Cell-free translation of mitochondrial nicotinamide nucleotide transhydrogenase

Mammalian nicotinamide nucleotide transhydrogenase is translated as a 5000 daltons larger molecular weight precursor in a cell-free system programmed with rat liver polysomes. The mature rat liver enzyme had the same molecular weight as the purified beef heart enzyme, 115 000 daltons. The precursor was not processed in vitro by liver mitochondria or by a rat liver mitochondrial matrix fraction, nor did it appear to bind to mitochondria. In contrast, pre-FeS protein of the cytochrome bc₁ complex was processed in the same samples by both mitochondria and matrix, suggesting an important difference in the processing mechanisms or in the efficiency of processing of the two precursors.     

Intramitochondrial proteolytic activity of rat liver]

Highly purified mitochondria and lysosomes are isolated from rat liver homogenate. pH optimum of proteolytic activity with respect to proteins of own structures and to mitochondrial structural protein is investigated. The purification of mitochondria from lysosomes is found to be accompanied by the change of proteolytic activity pH optimum from 5.0 to 6.0 in coarse and purified mitochondria respectively. Comparative study of structural protein hydrolysis products with enzyme preparations from purified mitochondria and lysosomes has revealed differences in the spectrum of the reaction products. The data obtained suggest a presence of a proteolytic enzyme in rat liver mitochondria.     

The induction of mitochondrial L-3-glycerophosphate dehydrogenase by thyroid hormone

L-3-Glycerophosphate dehydrogenase was purified from porcine brain mitochondria by a shorter and simpler procedure than previously reported. Immunoblotting with antiserum to the porcine enzyme established that rat liver L-3-glycerophosphate dehydrogenase has the same Mr (76 000) by SDS-polyacrylamide gel electrophoresis. In liver mitochondria from normal and hyperthyroid rats, changes in L-3-glycerophosphate dehydrogenase activity were parallelled by changes in enzyme content assayed by immunoblotting. Similar changes were found in the amount of enzyme synthesised in vitro by reticulocyte lysate programmed with rat liver mRNA, suggesting that thyroid hormone causes specific induction of L-3-glycerophosphate dehydrogenase mRNA.     

Biochemical properties of porcine white adipose tissue mitochondria and relevance to fatty acid oxidation

The capacity of white adipose tissue mitochondria to support a high beta-oxidative flux was investigated by comparison to liver mitochondria. Based on marker enzyme activities and electron microscopy, the relative purity of the isolated mitochondria was similar thus allowing a direct comparison on a protein basis. The results confirm the comparable capacity of adipose tissue and liver mitochondria for palmitoyl-carnitine oxidation. Relative to liver, both citrate synthase and α-ketoglutarate dehydrogenase were increased 7.87- and 10.38-fold, respectively. In contrast, adipose tissue NAD-isocitrate dehydrogenase was decreased (2.85-fold). Such modifications in the citric acid cycle are expected to severely restrict citrate oxidation in porcine adipose tissue. Except for cytochrome c oxidase, activities of the enzyme complexes comprising the electron transport chain were not significantly different. The decrease in adipose cytochrome c oxidase activity could partly be attributed to a decreased inner membrane as suggested by lipid and enzyme analysis. In addition, Western blotting indicated that adipose and liver mitochondria possess similar quantities of cytochrome c oxidase protein. Taken together these results indicate that not only is the white adipose tissue protoplasm relatively rich in mitochondria, but that these mitochondria contain comparable enzymatic machinery to support a relatively high beta-oxidative rate.     

The influence of fasting on chicken liver metabolites, enzymes and mitochondrial respiration

Chicken liver mitochondria were isolated in relatively pure form as indicated by electron microscopy and marker enzyme assay. The rate of respiration, respiratory control index and ADP/O ratios with several different substrates indicated that chicken liver mitochondria are more uncoupled than rat liver mitochondria. Chickens have ten-fold higher malate concentrations in liver than do rats, 2-oxoglutarate was also more abundant in chicken livers. Fasted birds had a five-fold increase in beta-hydroxybutyrate as compared with fed birds; whereas malate and lactate concentrations decreased. Fasted birds had increased levels of isocitrate dehydrogenase (NADP dependent) and lactate dehydrogenase in the cytosol, and increased malate dehydrogenase (NAD dependent), isocitrate dehydrogenase (NADP dependent) and malic enzyme activities in the mitochondria.     

