Phosphatidate biosynthesis in mitochondrial subfractions of rat liver

1. After conventional fractionation of rat liver homogenates in 0.88m-sucrose the mitochondrial fraction was subjected to short-term water lysis followed by separation of the resulting membrane preparations. 2. Phosphatidate formation was measured in all subcellular fractions and subfractions and was compared with the distribution of succinate dehydrogenase, monoamine oxidase, rotenone-insensitive NADH cytochrome c reductase, arylsulphatase, urate oxidase, arylesterase and glucose 6-phosphatase. 3. The results obtained indicated that mitochondria were capable of synthesizing phosphatidate, though this activity was only about one-third of the total homogenate activity. 4. Mitochondrial phosphatidate formation was located predominantly in the outer mitochondrial membrane. Although this membrane preparation was found to be significantly contaminated by the microsomal fraction, this contamination was estimated to account for not more than about 20% of the total phosphatidate formation observed in preparations of outer mitochondrial membrane.     

The biosynthesis of tyramine glucuronide by liver microsomal fractions.

Labelled tyramine glucuronide was synthesized in vitro from UDP-[14C]glucuronic acid, [14C]tyramine or [3H]tyramine. The glucuronidation was carried out at pH9.2 in the presence of a monoamine oxidase inhibitor, trans-2-phenylcyclopropylamine. The Km values for tyramine were 69 and 125 micrometer and those for UDP-glucuronic acid were 260 and 290 micrometer respectively for guinea-pig and rat liver microsomal preparatons. The specific activities of microsomal glucuronyltransferase measured in fresh hepatic preparations of guinea pig, mouse and rat were respectively 601, 251 and 235 pmol of [14C]tyramine glucuronide/min per mg of protein. Increase in activity ranged from 2- to 6-fold in preparations which were frozen and thawed once and 5.4- to 10-fold when the freezing and thawing was repeated. Rabbit liver has very low activity, and monkey liver and intestine were completely devoid of this conjugating capacity.     

Effect of tri-iodothyronine treatment on electrophoretic mobilities of rat liver mitochondrial fractions

Heterogeneity of mitochondrial fractions, observed earlier in respect of membrane-bound enzyme profiles and protein turnover, is not reflected in their electrophoretic mobilities.     

Kinetics of the biosynthesis and distribution of labelled actin-like proteins in rat liver submitochondrial fractions

Affinity adsorption on immobilized DNAase I and the measurements of the protein mobility upon SDS-PAGE electrophoresis were used for the identification of the actin-like protein as well as for the study of its biosynthesis is liver mitochondria of hepatectomized rats. The kinetics of biosynthesis showed a maximum on the 10th min after intraperitoneal injection of the label. Fractionation of mitochondria demonstrated that more than 50% of the whole amount of the "de novo" synthesized protein is localized in the intermembrane space, approximately 30%--in the mitochondrial matrix. The purity of the fractions was controlled by analyzing the polypeptide content of the samples and by measuring the marker enzyme activity. Besides, additional identification of the actin-like protein was carried out directly in the mitochondrial matrix and intermembrane space by two-dimensional electrophoresis in polyacrylamide gel performed by the O'Farrell method. The subsequent staining of the gels with silver revealed the presence of two basic isoforms of non-muscle action (beta- and gamma-actins). The presence of the actin-like protein in the inner mitochondrial compartments characterized by a high rate of metabolism may be regarded as compelling evidence of its mitochondrial localization.     

Effect of vitamin E on characteristics of liver mitochondrial fractions from cold-exposed rats

In cold exposed rats, it is known that vitamin E induces an increase in the respiration of the whole mitochondrial population isolated from liver. To obtain information on the effects of cold exposure and vitamin E treatment on the dynamics of mitochondrial population, we determined characteristics of rat liver mitochondrial fractions, resolved at 1,000 (M₁), 3,000 (M₃), and 10,000 g (M₁₀). We found that cold exposure increased the liver content of total mitochondrial proteins irrespective of vitamin E treatment. Conversely, protein distribution among the mitochondrial subpopulations was differentially affected by cold and antioxidant integration. In a cold environment, the M₁ fraction, characterized by the highest O₂ consumption and H₂O₂ production rates, underwent a remarkable protein content reduction, which was attenuated by vitamin E. These changes were dependent on the opposite effects of the two treatments on mitochondrial oxidative damage and susceptibility to swelling. The proteins of the other fractions, in which the above effects were lower, underwent smaller (M₃) or no change (M₁₀) in the treatment groups. The cold also led to an increase in O₂ consumption of the M₁ fraction which was accentuated by vitamin E treatment. This phenomenon and the vitamin-induced recovery of the M₁ proteins supply an explanation of the previously reported increase in the respiration of the whole mitochondrial population induced by vitamin E in the liver from cold exposed rats.     

