Anna Maria Sechi

Activation energies of different mitochondrial enzymes: Breaks in Arrhenius plots of membrane-bound enzymes occur at different temperatures

A series of mitochondrial enzymes exhibit breaks in Arrhenius plots from low to high activation energies upon temperature decrease. All membrane-bound enzymes tested except cytochrome c oxidase have shown these transitions, while soluble matrix enzymes (malic dehydrogenase and fumarase) did not have breaks or they had different characteristics. Triton-X-100 which solubilizes the mitochondrial membrane, induces a straightening of the plot for ATPase. The observation that the breaks in the Arrhenius plots for membranous enzymes fall at different temperatures suggests caution in their correlation with phase transitions of membrane phospholipids.

Inhibition of phospholipase A2 and phospholipase C by polyamines.

Abstract The polyamines spermine, spermidine, and putrescine inhibit the activity of phospholipase A2 (Naja naja) and phospholipase C (Clostridium welchii) on phospholipid vesicles and mitochondrial membranes as sources of substrate phospholipids. The inhibitory effect is highest for spermine and lowest for putrescine. With both enzymes, inhibition is stronger when phospholipid vesicles rather than mitochondrial membranes are used as the substrate. No clear competition of polyamines with Ca2+, which is required for the activity of both enzymes, has been observed. The inhibition appears to be due to steric hindrance of enzyme-substrate interaction due to the binding of the organic polycations to the phospholipid bilayer.

Lipid-protein interactions in mitochondria. I. Conditions affecting binding of phospholipids to lipid-depleted mitochondria.

Abstract The interaction of lipid-depleted mitochondria with mixed micellar phospholipids is not inhibited by increasing the ionic strength of the medium with NaCl or certain other salts. The graph describing binding as function of ionic strength has a diphasic character. The extent of the binding increases with temperature. Mitochondrial membranes freed of soluble and detachable proteins can also be depleted of their lipids and reconstituted by readdition of phospholipid micelles: this interaction also is not impaired by NaCl. The interaction is, however, inhibited by alcohols and by lyotropic agents inducing disorder in the structure of water (such as certain salts at high concentrations). From these data and other characteristics of the binding we conclude that the binding of phospholipids to the proteins of the mitochondrial membranes is largely hydrophobic in nature.

Lipid-protein interactions in mitochondria. Changes in mitochondrial adenosine triphosphatase activity induced by n-butyl alcohol.

Abstract Butanol at a concentration of 0.35 m decreases the oligomycin sensitivity of the mitochondrial ATPase; at the same concentration of butanol the activation energy of enzyme is increased threefold. Butanol does not detach the ATPase from the membrane of either mitochondria or submitochondrial particles. The same effect is exerted by butanol on the sensitivity of the ATPase to DCCD, which is covalently bound to the ATPase complex in the oligomycin inhibition site. Diethyl ether also makes the ATPase oligomycin- and DCCD-insensitive; however, its effect on the activation energy of the enzyme is different from that of butanol, since ether does not increase the activation energy but lowers the temperature where a transition occurs in an Arrhenius plot of ATPase. The effect of both organic solvents on ATPase may be closely related to changes occurring in the lipid environment which might be transferred to the enzymic activity via a conformational change of the enzymic protein.

Antioxidant behaviour of Ubiquinone and β-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.

Lipid protein interactions in mitochondria. VII. A comparison of the effects of lipid removal and lipid perturbation on 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.

Lipid-protein interactions in mitochondria: II. On the nature and biochemical significance of the interaction between phospholipids and lipid-depleted mitochondria

Abstract The binding of phospholipid micelles to lipid-depleted mitochondrial membranes has been studied in further detail. The diphasic character of the curve of binding plotted against the concentration of salt has been explained on the basis of an enhancement of hydrophobic binding by increasing ionic strengths and subsequent impairment of the binding due to the lyotropic effects of high salt concentrations. The amount of phospholipid bound in certain conditions is above the natural levels in mitochondrial membranes, because sites normally buried become exposed and available for interaction with phospholipid. The composition of the bound phospholipids is the same when the interaction is done in the absence or in the presence of 1 m NaCl. Succinoxidase activity is restored in lipid-depleted mitochondria by phospholipid addition either in salt-free media or at low ionic strengths, but is decreased when the incubation is carried out in 1 m NaCl, in 0.5 m NaSCN, and also in 3 m urea. From these results we conclude that the interactions we have studied appear to be relevant to the interactions in the native membranes and that the bonds formed between protein and lipids must be primarily hydrophobic, although a contribution of polar forces to the binding cannot be excluded.