Acetate generation in rat liver mitochondria; acetyl-CoA hydrolase activity is demonstrated by 3-ketoacyl-CoA thiolase

Acetate has been found as an endogenous metabolite of beta-oxidation of fatty acids in liver. In order to investigate the regulation of acetate generation in liver mitochondria, we attempted to purify a mitochondrial acetyl-CoA hydrolase in rat liver. This acetyl-CoA-hydrolyzing activity in isolated mitochondria was induced by the treatment of rats with di(2-ehtylhexyl)phthalate (DEHP), a peroxisome proliferator which induces expression of several peroxisomal and mitochondrial enzymes involved in beta-oxidation of fatty acids. The purified enzyme was 43-kDa in molecular mass by SDS/PAGE. Internal amino acid sequencing of this enzyme revealed that it was identical with mitochondrial 3-ketoacyl-CoA thiolase, suggesting that this enzyme has two kinds of activities, 3-ketoacyl-CoA thiolase and acetyl-CoA hydrolase activities. Kinetic studies clearly indicated that this enzyme had the both activities and each activity was inhibited by the substrates of the other activity, that is, 3-ketoacyl-CoA thiolase activity was inhibited by acetyl-CoA, on the other hand, acetyl-CoA hydrolase activity was inhibited by acetoacetyl-CoA in a competitive manner. These findings suggested that acetate generation in liver mitochondria is a side reaction of this known enzyme, 3-ketoacyl-CoA thiolase, and this enzyme may regulate its activities depending on each substrate level.     

Increased content of natural ATPase inhibitor in tumor mitochondria

The ATPase activity of Zajdela hepatoma and Yoshida sarcoma submitochondrial particles was several times lower than the enzyme activity in rat heart and rat liver submitochondrial particles. The content of F1-ATPase in the tumor mitochondria was found not to be very different from that in mitochondria of rat liver. Immunochemical determination of the amount of the natural ATPase inhibitor revealed that the tumor mitochondria contain 2-3-times more ATPase inhibitor than control mitochondria. It is concluded that the low ATPase activity of the tumor mitochondria results from the inhibition of the enzyme activity by the natural ATPase inhibitor.     

Pathway of glutamate oxidation and its regulation in the HuH13 line of human hepatoma cells.

Well-coupled mitochondria were isolated from a HuH13 line of human hepatoma cells and human liver tissue. The liver mitochondria showed a feeble glutamine oxidation activity in contrast to the hepatoma mitochondria, whereas they utilized glutamate well for the oxidative phosphorylation. In the liver mitochondria, glutamate was oxidized via the routes of transamination and deamination. On the other hand, glutamate oxidation was initiated preferentially via transamination pathway in the tumor mitochondria. In the liver mitochondria, bicarbonate nearly at a physiological concentration inhibited oxygen uptake with glutamate as substrate. The interaction of bicarbonate with the pathway of glutamate oxidation occurred primarily at the level of succinate dehydrogenase, due to competitive inhibition of the enzyme by the compound. In contrast to the liver mitochondria, glutamate oxidation was not affected by bicarbonate in the tumor mitochondria. These results indicate that the aberrations in the glutamate metabolism and its regulation observed in the hepatoma mitochondria may be favorable to the respiration utilizing glutamine and/or glutamate as an energy source.     

Radiation-induced damage to mitochondrial D-beta-hydroxybutyrate dehydrogenase and lipid peroxidation

Radiation-induced damage to the reconstituted system of membrane-bound enzyme, D-beta-hydroxybutyrate dehydrogenase obtained from rat liver mitochondria, was investigated in relation to the lipid peroxidation of membranes. The activity of D-beta-hydroxybutyrate dehydrogenase in fresh mitochondria was very low in general and was not affected by irradiation because of little incorporation of substrates into mitochondria. However, the enzyme activity in one-day-aged mitochondria or submitochondrial particles was five times higher than that of fresh mitochondria and decreased with increasing radiation dose accompanying the increase in peroxidation of membrane lipids. The activity of D-beta-hydroxybutyrate dehydrogenase in the reconstituted system of the purified enzyme with irradiated liver microsomes or irradiated liposomes was decreased considerably in comparison with either unirradiated control or irradiated enzyme. Therefore, the radiation-induced decrease in the enzyme activity was thought to be caused mainly by peroxidation of membrane lipids and not to be due to direct damage by radiation to the enzyme molecule itself. Irradiation of microsomes, a component of the reconstituted system, caused decreases in phosphatidylcholine and phosphatidylethanolamine content and an increase in lysophosphatidylcholine content. In addition, arachidonic acid contents in phosphatidylcholine, phosphatidylinositol and phosphatidylethanolamine were also markedly decreased with increasing radiation dose. These results are discussed in terms of a mechanism involving radiation-induced damage to membrane function and structures.     

