Membrane Of Rat Liver Mitochondria Research Articles

Substrate specificity of rat liver mitochondrial carnitine palmitoyl transferase I: evidence against α-oxidation of phytanic acid in rat liver mitochondria

The two branched chain fatty acids pristanic acid (2,6,10,14-tetramethylpentadecanoic acid) and phytanic acid (3,7,11,15-tetramethylhexadecanoic acid) were converted to co-enzyme A thioesters by rat liver mitochondrial outer membranes. However, these branched chain fatty acids could not be converted to pristanoyl and phytanoyl carnitines, respectively, by mitochondrial outer membranes. As expected, the unbranched long chain fatty acids, stearic acid and palmitic acid, were rapidly converted to stearoyl and palmitoyl carnitines, respectively, by mitochondrial outer membranes. These observations indicate that the branched chain fatty acids could not be transported into mitochondria. The data presented strongly suggest that in rat liver, α-oxidation of phytanic acid occurs in organelles other than mitochondria.

Characteristics of Fe(II)ATP complex-induced damage to the rat liver mitochondrial membrane.

It is well established that several iron complexes can induce oxidative damage in hepatic mitochondrial membranes by catalyzing the formation of OH radicals and/or by promoting lipid peroxidation. This is a relevant process for the molecular basis of iron overload diseases. The present work demonstrates that Fe(II)ATP complexes (5-50 microM) promote an oxygen consumption burst in a suspension of isolated rat liver mitochondria (either in the absence or presence of Antimycin A), caused mainly by lipid peroxidation. Fe(II)ATP alone induced small levels of oxygen uptake but no burst. The time course of Fe(II)ATP oxidation to Fe(II)ATP in the extramitochondrial media also reveals a simultaneous 'burst phase'. The iron chelator Desferal (DFO) or the chain-break antioxidant butylated hydroxytoluene (BHT) fully prevented both lipid peroxidation (quantified as oxygen uptake burst) and mitochondrial swelling. DFO and BHT were capable of stopping the ongoing process of peroxidation at any point of their addition to the mitochondrial suspension. Conversely, DFO and BHT only halted the Fe(II)ATP-induced mitochondrial swelling at the onset of the process. Fe(II)ATP could also cause the collapse of mitochondrial potential, which was protected by BHT if added at the onset of the damaging process. These results, as well as correlation studies between peroxidation and mitochondrial swelling, suggest that a two phase process is occurring during Fe(II)ATP-induced mitochondrial damage: one dependent and another independent of lipid peroxidation. The involvement of lipid peroxidation in the overall process of mitochondrial membrane injury is discussed.

Purification and characterization of glycerophosphate acyltransferase from rat liver mitochondria.

Glycerophosphate acyltransferase (GAT) catalyzes the conversion of sn-glycerol 3-phosphate to lysophosphatidic acid (LPA), the first and committed step of triacylglycerol and phospholipid synthesis. In spite of the important regulatory roles GAT may play in this biosynthetic pathway, little information is available on the structure, biochemical properties, and regulation of GAT from eukaryotic cells. We solubilized GAT from rat liver mitochondrial membranes and purified it to an apparent homogeneity by hydroxylapatite chromatography, preparative isoelectric focusing, and gel filtration. The enzyme is composed of a single polypeptide of 85 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration chromatography of the native protein. The GAT activity was completely lost during the purification procedure and required addition of exogenous phospholipids for its reconstitution. Since a high phospholipid to detergent ratio was needed for full reactivation, it is concluded that GAT requires "lipid boundary" for reconstitution. The ability of different phospholipids to reactivate GAT decreased in the following order: phosphatidylglycerol (PG), phosphatidylethanolamine (PE), phosphatidylcholine (PC), asolectin, phosphatidylinositol (PI), phosphatidylserine (PS), and cardiolipin. 1,2-Dioleoyl derivatives of PG and PE were more effective in reconstituting the GAT activity than corresponding dipalmitoyl derivatives. The GAT activation was further increased by using a combination of PG and PE or PG and PC. Regardless of the phospholipid used for reconstitution, palmitoyl-CoA was the best acyl donor and LPA was the only reaction product.

