Lipid protein interactions in mitochondria. VII. A comparison of the effects of lipid removal and lipid perturbation of the kinetic properties of mitochondrial ATPase.

We investigated the kinetics of mitochondrial ATPase in bovine heart mitochondria and submitochondrial particles upon treatment with phospholipase A2, or upon addition of n-butanol to perturb the lipid protein interactions. The changes observed are the following: (1) Lipid removal or perturbation with butanol is accompanied by loss of ATPase activity with decrease of both V and of the KM for ATP. (2) There are changes of activation energy of ATPase activity at temperatures above the discontinuity normally observed for membrane-bound enzymes in mitochondria. In particular, butanol abolishes the discontinuity, and induces a constant activation energy of about 32 kcal/mol in the range 8--37 degrees C. (3) Butanol modifies the pH dependence of ATPase shifting the pH optimum from around 10 to less alkaline values. The optimum for Mg2+ concentrations is increased by the solvent. (4) Treatment with phospholipase A2 results in a removal of oligomycin-sensitive ATPase, whereas butanol addition prevents oligomycin inhibition of ATPase. (5) In beef heart mitochondria, a spin-labelled analog of the inhibitor, dicyclohexyl carbodiimide, did not show any change in environment upon butanol addition, unlike that found in mitochondria from Saccharomyces cerevisiae.

Autoxidation and antioxidant activity of ubiquinol homologues in large unilamellar vesicles.

The antioxidant activity of ubiquinol homologues with different side-chain length such as ubiquinol-3 and ubiquinol-7 was compared with that of alpha-tocopherol when peroxidation was induced by the water-soluble initiator 2,2'-azobis-(2-amidinopropane hydrochloride). In large unilamellar vesicles containing equal amounts of alpha-tocopherol, ubiquinol-3 and ubiquinol-7 the rates of inhibition were very similar but the stoichiometric factor of quinols was approximately 1. To explain this low value, which is one-half of that found when the autoxidation was performed in apolar solvents (Chem. Phys. Lipids (1992) 61, 121-130), the oxidation of alpha-tocopherol and ubiquinol-3 initiated by the azocompound was studied both in methanol and in dimiristoyl-lecithin vesicles. The results obtained show that the ubiquinol homologues undergo a radical chain reaction taking place at the polar interface and suggest that the average preferred location of both quinol headgroups is near to the outer surface of the bilayer.

Reactions of oxygen radicals with the quinone ring of coenzyme Q.

Coenzyme Q, besides its role in electron transfer reactions, may act as a radical scavenger. The effect of oxygen radicals produced by ultrasonic irradiation on the quinone ring was investigated. Aqueous solutions of a Q homologue, completely lacking the side chain, were irradiated and the modifications were spectrophotometrically followed. The experimental results show that both degradation and reduction of the benzoquinone ring took place when the irradiation was performed in water. Data obtained when ultrasonic irradiation was carried out in the presence of OH. scavengers, as formate, organic and inorganic buffers, suggest: a) the responsible species for most the ubiquinol generated by sonication appeared to be the superoxide radical b) addition reactions of OH. radicals with the aromatic ring led probably to the degradation of Coenzyme Q molecules.

Incorporation of ubiquinones into lipid vesicles and inhibition of lipid peroxidation.

Ubiquinone incorporation into vesicles to evaluate its antioxidative effect on lipid peroxidation has been studied. Only sonication and not vortication allows comparable incorporation patterns of the various ubiquinone homologues into lipid vesicles. The measure of malondialdehyde, a convenient index for determining the extent of autoxidation, shows that both the naturally occurring homologues and synthetic shorter-chain ones, also in the oxidized form, possess similar antioxidant efficiency.

A conformational model of the action of general anesthetics at the membrane level. III. Anesthetics and the properties of membrane-bound enzymes: mitochondrial ATPase.

We have studied the effect of general anesthetics on the kinetic properties of the mitochondrial Mg2+-dependent ATPase. The enzyme is inhibited by anesthetics (alcohols, halotane, pentrane, ketamine) at concentrations of the order of those found to affect lipid-protein interactions. The inhibition appears usually uncompetitive with respect to the substrate, ATP, with a decrease of both Vmax and KM, indicating a possible stabilization of the enzyme-substrate complex. Arrhenius plots of ATPase activity show a striking increase in activation energy below 17-20 degrees C. Anesthetics affect the temperature dependence by increasing the activation energy above the break or abolishing the break whatsoever. An exception is diethyl ether, that induces a decrease in activation energy and a shift of the break to lower temperatures. Anesthetics make the ATPase insensitive to energy transfer inhibitor, oligomycin and dicyclohexyl carbodiimide. At low anesthetic concentration the oligomycin inhibition curve is changed from sigmoidal to hyperbolic, showing a loss of cooperativity in the inhibition.

