P. Pasquali

Polyamine binding to phospholipid vesicles and inhibition of lipid peroxidation

A study of the possible mechanism of inhibition by polyamines of lipid peroxidation was made utilizing vesicles prepared with mixed soy bean phospholipids. The results obtained can be summarized as follows: 1) Polyamines inhibit lipid peroxidation only when bound to the negative charges on vesicle surface. 2) Polyamines inhibit lipid peroxidation at concentrations lower than those required to cause precipitation of the vesicles and similar to those required for formation of the polyamine/phospholipid vesicle complex. 3) Spermine bound to vesicles, in contrast to free spermine, highly decreases the reactivity of both Fe2+ and Fe3+ versus superoxide.

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.

The inhibition of NADH oxidase by the lower homologs of coenzyme Q

Abstract The Coenzyme Q homologs having short isoprenoid chains are much less efficient than the higher homologs in restoring NADH oxidation in pentane-extracted lyophilized beef heart mitochondria; they have however high restoring activity for succinate oxidation. The same pattern is observed in pentane extracted submitochondrial particles ETP only if the quinones are added to detergent-treated membranes, showing that in ETP there is a decreased accessibility of the long chain quinones in comparison with the lower homologs. In intact mitochondria and ETP, CoQ 3 inhibits NADH oxidation while leaving succinate oxidation unaffected; the inhibition of NADH oxidation by CoQ 3 is not reversed by serum albumin but is reversed by CoQ 7 , particularly when the membrane has been previously “opened” with deoxycholate. CoQ 3 may accept electrons from NADH in cyanide-inhibited ETP, allowing coupling at the first phosphorylation site as shown by the quenching of the fluorescence of atebrine. The mechanism of CoQ 3 inhibition is probably related to its insufficient rate of reoxidation by the following segment of the respiratory chain when it has been reduced by NADH dehydrogenase.

Effect of endogenous ubiquinone on the interaction of exogenous Ubiquinone-1 with the respiratory chain of bovine heart mitochondria.

Abstract Pentane extraction of lyophilized mitochondria with depletion of up to 92% of endogenous ubiquinone (UQ) does not affect ubiquinol oxidase activity in terms of K m ; in certain preparations the V is decreased probably because the extraction is harmful to the membrane integrity. In such case dl -α-tocopherol is able to maintain enzymatic activity up to the normal values found in control mitochondria. On the other hand, NADH-UQ-1 reductase activity is greatly affected by pentane extraction with a large decrease in V but no change in K m , but this activity is protected by addition of dl -α-tocopherol to the extraction medium. The same conclusions can be drawn for succinate-UQ-1 reductase activity. In conclusion it appears that endogenous UQ does not mediate the interaction of exogenous UQ-1 with the redox sites for UQ in the respiratory chain.

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.

Effect of protein binding on phospholipase C hydrolysis of aqueous phospholipid dispersions

Abstract Formation of ionic complexes with basic proteins (lysozyme, cytochrome c ) does not allow phospholipid hydrolysis by phospholipase C. On the other hand phospholipids in hydrophobic complexes with lipid-depleted mitochondria (LDM) and intact submitochondrial membranes (ETP) are rapidly hydrolysed by the phospholipase. Phospholipids in ternary complexes of basic proteins with LDM-phospholipid complexes or with ETP are however also hydrolysed. These results suggest that the lipid bilayer has a greater dynamic character in membranes than in phospholipid vesicles.

Incorporation of a lipophilic series, as coenzyme Q Homologs, din phospholipid vesicles

The incorporation of Coenzyme Q† homologs into phospholipid vesicles to achieve a concentration close to the quinone content of mitochondria has been studied with different methods. The results obtained can be summarized as follows: (a) stirring does not lead to comparable incorporation of the various CoQs tested; (b) ultrasonic irradiation results in a homogeneous incorporation both of the naturally occurring homologs of Coenzyme Q (polyisoprenoid chainlength ranging from ten to six units) and of the nonphysiological shorter chain homologs; (c) ethanol injection, which is a simple, rapid, and inexpensive technique, gives results comparable to those obtained by ultrasonication.

Effect of ubiquinone extraction on ubiquinol-1 oxidase activity in beef heart mitochondria.

Extraction of endogenous ubiquinone with different methods does not influence ubiquinol oxidase activity in lyophilized mitochondria in terms ofKM, although a decrease ofVmax is sometimes observed. Experiments with submitochondrial particles from a UQ-deficient mutant ofS. cerevisiae confirm the results with UQ-depleted mitochondria and support the idea that endogenous ubiquinone is not required for the oxidation of exogenous ubiquinols by complex III.

Lipid—Protein interactions in mitochondria: The effect of protein interaction on susceptibility of phospholipids to phospholipase A2 and C hydrolysis☆

Abstract Phospholipase A2 (Naja naja) and phospholipase C (from either Clostridium welchii or Bacillus cereus) have been tested on phospholipid dispersions and natural or reconstituted membranes; notwithstanding the different substrate specificities, the different enzymes gave comparable behaviors, suggesting that the results were the expression of sterical features in the lipid bilayers, i.e., availability of the phospholipids to enzymatic attack. The hydrolysis of phospholipids (Asolectin) in sonic protein-free vesicles is hindered by ionic interaction with basic proteins (cytochrome c or lysozyme). On the other hand binding of Asolectin to lipid-depleted mitochondria to obtain reconstituted mitochondria does not prevent phospholipase action on the phospholipids; similarly, phospholipids are hydrolyzed at maximal rates in natural membranes (mitochondria or submitochondrial particles). Surprisingly, ionic interaction of RM or natural membranes with basic proteins does not prevent phospholipase hydrolysis of the membrane phospholipids. The interpretation of this phenomenon may be related to the heterogeneity of phospholipid distribution in protein-containing membranes.

On the mechanism of inhibition of NADH oxidase by ubiquinone-3

The combined effects of rotenone and ubiquinone-3 on the kinetics of NADH dehydrogenase and NADH oxidase have been investigated. The two inhibitors do not show additivity; on the other hand, ubiquinone-3, when preincubated with the enzyme, partially removes rotenone sensitivity. The inhibition of NADH oxidase by ubiquinone-3 is the result of at least two combined effects: the competition of the less active ubiquinone-3 with endogenous ubiquinone-10 in the acceptor site of the dehydrogenase, and a nonspecific action on the structure of complex I. The latter effect is perhaps mediated by a physical change of the phospholipid bilayer similar to that observed with agents such as butanol, perturbing lipid-protein interactions in the membrane.