Catecholamine control of enzymes involved in isocitrate oxidation of rat liver mitochondria

Treatment of rats or liver homogenates with catecholamines (isoproterenol or noradrenaline) increased activities of both NAD+ -dependent isocitrate dehydrogenase and NAD(P)+-transhydrogenase (in the direction of hydrogen transfer NADPH----NAD+) with no change in NADP+ -dependent isocitrate dehydrogenase. These effects were realized via beta-adrenoceptors. Cyclic AMP mimicked the catecholamine action on incubation with liver homogenate. The effects of catecholamines and cyclic AMP were not additive.     

The inhibition of isocitrate oxidation by palmitoyl-l-carnitine and palmitoyl-C0 A in rat liver mitochondria.

Palmitoyl-L carnitine decreases the oxidation of isocitrate in rat liver mitochondria in state 3 by 25-30%. Palmitoyl-L-carnitine acts as an additional substrate raising the rate of oxidative phosphorylation, NAD reduction and ATP/ADP ratio in mitochondria. Palmitoyl-CoA added to mitochondria oxidizing isocitrate in state 3 causes a strong inhibition of isocitrate oxidation and of oxidative phosphorylation and a considerable elevation of intramitochondrial NADH/NAD and ATP/ADP ratios. The effect of palmitoyl-CoA is dependent on its concentration and is competitive with ADP. Carnitine restores only oxidative phosphorylation, but the oxidation of isocitrate remains inhibited. Evidence is presented that the transport of isocitrate is not affected by palmitoyl-CoA is due to the inhibition of adenine nucleotide translocation. The kinetic studies of NAD-dependent isocitrate dehydrogenase in the soluble fraction of sonicated mitochondria revealed that the enzyme is very sensitive towards the inhibition by NADH and only very slightly affected by ATP (Ki for NADH and ATP are 0.017 and 3.6 mM respectively). On the basis of the kinetic data the relative contribution of NADH and ATP in the inhibition of isocitrate oxidation by fatty acids was calculated. It is concluded that the inhibition of isocitrate oxidation caused by palmitoyl-L-carnitine and palmitoyl-CoA is primarily due to the increased reduction of NAD, whereas the increase of ATP/ADP ratio is much less important.     

Choline and betaine aldehyde oxidation by rat liver mitochondria

1. The question of the ability or inability of rat liver mitochondria to oxidize externally added or internally generated betaine aldehyde has been reexamined. Well washed mitochondria were demonstrated to contain approx. 7% of the post-nuclear betaine aldehyde dehydrogenase as an integral component. The enzyme is approximately equally distributed between the inner membrane and the intermembrane plus matrix fractions. Significantly, none was found in the outer membrane fraction. The mitochondrial enzyme was shown to be functional under all the conditions tested; betaine aldehyde generated within the mitochondria by choline oxidation or added externally was oxidized to betaine in significant amounts.

2. The stoichiometry for the complete oxidation of choline or externally added betaine aldehyde was confirmed to be 2 and 1 moles, respectively, of O₂ utilized per mole of substrate added. Depending on the reaction conditions employed, considerable variation in the relative amount of choline oxidase and betaine aldehyde oxidase activities of mitochondria was observed when they were allowed to oxidize only a portion of the choline added. The necessity of measuring the contribution of betaine aldehyde oxidase in studies of choline oxidase is discussed.

3. Reasons for the discrepancies in the literature concerning the ability of mitochondria to oxidize betaine aldehyde are discussed.     

The effect of acetoacetate on 3-hydroxybutyrate oxidation by rat liver mitochondria

Effect of acetoacetate on 3-hydroxybutyrate oxidation by rat liver mitochondria is described. State 3 respiration is inhibited by acetoacetate, while state 4 respiration is not inhibited, though cytochrome c reduction was decreased. Acetoacetate is also non-competitive inhibitor of 3-hydroxybutyrateoxidase and 3-hydroxybutyrate dehydrogenase activity in frozen-thawed mitochondria. The results are discussed in terms of the thermodynamic hypothesis and control strength method.     