Fromation and stoichiometry of a lysozyme-phospholipid-mitochondrial protein ternary complex.

Mitochondrial membranes reconstituted from lipid-depleted mitochondria and aqueous phospholipid dispersions still have the phospholipid negative charges available for ionic interaction with the basic protein, lysozyme. The stoichiometry of the binding is of about 6 nmoles of lysozyme per 100 nmoles of phospholipid in membranes reconstituted with Asolectin, and of 10 nmoles of phospholipid phosphorus in membranes reconstituted with cardiolipin. Unextracted submitochondrial particles ETP also bind lysozyme (about 3 nmoles per 100 nmoles of phospholipid). These observations indicate that the phospholipid anionic groups are not completely shielded by the mitochondrial proteins, which might occupy areas between the nonpolar groups of the lipid molecules.

Representative applications of heparin-sepharose in the removal of polyamines from biological materials.

Many experimental systems would greatly benefit from the availability of a simple and effective technique to remove polyamines from biological materials. We have examined the possibility of utilizing heparin-sepharose in the removal of polyamines from rat heart mitochondria, DNA-spermine complex, and fetal calf serum. Heparin-sepharose removes 90% of spermine adsorbed to the cytoplasmic surface of rat heart mitochondria. Heparin-sepharose almost totally removes spermine from DNA-spermine complex, leaving less than 0.003 mol spermine/mol DNA phosphorus. Heparin-sepharose is highly effective in removing spermine and spermidine (99.5 and 95% adsorbed, respectively) from fetal calf serum. Under the same experimental conditions only 50% of putrescine is adsorbed. A higher amount of resin corresponding to an increased capacity for putrescine must be used to achieve a satisfactory removal of putrescine.

Determination of the polyamine content of rat heart mitochondria by the use of heparin-sepharose.

Heparin-sepharose has been utilized to remove polyamines adsorbed to the cytoplasmic surface of rat heart mitochondria. The results obtained can be summarized as follows: Heparin-sepharose removes 90% of the spermine, 98% of the spermidine, and 98% of the putrescine adsorbed. Polyamine contents of chromatographed mitochondria amount to 2.66 and 0.36 nmol spermine and spermidine, respectively, per mg of mitochondrial protein.

Iron oxidation in Mops buffer. Effect of phosphorus containing compounds.

Fe2+ autoxidation in Mops buffer both in absence and presence of substoichiometric concentrations of EDTA, H2O2 and of Fe3+ is greatly affected by phosphorus containing compounds. They increase the lag phase, characteristic of Fe2+ oxidation in this buffer, and decrease the rate of the reaction. This effect is due to the phosphates of the molecule. The ability of the different compounds tested to affect Fe2+ oxidation, however, appears to be influenced also by the rest of the molecule. The concentration of the different phosphorus containing compounds that inhibits 50% of Fe2+ oxidation is rather different. The effect exerted appears to be the result of an equilibrium between an inhibitory effect on the pathway of Fe2+ oxidation that occurs in Mops buffer and the onset of a different oxidation pathway of Fe2+ similar to that occurring in Na phosphate buffer. A hypothesis is proposed that the phosphorus containing compounds inhibit Fe2+ oxidation by binding Fe3+ and decreasing its ability to accelerate Fe2+ autoxidation. It is suggested that the presence in vitro and in vivo of phosphorus containing compounds may modify Fe2+ autoxidation and thus the production of oxygen active species.

Phospholipid polar heads affect the generation of oxygen active species by Fe2+ autoxidation.

