Solubilization of inner mitochondrial membranes by triton X-100. Effect of ionic strength and temperature.

Rat liver inner mitochondrial membranes have been subjected to the solubilizing action of the non-ionic detergent Triton X-100 under a variety of ionic strength and temperature conditions. Increasing ionic strength has little influence on the amount of solubilized membrane protein and lipid phosphorus. Calcium chloride actually increases the proportion of solubilized protein. This effect is preserved by 1 mM EDTA. Increasing temperatures tend to decrease the proportion of protein solubilized by the detergent. SDS-polyacrylamide gel electrophoresis fails to reveal any difference in polypeptide composition of the membrane fraction solubilized under the various conditions. However, differences are observed in the solubilization of individual cytochromes. The data are interpreted in terms of changes in membrane architecture induced by the various conditions of the incubation medium.

The impairment of mitochondrial function by Triton X-100. A study of mitochondrial respiration and energy-dependent swelling.

Coupled and uncoupled respiration, and energy-dependent phosphate swelling have been studied in rat liver mitochondria in the presence of various concentration of Triton X-100. Detergent concentrations up to 10(-5) M do not affect any of the processes under study. At 10(-5) M, Triton X-100 produces a slight decrease of coupled respiration and a considerable inhibition of mersalyl-induced shrinking in swollen mitochondria. Increasing the surfactant concentration to 10(-4), coupled as well as uncoupled O2 consumption is decreased, succinate-dependent phosphate swelling is inhibited and an energy-dependent phosphate swelling in the absence of valinomycin is observed. At 2 X 10(-4) M. Triton X-100, ATP- dependent phosphate swelling is abolished, and passive swelling may be induced by various ions. Higher detergent concentrations do not allow observation of any of these events. On the basis of these results, a model of membrane-detergent interaction is proposed.

Assay of mitochondrial membrane-bound enzyme activities in the presence of triton X-100.

Mitochondrial ATPase and cytochrome c oxidase activities are not severely affected by Triton X-100 concentrations between 0.1 and 2.0% (w/v). The former is solubilized by the effect of the detergent, while the latter is not. Succinate: cytochrome c reductase and rotenone-sensitive NADH: cytochrome c reductase activities are destroyed even a low detergent concentrations. Succinate:coenzyme Q oxidoreductase is affected by the surfactant in a more complex way, so that selective solubilization of some subunit(s) could be involved.

Fractionation of rat liver mitochondrial components after short treatments with Triton X-100.

1. When rat liver mitochondria are treated with the non-ionic detergent Triton X-100, the solubilizing effects of the latter are already maximal after a few seconds. 2. We have developed a filtration technique that allows a fast separation of the solubilized and non-solubilized fractions. By means of this technique, we have been able to show differences in the solubilizing effect of Triton X-100 according to the physiological state of mitochondria. 3. In addition, the filtrate may be subjected to further fractionation by ultracentrifugation, by which two filtrate subfractions, supernatant and pellet, may be obtained. 4. Filtrates, supernatants and pellets differ from each other, and from intact mitochondria, in phospholipid and polypeptide composition, and possess a characteristic distribution of enzyme activities; they are also different from the ultrastructural point of view. 5. It is concluded that the combined techniques of short detergent treatments and fast filtration are useful in structural and functional studies of mitochondria and in the selective solubilization of mitochondrial components.

The interaction of Triton X-100 with purple membranes. Detergent binding, spectral changes and membrane solubilization.

The interaction of the non-ionic surfactant Triton X-100 with Halobacterium purple membranes has been examined at sublytic and lytic surfactant concentrations. These membranes present a number of important peculiarities in their behaviour towards the surfactant. Although solubilization is a very slow process, with a half-time of the order of hours, detergent binding appears to occur at the same fast rate as that found in other membranes. Lipids are solubilized more easily than proteins, so that hardly any protein is solubilized at surfactant concentrations at which about 75% of the lipid is in the form of detergent-mixed micelles; once started, protein solubilization takes place within a narrow range of surfactant concentrations. Retinal provides a built-in probe to monitor detergent-induced conformational changes by spectroscopy in the visible range. No spectral variation is detected at the prelytic stage, i.e. when detergent is incorporated into the membrane in monomeric form. Membrane disruption is accompanied by a blue shift in the absorption maximum, retinal isomerization (from all-trans to 13-cis), and a decrease in specific absorbance (bleaching). Increasing detergent concentrations after solubilization is completed do not produce further shifts in the spectral maximum, but the specific absorbance is progressively decreased. It is shown that Triton X-100 has a complex effect on the retinal chromophore, modifying its configuration and microenvironment (changes in maximum wavelength) and promoting hydrolysis of the retinal-bacteriorhopsin Schiff's base (bleaching).