The oxidation of choline by liver slices and mitochondria during liver development in the rat

Betaine is the major oxidation product of [Me-¹⁴C] choline produced by rat liver slices. Liver slices from adult rats rapidly oxidize [Me-¹⁴C] choline to betaine and the bulk of the betaine produced is recovered in the incubation medium. Considerably more choline is oxidized to betaine than is phosphorylated to phosphorylcholine. The rate of phosphorylation of choline appears to be independent of the rate of choline oxidation. Liver slices from fetal and young rats oxidize choline to betaine at a lower rate than adult liver slices.The ability of mitochondria to oxidize [Me-¹⁴C] choline to betaine aldehyde and betaine is considerably lower in fetal liver than in adult liver. The major product with both fetal and adult mitochondria is betaine aldehyde. Choline oxidation by mitochondria begins to increase 1 day prior to birth and increases progressively to adult levels by 18 days. The developmental pattern for choline oxidation is similar to the pattern for succinic dehydrogenase activity.     

The oxidation of fatty acids combined with albumin by isolated rat liver mitochondria

Long chain fatty acids at concentrations inhibiting mitochondrial respiration were, in the presence of serum albumin, found to produce almost as high a rate of oxygen uptake as alpha-ketoglutarate, succinate, or acetate. This oxidation was characterized in terms of its coupling to phosphorylation, need for cofactors, and production of different metabolites during the reactions. Fatty acids were oxidized to carbon dioxide, acetoacetate, beta-hydroxybutyrate, and other water-soluble metabolites, tentatively identified as intermediates of the citric acid cycle. An agent to spark the citric acid cycle and adenosine tri- or monophosphate were necessary for optimal oxidation rate, as described for other fatty acid oxidation systems. Balance experiments with different amounts of malate were performed with incubations lasting as long as oxygen uptake took place. In the presence of 1 mumole of malate, practically all added palmitic acid was used up and found to be converted primarily to carbon dioxide, acetoacetate, and other water-soluble metabolites of which the major part was tentatively identified as succinate. A significant portion was found in mitochondrial phospholipids. With 10 mumoles of malate some palmitic acid remained in the system, while a comparatively small amount was converted to carbon dioxide, and a major part was found as succinate. Here also incorporation into phospholipids occurred. With no malate added, fatty acid oxidation was much smaller than with malate, although significant conversion to carbon dioxide took place. Only a little succinate and phospholipid were found. Oxygen uptake was greater than a theoretical value calculated from radioactive balance experiments. It was concluded that albumin contains oxidizable material even after extraction and dialysis. Albumin at high concentrations inhibited both fatty acid and alpha-ketoglutarate oxidation. The oxidation of long chain fatty acids in high concentrations in the form of albumin-fatty acid complex was coupled to phosphorylation. Thus P:O ratios above 2 were found as well as evidence for respiratory control. It was concluded that oxidation of long chain fatty acids by isolated mitochondria occurs from their albumin complex. This process can also be studied at high concentrations of fatty acids, where high rates of oxygen uptake are obtained from oxidation which is coupled to phosphorylation.     

The inhibition of pyruvate and ls(+)-isocitrate oxidation by succinate oxidation in rat liver mitochondria

1. The effects of succinate oxidation on pyruvate and also isocitrate oxidation by rat liver mitochondria were studied. 2. Succinate oxidation was without effect on pyruvate and isocitrate oxidation when respiration was maximally activated with ADP. 3. When respiration was partially inhibited by atractylate, succinate oxidation severely inhibited the oxidation of pyruvate and isocitrate. 4. This inhibitory effect of succinate was associated with a two- to three-fold increase in the reduction of mitochondrial NAD(+) but no change in the reduction of cytochrome b. 5. It is concluded that, in the partially energy-controlled state, respiration is more severely inhibited at the first phosphorylating site than at the other two. 6. The effects of succinate oxidation are compared with those of palmitoylcarnitine oxidation. It is concluded that a rapid flow of electrons directly into the respiratory chain at the level of cytochrome b is in itself inadequate to inhibit the oxidation of intramitochondrial NADH. 7. The effects of succinate oxidation on pyruvate oxidation were similar in rat heart and liver mitochondria.     

