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Oxidation of [1-14C]-α-ketoisocaproate to yield 14CO2 has been studied in respiring rat liver mitochondria. So long as oxidative phosphorylation proceeds the rate of decarboxylation is low. When ATP formation from added ADP is completed the rate of oxidative decarboxylation of α-ketoisocaproate increases abruptly. These observations indicate an inverse correlation between branched chain α-keto acid oxidation and oxidative phosphorylation in liver mitochondria.

1. There is a more than 2-fold increase in phospholipase A2 activity (EC 3.1.1.4) of liver mitochondria isolated from vitamin E-deficient rats compared with that in normal rats. 2. α-Tocopherol in lipoprotein-bound form is more effective than free α-tocopherol in restoring the enzyme activity to normal.

A study was made of the uncoupling action of polychlorinated biphenyls (Kanechlor®-400) on oxidative phosphorylation in rat liver mitochondria. Kanechlor-400 (KC-400) at 20 μg/ml stimulated state 4 respiration of rat liver mitochondria more than 4-fold withα-ketoglutarate/malate as a substrate, and released the oligomycin-inhibited state 3 respiration. KC-400 also dissipated the membrane potential across the mitochondrial membranes; thus, it acts as an uncoupler of oxidative phosphorylation in rat liver mitochondria. KC-400 altered the permeability properties of mitochondrial membranes as evidenced by the release of endogenous K+ and the oxidation of exogenously supplied NADH. It is concluded that KC-400 produces a nonspecific increase in mitochondrial ion permeability, thereby dissipating membrane potential, which leads to the uncoupling.

1. 1. Acetoacetate reduction by rat liver mitochondria in the presence of different succinate concentrations has been studied. 2. 2. At low succinate concentrations (0.5-1 mM) the oxidation of mitochondrial free fatty acids, sparked by succinate, plays a prominent role in the acetoacetate reduction. 3. 3. At higher succinate concentrations the reducing equivalents for acetoacetate reduction derive from succinate via the ‘ reversal’ pathway. 4. 4. Furthermore in the presence of high succinate concentrations (lomM) fatty acid oxidation is almost completely inhibited.

The decarboxylation of α-keto-γ-methiolbutyrate, the α-keto acid of methionine has been studied in rat liver. Most (70%) of the decarboxylation activity is in the mitochondrial fraction. The Km for the decarboxylation of α-keto-γ-methiolbutyrate in intact mitochondria is 0.6 mM. Pyruvate, α-keto-butyrate and α-ketoisocaproate significantly inhibit this activity. The inhibition due to α-ketoisocaproate is 3.3 fold that of pyruvate and 1.1 fold that of α-ketobutyrate at a concentration of 0.4 mM.

The effect of halothane on oxidation of exogenous NADH was evaluated in intact and disrupted rat liver mitochondria. A change in oxygen uptake was observed which represented an interplay between inhibition of NADH dehydrogenase (tending to decrease respiratory rate) and loss of impermeability to NADH (tending to increase respiratory rate).

The adenine nucleotide content of rat liver mitochondria was shown to increase significantly after birth. On the other hand, it was found that the ligand-binding properties of the adenine nucleotide translocator were essentially the same in foetal, suckling and adult rat liver mitochondria. These results are compatible with the proposal that the accumulation of adenine nucleotides which occurs during mitochondrial biogenesis and maturation is effected by a pathway different from the adenine nucleotide translocator.

A differential centrifugation method for the preparation of rat liver mitochondria is described, which results in final mitochondrial yields of at least 35 to 40 mg of mitochondrial protein per gram wet weight of liver. These mitochondria are shown to be functionally and ultrastructurally intact. They exhibit acceptor control ratios of 6 to 8 and ADP O ratios near 2.0 with succinate as a substrate. In addition, they appear homogeneous by electron microscopy criteria. This method can be used to prepare rat liver mitochondria in high yield on either a large- or a small-scale basis.

