Highly efficient DNA synthesis in isolated mitochondria from rat liver.

We have developed a highly efficient DNA-synthesizing system with isolated intact rat liver mitochondria. The ATP requirements for this in organello DNA synthesis are provided by endogenous synthesis in the presence of exogenous ADP and an oxidizable substrate. In this system, mitochondrial DNA synthesis strikingly proceeds at a constant rate for about 5 h at 37 degrees C. Gel electrophoresis, hybridization and restriction enzyme analyses show that intact mitochondria synthesize nucleic acids with a size of 16.5 kb, that correspond to mitochondrial DNA, and that both DNA strands are replicated. This in organello DNA synthesis requires the supply of dNTPs and decreases at high ADP concentration in the incubation medium.     

Proline transport in rat liver mitochondria.

Proline transport across the inner membrane of rat liver mitochondria shows the following properties: (a) It is stereospecific; the penetration of l-proline is two times faster than the penetration of dl-proline. (b) Proline is accumulated against a concentration gradient, (c) The transport of proline is enhanced in the presence of respiratory substrates such as succinate or tetramethylphenylenediamine + ascorbate; it is inhibited by uncouplers of oxidative phosphorylation. (d) Proline transport is inhibited by mersalyl and p-chloromercuribenzoate, but not by hydrophobic thiol blocking reagents; thus, proline transport involves thiol groups located in a very hydrophilic environment. The penetration of several other neutral amino acids (alanine, glycine, serine) is almost insensitive to mersalyl. These results suggest that proline does not travel across the mitochondrial membrane by free diffusion, but that its transport is mediated by a specific carrier. The rate of proline transport has been compared with the rates of the first two steps of proline oxidation: All of these rates are very similar, indicating that proline transport is not a limiting factor of proline metabolism in rat liver mitochondria.     

Primer directed poly(A) synthesis in rat liver mitochondria

Rat liver mitochondria contain an endogenous factor highly specific in stimulating the homologous poly(A) polymerase. By using an $\text{in vivo}$ labelling with [³²P] orthophosphate it is possible to prepare a labelled factor and to demonstrate that it is stably incorporated in an acid insoluble molecule. This suggests that the factor probably acts as a primer in the polymerization of ATP molecules, being involved in the recognition between the mitochondrial poly(A) polymerase and the homologous RNA molecules which have to be polyadenylated.     

The stimulation of rat liver microsomal CDP-diacylglycerol formation by guanosine triphosphate

GTP has been found to markedly enhance the formation of CDP-diacylglycerol in rat liver microsomes. Neither GDP, GMP nor the nonhydrolyzable analogues of GTP increased the synthesis of the liponucleotide. The GTP stimulation of phosphatidate cytidylyltransferase activity is inhibited by EDTA and NaF. GTP enhances the activity of the enzyme in a concentration-, time-, and temperature-dependent manner and preincubation of rat liver microsomes with GTP produces a persistently activated phosphatidate cytidylyltransferase. GTP reduces the Km for phosphatidic acid, but has no effect on either the Km for CTP or the Vmax of the reaction. GTP, by stimulating the activity of the phosphatidate cytidylyltransferase, enhances the formation of phosphatidylinositol from CTP, phosphatidic acid, and inositol. Evidence is presented suggesting that the mechanism by which GTP stimulates the activity of the phosphatidate cytidylyltransferase involves a covalent modification of the enzyme itself or a protein intimately associated with the phosphatidate cytidylyltransferase.     

The synthesis of proteolipid protein by isolated rat liver mitochondria

About 15% of the total (³H)leucine incorporated into protein by isolated rat liver mitochondria $\text{in}\text{vitro}$ could be extracted by chloroform:methanol. This incorporation was inhibited by chloramphenicol and carbomycin, both specific inhibitors of mitochondrial protein synthesis. SDS-gel electrophoresis of the mitochondrial membrane revealed 6–7 labeled bands. Label in the proteolipid fraction was present mainly in a band of 40,000 molecular weight. Several labeled bands observed in gels of the mitochondrial membrane were not removed or changed by extraction with chloroform:methanol suggesting that some, but not all, of the proteins synthesized by rat liver mitochondria are proteolipids.     