Recruitment of mitochondrial cyclophilin to the mitochondrial inner membrane under conditions of oxidative stress that enhance the opening of a calcium-sensitive non-specific channel

Binding of mitochondrial matrix cyclophilin (CyP) to the rat liver mitochondrial membranes was detected by SDS/PAGE and Western blotting with suitable antipeptide antibodies. Binding was not affected by prior exposure of mitochondria to Ca2+, adenine nucleotides or inhibitors of the adenine nucleotide translocase, but was greatly increased by t-butyl hydroperoxide (tBH), phenylarsine oxide or diamide. These all sensitized the opening of the non-specific mitochondrial pore to [Ca2+], and the effect of tBH was shown to be maintained after washing away the tBH, consistent with it being caused by the enhanced CyP binding. The bound CyP did not demonstrate peptidyl-prolyl cis-trans isomerase activity. CyP-binding was prevented by 5 microM cyclosporin A, but not reversed by cyclosporin treatment of the membranes. The effect of tBH on binding was concentration-dependent and maximal within 30 s.

Cytochrome b5-Like Hemoprotein/Cytochrome b5 Reductase Complex in Rat Liver Mitochondria Has NADH-Linked Aquacobalamin Reductase Activity

Rat liver mitochondrial NADH-linked aquacobalamin reductase was characterized to clarify its enzymological properties. Most of the enzyme was solubilized with 10 g/L Triton X-100 from rat liver mitochondrial membranes. The elution behavior of the solubilized enzyme was identical to that of NADH-cytochrome c reductase (b-type cytochromes/cytochrome b5 reductase complex) during DEAE-Sepharose Fast Flow column chromatography. By mixing both purified cytochrome b5-like hemoprotein (outer membrane-cytochrome b) and cytochrome b5 reductase, cob(II)alamin was formed from aquacobalamin and NADH. These results provide evidence that the outer membrane-cytochrome b/cytochrome b5 reductase complex has the activity of the NADH-linked aquacobalamin reductase in rat liver mitochondria. Some properties of the NADH-linked aquacobalamin reductase were studied using the function of rat liver mitochondrial membranes. The specific activity (109.5 +/- 14.3 nmol.min-1.mg protein-1) of the enzyme was shown under physiological conditions (pH 7.1 at 40 degrees C). The optimal pH and temperature for activity were 7.1 and 40 degrees C, respectively. The apparent Km values were 41.9 mumol/L for aquacobalamin in the presence of 0.2 mmol/L NADH and 14.4 mumol/L for NADH in the presence of 0.1 mmol/L aquacobalamin. The enzyme was specific for aquacobalamin, because cyanocobalamin could not be reduced by the enzyme.

Effects of amphipathic peptides, including presequences, on the functional integrity of rat liver mitochondrial membranes

A number of amphipathic peptides were tested for their effects on structural and functional properties of isolated rat liver mitochondria. The peptides included the matrix targeting sequence of subunit IV of (yeast) cytochrome c oxidase. Titration experiments in which the mitochondria were incubated with increasing concentrations of the peptides revealed two major stages in the interaction. First, at low peptide/mitochondria ratios, peptide binding to the outer membrane occurred which was accompanied by gradual lysis of the outer membrane at higher ratios. The latter was deduced from the release of adenylate kinase, the classical marker enzyme of the intermembrane space. Secondly, at still higher peptide/mitochondria ratios, the permeability of the inner membrane progressively increased, as evidenced by measurements of respiratory control and of the membrane potential. Complete uncoupling of respiration seemed to precede dissipation of the membrane potential.

Spermine-mediated casein kinase II-uptake by rat liver mitochondria

Spermine, ubiquitous intracellular polyamine, is able to promote the transmembrane translocation of casein kinase CKII through the outer membrane of rat liver mitochondria and its binding to more internal mitochondrial structures. These findings suggest taht spermine may play a critical role in regulating the subcellular distribution of casein kinase CKII.

