Thermal adaptation of Tetrahymena membranes with special reference to mitochondria Role of cardiolipin in fluidity of mitochondrial membranes

During temperature acclimation of Tetrahymena pyriformis, the changes in fluidity and composition of total lipids from three membrane fractions, mitochondria, pellicles and microsomes were studied by a spin-label technique using a stearate probe and thin-layer and gas-liquid chromatography. The increase of fluidity observed in microsomal and pellicular lipids following the temperature shift from 39 to 15°C corresponds with the increase of the ratio of total unsaturated to saturated fatty acid content. However, despite the increase of this ratio, the fluidity of mitochondrial lipids was found to be constant up to 10 h after the temperature shift. The fluidity of total lipids of mitochondria isolated from Tetrahymena cells grown at 39°C was not changed by removal of cardiolipin, whereas cardiolipin-depleted lipids of mitochondria from 15°C-acclimated cells showed a decrease in fluidity. The re-addition of cardiolipin to the mitochondrial lipids depleted of cardiolipin restored the fluidity to the initial leve, thereby confirming the rigidifying effect of cardiolipin in cold-acclimated cells. These results suggest that cardiolipin may be implicated in maintaining consistent fluidity of mitochondrial membranes against change in thermal environment.     

Thermal adaptation of Tetrahymena membranes with special reference to mitochondria II. Preferential interaction of cardiolipin with specific molecular species of phospholipid

A specific effect of cardiolipin on fluidity of mitochondrial membranes was demonstrated in Tetrahymena cells acclimated to a lower temperature in the previous report (Yamauchi, T., Ohki, K., Maruyama, H. and Nozawa, Y. (1981) Biochim. Biophys. Acta 649, 385–392). This study was further confirmed by the experiment using fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene (DPH). Anisotropy of DPH for microsomal and pellicular total lipids from Tetrahymena cells showed that membrane fluidity of these lipids increased gradually as the cells were incubated at 15°C after the shift down of growth temperature from 39°C. However, membrane fluidity of mitochondrial total lipids was kept constant up to 10 h. This finding is compatible with the result obtained using spin probe in the previous report. Additionally, the break-point temperature of DPH anisotropy was not changed in mitochondrial lipids whereas those temperatures in pellicular and microsomal lipids lowered during the incubation at 15°C. Interaction between cardiolipins and various phospholipids, which were isolated from Tetrahymena cells grown at 39°C or 15°C and synthesized chemically, was investigated extensively using a spin labeling technique. The addition of cardiolipins from Tetrahymena cells grown at either 39°C or 15°C did not change the membrane fluidity (measured at 15°C) of phosphatidylcholine from whole cells grown at 39°C. On the other hand, both cardiolipins of 39°C-grown and 15°C-grown cells decreased the membrane fluidity of phosphatidylcholine from Tetrahymena cells grown at 15°C. The same results were obtained for phosphatidylcholines of mitochondria and microsomes. Membrane fluidity of phosphatidylethanolamine, isolated from cells grown at 15°C, was reduced to a small extent by Tetrahymena cardiolipin whereas that of 39°C-grown cells was not changed. Representative molecular species of phosphatidylcholines of cells grown at 39°C and 15°C were synthesized chemically; 1-palmitoyl-2-oleoylphosphatidylcholine for 39°C-grown cells and dipalmitoleoylphosphatidylcholine for 15°C-grown ones. By the addition of Tetrahymena cardiolipin, the membrane fluidity of 1-palmitoyl-2-oleoylphosphatidylcholine was not changed but that of dipalmitoleoylphosphatidylcholine was decreased markedly. These phenomena were caused by Tetrahymena cardiolipin. However, bovine heart cardiolipin, which has a different composition of fatty acyl chains from the Tetrahymena one, exerted only a small effect.     

Thermal adaptation of Tetrahymena membranes with special reference to mitochondria. II. Preferential interaction of cardiolipin with specific molecular species of phospholipid

