Temperature-induced changes of spin-labelled radioactive lipids in isolated guinea pig liver microsomal membranes before and in mitochondrial membranes after their translocation.

Lipids of isolated guinea pig liver microsomal membranes were labelled biosynthetically with isomeric doxyl stearic acid and temperature-induced changes of these membranes were studied by electron spin resonance. A noticeable discontinuity was detected at 10--12 degree C with 12- or 16-doxyl stearic acid containing membrane lipids which was attributed to the spin-labelled lipid--microsomal membrane protein interactions since no such discontinuity was detected in liposomes prepared from total lipid extracts of microsomal membranes. When microsomal membranes containing radioactive isomeric spin-labelled lipids were incubated with unlabelled mitochondria, reisolated mitochondrial membranes contained translocated radioactive isomeric spin-labelled lipids. Temperature-induced changes in these membranes showed no discontinuity with either isomeric doxyl stearic acid derivative, establishing a difference in the environment of translocated lipids in the membrane donor compared with that in the membrane acceptor. Microsomal membranes recovered from translocation experiments showed the same behaviour as the original membranes and exhibited the same discontinuity at 10--12 degree C, establishing that the translocation incubation itself did not alter the spin-labelled lipid interaction within these membranes. Studies of the loss of paramagnetism of spin-labelled lipids in microsomal membranes before and in mitochondrial membranes after their translocation showed a significant difference and suggested that both the outer and the inner mitochondrial membranes might have been involved.     

Biogenesis of mitochondrial membranes in Neurospora crassa during cellular differentiation: changes in oxidative phosphorylation and synthesis of mitochondrial phospholipids.

Changes in the capacity of mitochondria to carry out oxidative phosphorylation and in the rate of synthesis and incorporation of phospholipids into mitochondria were measured during the germination of conidiospores of Neurospora crassa. The competence of isolated mitochondria to carry out coupled respiration was very low during the first 3 h growth, but it increased rapidly, reaching maximal levels at 5 to 6 h growth. Changes in mitochondrial function were the same in cells grown in 2% sucrose- or 15% glucose-supplemented medium. The rate of synthesis of mitochondrial phospholipids was very low during the first 2 h growth and increased to maximal levels between 3 and 5 h. The rate of synthesis of mitochondrial phospholipids was approximately three times higher in cells grown in 15% glucose than in those grown in 2% sucrose. The maximal rate of synthesis of mitochondrial phospholipids occurred during spore germination and preceded attainment of full competence for oxidative phosphorylation. The lipid-rich condition of the mitochondrial resulting from the high rate of synthesis of phospholipids in glucose-grown cells is postulated to be related to the whorled inclusions observed in thin sections of Neurospora cells.     

Selective degradation with phospholipases D and C of radioactive isomeric spin-labelled lipids bound to guinea pig liver microsomal membranes.

Membrane-bound lipids of isolated guinea pig liver microsomal membranes were selectively enzymatically labelled with isomeric (5-, 12-, and 16-)doxyl stearic acid. After reisolation, the membranes were degraded with phospholipases D and C under conditions not requiring detergents or organic solvent activators. The degradation of membrane-bound lipids occurred according to the recognized specificity of phospholipases D and C. Temperature-induced changes of degraded membranes containing radioactive spin-labelled isomeric lipids were followed by the electron spin resonance and spectral changes correlated with the lipid composition of membranes. Discontinuities in plots of experimental spectral parameters versus temperature detected in the case of microsomal membranes before and after degradation with phospholipases D and C were attributed to lipid-protein and lipid-lipid interaction(s). On the basis of these and control experiments, discontinuity at around 10-12 degrees C was attributed to the microsomal membrane phosphatidylcholine intrinsic microsomal membrane protein interaction(s), while discontinuities detected at 19-21 degrees C approximately and at 20-30 degrees C approximately were attributed to the phase separation of Ca or Zn salts of membranous phosphatidic acid and to the similar phenomenon involving membrane-bound diglycerides respectively.     