A study of glycerolphosphate acyltransferase in rat liver mitochondria-submitochondrial localization and some properties of the solubilized enzyme

1. 1. Glycerolphosphate acyltransferase (GPAT) was solubilized from the rat liver mitochondrial membranes using sodium cholate. Dithiothreitol was necessary to stabilize the solubilized enzyme on storage. 2. 2. Unlike the enzyme in situ in mitochondrial membranes, the solubilized mitochondrial GPAT was susceptible to inhibition by N-ethylmaleimide; a property more characteristic of the distinct microsomal form of GPAT. 3. 3. Solubilized mitochondrial GPAT retained its very high preference for saturated acyl-CoA substrate (palmitoyl-CoA) and had no activity whatever with any tested concentration of the unsaturated substrate oleoyl-CoA. 4. 4. Solubilization increased the affinity of mitochondrial GPAT for palmitoyl-CoA whilst decreasing the K m for glycerol phosphate. 5. 5. After separation of liver mitochondrial outer and inner membranes and estimation of cross-contamination by appropriate markers it was concluded that the mitochondrial inner membrane contains significant GPAT activity. This was established with preparations from fed, 48 hr-starved and streptozotocin-diabetic rats.

Interactions between ubiquinones and vitamins in membranes and cells

The interaction between ubiquinones and vitamin E was studied in the inner membranes of rat liver mitochondria, liposomes and human erythrocyte plasma membranes. Free radicals were produced by addition of exogenous oxidants, and their reaction with chromanols and ubiquinone was followed by ESR and HPLC. Membranes were made deficient in ubiquinone but sufficient in α-tocopherol and were reconstituted with added ubiquinone. With these membrane preparations it was shown that (i) in the inner mitochondrial membranes there is a requirement for ubiquinone in the enzymatic recycling of vitamin E; (ii) succinate-ubiquinone reductase incorporated in liposomes cannot protect vitamin E in the absence of ubiquinone and (iii) in human erythrocyte plasma membranes protection against the loss of vitamin E can be provided by NADH-cytochrome-b 5-dependent enzymatic recycling. We conclude that ubiquinonols (ubisemiquinones) reduce vitamin E through electron transport.

Demonstration of argininosuccinate synthetase activity associated with mitochondrial membrane: characterization and hormonal regulation.

Argininosuccinate synthetase (AS) is the third enzyme in ureogenesis, it catalyses the reaction of condensation of citrulline and aspartate into argininosuccinate. In the present report, we described the first characterization of AS within the outer membrane of rat liver mitochondria. Mitochondria-associated AS displayed the same kinetic characteristics as the cytoplasmic enzyme, but was found to be thermostable while cytoplasmic AS was not. The evolution of the co-location of AS was analyzed during ontogenesis. Total AS activity increased throughout rat fetal development. Simultaneously, the subcellular distribution of the enzyme has changed. AS activity was mainly mitochondrial in fetal and new-born liver liver and cytoplasmic in adult rat liver. The variation in subcellular distribution of AS may be due to the dramatic changes in hormonal levels that occur during this period. The role of corticosteroid and pancreatic hormones was studied. During fetal period, corticosteroid hormones induced an increase in mitochondria-associated AS activity. This was prevented by insulin. Glucagon did not modify total AS activity but reduced mitochondrial AS activity, meanwhile, a comparable increase in cytoplasmic AS activity was observed. One may hypothesize that glucagon may participate in the transfer of mitochondrial enzyme into the cytosol.

