Non-protein-mediated transfer of phosphatidic acid between microsomal and mitochondrial membranes

The transfer of phosphatidic acid between rat liver microsomes loaded with [32P]-phosphatidic acid and rat liver mitochondria was studied in the absence of added lipid transfer proteins. It was found that during 1 h at 37 degrees C in the medium containing 100 mM KCl, 20-30% of phosphatidic acid but only 2.5% of phosphatidylcholine were transferred. This spontaneous transfer of phosphatidic acid remained the same after pretreatment of microsomes and mitochondria with 125 mM KCl or microsomes alone with 1 mM Tris, pH 8.6, procedures reported to remove adsorbed lipid transfer proteins. This transfer was insensitive to thiol-blocking reagents. The initial rate of this non-protein-mediated transfer of phosphatidic acid was virtually independent of the concentration of the acceptor membranes (mitochondria), thus indicating that it occurs by diffusion of the phospholipid through the aqueous phase rather than by membrane collision. About 80% of phosphatidic acid synthesized in the outer mitochondrial membrane was recovered in the inner membrane after a 1-h incubation, pointing to a high rate of the intermembrane transfer of this phospholipid within intact mitochondrion.     

Effect of liposomal phospholipid composition on cholesterol transfer between microsomal and liposomal vesicles.

Preincubation of rat liver microsomal vesicles at 37 degrees C in the presence of [3H]cholesterol/phospholipid liposomes results in a net transfer of cholesterol from liposomes to microsomal vesicles. This transfer follows first-order kinetics. For similar concentrations of the donor vesicles, rates of transfer are about 6-8 times lower with cholesterol/sphingomyelin liposomes compared with cholesterol/phosphatidylcholine liposomes. Also, transfer of cholesterol from cholesterol/sphingomyelin liposomes to microsomal vesicles reveals a larger activation energy than for the process from cholesterol/phosphatidylcholine liposomes. There is a significant correlation between the amount of liposomal cholesterol transferred to microsomal vesicles during preincubation and the increase found with acyl-CoA:cholesterol acyltransferase activity in these microsomes over their corresponding controls. If, however, liposomes made solely of phospholipids are substituted for the cholesterol/phospholipid liposomes in the preincubation system containing microsomal vesicles, then the acyl-CoA:cholesterol acyltransferase activity is decreased compared with the corresponding control system. Both sphingomyelin and phosphatidylcholine liposomes are equally effective in decreasing the enzyme activity. These results offer direct kinetic evidence for the positive correlation between cholesterol and sphingomyelin found in vivo in biological membranes.     

Sedimentation field flow fractionation of mitochondrial and microsomal membranes from corn roots

Sedimentation field flow fractionation (sed-FFF) is shown to be a valuable procedure for analysis of a wide variety of subcellular particle preparations. The principles underlying this relatively new separation procedure are described. Separation is based on differences between particles in mass and/or density. As in chromatography, the procedure involves relating on-line or off-line measurements made on the effluent from the separation chamber to the elution (retention) time. In this work effluents were monitored for absorbance at 254, 280, and/or 320 nm; collected fractions were assayed for protein content, total ATPase activity, and/or marker enzyme activities and, when appropriate, were examined by electron microscopy. The ratio of the absorbances at 254 and 320 nm was found to provide a sensitive measure of partial resolution of subcellular particles. Preparations containing all of the subcellular particles of corn roots (exclusive of nuclei, cell walls, and ribosomes), and fractions thereof enriched in mitochondria, microsomes, Golgi membranes, or plasma membranes, were examined by sed-FFF. The subcellular particles appear to remain largely intact. All of the particles observed had a mass less than 2 X 10(11) g/mol. All of the preparations were grossly heterogeneous with respect to effective mass distribution. This is due in part to heterogeneity with respect to the organelle of origin. In microsome preparations, components of low, medium, and high density were present in the unretained peak; the retained region had comparatively more high density particles. Plasma membrane preparations had a very wide effective particle mass distribution. The observations suggest that, in addition to its utility for analytic purposes, sed-FFF is likely to prove useful for micro-preparative fractionation of some subcellular particle preparations. Sed-FFF and density gradient centrifugation can be utilized as complementary methods.     