Cytosol protein-independent translocation of isomeric spin-labelled radioactive lipids from isolated guinea pig liver microsomal to mitochondrial membranes

Intermembranous translocation of membrane-bound radioactive lipids covalently labelled with 5-, 12, and 16-doxyl stearic acid was studied. Guinea pig liver microsomal membranes containing known amounts of isomeric spin-labelled radioactive phosphatidic acid, phosphatidylcholine, and diglycerides were incubated with unlabelled mitochondria; reisolated mitochondria contained around 28-31% of microsomal labelled lipids above the microsomal contamination. The effect of adding crude or 'pH 5.1' 105 000 X g cytosol supernatant on the amount and composition of translocated labelled lipids was studied. While the translocation of labelled phosphatidylcholine was slightly stimulated by the addition of these cytosol supernatants, no significant increase of the amount of translocated labelled phosphatidic acic and diglycerides was observed by this addition. In view of these results, a probable mechanism for the cytosol protein-independent translocation of lipids between biological membranes is proposed.     

Study of the transverse diffusion of spin labelled phospholipids in biological membranes. I. Human red blood cells

Spin labeled analogs of phosphatidylcholine were used to study the transverse diffusion (flip-flop) of phospholipids in the erythrocyte membrane. The nitroxide spin label was placed either on the β acyl chain or on the choline group. These labeled phosphatidylcholine molecules were incorporated into the membrane by incubation of the red cells at 22°C with sonicated spin-labeled phosphatidylcholine vesicles from which all traces of free fatty acids and lyso derivatives were carefully removed by bovine serum albumin treatment. This incorporation did not provide any change in the morphology of the cell as indicated by scanning electron microscopy. When spin-labeled phosphatidylcholine, having a nitroxide on the β chain but near the polar head-group, was incorporated into the erythrocyte membrane, ascorbate treatment at 0dgC allows selective reduction of the signal coming from the outer layer of the membrane. When the label was on the polar head-group, the inner content of the erythrocyte rapidly reduced the label facing the cytoplasm, thus creating a spontaneous anisotropy of the labeling. The anisotropic distribution of spin-labeled phosphatidylcholine in the erythrocyte membrane was found to be stable at 22 and 37°C for more than 4 h. It is therefore concluded that the rate of outside-inside and inside-outside transition is so slow that the anisotropic distribution of the phospholipids in the erythrocyte membrane can be maintained during cell life.     

Study of the transverse diffusion of spin labeled phospholipids in biological membranes. I. Human red bloods cells.

Spin labeled analogs of phosphatidylcholine were used to study the transverse diffusion (flip-flop) of phospholipids in the erythrocyte membrane. The nitroxide spin label was placed either on the beta acyl chain or on the choline group. These labeled phosphatidylcholine molecules were incorporated into the membrane by incubation of the red cells at 22 degrees C with sonicated spin-labed phosphatidylcholine vesicles from which all traces of free fatty acids and lyso derivatives were carefully removed by bovine serum albumin treatment. This incorporation did not provide any change in the morphology of the cell as indicated by scanning electron microscopy. When spin-labeled phosphatidylcholine, having a nitroxide on the beta chain but near the polar head-group, was incorporated into the erythrocyte membrane, ascorbate treatment at 0 degrees C allows selective reduction of the signal coming from the outer layer of the membrane. When the label was on the polar head-group, the inner content of the erythrocyte rapidly reduced the label facing the cytoplasm, thus creaging a spontaneous anisotropy of the labeling. The anisotropic distribution of spin-labeled phosphatidylcholine in the erythrocyte membrane was found to be stable at 22 and 37 degrees C for more than 4 h. It is therefore concluded that the rate of outside-inside and inside-outside transition is so slow that the anisotropic distribution of the phospholipids in the erythrocyte membrane can be maintained during cell life.     