Mitochondrial NADPH-Linked Aquacobalamin Reductase Is Distinct from the NADPH-Linked Enzyme from Microsomal Membranes in Rat Liver

Mitochondrial NADPH-linked aquacobalamin reductase was purified and characterized to clarify its enzymatic properties. The enzyme was purified about 360-fold over rat liver mitochondrial membranes in a yield of 7.5%. The purified enzyme was homogenous in SDS-PAGE. The molecular mass (M(r)) of the enzyme was calculated to be 65 kDa by SDS-PAGE and by Toyopearl HW55 gel filtration, indicating that the enzyme is a monomeric polypeptide with M(r) of 65 kDa. The enzyme was a flavoprotein containing 1 mol of FAD and FMN per mole of the enzyme. The enzyme was specific for NADPH as electron donor and had the ability to reduce cytochrome c (15.4 mumol.min-1 x mg protein-1), potassium ferricyanide (4.9 mumol.min-1 x mg protein-1) and 2,6-dichlorophenolindophenol (16.8 mumol.min-1.mg protein-1) as well as aquacobalamin (6.4 mumol.min-1 x mg protein-1). Although the enzyme immunoreacted with an antibody against NADPH-cytochrome P-450 reductase, which had the activity of the NADPH-linked aquacobalamin reductase in rat liver microsomes, the mitochondrial enzyme and the microsomal enzyme had different enzymological properties.

The bcl-2 Protein Is Inserted into the Outer Membrane but Not into the Inner Membrane of Rat Liver Mitochondria in Vitro

The bcl-2 gene encodes a 26 kDa protein, overexpression of which blocks cell apoptosis. Since conflicting data have been reported concerning the localization of the Bcl-2 protein with mitochondrial inner and outer membranes, we analyzed in vitro import of the human Bcl-2 protein into isolated rat liver mitochondria. The Bcl-2 protein translated in rabbit reticulocyte lysate was efficiently inserted into the mitochondrial outer rather than the inner membrane in a membrane potential-independent manner. The carboxyl-terminal hydrophobic stretch is essential for the Bcl-2 protein to integrate into the outer membrane. Binding of the Bcl-2 protein to the mitochondria was not affected by pretreatment of the mitochondria with trypsin, indicating that the Bcl-2 protein does not require protease-sensitive mitochondrial surface components for its membrane insertion.

Structure, function and regulation of the tricarboxylate transport protein from rat liver mitochondria

Recent progress is summarized on the structure, function, and regulation of the tricarboxylate (i.e., citrate) transport protein (CTP) from the rat liver mitochondrial inner membrane. The transporter has been purified and its reconstituted function characterized. A cDNA clone encoding the CTP has been isolated and sequenced, thus enabling a deduction of the complete amino acid sequence of this 32.6 kDa transport protein. Dot matrix analysis and sequence alignment indicate that based on structural considerations the CTP can be assigned to the mitochondrial carrier family. Hydropathy analysis of the transporter sequence indicates six putative membrane-spanning alpha-helices and has permitted the development of an initial model for the topography of the CTP within the inner membrane. The questions as to whether more than one gene encodes the CTP and whether more than one isoform is expressed remain unanswered at this time. Studies documenting a diabetes-induced alteration in the function of several mitochondrial anion transporters, which can be reversed by treatment with insulin, provide a physiologically/pathologically relevant experimental system for studying the molecular mechanism(s) by which mitochondrial transporters are regulated. Potential future research directions are discussed.

Phospholipid asymmetry of the outer membrane of rat liver mitochondria: Evidence for the presence of cardiolipin on the outside of the outer membrane

The phospholipid topology of the outer membrane of intact rat liver mitochondria and derived outer membrane vesicles was investigated by determining the accessible pool of the various phospholipid classes towards phospholipase A 2, a phosphatidylcholine-specific transfer protein and by chemical labeling using trinitrobenzene sulfonic acid. The outer membrane vesicles are sealed and have a right-side-out topology with a proposed localization of 55%, 77%, 100%, and at least 30% of the phosphatidylcholine, phosphatidylethanolamine, cardiolipin, and phosphatidylinositol plus phosphatidylserine in the outer leaflet, respectively. The outer membrane in intact mitochondria appears to have a similar phospholipid distribution.

