites, and is electrogenic. The existence of an electrogenic glutamine-glutamate anti-porter is proposed.     

Transport of glutamate and aspartate across the membranes of rat liver mitochondria

Chromatographic analyses have indicated that aspartate and glutamate constitute from 50–70% of the total free amino acids in freshly isolated mitochondria. Radioactive tracer studies indicate that while the l-isomers of glutamate and aspartate are rapidly accumulated by mitochondria, the d-isomers of these amino acids do not penetrate the mitochondrial membrane. The action of two inhibitory compounds, 1-fluoro-2,4-dinitrobenzene (Sanger's reagent) and tannic acid, on the transport of l-glutamate and l-aspartate has been examined. A marked inhibition of l-glutamate transfer by 1-fluoro-2,4-dinitrobenzene is observed. A corresponding effect on the transport of either l-aspartate or the anionic substrate, succinate has not been found. Tannic acid, an agent previously known to inhibit certain carrier-mediated solute fluxes in mitochondria, is shown also to inhibit the uptake of both l-glutamate and l-aspartate. These findings are consistent with the view that the mitochondrial membranes of rat liver cells contain distinct, stereospecific transport mechanisms for aspartate and glutamate.     

Control of choline oxidation by rat-liver mitochondria

The steady-state concentrations of choline and its reaction products in intact rat-liver mitochondria were determined under different conditions. From these measurements, it is concluded that in a sucrose medium choline dehydrogenation and betaine aldehyde dehydrogenation are the rate-limiting steps in overall choline oxidation under “State-3” or uncoupled conditions, respectively.Ageing of the mitochondria leads to changes in the mitochondrial membrane, resulting in a markedly different pattern of oxidation products. This finding explains why rotenone inhibits oxygen uptake with choline as substrate in fresh but not in aged mitochondria.     

Uncoupler-stimulated oxidation of choline by rat-liver mitochondria

The main product of uncoupler-stimulated oxidation of choline by rat-liver mitochondria is betaine, which is found almost exclusively extramitochondrially. The uncoupled oxidation of choline is stimulated by intramitochondrial phosphate. The effect of intramitochondrial phosphate is to induce adenine nucleotide efflux, which in its turn allows the efflux of betaine from the mitochondria.