Calcium efflux from isolated mitochondria on ruthenium red addition was shown to be biphasic. The rate of efflux from a slowly releasable pool was independent of preincubation. It could be saturated and in extrapolation revealed a maximal rate of 3.6 nmol/(min X mg protein). The efflux from a second, rapidly dischargeable pool was related to calcium added up to 300 nmol/mg protein when a final rate of 15 nmol/(min X mg protein) was reached. The magnitude of the latter pool depended on the time of preincubation in the presence of calcium and correlated with mitochondrial swelling. After ruthenium red addition, a further increase of this pool and spontaneous, destructive calcium release was prevented. Three conclusions are drawn from these results: On preincubation with calcium, part of the mitochondrial calcium develops into a rapidly dischargeable pool. This pool is responsible for mitochondrial alterations resulting in a spontaneous, destructive release of total calcium. Ruthenium red inhibits calcium release by discharging mitochondria from this destructive calcium pool. To avoid artefacts, mitochondrial parameters should be carefully controlled when ruthenium red-insensitive calcium efflux is studied.

2-Phenylpropylguanidino-4,6-dimethylpyrimidine (PPG–2) inhibited completely the respiration of Ehrlich ascites tumor cells (E-cells) at 3,5 × 10−4 m. Against Esherichia coli, the inhibitory effect was observed at 1.0 × 10−4 m; at a lower concentration, 3.3 × 10−6 m, the oxygen uptake was remarkably stimulated. In the polarographic studies of rat liver mitochondria oxidation PPG–2 stimulated the state 4 oxidation of succinate and released the inhibition by oligomycin. Therefore, it is supposed that PPG-2 acts like as an uncoupling agent. The ATPase activation by PPG–2, however, was not observed.

Summary ATP-driven, rotenone-sensitive, reverse electron transfer from succinate to acetoacetate was measured in rat liver mitochondria in the presence of cyanide. In the approach to equilibrium, the absolute ratio of the free energy change of electron transfer to that of ATP hydrolysis exceeded 1, tending towards about 1¼. The data support an H + /2e − stoichiometry of 5 for Site 1 as predicted by a thirteen-proton model of chemiosmotic coupling.

Fulvic and humic acids extracted from a podzol were shown to stimulate respiration in rat liver mitochondria, when present at concentrations of between 40 and 360 mg L-1. Low molecular weight fractions induced a more significant increase in respiration than high molecular weight material. Humic substances, at concentrations of between 40 and 400 mg L-1, normally also increased the efficiency of the process of oxidative phosphorylation in vitro, particularly after contact periods with the mitochondria of over 1 hour.

Summary Reye's Syndrome serum stimulates O2 utilization in preparations of isolated rat liver mitochondria and causes mitochondria to swell. The substance responsible for the respiratory activity has been purified by ion exchange chromatography and isolated in one of three ways: crystallization, high voltage electrophoresis, or high pressure liquid chromatography. All three methods yielded active material with identical UV spectra. Further analysis by GC-mass spectrometry and infrared spectroscopy revealed the active substance to be uric acid. Uric acid probably does not cause mitochondrial injury, but it may deserve attention as a possible prognostic marker in Reye's Syndrome.

An ATP-dependent DNA ligase has been demonstrated in extracts of rat liver mitochondria. The activity may be released from the mitochondria by treatment with hypotonic solutions or a detergent, indicating an intramitochondrial localization. The properties of the partially purified enzyme are similar to those of the nuclear DNA ligase from rat liver.

