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.     

Species pattern of phosphatidic acid, diacylglycerol, CDP-diacylglycerol and phosphatidylglycerol synthesized de novo in rat liver mitochondria

Rat liver mitochondria were incubated with [3H]glycerol 3-phosphate, ATP, CTP and coenzyme A allowing acylatin of glycerophosphate with endogenous fatty acids and the further conversion of labelled phosphatidic acid (PA) to diacylglycerol (DG), CDP-diacylglycerol (CDP-DG) and phosphatidylglycerol (PG). In these glycerolipids, the distribution of label among the individual molecular species was found to be similar, with 16:0-18:1, 16:0-18:2 and 18:0-18:2/16:0-16:0 being the main species. It was concluded that mitochondrial enzymes involved in the de novo synthesis of these glycerolipids exhibited no acyl selectivity for their substrates. The pattern of molecular species of mitochondrial PA, DG and CDP-DG closely approached that of the same glycerolipids synthesized de novo in isolated rat liver microsomes.     

The structural organization of haem synthesis in rat liver mitochondria

1. Anaerobic conditions are normally necessary for incorporation of iron into haems and only ferrous iron is used. After addition of succinate to an incubation mixture containing intact or ultrasonically treated mitochondria, Fe(3+) is used, but only if no inhibitors prevent the transfer of electrons from the mitochondrial respiratory chain to oxygen. 2. A dual-wavelength spectrophotometric assay for ferrochelatase is described that has been used for the continuous assay of incorporation of metal ions into porphyrins. Constants are given for the determination of rates of formation of protohaem and cobalt protoporphyrin, mesohaem, cobalt mesoporphyrin and zinc mesoporphyrin. For cobalt mesoporphyrin formation the K(m) for Co(2+) is 11x10(-6)m and that for mesoporphyrin is 5x10(-6)m. 3. An improved method for the separation of inner and outer membranes of mitochondria is described. Mitochondria swollen in hypo-osmotic media were contracted in hyperosmotic potassium chloride solution containing ATP and the outer membranes detached by mild ultrasonic treatment. Sucrose inhibited the ATP-induced contraction and decreased the yield of outer membranes. 4. Ferrochelatase is associated with cytochrome oxidase, which is used as a marker for inner mitochondrial membranes. 5. By using as substrate porphyrin dissolved in phospholipid micelles, ferrochelatase activity of intact mitochondria was shown to be latent, and to be liberated by ultrasonic treatment. 6. No ferrochelatase was detectable in microsomes or soluble cell components.     

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.     

Influence of mitochondria on phospholipid synthesis in preparations from rat liver

1. The addition of mitochondria to an incubation system containing the soluble and microsomal fractions of rat liver enhances severalfold the incorporation of each of ethanolamine, phosphorylethanolamine and CDP-ethanolamine into phosphatidylethanolamine. 2. In the presence of microsomal, mitochondrial and soluble fractions, CDP-ethanolamine exhibits the greatest initial rate of incorporation (approx. 6nmol/h per mg of protein), being slightly faster than that of phosphorylethanolamine (approx. 5nmol/h per mg of protein). Incorporation of ethanolamine proceeds very slowly for the first 20min and only after 30min gives rates approaching those of the other two precursors. 3. By using a substrate ;dilution' technique it was shown that in the reconstituted system the affinity of each of the enzymes for their respective substrates is very high: 10mum for ethanolamine, 25mum for phosphorylethanolamine and 5mum for CDP-ethanolamine. 4. Isolation of the mitochondrial and microsomal fractions from the medium after incubation together with phosphorylethanolamine showed that about 70% of the total radioactivity was present in the microsomal fraction and about 30% in the mitochondria after only 20min. Similar experiments with ethanolamine as precursor revealed that after 20min only about 15% of the total radioactivity was present in the mitochondria but that after 40min about 30% was present in this fraction. 5. Heating and phospholipase treatment of mitochondria, but not freeze-thawing, eliminated the stimulatory effect of mitochondria on phospholipid synthesis. 6. The reconstituted system exhibits an absolute requirement for Mg(2+) (2mm gave maximal rates) and is inhibited by very low concentrations of Ca(2+) (100mum-Ca(2+) produced half-maximal inhibition with 3mm-Mg(2+)). Further addition of Mg(2+) overcame the Ca(2+) inhibition, suggesting that the inhibitory effect is readily reversible. 7. The concept that modification of the Mg(2+)/Ca(2+) ratio is a means of controlling the rate of cellular phospholipid synthesis is introduced.     

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.     

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.     

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.