Mitochondrial Membrane Of Rat Liver Research Articles

Inhibition of Lycium barbarum polysaccharides and Ganoderma lucidum polysaccharides against oxidative injury induced by γ-irradiation in rat liver mitochondria

Lycium barbarum and Ganoderma lucidum, the two precious traditional Chinese medicines, possesses the antitumor activity, antioxidant activity and the capability of modulating the immune system. In the present study, the possible antioxidant effects of L. barbarum polysaccharides (LBP) isolated from dried L. barbarum fruits and Ganoderma lucidum polysaccharides (GLP) isolated from dried G. lucidum against membrane damage induced by the free radicals generated during γ-irradiation were examined in rat liver mitochondria. Using an effective dose of 450 Gy, antioxidant effects of GLP and LBP were studied against oxidative damage in terms of protection against lipid peroxidation, protein oxidation, depletion of protein thiols and the levels of the antioxidant enzyme, such as superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px). The results showed that both GLP and LBP significantly protects against irradiation-induced loss of protein thiols and inactivation of SOD, CAT and GSH-Px in a dose-dependent manner. The inhibitory effects of GLP, at the entire concentration range, are stronger than LBP. Moreover, the two polysaccharides are more effective than α-tocopherol (VE) in inhibiting irradiation-induced oxidative injury. Hence our results indicate that GLP and LBP have potent antioxidant properties in vitro in mitochondrial membranes of rat liver.

The F 1-ATPase β-subunit is the putative enterostatin receptor

It has been suggested that the F 1-ATPase β-subunit is the enterostatin receptor. We investigated the binding activity of the purified protein with a labeled antagonist, β-casomorphin 1–7, in the absence and presence of cold enterostatin. 125I-β-casomorphin 1–7 weakly binds to the rat F 1-ATPase β-subunit. Binding was promoted by low concentrations of cold enterostatin but displaced by higher concentrations. To study the relationship between binding activity and feeding behavior, we examined the ability of a number of enterostatin analogs to affect β-casomorphin 1–7 binding to the F 1-ATPase β-subunit. Peptides that suppressed food intake promoted β-casomorphin 1–7 binding whereas peptides that stimulated food intake or did not affect the food intake displaced β-casomorphin 1–7 binding. Surface plasmon resonance measurements show that the β-subunit of F 1-ATPase binds immobilized enterostatin with a dissociation constant of 150 nM, where no binding could be detected for the assembled F 1-ATPase complex. Western blot analysis showed the F 1-ATPase β-subunit was present on plasma and mitochondrial membranes of rat liver and amygdala. The data provides evidence that the F 1-ATPase β-subunit is the enterostatin receptor and suggests that enterostatin and β-casomorphin 1–7 bind to distinct sites on the protein.

Single-Channel Currents of the Permeability Transition Pore from the Inner Mitochondrial Membrane of Rat Liver and of a Human Hepatoma Cell Line

Single-channel currents were recorded from inner mitochondrial membranes of HepG2 hepatoma cells and of normal rat liver cells by means of patch-clamp techniques. The current events showed variable amplitudes of up to 1.1 ± 0.2 nS (n = 35) at room temperature (24 °C) and of up to 1.5 ± 0.2 nS (n = 10) at 34 °C including large numbers of subconductance states. Voltages of -40 mV and below closed the channels usually with a delay of about 2 min. Increasing Ca²⁺ concentrations activated the channels, whereas cyclosporin A (100 nM) blocked. The concentration-response relation for the Ca²⁺-effect could be fitted best using an EC₅₀ of 10 µM and a Hill coefficient of 1.5. Taken together the results indicate that we recorded from the permeability transition pore (PTP). As PTP activity may be related to apoptosis we tested if lysate from differently treated T-lymphocytes (Jurkat cells) was able to induce PTP activity in HepG2 cells. Lysate of untreated cells completely abolished the activity at a Ca²⁺ concentration of 18 nM (buffered by EGTA), i.e. three orders of magnitude below the EC₅₀. Under these conditions the lysate is not likely to contain stable factors that could open the PTP. Preliminary experiments show PTP activity in CD95-activated lysate.