A specific effect of cardiolipin on fluidity of mitochondrial membranes was demonstrated in Tetrahymena cells acclimated to a lower temperature in the previous report (Yamauchi, T., Ohki, K., Maruyama, H. and Nozawa, Y. (1981) Biochim. Biophys. Acta 649, 385-392). This study was further confirmed by the experiment using fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene (DPH). Anisotropy of DPH for microsomal and pellicular total lipids from Tetrahymena cells showed that membrane fluidity of these lipids increased gradually as the cells were incubated at 15 degrees C after the shift down of growth temperature from 39 degrees C. However, membrane fluidity of mitochondrial total lipids was kept constant up to 10 h. This finding is compatible with the result obtained using spin probe in the previous report. Additionally, the break-point temperature of DPH anisotropy was not changed in mitochondrial lipids whereas those temperatures in pellicular and microsomal lipids lowered during the incubation at 15 degrees C. Interaction between cardiolipins and various phospholipids, which were isolated from Tetrahymena cells grown at 39 degrees C or 15 degrees C and synthesized chemically, was investigated extensively using a spin labeling technique. The addition of cardiolipins from Tetrahymena cells grown at either 39 degrees C or 15 degrees C did not change the membrane fluidity (measured at 15 degrees C) of phosphatidylcholine from whole cells grown at 39 degrees C. On the other hand, both cardiolipins of 39 degrees C-grown and 15 degrees C-grown cells decreased the membrane fluidity of phosphatidylcholine from Tetrahymena cells grown at 15 degrees C. The same results were obtained for phosphatidylcholines of mitochondria and microsomes. Membrane fluidity of phosphatidylethanolamine, isolated from cells grown at 15 degrees C, was reduced to a small extent by Tetrahymena cardiolipin whereas that of 39 degrees C-grown cells was not changed. Representative molecular species of phosphatidylcholines of cells grown at 39 degrees C and 15 degrees C were synthesized chemically; 1-palmitoyl-2-oleoylphosphatidylcholine for 39 degrees C-grown cells and dipalmitoleoylphosphatidylcholine for 15 degrees C-grown ones. By the addition of Tetrahymena cardiolipin, the membrane fluidity of 1-palmitoyl-2-oleoylphosphatidylcholine was not changed but that of dipalmitoleoylphosphatidylcholine was decreased markedly. These phenomena were caused by Tetrahymena cardiolipin. However, bovine heart cardiolipin, which has a different composition of fatty acyl chains from the Tetrahymena one, exerted only a small effect.     

Tetrahymena strives to maintain the fluidity interrelationships of all its membranes constant. Electron microscope evidence

When cells of Tetrahymena pyriformis, strain NT-1, were chilled from their growth temperature of 39.5 degrees C to lower temperatures, the plasma membrane, outer alveolar, nuclear, outer mitochondrial, food vacuolar, and endoplasmic reticulum membranes each responded in a fashion quite characteristic of the membrane type. In most cases a distinctive rearrangement of intramembrane particles, as discerned by freeze-fracture electron microscopy, began abruptly at a definitive temperature. By comparing the freeze-fracture patterns of membranes in cells grown at 39.5, 27, and 15 degrees C, it was shown that the initial particle rearrangement in a given membrane always occurred at a fixed number of degrees below the growth temperature of the cell. Gradual chilling of a cell grown at constant temperature induced these membrane changes first in the outer alveolar membrane, then, in order of decreasing response to temperature, in the endoplasmic reticulum, outer mitochondrial membrane, nuclear envelope, and vacuolar membrane. The normally stable relationships between the physical properties of the several membrane types could in some cases be reversed, but only temporarily, by fatty acid supplementation or during the initial phases of acclimation to growth at a different temperature. The system provides a unique opportunity to study the effects of environmental change upon the physical properties of several functionally distinct but metabolically interrelated membranes within a single cell.     

Interaction of carnitine with mitochondrial cardiolipin.

The physiological role of L-carnitine is to determine the transport of acyl-CoA through the mitochondrial membrane. However, some observations may also suggest a direct effect of the molecule per se on the physical properties of the membrane, most probably at the level of the binding site. This possibility has been investigated by studying the influence of adriamycin, a drug that binds to cardiolipin, on the effect of carnitine on isolated rat liver mitochondria. It has been found that adriamycin almost abolishes the activating effect of carnitine on state 2 respiration. The effect and its inhibition is seen by using either the L-form of carnitine or the D-form or both. Cardiolipin removes the effect of adriamycin and restores the activation by carnitine. It is proposed that some effects of carnitine on mitochondrial properties may be the result of interaction of carnitine with cardiolipin at the membrane level.     

Role of cardiolipin alterations in mitochondrial dysfunction and disease

Cardiolipin (CL) is a structurally unique dimeric phospholipid localized in the inner mitochondrial membrane where it is required for optimal mitochondrial function. In addition to its role in maintaining membrane potential and architecture, CL is known to provide essential structural and functional support to several proteins involved in mitochondrial bioenergetics. A loss of CL content, alterations in its acyl chain composition, and/or CL peroxidation have been associated with mitochondrial dysfunction in multiple tissues in a variety of pathological conditions, including ischemia, hypothyroidism, aging, and heart failure. Recently, aberrations in CL metabolism have been implicated as a primary causative factor in the cardioskeletal myopathy known as Barth syndrome, underscoring an important role of CL in human health and disease. The purpose of this review is to provide an overview of evidence that has linked changes in the CL profile to mitochondrial dysfunction in various pathological conditions. In addition, a brief overview of CL function and biosynthesis, and a discussion of methods used to examine CL in biological tissues are provided. phospholipid; metabolism; heart failure; aging; hypothyroidism; lipid peroxidation; oxidative stress; diet; ischemia     

The interaction of adriamycin with cardiolipin in model and rat liver mitochondrial membranes