Interacting effects of L-carnitine and malonyl-CoA on rat liver carnitine palmitoyltransferase.

Malonyl-CoA significantly increased the Km for L-carnitine of overt carnitine palmitoyltransferase in liver mitochondria from fed rats. This effect was observed when the molar palmitoyl-CoA/albumin concentration ratio was low (0.125-1.0), but not when it was higher (2.0). In the absence of malonyl-CoA, the Km for L-carnitine increased with increasing palmitoyl-CoA/albumin ratios. Malonyl-CoA did not increase the Km for L-carnitine in liver mitochondria from 24h-starved rats or in heart mitochondria from fed animals. The Km for L-carnitine of the latent form of carnitine palmitoyltransferase was 3-4 times that for the overt form of the enzyme. At low ratios of palmitoyl-CoA/albumin (0.5), the concentration of malonyl-CoA causing a 50% inhibition of overt carnitine palmitoyltransferase activity was decreased by 30% when assays with liver mitochondria from fed rats were performed at 100 microM-instead of 400 microM-carnitine. Such a decrease was not observed with liver mitochondria from starved animals. L-Carnitine displaced [14C]malonyl-CoA from liver mitochondrial binding sites. D-Carnitine was without effect. L-Carnitine did not displace [14C]malonyl-CoA from heart mitochondria. It is concluded that, under appropriate conditions, malonyl-CoA may decrease the effectiveness of L-carnitine as a substrate for the enzyme and that L-carnitine may decrease the effectiveness of malonyl-CoA to regulate the enzyme.     

Immunochemical studies on induction of rat liver mitochondrial serine: pyruvate aminotransferase by glucagon.

The 7- to 10-fold increase in the rat liver serine:pyruvate aminotransferase activity after glucagon administration was shown to occur mainly in the mitochondrial matrix of parenchymal cells. The enzyme was purified from glucagon-treated rat liver mitochondria to apparent homogeneity as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A specific rabbit antibody was prepared against the purified enzyme. Upon Ouchterlony double diffusion analysis, the mitochondrial extracts of glucagon-treated rat liver produced a single and fused precipitin line between the purified enzyme against the antibody. The supernatant fraction of glucagon-treated rat liver and the mitochondrial extracts of normal liver were also shown to make a single and fused precipitin line with the purified enzyme, when applied in large quantities. The quantitative immunotitration demonstrated that the glucagon-induced increase in the activity of liver serine:pyruvate aminotransferase were accompanied by the parallel increase in the amount of the enzyme antigen. Isotopic leucine incorporation studies showed that the relative rate of synthesis of the enzyme was increased approximately 10-fold by glucagon administration under the conditions employed. The rate of the degradation of the aminotransferase in the normal rat liver was a relatively slow process with a half-life of approximately 30 h. Thus the accumulation of serine:pyruvate aminotransferase in rat liver mitochondria by glucagon treatment can be ascribed mainly to the rise in the rate of enzyme synthesis.     

Comparison of 3-hydroxybutyrate dehydrogenase from bovine heart and rat liver mitochondria

3-Hydroxybutyrate dehydrogenase is a lipid-requiring enzyme with an absolute requirement of phosphatidylcholine for enzymatic activity. Purification of the enzyme to homogeneity from bovine heart mitochondria was described more than a decade ago [H. G. Bock and S. Fleischer (1975) J. Biol. Chem. 250, 5774-5781]. We have modified the purification procedure so that it is faster, the yield has been improved, and the specific activity is greater by approximately 50%. The updated procedure has also been applied to isolate the enzyme from rat liver mitochondria. Characteristics of the enzyme from bovine heart and rat liver mitochondria have been compared and found to be similar with respect to: (1) purification characteristics; (2) amino acid composition; (3) pH optimum for enzymatic activity; (4) kinetic characteristics; (5) molecular weight as determined by sedimentation equilibrium in guanidine hydrochloride; (6) peptide maps; (7) immunological cross-reactivity. These studies show that 3-hydroxybutyrate dehydrogenase from bovine heart and rat liver mitochondria, though similar, are not identical.     