Adaptation of biological membranes to temperature: molecular species compositions of phosphatidylcholine and phosphatidylethanolamine in mitochondrial and microsomal membranes of liver from thermally-acclimated rainbow trout

Summary Molecular species profiles were determined for both phosphatidylcholine (PC) and phosphatidylethanolamine (PE) of mitochondrial and microsomal membrane fractions from liver tissue of thermally-acclimated rainbow trout,Salmo gairdneri. The predominant molecular species of PC were 16:0/22:6, 16:0/18:1, 16:0/20:3 and 16:0/22:5, whereas predominant molecular species of PE were 18:1/20:4, 14:0/16:0, 18:0/22:6 and 18:1/22:6. PE possessed short chain saturates (primarily 14:0/16:0) and monoenes (primarily 14:0/16:1) not present in PC and larger proportions of polyunsaturated (18:0/22:6, 18:0/22:5 and 18:1/22:6. and diunsaturated molecular species than PC. Differences between membrane fractions were most evident in warm (20°C)-acclimated trout. Mitochondria contained higher proportions of long-chain, polyunsaturated molecular species of PE, but less of the corresponding species of PC than other membrane fractions. Rankings based on unsaturation index were accordingly: mitochondria heavy microsomes>light microsomes for PE, but heavy microsomes>light microsomes>-mitochondria for PC. Mitochondria were notable for high proportions of diunsaturated molecular species of both phosphatides. Growth at cold temperatures (5°C) was generally associated with a replacement of shorter chain mono- and dienoic molecular species (16:0/18:1, 16:1/18:1, 14:0/16:2 and 18:1/18:1 in the case of PC and 14:0/16:1, 14:0/16:2 and 16:1/18:1 for PE), and occasionally saturates, with long-chain, polyunsaturated molecular species (for PC, C_36–38: 16:0/22:6, 16:1/22:6, 16:0/20:3 and 16:0/20:5; for PE, C_38–40: 18:1/20:4, 16:1/22:6, 18:0/20:5, 18:2/20:4, 18:0/22:5 and 18:0/22:6). However, compositions of mitochondrial PE and PC from heavy microsomes were not significantly influenced by acclimation temperature. The role of phospholipase A₂, in addition to other metabolic processes, in mediating these changes is discussed.Abbreviations ACL average chain length - UI unsaturation index     

Modification of the biosynthesis and composition of polyglycerophosphatides in outer and inner mitochondrial membranes by cytidine liponucleotides

The biosynthesis of [3H]polyglycerophosphatides ([3H]phosphatidylglycerophosphate and [3H]phosphatidylglycerol) in mitochondrial and submitochondrial (outer and inner) membranes isolated from guinea pig liver was examined. Experimental results have established that the amount of biosynthesized [3H]polyglycerophosphatides and the relative amounts of biosynthesized [3H]phosphatidylglycerol and [3H]phosphatidylglycerolphosphate can be influenced by varying the composition of fatty acids in CDP-diglycerides and by altering the incubation time of the mixture containing CDP-diglycerides (obligatory precursor), sn-[2-3H]glycerol-3-phosphate and mitochondria or submitochondrial membranes. The changes thus obtained in respect to the amount and composition of biosynthesized [3H]polyglycerophosphatides are different in mitochondria and submitochondrial membranes. The highest amount of biosynthesized [3H]polyglycerophosphatides was obtained with CDP-didecanoin and inner mitochondrial membranes. The greatest accumulation of [3H]phosphatidylglycerol with CDP-didecanoin was obtained in mitochondria and outer mitochondrial membranes, while in inner mitochondrial membranes the amounts of [3H]phosphatidylglycerol and [3H]phosphatidylglycerolphosphate accumulated were approximately the same. In general, prolongation of the incubation time decreased the relative amounts of [3H]phosphatidylglycerolphosphate and increased the amount of accumulated [3H]phosphatidylglycerol, but the absolute amounts of these [3H]polyglycerophosphatides were more dependent on fatty acid composition of CDP-diglycerides tested. The following cytidine liponucleotides were tested: CDP-didecanoin, CDP-dipalmitin, CDP-diolein, and CDP-diglycerides containing saturated and unsaturated fatty acids similar to those in egg yolk lecithin. The formation of [3H]cardiolipin from [3H]phosphatidylglycerol in the presence of CDP-didecanoin and Mn2+ was found in both the outer and inner mitochondrial membranes.     

Enzymatic degradation and partial biosynthetic reconstitution of microsomal and mitochondrial membranes.