It has been shown previously by the author that the structure of rat liver mitochondria is changed by a low casein diet. In the present experiments, using the free fatty acid producing substances Ca2+ and thyroxine, and the free fatty acid sodium oleate, the influence of a 4% casein diet on the in vitro swelling process of purified rat liver mitochondria was investigated by following the decrease in optical density at 520 nm after addition of one of the swelling agents. Swelling due to hypotonicity was also investigated. Rats were fed the test diets for 80 to 100 days. Mitochondria were purified by washing three times; after each wash the isotonic suspension was centrifuged at 3,000 X g for 6 minutes. The results show that the in vitro swelling of mitochondria induced by Ca2+ or thyroxine was inhibited greatly in the early period of swelling in rats fed a low casein diet. By comparison to this, there was not a notable effect of a low casein diet on the rate of swelling induced by sodium oleate. But in the case of sodium oleate, the values of optical density were always higher in rats fed a low casein diet, and the differences between two groups were always significant. Swelling due to hypotonicity was not affected at all by a low casein diet.

The presence of inorganic phosphate and Ca2+ in the external medium induces a closely parallel efflux of both endogenous adenine nucleotides and Mg2+ from rat liver mitochondria. These effluxes are (a) pH-dependent and inhibited by uncouplers, respiration inhibitors and external Mg2+; (b) completely prevented by bongkrekate, but stimulated by atractylate. ATP, ADP or AMP each inhibit the release of Mg2+ promoted by Ca2+ and phosphate: however, in the presence of oligomycin and P1,P5-di(adenosine-5′)-pentaphosphate (an inhibitor of adenylate kinase) only ADP is effective. Also the release of accumulated Ca2+ observed when ∼ 50% Mg2+ is discharged is retarded by bongkrekate and added Mg2+ whereas it is accelerated by atractylate. All adenine nucleotides have a significant effect in retarding the efflux of accumulated Ca2+ but, in the presence of oligomycin and P1,P5-di(adenosine-5′)-pentaphosphate only ADP is active. From these results we conclude that effluxes of Mg2+, Ca2+ and adenine nucleotide from rat liver mitochondria induced by external phosphate are interconnected and regulated by external ADP and Mg2+ levels.

Effects of pantothenic acid deficiency on rat hepatocytes and rat liver mitochondria are reported. Further studies on mitochondrial structure and function and liver function in pantothenic acid deficiency are likely to yield useful data on malnutrition and disease.s

Transmembrane potential of liver mitochondria in hyperthyroid rats was higher than that in euthyroid animals. Besides, a lower resistance to Ca2+ of the system responsible for the maintenance of the transmembrane potential was observed in the preparations from hyperthyroid rats. This was manifested in a decrease of the calcium dose required for the decay of the transmembrane potential and in a shorter period of Ca2+ retention by mitochondria. The observed decrease in the resistance to Ca2+ ions of the potential-maintaining system in hyperthyroid mitochondria is supposed to be determined by the increased activity of mitochondrial endogenous phospholipase.

One of the major polypeptide bands seen after rat liver mitochondria are subjected to dodecyl sulfate gel electrophoresis is a component with a mass of 36,000 daltons that makes up 3 to 4% of the total mitochondrial protein. This band, designated Va (1), purifies with an enzyme of the urea cycle, ornithine transcarbamylase (E.C. 2.1.3.3). Evidence is presented that the band Va polypeptide is a single molecular species corresponding to the polypeptide chain of this enzyme.

The mechanism by which metalloporphyrins escape from mitochondria has been studied in isolated rat-liver mitochondria using Co-deuteroporphyrin as the model compound. During the first 10–15 min of incubation the efflux is about 10% of the total amount of Co-deuteroporphyrin synthesized. The efflux then increases to a second steady-state leve of 25–35% after 30–45 min of incubation. The efflux is inversely correlated to the energy state of the mitochondria. Globin at concentrations less than 0.4 mumol/l enhances the efflux of Co-deuteroporphyrin, but has no effect on the degree of energy coupling or on the rate of Co-deuteroporphyrin synthesis. The effect of globin can be competitively inhibited by adding haemin. Haemin (0.5–1.0 mumol/l) when added to the medium in the absence of globin reduces the efflux of Co-deuteroporphyrin by 20–30%, but has no effect on the metal-chelatase activity. Neither albumin nor haemoglobin increases the efflux of Co-deuteroporphyrin from intact mitochondria. The results suggest that the efflux of metalloporphyrin is regulated in part by the energy state of the mitochondria and in part by the presence of metalloporphyrin-binding ligants and unattached haemin in the incubation medium.