Immunological study of the tissue expression of D-beta-hydroxybutyrate dehydrogenase, a ketone body-converting enzyme

In rats, as in most mammal, ketone bodies are mainly produced in liver while they are metabolized in extrahepatic tissues. The expression of mitochondrial membrane-bound D-beta-hydroxybutyrate dehydrogenase (BDH), a ketone body-converting enzyme, has been estimated by two immunological techniques: immunohistofluorescence and Western blotting. The in situ labeling with anti-BDH antibody shows that the enzyme is expressed differently among the organs. Furthermore, within a given organ there are strong differences according to the cell type. The quantification of the enzyme by immunoblotting reveals that liver mitochondria have the highest content (more than 3% in protein mass). This content is 3,5 and 10 times lower in kidney, heart and brain mitochondria, respectively. Parallel D-beta-hydroxybutyrate dehydrogenase activity measurements on isolated mitochondria show differences in molecular activity of this enzyme according to the tissue origin. Due to the phospholipid requirement of this enzyme these differences in molecular activity are related to specific membrane lipid composition.     

Monamine oxidase in outer membrane of skeletal muscle mitochondria.

(1) Monoamine oxidase (EC 1.4.3.4) is present in rat skeletal muscle mitochondria. (2) A radioassay procedure for the assay of monoamine oxidase in muscle mitochondria is described. It is based on teh procedure using side-chain [2-14C]-tryptamine as substate described by Wurtman, R.J. and Axelrod, J. (1963) Biochem. Pharmacol. 12, 1439--1441 and employs a pH of 8.0 and a substrate concentration of 0.25 mM. (3) The Km of the muscle mitochondrial enzyme at pH 8.0 is 1.34 - 10(-5) M and that of the liver enzyme under the same conditions is 2.5 - 10(-5) M. Muscle mitochondria contain only one quarter of the activity of enzyme present in liver mitochondria. (4) Monoamine oxidase is shown to be in the outer membrane of skeletal muscle mitochondria and thus to be a suitable marker enzyme for use in the fractionation of these mitochondria.     

Side-chain metabolism of propranolol: involvement of monoamine oxidase and mitochondrial aldehyde dehydrogenase in the metabolism of N-desisopropylpropranolol to naphthoxylactic acid in rat liver

We examined the metabolism of N-desisopropylpropranolol (NDP), which is generated from propranolol (PL) by side-chain N-desisopropylation, to naphthoxylactic acid (NLA) in rat liver. S(-)-NDP (S-NDP) and R(+)-NDP (R-NDP) were enantioselectively metabolized to NLA in isolated rat hepatocytes and in an enzyme reaction system of rat liver mitochondria with cofactor NAD+. Furthermore, the clearance profiles of NDP enantiomers were examined in an enzyme reaction system of rat liver mitochondria without NAD+. The amounts of S-NDP remaining in the incubation medium were similar to those of R-NDP, suggesting that monoamine oxidase (MAO) catalyzes the deamination of NDP to the aldehyde intermediate, but fails to deaminate enantioselectively S-NDP or R-NDP. Cyanamide, a potent inhibitor of aldehyde dehydrogenase (ALDH), markedly decreased the formation of NLA from racemic NDP in the enzyme reaction system of rat liver mitochondria with NAD+. When rat liver cytosol and microsomes were added to this enzyme reaction system, no significant alterations were observed in the amount of NLA generated from racemic NDP. We concluded that MAO deaminates NDP to an aldehyde intermediate, and that mitochondrial ALDH subsequently catalyzes the enantioselective metabolism of the aldehyde intermediate to NLA in rat liver.     

A DNA ligase from mitochondria of rat liver.

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.     

Leaky beta-oxidation of a trans-fatty acid: incomplete beta-oxidation of elaidic acid is due to the accumulation of 5-trans-tetradecenoyl-CoA and its hydrolysis and conversion to 5-trans-tetradecenoylcarnitine in the matrix of rat mitochondria

The degradation of elaidic acid (9-trans-octadecenoic acid), oleic acid, and stearic acid by rat mitochondria was studied to determine whether the presence of a trans double bond in place of a cis double bond or no double bond affects beta-oxidation. Rat mitochondria from liver or heart effectively degraded the coenzyme A derivatives of all three fatty acids. However, with elaidoyl-CoA as a substrate, a major metabolite accumulated in the mitochondrial matrix. This metabolite was isolated and identified as 5-trans-tetradecenoyl-CoA. In contrast, little or none of the corresponding metabolites were detected with oleoyl-CoA or stearoyl-CoA as substrates. A kinetic study of long-chain acyl-CoA dehydrogenase (LCAD) and very long-chain acyl-CoA dehydrogenase revealed that 5-trans-tetradecenoyl-CoA is a poorer substrate of LCAD than is 5-cis-tetradecenoyl-CoA, while both unsaturated acyl-CoAs are poor substrates of very long-chain acyl-CoA dehydrogenase when compared with myristoyl-CoA. Tetradecenoic acid and tetradecenoylcarnitine were detected by gas chromatography/mass spectrometry and tandem mass spectrometry, respectively, when rat liver mitochondria were incubated with elaidoyl-CoA but not when oleoyl-CoA was the substrate. These observations support the conclusion that 5-trans-tetradecenoyl-CoA accumulates in the mitochondrial matrix, because it is less efficiently dehydrogenated by LCAD than is its cis isomer and that the accumulation of this beta-oxidation intermediate facilitates its hydrolysis and conversion to 5-trans-tetradecenoylcarnitine thereby permitting a partially degraded fatty acid to escape from mitochondria. Analysis of this compromised but functional process provides insight into the operation of beta-oxidation in intact mitochondria.     