Triton X-100 solubilization of mitochondrial inner and outer membranes.

Rat liver mitochondrial inner and outer membranes were subjected to the solubilizing effect of the nonionic detergent Triton X-100 under various conditions. After centrifugation, the supernatants (containing the solubilized fraction) and pellets were characterized chemically and/or ultrastructurally. The detergent seems to act by inducing a phase transition from membrane lamellae to mixed protein-lipid-detergent micelles. Different electron-microscopy patterns are shown by the inner membranes after treatment with different amounts of surfactant, whereas the corresponding images from outer membranes vary but slightly. Selective solubilization of various components is observed, especially in the case of the inner membrane. Some membrane lipids (e.g., cardiolipin) are totally solubilized at detergent concentrations when others, such as sphyngomyelin, remain in the membrane. Other inner-membrane components (flavins, cytochromes, coenzyme Q) show different solubilization patterns. This allows the selection of conditions for optimal solubilization of a given membrane component with some degree of selectivity. The influence of Triton X-100 on various mitochondrial inner-membrane activities was studied. The detergent seems to act especially through disruption of the topology of the functional complexes, although the activity of the individual enzymes appears to be preserved. Relatively simple enzyme activities, such as ATPase, are more or less solubilized according to the detergent concentration, whereas the more complex succinate-cytochrome c reductase activity practically disappears even at low Triton X-100 concentrations.

Effect of the nonionic detergent Triton X-100 on mitochondrial succinate-oxidizing enzymes.

Specific activities of succinate:coenzyme Q reductase, ubiquinone:cytochrome c reductase, cytochrome oxidase, succinate:cytochrome c reductase, succinate oxidase, and ubiquinol oxidase have been measured in rat liver mitochondria in the presence of Triton X-100. The last three activities are much more sensitive to Triton X-100 than the first ones; the data suggest that the electron transport chain components cannot react with each other in the presence of the detergent. At least in the case of succinate:cytochrome c reductase, reconstitution of the detergent-treated membranes with externally added phospholipids reverses the inhibition produced by Triton X-100. These results support the idea that the respiratory chain components diffuse at random in the plane of the inner mitochondrial membrane; the main effect of the detergent would be to impair lateral diffusion by decreasing the area of lipid bilayer. When detergent-treated mitochondrial suspensions are centrifuged in order to separate the solubilized from the particulate material, only the first three enzyme activities mentioned above are found in the supernatants. After centrifugation, a latent ubiquinol:cytochrome c oxidase activity becomes apparent, whereas the same centrifugation process produces inhibition of cytochrome c oxidase in the presence of certain Triton X-100 concentrations. These effects could be due either to a selective solubilization of regulatory or catalytic subunits or to a conformational change of the enzyme-detergent complex.

Physiological state of submitochondrial particles and their susceptibility to Triton X-100.

The solubilizing effect of Triton X-100 on beef heart submitochondrial particles (ETPH) has been studied under various physiological conditions. Coupled, uncoupled and azide-inhibited ETPH particles have been studied. Quantitative and qualitative differences are found in the proteins solubilized by the detergent from ETPH particles under the various conditions tested.

Membrane solubilization by the non-ionic detergent triton X-100. A comparative study including model and cell membranes.

The solubilizing effects of the non-ionic detergent Triton X-100 have been examined on three membranous systems, namely rabbit sarcoplasmic reticulum, Halobacterium purple membrane and gramicidin A-phosphatidylcholine liposomes. The loss of membrane structure has been assessed through changes in suspension turbidity, while chemical analysis has revealed the differential solubilization of proteins and lipids. Solubilization data obtained on the above three systems are compared with previously published values concerning other membrane preparations. Also, solubilization of sarcoplasmic reticulum by Triton X-100 is monitored by Fourier-transform infrared spectroscopy and, similarly, purple membrane-surfactant interaction is studied using visible spectroscopy. The biochemical and spectroscopic data may be rationalized assuming a three-stage model of membrane-detergent interaction, incorporation of surfactant monomers into the membrane; disruption of the bilayer into mixed micelles, and separation of lipid and protein.