Inhibitors of cytoplasmic protein synthesis purified from rat liver mitochondria

Summary Purified mitochondria from rat liver were found to contain protein synthesis inhibitors, that could be extracted by disruption of mitochondrial membranes and fractionated by gel filtration into two fractions of low and high molecular weight. Small size inhibitors were also released from the latter peak by high ionic strength followed by gel filtration. Both types of factors inhibit incorporation of radioactive amino acids into protein by liver cytoplasmic polysomes programmed with endogenous mRNA or poly U, and by rabbit reticulocyte lysates programmed with added globin mRNA and by incubations of Walker carcinoma cells. They decrease to the same level the cytoplasmic synthesis of proteins for the mitochondrial and extra-mitochondrial compartments in intact cells, but do not appear to inhibit substantially endogenous mitochondrial protein synthesis. Inhibitors were purified by paper chromatography and reverse phase high performance liquid chromatography into fractions which block with the same kinetics the incorporation of [¹⁴]leucine and [³⁵]methionine into protein in systems able to initiate protein synthesis, such as reticulocyte lysates or intact cells, but differ in this respect in incubations of liver ribosomes where re-binding of mRNA is a limiting step. Some of these factors behave as oligopeptides that are assumed to inhibit in vitro primarily the initiation stage but whose function in vivo is still undetermined.     

Mitochondrial metabolism of hydroxocobalamin: synthesis of adenosylcobalamin by intact rat liver mitochondria.

When free hydroxocobalamin (vitamin B₁₂) is added in vitro to a suspension of intact rat liver mitochondria in the presence of a source of both reducing equivalents and ATP, adenosylcobalamin synthesis is observed. This synthetic process is not dependent on electron transport or oxidative phosphorylation and is not detected when cyanocobalamin is substituted for hydroxocobalamin. Adenosylcobalamin synthesis is linear with time for at least 10 min and with hydroxocobalamin concentration up to 37 nm. At the latter concentration of hydroxocobalamin, the rate of synthesis at 37 °C is 0.26 pmol/min/mg of protein. Only part (<30%) of the newly synthesized adenosylcobalamin is bound to the mitochondrial cobalamin binding protein, whereas most (90%) of the concurrently accumulated hydroxocobalamin is bound. On the other hand, when adenosylcobalamin is added to a suspension of intact mitochondria, it is accumulated at a rate similar to that for hydroxocobalamin, and is bound to the mitochondrial binding protein to a similar extent. These findings indicate that rat liver mitochondria contain all of the enzymatic components necessary to convert hydroxocobalamin to adenosylcobalamin, the coenzyme for the mitochondrial enzyme methylmalonyl CoA mutase.     

tert-Butyl hydroperoxide-induced radical production in rat liver mitochondria.

When rat liver mitochondria are treated with tert-butyl hydroperoxide (TBHP) in the presence of the spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO), electron paramagnetic resonance (EPR) signals are detected attributable to spin adducts resulting from the trapping of methyl, tert-butoxyl, and tert-butylperoxyl radicals. The addition of respiratory substrate results in a 3- to 7.5-fold increase in the signal intensity of the DMPO/methyl adduct, no change in the signal intensity of the DMPO/tert-butoxyl adduct, and complete loss of the DMPO/tert-butylperoxyl adduct signal. The magnitude of increase of methyl radical production in the presence of respiratory substrate is related to the respiratory control ratio (RCR) of the mitochondrial preparation. In the presence of antimycin A, which blocks electron flow between cytochromes b and c1, no stimulation of methyl radical production is detected with respiratory substrate. Stimulation of methyl radical production by the addition of respiratory substrate is detected in cytochrome c-depleted mitochondria. A similar increase in methyl radical production is detected when ferrous cytochrome c is treated with TBHP in the presence of DMPO (as compared to when ferricytochrome c is used). These results indicate that TBHP is reduced directly by either cytochrome c1, cytochrome c, or by both of these electron transport chain components in mitochondria undergoing state 4 respiration.     

Arsenite inhibits beta-oxidation in isolated rat liver mitochondria.

A partial inhibition of acylcarnitine oxidation by arsenite in rat liver mitochondria has been studied. This inhibition is confined to the thiolase(s). The inhibition was observed also in the presence of malate, indicating no selective effect on ketogenesis. Ketogenesis from acetyl-CoA was inhibited by arsenite. Mitochondrial CoA was acylated by acylcarnitine nearly as rapidly in the presence of arsenite as in its absence. Thus, arsenite did not interfere with the availibility of CoA in the mitochondria. No effect of arsenite on enzymes of beta-oxidation other than the thiolase(s) was observed. When arsenite and acylcarnitine were added simultaneously to mitochondria, there was a delay before maximal inhibition of oxygen uptake occurred. When the mitochondria were preincubated with arsenite before addition of acylcarnitine, the inhibitory effect on oxygen utilization was initially large, but then partially repealed. Similar time delays were observed in the activity of acetoacetyl-CoA thiolase of disrupted mitochondria depending on the sequence of arsenite and acetoacetyl-CoA addition. It is suggested that substrate and arsenite complete for the reactive sulfhydryl group at the active site of the thiolase(s).     