5-Aminolevulinic Acid Induces Lipid Peroxidation in Cardiolipin-Rich Liposomes

5-Aminolevulinic acid (ALA), a heme precursor accumulated in lead poisoning and acute intermittent porphyria, is known to undergo metal-catalyzed aerobic oxidation to yield reactive oxygen species. In phosphatidylcholine:cardiolipin (80:20) liposomes ALA (0.1-3.0 mM) promoted lipid peroxidation as evaluated by the formation of conjugated dienes and 2-thiobarbituric-reactive substances (TBARS). TBARS formation was dependent on ALA concentration and incubation time. ALA-induced lipid peroxidation was associated with an increase in liposome permeability as measured by the release of encapsulated carboxyfluorescein. α-Tocopherol (0.1-0.5 mol %), an efficient oxyradical scavenger, inhibits lipid peroxidation and prevents carboxyfluorescein rejease, suggesting that the permeabilization of liposomes is mainly due to lipid peroxidation. Cardiolipin, a major component of mitochondrial inner membrane, was particularly susceptible to ALA-induced lipid peroxidation. These results may be relevant to the previously observed Ca 2+-dependent permeabilization of the inner membrane of rat liver mitochondria promoted by external 0.1-1.0mM ALA; this mechanism has been implicated in the pathophysiology of acute intermittent porphyria and lead poisoning.

Mature carnitine palmitoyltransferase I retains the N-terminus of the nascent protein in rat liver

Carnitine palmitoyltransferase I was isolated from octylglucoside extracts of rat liver mitochondrial outer membranes. This native enzyme was digested proteolytically with V8 protease. Five major peptides were obtained all of which were found in the amino acid sequence predicted from the full-length cDNA sequence of the protein. One peptide was found to correspond to the extreme N-terminus of the deduced amino acid sequence. Therefore, the mature protein retains the N-terminus of the nascent protein after import into the mitochondrial membrane. Knowledge of the identity of the N-terminus of the mature protein allows a reappraisal of the role of the two main, N-terminal hydrophobic domains of the protein and of the possible topology of the protein within the membrane.

Visualization of multiple imidazoline/guanidinium-receptive sites

Compounds with an imidazoline or guanidinium moiety elicit a variety of stimulatory and inhibitory cell responses in both central and peripheral tissues. Many of these effects are mediated by interaction with alpha-adrenergic receptors, but these molecules also selectively recognize other membrane-bound proteins with high affinity. We used a functionalized derivative of the imidazoline molecule cirazoline to visualize the imidazoline/guanidinium-receptive site (IGRS). 2-[3-Aminophenoxy]methyl imidazoline was radioiodinated and subsequently converted to the arylazide to generate the photoaffinity adduct 2-[3-azido-4-[125I]iodophenoxy]methyl imidazoline ([125I]AZIPI). Both 2-[3-amino-4-[125I]iodophenoxy]methyl imidazoline and [125I]AZIPI exhibited saturable high affinity binding in rat liver membrane preparations (Ki = 2-5 nM). In rat liver mitochondrial membranes, [125I]AZIPI photoincorporates into two peptides with apparent molecular weights of approximately 55,000 and approximately 61,000 as determined by SDS-polyacrylamide gel electrophoresis. The labeling of these two species is blocked by various competing ligands (10 microM) with a potency order expected for an IGRS. The photolabeling of both peptides is blocked by the imidazolines cirazoline and idazoxan or by the guanidinium guanabenz, but it is not altered by the alpha 2-adrenergic receptor antagonist rauwolscine or by the adrenergic receptor agonist epinephrine. Photoincorporation of [125I]AZIPI is minimally inhibited by the imidazoline clonidine or by the alpha 1-adrenergic receptor antagonist prazosin. However, the guanidinium ligand amiloride exhibits higher affinity for the M(r) = 61,000 peptide as compared with the M(r) = 55,000 peptide, suggesting that the two labeled species differ in their ligand recognition properties. An additional IGRS was identified by photolabeling in membranes prepared from PC-12 pheochromocytoma cells. In PC-12 membranes, [125I]AZIPI photolabels a major M(r) = approximately 61,000 peptide; the photoincorporation is blocked by cirazoline, guanabenz, and amiloride but not by idazoxan (competing ligands = 10 microM). These data indicate the existence of at least three subtypes of IGRS that differ in their ligand recognition properties, their apparent molecular weight, and their tissue distribution.