Antioxidant properties of Asparagus racemosus against damage induced by γ-radiation in rat liver mitochondria

The possible antioxidant effects of crude extract and a purified aqueous fraction of Asparagus racemosus against membrane damage induced by the free radicals generated during γ-radiation were examined in rat liver mitochondria. γ-Radiation, in the dose range of 75–900 Gy, induced lipid peroxidation as assessed by the formation of thiobarbituric acid reactive substances (TBARS) and lipid hydroperoxides (LOOH). Using an effective dose of 450 Gy, antioxidant effects of A. racemosus extract were studied against oxidative damage in terms of protection against lipid peroxidation, protein oxidation, depletion of protein thiols and the levels of the antioxidant enzyme, superoxide dismutase. An active fraction consisting of polysaccharides (termed as P3) was effective even at a low concentration of 10 μg/ml. Both the crude extract as well as the P3 fraction significantly inhibited lipid peroxidation and protein oxidation. The antioxidant effect of P3 fraction was more pronounced against lipid peroxidation, as assessed by TBARS formation, while that of the crude extract was more effective in inhibiting protein oxidation. Both the crude extract and P3 fraction also partly protects against radiation-induced loss of protein thiols and inactivation of superoxide dismutase. The inhibitory effects of these active principles, at the concentration of 10 μg/ml, are comparable to that of the established antioxidants glutathione and ascorbic acid. Hence our results indicate that extracts from A. racemosus have potent antioxidant properties in vitro in mitochondrial membranes of rat liver.

Hemin Is Kinetically Trapped in Cytochrome b5 from Rat Outer Mitochondrial Membrane

Cytochrome b5 from the outer mitochondrial membrane of rat liver (OM cyt b5) is substantially more stable to thermal and chemical denaturation than cytochrome b5 from the endoplasmic reticulum of bovine liver (microsomal, or Mc cyt b5). In contrast, the corresponding apoproteins have similar stability, suggesting stronger interactions between hemin and the polypeptide in OM cyt b5. Whereas complete transfer of hemin from bovine Mc cyt b5 to apomyoglobin at pH 5.2 takes less than 1 h, hemin transfer from OM cyt b5 is unmeasurably slow. Coupled with the previously reported 1:1 ratio of hemin orientational isomers in OM cyt b5, this finding suggests that the cofactor is kinetically trapped under physiologically relevant conditions. This conclusion is confirmed by 1H NMR studies which show that the hemin isomeric ratio changes when the protein is incubated for several hours at 68°C. Interestingly, the orientational isomer favored in OM cyt b5 is the form less favored in all other known cytochromes b5.

Integration of porin synthesized in vitro into outer mitochondrial membranes.

Porin, an intrinsic protein of outer mitochondrial membranes of rat liver, was synthesized in vitro in a cell-free in a cell-free translation system with rat liver RNA. The apparent molecular mass of porin synthesized in vitro was the same as that of its mature form (34 kDa). This porin was post-translationally integrated into the outer membrane of rat liver mitochondria when the cell-free translation products were incubated with mitochondria at 30 degrees C even in the presence of a protonophore (carbonyl cyanide m-chlorophenylhydrazone). Therefore, the integration of porin seemed to proceed energy-independently as reported by Freitag et al. [(1982) Eur. J. Biochem. 126, 197-202]. Its integration seemed, however, to require the participation of the inner membrane, since porin was not integrated when isolated outer mitochondrial membranes alone were incubated with the translation products. Porin in the cell-free translation products bound to the outside of the outer mitochondrial membrane when incubated with intact mitochondria at 0 degrees C for 5 min. When the incubation period at 0 degrees C was prolonged to 60 min, this porin was found in the inner membrane fraction, which contained monoamine oxidase, suggesting that porin might bind to a specific site on the outer membrane in contact or fused with the inner membrane (a so-called OM-IM site). This porin bound to the OM-IM site was integrated into the outer membrane when the membrane fraction was incubated at 30 degrees C for 60 min. These observations suggest that porin bound to the outside of the outer mitochondrial membrane is integrated into the outer membrane at the OM-IM site by some temperature-dependent process(es).