Kinetics of ganglioside transfer between liposomal and synaptosomal membranes

The transfer of ganglioside GM1 from micelles to membranes and between different membrane populations has been examined by using a pyrene fatty acid derivative of the ganglioside. The transfer of gangliosides from micelles to membranes depends on the physical state as well as the molecular composition of the acceptor vesicles. At 30 degrees C, the transfer of micellar gangliosides to dipalmitoylphosphatidylcholine (DPPC) large unilameller vesicles (Tm = 41.3 degrees C) is characterized by a rate constant of 0.01 min-1; at 48 degrees C, however, the rate constant is 0.11 min-1. Below the phase transition temperature, the activation energy is 25 kcal/mol whereas above the phase transition it is 17 kcal/mol. Similar experiments performed with synaptic plasma membranes yielded a rate constant of 0.05 min-1 at 37 degrees C. The rate of transfer of ganglioside molecules, asymmetrically located on the outer layer of donor vesicles, to acceptor vesicles lacking ganglioside depends on the physical state of both the donor and acceptor vesicles. For the transfer of ganglioside from DPPC (donor) vesicles to dimyristoylphosphatidylcholine (DMPC) (acceptor) vesicles, the rates were essentially zero at 15 degrees C in which both vesicle populations were in the gel phase, 0.008 min-1 at 30 degrees C in which DPPC is in the gel phase and DMPC is in the fluid phase, and 0.031 min-1 at 48 degrees C in which both vesicle populations are in the fluid phase. The transfer of ganglioside from DPPC vesicles to synaptic plasma membranes was also dependent on the physical state of the donor vesicles and showed an inflection point at the phase transition temperature of DPPC.(ABSTRACT TRUNCATED AT 250 WORDS)     

Electrostatic interaction between merocyanine 540 and liposomal and mitochondrial membranes

Summary The fluorescence of merocyanine 540 (MC) in liposomal and mitochondrial suspensions was measured under various conditions. Under a given condition, both the amount of dye bound to the membrane and the zeta potential were determined simultaneously. It was found that the fluorescence intensity was proportional to the amount of bound dye and correlated with the zeta potential of particles. The fluorescence intensity was represented quantitatively in terms of the Langmuir adsorption isotherm, when the electrostatic interaction acting between MC and membrane surface was properly taken into account. It was concluded that the changes in MC fluorescence in the liposomal and mitochondrial suspensions are mainly attributed to the changes in the surface potential of the membranes.     

Cholesterol transfer from mitochondrial membranes and cells to human and rat serum lipoprotein fractions

We have investigated the transfer of [14C]cholesterol from labeled bovine heart mitochondria and Friend erythroleukemic cells to high density lipoprotein (HDL), low density lipoprotein (LDL), and very low density lipoprotein (VLDL) fractions from human and rat plasma. The lipoprotein fractions were obtained by molecular sieve chromatography of plasma on agarose A-5m columns. For either membrane system, the highest rate of [14C]cholesterol transfer was observed with the human and the rat HDL fraction. Since the mitochondria lack the receptors for HDL, one may conclude that the observed preferential transfer is not governed by a receptor-controlled interaction of HDL with the membrane. Under conditions where the pool of free cholesterol in the lipoprotein fractions was the same, HDL was a much more efficient acceptor of [14C]cholesterol from mitochondria than LDL or VLDL. Similarly, transfer of [14C]cholesterol proceeded at a higher rate to HDL than to sonicated egg phosphatidylcholine (PC) vesicles, even under conditions where there was a tenfold excess of the vesicle-PC pool over the HDL phospholipid pool. This preferred transfer of [14C]cholesterol to HDL cannot be explained by a random diffusion of monomer cholesterol molecules. Rather, it shows that HDL has a specific effect on this process in the sense that it most likely enhances the efflux of cholesterol from the membrane. Treatment of HDL with trypsin reduced the rate of [14C]cholesterol transfer by 40-50%, indicating that protein component(s) are involved. One of these components appears to be apoA-I, as this protein was shown to enhance the transfer of [14C]cholesterol from mitochondria to lipid vesicles.     

Calcium and lipid peroxidation in the heart mitochondrial and microsomal membranes

Effect of the lipid peroxidation (LP) on the Ca2+-transport and the effect of different Ca2+-concentrations on the LP activation were studied in microsomes and mitochondria of the heart. A slight accumulation of LP-products in the microsomal fraction results in a complete inhibition of the membrane calcium-transport activity. Preliminary administration of antioxidants (4-methyl 2,6-ditretbutylphenol and alpha-tocopherol) prevents both the accumulation of LP-products and damage of the Ca2+-transport system. Calcium at 10(-6) M to 5 X 10(-5) M concentrations stimulates LP and while being increased to 2 X 10(-3) M it inhibits LP. The data obtained evidence an interrelation between alterations of the Ca2+-concentrations and LP activation in cardiomyocytes.     