Hypotonic fragility of outer membrane and activation of external pathway of NADH oxidation in rat liver mitochondria are increased with age

Great importance is attached to structural and functional deterioration of mitochondria as a reason for ageing of an organism; the attention of many scientists has been concentrated on such questions as age changes in the system of oxidative phosphorylation, damage of mitochondrial DNA by free radicals generated in the respiratory chain and inclusion of some fragments of mitochondrial DNA into the nuclear genome. Mitochondrial high amplitude swelling in a cell under some extreme conditions can possibly play a very important role in mechanisms of deterioration of energy transformation function, in activation of lipid peroxidation and mitochondrial DNA damage as a result of outer membrane disruption and release of enzymes from the intermembrane space (e.g. superoxide dismutase and adenylate kinase). In this work the age changes of the hypotonic fragility of the outer membrane of rat liver mitochondria and the activation of the external, rotenone-insensitive pathway of NADH oxidation have been examined. It is shown that the obligatory condition for activation of rotenone-insensitive NADH oxidation is a break in the outer membrane and that the rate of NADH oxidation substantially increases in the presence of physiological concentrations of Mg 2+ which cause a multiple increase in the affinity of the inner membrane to cytochrome c. Research on the rate of rotenone-insensitive NADH oxidation with respect to the osmotic pressure, the ionic strength of the medium, the presence of Mg 2+ ions and cytochrome c in the medium has demonstrated a considerable increase in the hypotonic fragility of the outer membrane of liver mitochondria with age in male rats. In female rats the age changes were insignificant. It is supposed that the damage to the outer membrane of mitochondria in cells can serve as one of the possible explanations of both decrease in the reliability of an aged organism under extreme conditions and sex differences of life-span.

Transport of prepro-albumin into inverted vesicles prepared from the inner membrane of rat liver mitochondria

When inverted vesicles prepared from the inner membrane of rat liver mitochondria were incubated with prepro-rat serum albumin, considerable amounts of prepro-albumin and pro-albumin were recovered with the inverted vesicles re-isolated by centrifugation. Pro-albumin was resistant to trypsin, but prepro-albumin was completely digested by trypsin, indicating that prepro-albumin was transported into the vesicles and concomitantly converted to pro-albumin. This transport process required ATP, but not a membrane potential. These results suggest that some export machinery for a protein having an amino acid sequence in its N-terminal portion similar to the signal sequence of secretory protein exists in the inner mitochondrial membrane.

Cardiolipin is synthesized on the matrix side of the inner membrane in rat liver mitochondria.

In the mitochondrial inner membrane, cardiolipin is a specific lipid component associated with various protein complexes. The assembly of such complexes has been studied, and it seems that most protein subunits enter the inner membrane from the matrix side, but nothing is known about the path of cardiolipin. In this paper, the topography of cardiolipin biosynthesis is investigated. Cardiolipin synthase, a membrane-bound protein, could not be released by sonication or 1 M KCl. In sucrose density gradient subfractionation, cardiolipin synthase co-migrated with the inner membrane marker cytochrome oxidase. no indication was obtained for a preferential localization of this enzyme at contact sites between the outer and inner membranes. Protease digestion experiments showed that cardiolipin synthase exposed protease-susceptible domains mainly to the matrix side of the inner membrane. In intact mitochondria, the Mn(2+)-dependent stimulation of cardiolipin synthesis was abolished when the Mn2+ influx into the matrix was blocked by ruthenium red. 1-Decanoyl-sn-glycero-3-phosphorylcholine, a water-soluble inhibitor of cardiolipin synthase, was only effective after disintegration of mitochondria. The metabolic precursor of cardiolipin, CDP-diacylglycerol, was synthesized by an inner membrane enzyme whose protease-susceptible domains were mainly exposed to the matrix side. It is concluded that cardiolipin is synthesized in the inner leaflet of the mitochondrial inner membrane.