The orientation of transhydrogenase in the inner mitochondrial membrane of rat liver

The orientation of the transmembranous enzyme, pyridine dinucleotide transhydrogenase, in the inner mitochondrial membrane of rat liver has been determined by evaluating effects of proteases on the integrity of the enzyme in mitoplasts and submitochondrial particles. Following treatment of these membranes with the nonspecific protease, proteinase K, antigenic proteolytic products were detected by immunoblot analysis using polyclonal antibody prepared against purified bovine heart enzyme. Proteinase K treatment of mitoplasts converted the 110,000 transhydrogenase monomer into a single immunoreactive species having M r 75,000. This proteolytic product is stable to further incubation with the protease. Treatment of submitochondrial particles with proteinase K resulted in the disappearance of the 110,000 monomer and the transient formation of an intermediate product with M r 52,000. Information from these proteolysis studies was used to construct a model of the orientation of transhydrogenase in the inner mitochondrial membrane. This model indicates that transhydrogenase ( M r 110,000) contains a core of proteolytically inaccessible proteins within the membrane ( M r 23,000) bounded by extramembranous domains on the matrix ( M r 52,000) and cytoplasmic ( M r 35,000) face of the inner mitochondrial membrane.

Phospholipid topology of the inner mitochondrial membrane of rat liver

Phospholipid topology of the inner mitochondrial membrane of rat liver

Thyroid Hormone Action: The Mitochondrial Pathway

The subcellular compartments have been investigated to compare proteins capable of binding triiodothyronine and thyroxine; specific binders have been found in cytosol, nuclei, and mitochondria from rat liver and kidney. The binding protein from the inner mitochondrial membrane had the highest association constant (greater than 10(11) liters per mole), suggesting possible direct hormone action on the mitochondria. Binding of hormone analogs was found to be related to known physiological potency, and stereospecific discrimination between L- and D-thyroxine was observed. The saturable receptor was found in the mitochondrial membranes of rat liver, kidney, myocardium, and skeletal muscle but not in mitochondria from the unresponsive tissues: brain, spleen, and testis. Oxidative phosphorylation by mitochondrial vesicles from hypothyroid rats increased after the addition of physiological concentrations of triiodothyronine, which corroborated direct hormone action on mitochondria.

Age related changes in the lipids of the microsomal and the mitochondrial membranes of rat liver and kidney

The lipid contents of the microsomal and mitochondrial membrane fractions of liver and kidney were determined in 6 and 24 month old rats. A significant age related increase in the molar ratio of cholesterol/phospholipid was observed in all membrane fractions. A significant age related reduction of phospholipid was noted in the microsomal fractions of liver and kidney. The relative amount of phosphatidylethanolamine was found to decrease in all membrane fractions during aging. Membrane glyceride content, however, remained relatively constant with age. Significant increase in oleic acid was seen in the neutral lipid of both liver and kidney and in the polar lipid of kidney. Significant increase in docosahexaenoic acid and significant decrease in linoleic acid were seen in the polar lipid of the liver membrane fractions. Possible alterations in membrane physiochemical properties and in membrane function due to these age related lipid changes are discussed.

Study of the transverse diffusion of spin-labeled phospholipids in biological membranes. II. Inner mitochondrial membrane of rat liver: Use of phosphatidylcholine exchange protein

