CDP-diacylglycerol synthesis in rat liver mitochondria.

CDP-diacylglycerol for polyglycerophosphatide biogenesis can be synthesized within rat liver mitochondria. This membrane-associated enzyme was predominantly located in the inner mitochondrial membrane. GTP had a significant effect in activating the microsomal CDP-diacylglycerol synthase, especially if the microsomes were preincubated with GTP in the presence of phosphatidic acid. This stimulatory effect of GTP on the microsomal enzyme was not detected in the mitochondrial fractions. The enzymes could be solubilized from the membrane fractions using CHAPS, and the detergent-soluble activity partially restored by addition of phospholipids. Mitochondrial and microsomal CDP-diacylglycerol synthase activity could be completely separated by anion-exchange column chromatography. The mitochondrial and microsomal CDP-diacylglycerol synthases appear to be two distinct enzymes with different localization and regulatory characteristics.     

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.     

Calmodulin stimulates DNA synthesis by rat liver cells

Incubation in low (0.02 mM)-calcium medium prevented T51B rat liver cells from initiating DNA synthesis. Raising the calcium concentration in the medium from 0.02 to 1.25 mM caused these arrested cells to initiate DNA synthesis 1–2 hours later. The possibility of this rapid DNA-synthetic response to calcium addition being mediated through Ca-calmodulin complexes was suggested by the following observations: It was blocked by the putative Ca-calmodulin blockers chlorpromazine and trifluoperazine; the trifluoperazine-inhibited cells were stimulated by purified rat calmodulin; and purified rat calmodulin itself (10^?7 to 10^?6 moles/l) mimicked calcium action, unless the already low ionic calcium concentration in the calcium-deficient medium was reduced further by adding the specific calcium chelator EGTA.     

Phosphoenolpyruvate synthesis by fetal guinea-pig liver mitochondria

Liver mitochondria isolated from fetal and newborn guinea pigs synthesized phosphoenolpyruvate at 4–6 nmol/min per mg protein with 2 mM malate, succinate, and α-ketoglutarate as substrates. These rates were 90–110% of that by adult liver mitochondria and were not substantially altered in the second half of gestation or within 24 h after birth. Both palmitoyl- and octanoylcarnitine were inhibitory to phosphoenolpyruvate synthesis in adult and fetal preparations, but free octanoate was inhibitory only in adult liver mitochondria.     

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.     

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.     

Accumulation of D-arginine by rat liver mitochondria

The permeability of the inner mitochondrial membrane from rat liver to D-arginine was studied. By using safranin as a probe of the membrane potential, depolarization of energized liver mitochondria occurred in a dose-dependent fashion starting at 3.3 mmol/L of D- or DL-arginine. When ethidium bromide fluorescence was employed, a decrease in the membrane potential due to D- or DL-arginine was observed. A parallel significant change in succinate-induced respiration in rat liver mitochondria was found in response to osmotic swelling in 125 mmol/L of D-arginine salts. L-Arginine, L-glutamine, L-asparagine, L-ornithine, D-ornithine, and L-lysine did not modify the membrane potential at the concentrations tested. D-Arginine was not transformed into citrulline, but 1.0 mmol/L of the D-amino acid inhibited, by 42%, the state 3 of mitochondrial respiration using succinate as substrate. When D-arginine was used in combination with nigericin, a 40% inhibition of mitochondrial respiration in state 3 was recorded with succinate and with glutamate-malate as substrates.     

Arginine uptake by isolated rat liver mitochondria

The question of arginine uptake by mitochondria is important in that arginine is an allosteric effector of N-acetylglutamate synthetase. Thus, changes in mitochondrial arginine concentration have the potential for acutely modifying levels of N-acetylglutamate, a compound necessary for maximal activity of carbamyl phosphate synthesis. Mitochondria were isolated from chow-fed rats, incubated with [guanido-¹⁴C]arginine and were centrifuged through silicon oil into perchloric acid for determination of intramitochondrial metabolites. Arginine was separated from urea by cation-exchange resin. Mitochondrial water space was determined by [¹⁴C]urea arising from arginase activity associated with the mitochondrial preparations. Extramatrix space was determined by parallel incubations with [inulin-¹⁴C]carboxylic acid or [¹⁴C]sucrose There was considerable degradation of arginine by arginase associated with the mitochondrial preparation. This was inhibited by 7 mM ornithine and 7 mM lysine. Arginine was concentrated intramitochondrially to 4-times the extramitochondrial levels. The concentration ratio was decreased in the presence of ornithine and lysine but not with citrulline, NH₄Cl, glutamate, glutamate or leucine. No uptake was observed when mitochondria were incubated at 0°C. Mitochondria did not concentrate citrulline.