ATPase (ATP phosphohydrolase, EC 3.6.1.3) activity was shown in the soluble fraction of rat liver mitochondria. Two molecular forms (ATPase 1 and 2) were isolated. ATPase 1 has already been studied. The present paper deals with the purification method of ATPase 2 which was achieved by the following steps: (NH4)2SO4 precipitation, DEAE-cellulose chromatography, hydroxyapatite chromatography, Sephadex G100 filtration and AMP-Sepharose affinity chromatography. The purified protein was characterized by bidimensional polyacrylamide gel electrophoresis. Molecular weight evaluated by SDS-polyacrylamide gel electrophoresis and Sephadex G100 gel filtration was found to be 61 500 ± 3000.

By using clorgyline and deprenyl to block the types A and B active sites of monoamine oxidase, respectively, the remaining site could be selectively labeled radiochemically with [3H]pargyline. Titration of the A and B active sites in rat liver mitochondria indicated that they were present in a ratio of 1:3.3. Subunits containing the labeled A and B catalytic sites could not be separated by SDS-gel electrophoresis, and each had a molecular weight of 60,000 daltons.

The effect of agaric acid (α cetyl citric acid) on the transport of ADP and ATP has been examined in rat liver mitochondria. Respiration stimulated by ADP is progresively inhibited by agaric acid; maximal inhibition is attained at 40 μM agaric acid. ATPase activity is inhibited 30% by 20 μM agaric acid. The exchange of adenine nucleotides is competitively inhibited by agaric acid.

In rat liver mitochondria, membrane lipid unsaturation increases, the temperature limits of the membrane phase transition decrease and the E a of succinate oxidase increases following thyroidectomy. All three parameters change in the opposite direction within 12 h after thyroxine treatment. It is suggested that many effects of thyroid hormones can be explained by changes in membrane structure and function.

Rat-liver mitochondria oxidized [1-14C]palmitate or [U-14C]palmitate and unlabelled pyruvate in a medium containing fluorocitrate and L-carnitine. The oxidation products (acetyl-l-carnitine, ketone bodies and citrate) were separated by anion-exchange chromatography and their specific activities were determined. The distribution of radioactivity over the two halves of the ketone bodies was essayed. Significant differences between the specific activities of citrate, acetyl-L-carnitine and the carboxyl-half of the ketone bodies were not observed; this was consistently the case, even when pyruvate contributed for more than 80% to the acetyl-CoA pool. Our results argue against compartition of mitochondrial acetyl-CoA. Instead, they strongly suggest that the acetyl-CoA originating from the simultaneous oxidation of pyruvate and palmitate equilibrates before being distributed over the various pathways of further metabolism.

1. Estimates of the functional sizes of the molecular species responsible for the overt (I) and latent (II) activities of carnitine palmitoyltransferase (CPT) in 48 h-starved rat liver mitochondria were obtained from radiation inactivation experiments. 2. The decay in the activity of total CPT and that of CPT II only (after inhibition of CPT I) was measured in mitochondrial samples exposed to different doses of high-energy ionizing radiation. 3. The decay curves obtained by plotting residual activity of total CPT as a logarithm function of irradiation dose suggested the contribution of more than one target towards total CPT activity. 4. By contrast, in mitochondria in which CPT I activity was approximately 95% inhibited, the activity of CPT decayed in a simple mono-exponential manner. Target-size analysis yielded an approximate Mr of 69,700 for this component (CPT II). 5. This information, as well as that on the relative non-irradiated activities of CPT I and CPT II, was used in graphical and statistical methods to obtain the parameters of the decay curve for CPT I. These analyses yielded an approximate Mr of 96,700 for CPT I.