Rate control of phosphorylation-coupled respiration by rat liver mitochondria

Liver mitochondria provided with an oxidizable substrate, ATP, oxygen, and an ADP-generating system (soluble F₁-ATPase) were used to reevaluate the rate-controlling step(s) intrinsic to all of the processes of mitochondrial oxidative phosphorylation. The quantity termed “control strength” (C), previously defined as the fractional change in flux through a (system) induced by a fractional change in the concentration of an individual enzyme in the system, has been used to evaluate rate-influencing steps in this overall process by carefully defining the dimensions of the “system” under analysis. If the system is defined by a suspension of mitochondria provided with substrates, plus an extrinsic ADP-generating process (ATPase), the value of C of the latter for the overall process of phosphorylation-linked respiration is near 1.0 until the capacity of the mitochondria to phosphorylate ADP is approached, after which C for the soluble ATPase becomes zero as the maximum capacity for phosphorylation is attained. Carboxyatractyloside was found only marginally to inhibit respiration stimulated by ATPase, even when a large percentage of adenine nucleotide translocase molecules were immobilized. The relative lack of effect of carboxyatractyloside on phosphorylating respiration is explained by the readjustment of the concentration of one of the substrates (ADP) and an inhibitor (ATP), which results from inhibition of adenine nucleotide translocase. The residual blunted inhibition of respiration is explained by product inhibition of the ADP-regenerating ATPase, and not necessarily to any intrinsically mitochondrial intermediate process. The system being evaluated can be redefined to include only the processes intrinsic to mitochondria. This can be achieved by providing exactly comparable substrate concentrations to the mitochondria under comparable incubation conditions. Under these conditions, the adenine nucleotide translocase is the principal, if not the only, rate-controlling step in the overall process of oxidative phosphorylation until a new rate-limitation is attained (ATP synthesis). These data are consistent with the conclusion that, at intermediate rates of phosphorylation-coupled respiration, the extramitochondrial $\text{ATP}\text{ADP}$ ratio regulates this process through its kinetic effects on the catalytic properties of the adenine nucleotide translocase.     

The effect of calcium on the oxidation of acetaldehyde by rat liver mitochondria.

Isolated liver mitochondria oxidized acetaldehyde in the following order: State 4< state 3< valinomycin. Ca²⁺, in concentrations greater than 0.10 mM, inhibited the oxidation of acetaldehyde by isolated liver mitochondria under all conditions. Valinomycin-stimulated oxidation of acetaldehyde was more sensitive to inhibition by Ca²⁺ than were the state 3 or 4 rates of acetaldehyde oxidation. Acetaldehyde could support an energy-dependent uptake of Ca²⁺ at rates about 20 percent that found with succinate. Ruthenium red, an inhibitor of Ca²⁺ translocation, almost completely prevented the inhibition by Ca²⁺, under all conditions. The addition of externally added NAD⁺ or NADH provided complete relief against the inhibitions by Ca²⁺ of the state 4 and 3 rates of acetaldehyde oxidation. Although some relief was also observed with the valinomycin-stimulated system, significant inhibition persisted. Cations such as Zn²⁺, Cu²⁺, or Hg²⁺ also inhibited acetaldehyde oxidation, whereas Mg²⁺ and Mn²⁺ were without effect. These cations also blocked glutamate oxidation and presumably inhibit acetaldehyde oxidation by preventing reoxidation of NADH. The greater sensitivity of the ionophore-stimulated oxidation of acetaldehyde to inhibition by Ca²⁺ may reflect release of intramitochondria K⁺, which is known to occur in the presence of Ca²⁺, suggesting that acetaldehyde oxidation is influenced by the cation environment within the mitochondria.