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.     

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.     

Transfer of phosphatidic acid between microsomal and mitochondrial outer and inner membranes

A protein fraction from rat liver cytoplasm, precipitable at 50-95% saturation of ammonium sulphate, binds phosphatidic acid from mitochondrial and microsomal membranes. Protein-bound phosphatidic acid was eluted from Sephadex G-75 in fractions corresponding to a molecular weight of about 10 000. No such binding was observed with mitochondrial soluble proteins, either total or precipitated with ammonium sulphate between 50 and 95% saturation. The transfer of phosphatidic acid from microsomes to mitochondria was increased by liver cytoplasmic proteins precipitable at 50-95% saturation of ammonium sulphate but not with mitochondrial soluble proteins. This increase by cytoplasmic proteins was pronounced in 200 mM sucrose but was negligible in 100 mM KCI where the spontaneous transfer was quite high. Cytoplasmic proteins stimulated the synthesis of cardiolipin and phosphatidylglycerol in mitochondria deprived of the outer membrane but not in intact mitochondria when phosphatidic acid was supplied either by microsomes or liposomes. It is suggested that the transfer of phosphatidic acid from the outer to the inner mitochondrial membrane is not mediated by transfer proteins but occurs either by direct contact of the membranes or as free diffusion through the aqueous phase.     

Biogenesis of microsomal membrane glycoproteins in rat liver. III. Release of glycoproteins from the Golgi fraction and their transfer to microsomal membranes

The presence in the Golgi fraction of glycoproteins destined to be incorporated into the microsomal membrane was investigated. When incubated in sucrose, washed Golgi vesicles released four major, weakly acidic glycoproteins, some of which could be incorporated into microsomal membranes by incubation. Double labeling with [3H]glucosamine and [14C]leucine demonstrated the incorporation of both protein and oligosaccharide moieties, and the main peak of radioactivity was associated with the 70,000 mol wt region after SDS-gel electrophoresis. The proteins that could be incorporated into microsomes were probably associated to a large extent with the outer surface of the Golgi membrane. Centrifugation of the proteins released from the Golgi in a KBr solution (p = 1.24) resulted in a separation of glycoproteins, those in the top layer most actively incorporated into microsomes. The lipoglycoproteins in the top layer that could be incorporated appeared in the 70,000 mol wt region after SDS-gel electrophoresis, as did the corresponding proteins isolated from the supernate. These results suggest that glycoproteins with completed oligosaccharide chains are released from the Golgi system to the cytosol and are subsequently transferred to microsomes as constitutive membrane components.     

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     

Ethanol sensitivity and fatty acid composition of chick liver mitochondrial and microsomal membranes

Fatty acid composition of hepatic mitochondrial and microsomal membranes was studied in 2-day-old chicks exposed to ethanol for 60 h (short treatment) or 18 days (chronic treatment). Short ethanol treatment induced in mitochondria an increase in the 18:1/18:0 ratio as a consequence of both an increase in the percentage of oleic and a decrease in that of stearic acid. Likewise, a clear decrease in the polyunsaturated fatty acids and in the 20:4/18:2 ratio was found in mitochondria after short ethanol administration. Microsomal membranes were practically unaffected by this treatment. However, chronic ethanol exposure produced a significant increase in the percentages of polyunsaturated fatty acids in both mitochondria and microsomes as well as a decrease in the 18:1/18:0 ratio. These results suggest that delta 9 desaturase modifies its activity in response to ethanol treatment with a different pattern to those showed by delta 6 and delta 5 desaturase activities.     

Lipase catalysed isomerisation of 1,2-(2,3)-diglyceride into 1,3-diglyceride. The crucial role of water

Summary Enzyme catalyzed isomerisation of 1,2-dipalmitin into the 1,3-isomer is shown to occur with 1,3 regiospecific lipases. The mechanism of this transformation is elucidated and the crucial role of the water still present in the enzyme preparation is shown. Thus, the first step is the hydrolysis of the dipalmitin followed by the isomerisation of the 2-monopalmitin into the 1-monoester. The last is then esterified into the 1,3-dipalmitin.