Guinea pig liver microsomal and mitochondrial membranes were degraded with phospholipase C and D followed by partial biosynthetic reconstitution. Activities of phosphatidylinositol synthetase in microsomal membranes and NADPH-cytochrome c reductase were almost completely lost after phospholipase C and D treatment; almost complete restoration of the original activity was achieved after biosynthesis of phosphatidylcholine in degraded microsomes, but was not reparable after biosynthesis of cytidinediphosphodiglycerides (CDP-diglycerides). The mitochondrial biosynthesis of polyglycerophosphatides was completely retained after degradation of these membranes with phospholipase C, but after similar treatment with phospholipase D, only about one-quarter of the original activity remained, the relative composition of polyglycerophosphatides being significantly different. The activity of NADPH-cytochrome c reductase of microsomes represented about 76% of the original activity after phospholipase C treatment, but only approximately 1% after treatment with phospholipase D. Although this activity could not be restored with CDP-diglyceride synthesis, it was restored to about 75% of the original activity after the biosynthesis of phosphatidylcholine in these fragments. These and additional experimental findings are discussed in terms of the relation between structural organization of lipids and proteins and enzymatic activities of membrane-bound phospholipid-synthesizing enzymes in microsomal and mitochondrial membranes isolated from guinea pig liver.     

Copper deficiency-induced changes in the fatty acid composition of mitochondrial and microsomal membranes of rat liver

The effects of copper deficiency on the fatty acid composition of mitochondrial and microsomal phospholipids in rat liver were studied. Copper deficiency was induced by a milk powder diet. To evaluate the effect of the milk diet on the fatty acid pattern of mitochondrial and microsomal phospholipids, one group of rats was fed Cusupplemented powdered milk. A decrease in the relative proportion of linoleic acid and an increase in the level of oleic and docosahexaenoic acids in membrane phospholipids were found in this group. However, no changes in the fatty acid pattern characteristic of essential fatty acid deficiency were observed. Dietary copper deficiency produced a significant decrease in the relative amounts of linoleic and arachidonic acids, as well as an increase in the docosahexaenoic acid content in both mitochondrial and microsomal membranes compared to the nondeficient controls. The disproportionate quantities of polyunsaturated fatty acids are discussed with a view to the disturbances of membrane function in copper deficiency.     

Age-dependent changes in rat liver microsomal and mitochondrial NADPH-dependent lipid peroxidation.

Purified outer membrane proteins O-8 and O-9 were able to bind to the peptidoglycan sacculi in sodium dodecyl sulfate solution. Binding was stimulated by lipopolysaccharide, that of protein O-9 being stimulated more remarkably. Proteins which had been heated in sodium dodecyl sulfate solution did not bind to the peptidoglycan sacculi even in the presence of lipopolysaccharide, while heated lipopolysaccharide stimulated the binding of non-heated proteins. The removal by pronase of the lipoprotein covalently bound to the peptidoglycan sacculi did not change the protein binding ability of the sacculi.     

A new approach to the study of phospholipid-protein interactions in biological membranes. Synthesis of fatty acids and phospholipids containing photosensitive groups.

In a general approach to the study, in vivo and in vitro, of the interactions between phospholipids and proteins in biological membranes, a variety of fatty acids containing photosensitive groups in different positions in the alkyl chains has been synthesized. The fatty acids synthesized include: 16-azidopalmitelaidic acid, 12-azidooleic acid, 6-, 9-, 11-, and 12-azidostearic acid, 12-omicron-(ethyl-2-diazomalonyl)stearic and -oleic acids, 12-omicron-(4-azido-2-nitrophenyl)stearic and -oleic acids, and 12-oxo-10-octadecenoic acid. Some of the above synthetic fatty acids were also prepared in the radioactively labeled form. For in vitro studies, many of the above fatty acids were used to acylate the 2 position in the preparation of a number of mixed acylphosphatidylcholines and mixed acylphosphatidylethanolamines. On sonication, the synthetic phospholipids formed sealed vesicles. Intermolecular cross-linking of the fatty acyl chains in phospholipids was demonstrated on photolysis of the vesicles.