The possibility that phospholipid polar heads may influence Fe2+ reaction with molecular oxygen and, thus, the generation of oxygen active species was investigated. Dipalmitoyl phosphatidylcholine (DPPC) and DPPC/dipalmitoyl phosphatidic acid (DPPA) were utilized as model liposomes. Fe2+ oxidation, oxygen consumption, nitro blue tetrazolium reduction and 2-deoxyribose degradation were the parameters evaluated. Comparison of the results obtained clearly shows that the two types of polar heads differently affect iron chemistry. DPPC liposomes are ineffective. By contrast, Fe2+ oxidation by oxygen occurs in the presence of DPPC/DPPA liposomes. During this reaction, species able to reduce nitro blue tetrazolium and to degrade 2-deoxyribose are generated. The results obtained indicate that the polar heads of phospholipids, by influencing Fe2+ autoxidation, generate dangerous oxygen species which may exert an active role in the oxidation of the associated hydrophobic components of the phospholipids.

Lipid metabolism in fatty liver of lysine- and threonine-deficient rats.

Rats were fed a low protein diet deficient in and supplemented with lysine and threonine. Liver lipids contained more lecithin, sphingomyelin, and free fatty acids, and less amino phospholipids in the deficient rats. No variations in fatty acid composition of choline- and ethanolamine-containing phospholipids were found; only palmitic acid was increased in the serine-containing phospholipids of the deficient animals. The incorporation of acetate-(14)C into phospholipids, but not into other liver lipids, was lower in deficient rats. In the plasma of deficient rats the concentration of esterified fatty acids and phospholipids was lower, of free fatty acids higher, than in the controls. The fatty acid composition of depot fat differed from that of liver neutral fat both in deficient and supplemented animals. The results presented establish that multiple metabolic defects resulting from lysine and threonine deficiency accompany the fatty liver. The design of the experiments does not permit conclusions to be drawn regarding the causal relationship between the various alterations in lipid metabolism and the fatty liver.

Antioxidative effect of ubiquinones on mitochondrial membranes.

Peroxidation of mitochondria occurs extensively in ubiquinone-depleted membranes. Reincorporation into the membranes of either the physiological ubiquinone or a short-chain homologue protects mitochondria against peroxidation. The ability to prevent this phenomenon is more evident in mitochondria that have incorporated ubiquinone-3 and might be ascribed to an ordering structural effect on the lipid bilayer.

Uncoupling effect of a low homolog of unbiquinone (UQ-3) in rabbit heart mitochondria.

Short-chain ubiquinone (UQ-3) inhibits ADP-stimulated respiration (state 3) in intact rabbit heart mitochondria. This effect appears to be similar in all the three sites of oxidative phosphorylation by using different respiratory substrates. Ubiquinone-3 also immobilizes lipids into mitochondrial membranes indicating that the uncoupling effect might be a consequence of an altered physical state of membrane lipids.

A role of ubiquinone in energy conservation in mitochondria.

Short chain ubiquinones (Q-3) uncouple oxidative phosphorylation in rat heart mitochondria, as shown by polarimetric experiments, and abolish P:O ratios in succinate driven oxidative phosphorylaton. The uncoupling is reversed by long chain ubiquinones (Q-7). Furthermore, short chain ubiquinones abolish oligomycin sensitivity of ATPase; the inhibition is restored by Q-7. The extraction of endogenous ubiquinone from mitochondria reversibly lowers oligomycin sensitivity of ATPase.

Antioxidant behaviour of ubiquinone and beta-carotene incorporated in model membranes.

Experiments with model membranes, in which ubiquinone was incorporated, were performed in order to clarify the mechanism by which ubiquinone can prevent or control chain lipid peroxidation in biomembranes. Comparing the behavior of ubiquinone-containing vesicles with beta-carotene containing vesicles we suggest that a possible explanation of the ubiquinone antioxidant effect could be to scavenge singlet oxygen and to affect structurally the lipid bilayer inhibiting hydroperoxide decomposition.

The influence of phospholipid polar head on the lipid hydroperoxide dependent initiation of lipid peroxidation.

In a buffer (Mes) and at a pH (6.5) where Fe2+ is very stable, we have studied the peroxidation of liposomes catalyzed by FeCl2. The liposomes studied, prepared by sonolysis, contained either phosphatidylcholine or 1:1 molar ratio of phosphatidylcholine and phosphatidic acid. The presence of the negatively charged phospholipid causes: 1) rapid Fe2+ oxidation and oxygen consumption; 2) increased generation of lipid hydroperoxides; 3) decreased generation of thiobarbituric acid-reactive materials; 4) very low inhibition of Fe2+ oxidation and lipid hydroperoxide generation by BHT; 5) inhibition of the termination phase of lipid peroxidation at high FeCl2 concentrations. A hypothesis is proposed to explain the results obtained.