Immunological similarity of epoxide hydrolase activity in the mitochondrial and cytosolic fractions of mouse liver

The light and heavy mitochondrial fractions of mouse liver have relatively high levels of epoxide hydrolase (EH) activity when monitored with trans-stilbene oxide as substrate. Using double diffusion analysis and immunoprecipitation experiments it was shown that EH activity in the mitochondrial fractions is immunologically similar to cytosolic EH, but immunologically dissimilar from microsomal EH. The EHs in the mitochondrial and cytosolic fractions also have a similar pI.     

Regulation of biosynthesis of the rat liver inner mitochondrial membrane by thyroid hormone

Regulation of mitochondrial protein synthesis by thyroid hormone has been studied in isolated rat hepatocytes and liver mitochondria. Small doses (5 micrograms/100 g body wt) of triiodothyronine (T3) injected into hypothyroid rats increased both state 3 and 4 respiration by approximately 100%, while the ADP:O ratio remained constant. This suggests that T3 increases the numbers of functional respiratory chain units. T3 also induces mitochondrial protein synthesis by 50-100%. Analysis of the mitochondrial translation products show that all of the products were induced. No differential translation of the peptides involved in the respiratory chain was found. Regulation of the cytoplasmically made inner membrane peptides was also investigated in isolated hepatocytes. The majority of these peptides were not influenced by T3, in contrast to the finding with mitochondrial translation products. Those found to be regulated by T3 belong to two subsets, which were either induced or repressed by hormone. Thus, T3 stimulated a general increase in the synthesis of mitochondrially translated inner membrane peptides, but regulates selectively those inner membrane peptides translated on cytoplasmic ribosomes. The findings suggest that hormone regulation of the respiratory chain is exerted through a few selective proteins, perhaps those which require subunits made from both nuclear and mitochondrial genes.     

In vitro biosynthesis of plasminogen in a cell-free system directed by mRNA fractions isolated from monkey liver

mRNA was isolated from total RNA of monkey liver by oligo(dT)-cellulose chromatography and was translated in a rabbit reticulocyte cell-free system. Analysis of the translation products immunoprecipitated with specific antibodies to monkey plasma plasminogen revealed a molecule with characteristics similar to those of native plasminogen. The purification of the mRNA by centrifugation on sucrose gradients indicated the presence of plasminogen mRNAs in both the 23S and 18S RNA fractions. Both plasminogen mRNAs can be further purified by chromatography on Sepharose 4B. Affinity chromatography of the proteins synthesized in vitro by total mRNA from liver, as well as by the purified mRNAs, on L-lysine-substituted Sepharose revealed that both major plasma plasminogen forms (1 and 2) are synthesized, as precursors, in the system. The in vitro synthesized plasminogen is similar in its physical and chemical properties to native plasma plasminogen as determined by its ability to bind to L-lysine-substituted Sepharose and its molecular interaction with streptokinase. The purified mRNAs were also translated in the presence of dog pancreas microsomal membranes, and and fractionated on concanavalin A-Sepharose. The 23S mRNA directed the synthesis of a plasminogen molecule similar to the circulating plasma plasminogen form 1, whereas the 18S mRNA directed the synthesis of a molecule similar to the circulating plasma plasminogen form 2. Our evidence indicates that the synthesis of the two major circulating plasma plasminogen forms is directed in the liver by separate mRNAs.     

Heterogeneity of rat liver mitochondrial fractions and the effect of tri-iodothyronine on their protein turnover

1. Rat liver mitochondria were separated into heavy, light and fluffy fractions by differential centrifugation under standard conditions. 2. All mitochondrial fractions possessed soluble as well as membrane-bound enzymes typical of mitochondria. 3. The heavy fraction represented the stable mitochondrial structures and the fluffy particles appear to be loosely coupled. 4. The light mitochondrial fraction lacked the ability of coupled phosphorylation. 5. A study of mobility and isoelectric pH indicated a similarity in the basic membrane structure of all the mitochondrial fractions. 6. The turnover rates of proteins in the heavy and fluffy particles were almost identical; however, this rate was rapid for the light mitochondrial fraction. 7. On treatment with 3,3',5-tri-iodo-l-thyronine, succinoxidase activity was maximally stimulated much earlier in the light mitochondrial fraction than in the heavy fraction. The activity of the fluffy particles, however, remained almost unaffected. 8. Malate dehydrogenase activity in all the mitochondrial fractions was stimulated only at 40h after tri-iodothyronine treatment. 9. The pattern of incorporation of dl-[1-(14)C]leucine in vivo in the tri-iodothyronine-treated animals indicated a rapid initial incorporation and high synthetic ability of the light mitochondrial fraction. 10. The turnover pattern of proteins of the mitochondrial fractions from animals receiving repeated doses of tri-iodothyronine was remarkably different from the normal pattern and suggested that preformed soluble protein units may be incorporated in the light mitochondrial fraction during maturation to form the stable heavy mitochondria. 11. The amount of light-mitochondrial proteins decreased by 40% on thyroidectomy and increased by 160% on treatment with tri-iodothyronine. 12. The possible significance of these results is discussed in relation to mitochondrial genesis.