Phosphate transport in rat liver mitochondria

Experiments were carried out to define the kinetic parameters of the major phosphate transport processes of rat liver mitochondria, and to obtain information about the molecular properties of these systems.     

Pyruvate/malate antiporter in rat liver mitochondria.

To gain some insight into the process by which both acetylCoA and NADPH, needed for fatty acid synthesis, are obtained, in the cytosol, from the effluxed intramitochondrial citrate, via citrate lyase and malate dehydrogenase plus malic enzyme respectively, the capability of externally added pyruvate to cause efflux of malate from rat liver mitochondria was tested. The occurrence of a pyruvate/malate translocator is here shown: pyruvate/malate exchange shows saturation features (Km and Vmax values, measured at 20 degrees C and at pH 7.20, were found to be about 0.25 mM and 2.7 nmoles/min x mg mitochondrial protein, respectively) and is inhibited by certain impermeable compounds. This carrier, together with the previously reported tricarboxylate and oxodicarboxylate translocators proved to allow for citrate and oxaloacetate efflux due to externally added pyruvate.     

The transport of L-cysteinesulfinate in rat liver mitochondria.

1. The mechanism of L-cysteinesulfinate permeation into rat liver mitochondria has been investigated. 2. Mitochondria do not swell in ammonium or potassium salts of L-cysteinesulfinate in all the conditions tested, including the presence of valinomycin and/or carbonylcyanide p-trifluoromethoxyphenylhydrazone. 3. The activation of malate oxidation by L-cysteinesulfinate is abolished by aminooxyacetate, an inhibitor of the intramitochondrial aspartate aminotransferase, it is not inhibited by high concentrations of carbonylcyanide p-trifluoromethoxyphenylhydrazone (in contrast to the oxidation of malate plus glutamate) and it is decreased on lowering the pH of the medium. 4. All the aspartate formed during the oxidation of malate plus L-cysteinesulfinate is exported into the extramitochondrial space. 5. Homocysteinesulfinate, cysteate and homocysteate, which are all good substrates of the mitochondrial aspartate aminotransferase, are unable to activate the oxidation of malate. Homocysteinesulfinate and homocysteate have no inhibitory effect on the L-cysteinesulfinate-induced respiration, whereas cysteate inhibits it competitively with respect to L-cysteinesulfinate. 6. In contrast to D-aspartate, D-cysteinesulfinate and D-glutamate, L-aspartate inhibits the oxidation of malate plus L-cysteinesulfinate in a competitive way with respect to L-cysteinesulfinate. Vice versa, L-cysteinesulfinate inhibits the influx of L-aspartate. 7. Externally added L-cysteinesulfinate elicits efflux of intramitochondrial L-aspartate or L-glutamate. The cysteinesulfinate analogues homocysteinesulfinate, cysteate and homocysteate and the D-stereoisomers of cysteinesulfinate, aspartate and glutamate do not cause a significant release of internal glutamate or aspartate, indicating a high degree of specificity of the exchange reactions. External L-cysteinesulfinate does not cause efflux of intramitochondrial Pi, malate, malonate, citrate, oxoglutarate, pyruvate or ADP. The L-cysteinesulfinate-aspartate and L-cysteinesulfinate-glutamate exchanges are inhibited by glisoxepide and by known substrates of the glutamate-aspartate carrier. 8. The exchange between external L-cysteinesulfinate and intramitochondrial glutamate is accompanied by translocation of protons across the mitochondrial membrane in the same direction as glutamate. The L-cysteinesulfinate-aspartate exchange, on the other hand, is not accompanied by H+ translocation. 9. The ratios delta H+/delta glutamate, delta L-cysteinesulfinate/delta glutamate and delta L-cysteinesulfinate/delta aspartate are close to unity. 10. It is concluded that L-cysteinesulfinate is transported by the glutamate-aspartate carrier of rat liver mitochondria. The present data suggest that the dissociated form of L-cysteinesulfinate exchanges with H+-compensated glutamate or with negatively charged aspartate.