Spin-labeled phosphatidylcholine was incorporated into the membrane of isolated “inner membrane+matrix” particles of rat liver mitochondria by incubation with sonicated spin-labeled phosphatidylcholine vesicles at 22°C. When the spin label was on the acyl chain the incorporation of phosphatidylcholine into the membrane was stimulated by the presence of the phosphatidylcholine exchange protein extracted from rat or beef liver. On the other hand no stimulation was observed when the nitroxide was on the polar head-group. When spin-labeled phosphatidylcholine was incorporated into the mitochondrial membrane in the absence of phosphatidylcholine exchange protein, ascorbate treatment at O°C reduced the EPR signal of the spin-labeled membranes by approximately 50%, indicating that fusion incorporates molecules equally on both sides of the membrane. On the other hand when spin-labeled phosphatidylcholine was incoporated in the presence of the exchange protein most of the EPR signal could be destroyed by the ascorbate treatment at 0°C, indicating that the spin-labeled phosphatidylcholine had been selectively incorporated in the outer layer of the membrane. Finally when the label is on the polar head-group the inner content of mitochondria reduces the label facing the matrix, thus creating again an anisotropy of the labeling. The anisotropic distribution of spin-labeled phosphatidylcholine in the mitochondrial membrane was found to be stable at 25°C for more than 2 h. It is therefore concluded that the rate of outside-inside and inside-outside transitions are extremely slow (half-life greater than 24 h).

Purification and characterization of cytochrome b5-like hemoprotein associated with outer mitochondrial membrane of rat liver.

The cytochrome b5-like hemoprotein associated with the outer membrane of rat liver mitochondria (OM-cytochrome) was found to be more susceptible to solubilization with trypsin [EC 3.4.4.4] than microsomal bound cytochrome b5. Based on this finding, a condition for tryptic digestion was set up under which OM-cytochrome could be preferentially released from crude outer mitochondrial membrane preparations leaving most, if not all, cytochrome b5 of contaminating microsomes still attached to the membrane. The OM-cytochrome thus solubilized was highly purified by DEAE-cellulose column chromatography and Sephadex G-75 gel filtration. The purified cytochrome obtained in one experiment (Prep I) was separated into two hemoprotein bands upon polyacrylamide gel electrophoresis, whereas the sample purified in a second experiment (Prep II) gave three hemoprotein bands. Both preparations were, however, free of any other protein contaminants. Evidence was obtained to indicate that the extra band in Prep II was mainly due to cytochrome b5 derived from contaminating microsomes. The other two bands of Prep II as well as those of Prep I seemed to correspond to OM-cytochrome. The separation of OM-cytochrome into two bands suggested that the solubilized hemoprotein had suffered certain proteolytic modifications during tryptic digestion. The molecular weight (about 12,000) and behavior upon DEAE-cellulose column chromatography of purified OM-cytochrome were closely similar to those of trypsin-solubilized microsomal cytochrome b5. However, the two cytochromes differed from each other in immunological properties. Small differences were also detected in their absorption spectra. Furthermore, the tryptic peptide map of apo-OM-cytochrome was clearly different from that of apo-cytochrome b5

Exchange of phospholipids between microsomes and inner and outer mitochondrial membranes of rat liver

1. 1. Sixty minutes after injection of 32P-labelled phosphate into rats, the specific activities of phosphatidylcholine and phosphatidylethanolamine were determined in mitochondria, outer and inner mitochondrial membranes, and microsomes. The phospholipid specific activities of the outer membrane were lower than those of the microsomes and 3 to 4 times higher than those of the inner membrane. This is compatible with the idea that 32P-labelled phospholipids, synthesized in the endoplasmic reticulum, exchange first with the outer mitochondrial membrane followed by an exchange between outer and inner mitochondrial membrane. 2. 2. Mitochondria were incubated with microsomes in the presence of a 105 000 × g supernatant fraction (the pH 5.1 supernatant). It was shown that both inner and outer mitochondrial membranes were involved in the exchange of phospholipids with microsomes. The phospholipids of the outer membrane exchanged to a larger extent than did those of the inner membrane. 3. 3. Adenylate kinase activity was determined in mitochondria as an index for the integrity of the outer membrane. It was found that the integrity was not seriously affected by incubating mitochondria with or without microsomes and supernatant. Electron micrographs of the same mitochondria supported this conclusion. This endorses the view that in vitro phospholipids exchange between inner membrane and microsomes via the outer membrane. 4. 4. Mitochondria isolated 60 min after injection of 32P-labelled phosphate were incubated at 37° in 0.25 M sucrose—1 mM EDTA (pH 7.8). It was demonstrated that the 3- to 6-fold difference between the specific activities of outer and inner membrane phospholipids was partially eliminated. It is concluded that phospholipids exchange between inner and outer membrane inside the mitochondrion. 5. 5. In the 105 000 × g supernatant fraction, a protein is present which stimulates the exchange of phospholipids between microsomes and mitochondria. Such a stimulatory protein appears to be lacking in the mitochondrion. Differences in phospholipid exchange between microsomes and outer mitochondrial membrane as compared to phospholipid exchange between outer and inner mitochondrial membrane are discussed with respect to this observation.