Lipid-protein interactions in mitochondria: III. Solvent extraction of phospholipids from mitochondrial preparations☆

Abstract Monohydric alcohols extract phospholipids from beef heart mitochondria and submitochondrial particles with an efficacy which depends on the chain length of the alcohol. MgCl2 and CaCl2 prevent alcohol extraction of part of the phospholipids. All particles used are affected by alcohols to similar extents. Diethyl ether extracts phospholipids from sonicated phospholipid vesicles in presence of NaCl or at acid pH even when the vesicles were bound to lipid-depleted mitochondria; ether extracts little phospholipid from beef heart mitochondria or submitochondrial particles under similar conditions; NaSCN is however much more effective than NaCl in increasing the extractability of phospholipids by ether. Mitochondrial membranes treated with 10 m m HCl are freed of the soluble proteins and of the proteins of the ATPase complex; phospholipids can be extracted in large amounts from such membranes by ether in presence of salt. Detachment of the ATPase complex from mitochondria by other means also results in higher phospholipid extractability by ether. Lyophilized mitochondria or submitochondrial particles can be extracted of phospholipids by means of ether or pentane only after HCl treatment. Chloroform-methanol 2:1 extracts phospholipids and a small but significant portion of the protein from mitochondria and various mitochondrial preparations. The possible presence of proteolipids is discussed. The results of this investigation are discussed in relation to the importance of hydrophobic interactions between phospholipids and proteins in mitochondria; hydrophobic bonds however are not unique and the hypothesis is advanced that superficial proteins are linked to the mitochondrial membrane by other types of bonds.

On the sidedness of the ubiquinone redox cycle. Kinetic studies in mitochondrial membranes

Abstract The inhibition of NADH oxidation but not of succinate oxidation by the low ubiquinone homologs UQ-2 and UQ-3 is not due to a lower rate of reduction of ubiquinone by NADH dehydrogenase: experiments in submitochondrial particles and in pentane-extracted mitochondria show that UQ-3 is reduced at similar rates using either NADH or succinate as substrates. The fact that reduced UQ-3 cannot be reoxidized when reduced by NADH but can be reoxidized when reduced by succinate may be explained by a compartmentation of ubiquinone. Using reduced ubiquinones as substrates of ubiquinol oxidase activity in intact mitochondria and in submitochondrial particles we found that ubiquinol-3 is oxidized at higher rates in submitochondrial particles than in mitochondria. The initial rates of ubiquinol oxidation increased with increasing lengths of isoprenoid side chains in mitochondria, but decreased in submitochondrial particles. These findings suggest that the site of oxidation of reduced ubiquinone is on the matrix side of the membrane; reduced ubiquinones may reach their oxidation site in mitochondria only crossing the lipid bilayer: the rate of diffusion of ubiquinol-3 is presumably lower than that of ubiquinol-7 due to the differences in hydrophobicity of the two quinones.

Effects of free radicals produced by sonolysis on ubiquinone-containing vesicles

The aim of this study was to evaluate both the effect of oxygen free radicals produced by ultrasonic irradiation on ubiquinone-3 incorporated into egg phosphatidylcholine vesicles and lipid peroxidation of the same vesicles. Owing to water sonolysis, degradation of polyunsaturated fatty acid residues occurred when model membranes were sonicated in the absence of ubiquinone. Under these conditions, Hepes buffer, but not formate, was able to protect against HO − damage, probably because access to the site of HO − formation is easier for the buffer. Incorporation of quinone prevented peroxidation due to HO − radicals, with a concomitant decrease of ubiquinone content. Experiments performed to evaluate the effects of varying egg phosphatidylcholine concentration on conjugated diene formation and ubiquinone stability suggest that the antioxidant effect of this compound mostly resides in its capacity to act as a ‘chain-breaker’ antioxidant.