Characterization of a mitochondrial matrix protease catalyzing the processing of adrenodoxin precursor

Adrenodoxin (Ad) is synthesized as a larger precursor (preAd) by cytoplasmic polysomes and then transported into mitochondria concomitant with its proteolytic processing to the mature form. The protease in bovine adrenal cortex mitochondria, which converts preAd to the mature form, is a metalloprotease in the matrix (Sagara, Y., Ito, A. & Omura, T. (1984) J. Biochem. 96, 1743-1752). In this study, the protease was purified about 100-fold from the matrix fraction of bovine adrenal cortex mitochondria. The partially purified protease converted not only preAd, but also the precursors of malate dehydrogenase (MDH) and 27 kDa protein (P-27) to the corresponding mature forms. However, it was inactive toward the precursors of P-450(SCC) and of P-450(11 beta). Since isolated rat liver mitochondria can import and process preAd as efficiently as bovine adrenal cortex mitochondria, we partially purified a preAd-processing protease from rat liver mitochondria and compared its properties with those of the bovine adrenal cortex enzyme. The properties of the rat liver protease were indistinguishable from those of the bovine adrenal cortex enzyme in molecular weight determined from Sephadex G-150 gel filtration, metal requirement and ability to process preMDH and preP-27. The rat liver enzyme was also inactive toward the precursors of P-450(SCC) and P-450(11 beta). These results indicate the presence in both adrenal cortex and liver mitochondria of the same type of processing protease, which processes preAd and also the precursors of some other mitochondrial proteins.     

Studies on the effects of copper deficiency on rat liver mitochondria. I. Changes in mitochondrial composition

As part of an investigation of the lesions of copper (Cu) deficiency a study was undertaken of the copper, iron, cytochrome and fatty acid composition of liver mitochondria from Cu deficient and Cu-adequate control rats. Cu concentrations were significantly decreased in whole liver, liver mitochondria and in blood plasma. Total iron was significantly increased in whole liver but remained at the normal level in mitochondria. Cytochrome c oxidase (EC 1.9.3.1) and its component cytochromes a and a3 were significantly reduced in liver mitochondria from Cu-deficient rats, whereas there was no effect on the concentration of cytochromes b, c1 and c. Evidence from comparisons between cytochrome c oxidase activity and the amount of enzyme present, as assessed from the mitochondrial cytochrome a and a3 content, suggests that in addition to an absolute loss of enzyme, Cu-deficiency adversely affects the efficiency of the residual enzyme. Severe Cu deficiency had no effect on 'ageing' or 'swelling' properties of liver mitochondria, indicating no marked effects on fatty acid composition. Fatty acid analyses demonstrated a slight but significant increase in docosapentenoic acid (22:5) of Cu-deficient mitochondria, but since this represents a minor component there was no change observed in the 'unsaturation index'. It was concluded that, in contrast to previous reports, Cu deficiency of the severity reported did not have a deleterious effect on the integrity and permeability of the inner mitochondrial membrane as exemplified by any qualitative modification of fatty acid constitution per se.     

Increased sensitivity of carnitine palmitoyltransferase I activity to malonyl-CoA inhibition after preincubation of intact rat liver mitochondria with micromolar concentrations of malonyl-CoA in vitro.

Carnitine palmitoyltransferase I in rat liver mitochondria preincubated with malonyl-CoA was more sensitive to inhibition by malonyl-CoA than was the enzyme in mitochondria preincubated in the absence of malonyl-CoA. For carnitine palmitoyltransferase I in mitochondria from starved animals this increase also resulted in the enzyme becoming significantly more sensitive than that in mitochondria assayed immediately after their isolation. Concentrations of malonyl-CoA that induced half the maximal degree of sensitization observed were 1-3 microM.