Effect of vitamin D 3 on mitochondrial membrane of rat liver.

Mitochondrial membrane of the liver from vitamin D deficient rats was found to be more fragile, when exposed to calcium ion, than that from normal rats. Malate dehydrogenase (MDH: EC 1.1.1.37), released from isolated mitochondria, was used as a parameter to assess the disruption of mitochondrial membrane.Oral supplementation with vitamin D3 restored the fragility of mitochondrial membrane toward normal, suggesting that vitamin D3 or its active metabolites play a role in maintaining the integrity of mitochondrial membrane.

Synthesis of Fatty Acids in Outer and Inner Membranes of Mitochondria

Fatty acids can be synthesized in mitochondrial membranes of rat liver either by elongation or by synthesis de novo. The outer membranes incorporate greater amounts of substrate into fatty acids than do the inner membranes. Outer membranes synthesize 6- and 8-carbon fatty acids completely de novo, while smaller amounts of 10-carbon acids are produced by the mechanism de novo. The reactions de novo utilize acetyl coenzyme A more effectively than malonyl coenzyme A, and synthesis de novo occurs in the absence of exogenous ATP. Elongation, which produces fatty acids 14 carbon atoms long and longer, depends on added ATP, and acetyl-CoA and malonyl-CoA are incorporated about equally. Synthesis de novo of shorter chain fatty acids by outer membranes continues in the presence of ATP and the concomitant elongation reactions. Inner membranes synthesize only longer fatty acids by elongation. If synthesis de novo of fatty acids occurs in the inner membranes, these acids might be removed by oxidation. The ATP concentrations in these experiments were compared with the levels of ATP produced by actively respiring liver mitochondria. During metabolically adverse conditions within the cell, the capacity for manufacturing fatty acids by elongation may fluctuate with the ATP levels whereas synthesis de novo continues.

The turnover of the protein of the inner and outer mitochondrial membrane of rat liver

The relative turnover rate of proteins of the inner and outer mitochondrial membranes and of the microsomal fraction of rat liver have been estimated by a double isotope procedure. Outer mitochondrial membrane proteins turn over more rapidly than inner mitochondrial membrane proteins. Microsomal proteins have a turnover rate similar to that of the outer mitochondrial membrane.

Formation of membranes by repeating units

Electron microscopic evidence is presented for membrane formation by the repeating units of ( 1) the individual complexes of the electron transfer chain; ( 2) the outer membrane of beef heart mitochondria; ( 3) the mitochondrial membranes of rat liver; ( 4) the chloroplast membrane of spinach; ( 5) the outer segments of bovine photoreceptors; ( 6) a bovine liver microsomal membrane; and ( 7) the plasma membrane of bovine erythrocytes. Several hitherto unexplained phenomena, such as the reconstitution of the electron transfer chain and the requirement of lipid for certain enzymic activities, are rationalized in terms of this capability of the repeating units to form membranes. On the basis of the properties of lipid-containing and lipiddepleted membranes, a theory of how membranes are formed has been developed. This theory depends primarily on the restrictions, imposed by phospholipids, on interactions between repeating units. Lipid-containing repeating units can associate into a continuum, one unit or macromolecule thick that can be identified with a membrane. By contrast, lipid-depleted repeating units tend to form a bulk phase, which can be identified with a random three-dimensional polymeric aggregate. Enzymic activities cannot be fully demonstrated when the repeating units exist in the state of a bulk phase.