The interaction of adriamycin with cardiolipin in model membranes and in various membrane preparations derived from rat liver mitochondria was studied and the results are analyzed in the light of a possible specific interaction between adriamycin and cardiolipin. It was found that adriamycin binds to cardiolipin-containing model membranes with a fixed stoichiometry of two drug molecules per cardiolipin. Furthermore, the extent of drug complexation by mitochondria and mitoplasts (inner membrane plus matrix) is in reasonable agreement with their cardiolipin content. In contrast, adriamycin-binding curves of inner membrane ghosts and submitochondrial particles reveal considerable association to an additional site, presumably RNA. The evidence for the potential importance of RNA as a target comes from experiments on outer membranes and microsomes which both appear to bind substantial amounts of adriamycin. Removal of the major part of the RNA associated with these fractions by EDTA treatment is accompanied by a dramatic reduction of binding capacity. We propose that endogenous RNA present in mitochondria and mitoplasts is not accessible for adriamycin at low concentrations of the drug due to the presence of an intact lipid barrier. This potential site comes to expression in ghosts and submitochondrial particles, due to the absence of an intact lipid bilayer and due to the inside-out orientation of the limiting membrane, respectively. Electron microscopical studies show that adriamycin induces dramatic changes in mitochondrial morphology, similar to the uncoupler-induced effects described by Knoll and Brdiczka (Biochim. Biophys. Acta 733, 102-110 (1983). Adriamycin has an uncoupling effect on mitochondrial respiration and oxidative phosphorylation. The concentration dependence of this effect correlates with the adriamycin-binding curve for mitochondria which implies that only bound adriamycin actively inhibits respiration.     

The role of cardiolipin in the structural organization of mitochondrial membranes

Considerable progress has recently been made in understanding the role of cardiolipin in mitochondria. In this brief review, we discuss new data that show how cardiolipin specifically contributes to the lateral organization of mitochondrial membranes. We argue that the function of cardiolipin has to be understood in the context of dynamic membrane assembly rather than static membrane structure, and we propose that remodeling of cardiolipin, i.e. the formation of uniformly substituted molecular species, may reduce the energy barrier of the assembly process.     

Changes induced by cold adaptation in the brown adipose tissue from several species of rodents, with special reference to the mitochondrial components.

(1) The effects of cold adaptation upon the brown adipose tissue have been studied in rats, hamsters, mice, and guinea pigs. (2) Striking effects were found for total tissue as well as at the mitochondrial level, e.g., increases in protein and phospholipid contents, changes in phospholipid fatty acid composition (a decrease in the percentage of palmitic and palmitoleic acids and an increase in stearic and linoleic acids), and a change in the mitochondrial polypeptide composition (a marked increase in a 32000 molecular weight polypeptide, except for hamsters). (3) In situations where animals exhibit a greatly enhanced capacity for nonshivering thermogenesis (cold adaptation for rats, mice, and guinea pigs, birth for guinea pigs, and hibernation ability for hamsters, dormice, and garden dormice), brown fat mitochondria are characterized by the occurrence of large amounts of the 32000 molecular weight polypeptide characteristic of these mitochondria.     

Studies on Tetrahymena membranes. Palmitoyl-coenzyme a desaturase, a possible key enzyme for temperature adaptation in Tetrahymena microsomes.

(1) Microsomes from a thermotolerant Tetrahymena NT-1 catalyze the conversion of palmitoyl-CoA to palmitoleate. (2) Palmitoyl-CoA desaturase enzyme requires molecular oxygen and NADH or NADPH as cofactor and its activity is inhibited by cyanide. A pH optimum range 7.0--7.3 is observed. (3) There is a clear break at 30 degrees C and a slight bend around 15 degrees C in the Arrhenius plots of palmitoyl-CoA desaturase activity. (4) After quenching from 39.5 degrees C, at 26 degrees C microsomal membranes show small particle-free areas, when examined by freeze-fracture electron microscopy, indicating the onset of phase separation. Larger smooth areas devoid of membrane-intercalated particles are observed in microsomes at 23 and 15 degrees C. The results support evidence that the thermally induced transition of desaturase enzyme activity in related to the altered membrane properties due to temperature change.     

Changes of the fluidity of mitochondrial membranes induced by the permeability transition.

The dynamic properties of protein and lipid regions of mitochondrial membranes during the permeability transition (PT) process were studied by following the anisotropy changes of hematoporphyrin (HP) and 1,6-diphenyl-1,3,5-hexatriene (DPH), respectively. We show that opening of the PT pore is accompanied by a remarkable increase of mitochondrial membrane fluidity which is specifically localized to protein sites, while lipid domains are unaffected. The increased membrane fluidity is not related to the collapse of transmembrane potential that follows the PT, as demonstrated by a comparison between the anisotropy properties of permeabilized mitochondria and impermeable, depolarized organelles. Parameters such as osmotic swelling and temperature, which are shown to affect the mitochondrial membrane dynamics in the absence of permeability transition, cannot alone account for the pore dynamical properties. We suggest that the observed increase in fluidity is mainly due to a conformational change of pore-forming protein(s) during the "assembly" of the PT pore.