Investigation of lipid-exchange lipoproteins from rat liver and hepatoma 27]

A phospholipid exchange lipoprotein from the postmicrosomal supernatant of rat hepatoma 27, which stimulated in vitro the exchange of sphingomyelin between mitochondria and microsomes, was found. Sphingomyelin is incorporated into the mitochondria under incubation of this complex with rat liver mitochondria (in which sphingomyelin is absent) an microsomes. Under the same conditions the phospholipid exchange lipoproteins of rat liver do not transfer sphingomyelin form microsomes to mitochrondria.     

A universal lipid exchange protein from rat hepatoma.

The postmicrosomal protein fraction from rat hepatoma 27 adjusted to pH 5.1 stimulates phospholipid exchange between rat liver microsomes and mitochondria with higher rates and in a less specific way than the corresponding fraction from rat liver. A phospholipid exchange protein has been purified to homogeneity from the hepatoma pH-5.1 supernatant by gel filtration on Sephadex G-75 and ion-exchange chromatography on carboxymethylcellulose. The isolated protein had a molecular weight of 11200 as determined by electrophoresis on polyacrylamide in the presence of dodecyl sulfate and of 11168 as calculated from the amino acid composition. Isoelectric focusing showed a single band at pH 5.2. in the assay system rat liver microsomes leads to mitochondria the protein exhibits a complete lack of substrate specificity transferring all the major microsomal phospholipids to about the same extent. The possible role of the isolated phospholipid exchange protein in the chemical dedifferentiation of hepatoma cell membranes is discussed.     

Fatty acid composition and unsaturated of intracellular membrane phospholipids from rat liver and hepatoma 27]

The fatty acid composition of phospholipids of mitochondria and microsomes from rat liver and hepatoma 27 was investigated. Basing on the fatty acid and phospholipid composition the unsaturation of the lipid bilayer of the intracellular membranes was calculated. The unsaturation of the phospholipids of the hepatoma mitochondria and microsomes was found to be much lower than that of the corresponding rat liver membranes. The lipid bilayer of the rat liver and hepatoma plasma membranes was shown to be more saturated than that of the intracellular membranes.     

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.     

Abnormal membrane phospholipid content in subcellular fractions from the Morris 7777 hepatoma.

1. Mitochondrial and microsomal fractions were prepared from normal rat liver and the Morris 7777 hepatoma and characterized by the use of the marker enzymes, succinate dehydrogenase and rotenone-insensitive NADPH-cytochrome c reductase. 2. The phospholipid content per mg membrane protein of Morris 7777 hepatoma mitochondria was increased by 75% as compared with mitochondria from normal rat liver. Microsomes from this poorly-differentiated tumor were found to have a 45% decrease in the content of phospholipid. These abnormalities were independent of tumor size or age. 3. The percent phospholipid content of the subcellular fractions was determined, and revealed an increase in the percent sphingomyelin in both the microsomal and mitochondrial fractions of the tumor. Decreases in the percent phosphatidylcholine and phosphatidylethanolamine were noted in tumor microsomes as compared with normal liver. Diphosphatidylglycerol was not found in significant quantities in the microsomal fraction of this hepatoma line. 4. The content of the various phospholipid classes per mg protein in the respective mitochondrial and microsomal fractions was determined. Large increases in nearly all the major phospholipid classes were found in tumor mitochondria; tumor microsomes were characterized by an increased content of sphingomyelin but the content of nearly all other phospholipids was significantly decreased. These findings suggest the presence of disturbances in the regulation of phospholipid metabolism in subcellular organelle membranes of the Morris 7777 hepatoma.     

Phospholipid exchange reactions within the liver cell

1. Isolated rat liver mitochondria do not synthesize labelled phosphatidylcholine from CDP-[(14)C]choline or any phospholipid other than phosphatidic acid from [(32)P]phosphate. The minimal labelling of phosphatidylcholine and other phosphoglycerides can be attributed to microsomal contamination. However, when mitochondria and microsomes are incubated together with [(32)P]phosphate, the phosphatidylcholine, phosphatidylinositol and phosphatidylethanolamine of the reisolated mitochondria become labelled, suggesting a transfer of phospholipids between the two fractions. 2. When liver microsomes or mitochondria containing labelled phosphatidylcholine are independently incubated with the opposite un-labelled fraction, there is a substantial and rapid exchange of the phospholipid between the two membranes. Exchange of phosphatidylinositol also occurs rapidly, whereas phosphatidylethanolamine and phosphatidic acid exchange only slowly. There is no corresponding transfer of marker enzymes. The transfer of phosphatidylcholine does not occur at 0 degrees , and there is no requirement for added substrate, ATP or Mg(2+), but the omission of a heat-labile supernatant fraction markedly decreases the exchange. 3. After intravenous injection of [(32)P]phosphate, short-period labelling experiments of the individual phospholipids of rat liver microsomes and mitochondria in vivo give no evidence for a similar exchange process. However, the incubation of isolated microsomes and mitochondria with [(32)P]phosphate also fails on reisolation of the fractions to demonstrate a precursor-product relationship between the individual phospholipids of the two membranes. 4. The intraperitoneal injection of [(32)P]phosphate results in a far greater proportion of the dose entering the liver than does intravenous administration. After intraperitoneal administration of [(32)P]phosphate the specific radioactivities of the individual phospholipids are in the order microsomes > outer mitochondrial membrane > inner mitochondrial membrane. 5. The incorporation of (32)P into cardiolipin is very slow both in vivo and in vitro. After labelling in vivo the radioactivity in the cardiolipin persists compared with that of the other phospholipids, whose specific radioactivities in the microsomes and mitochondrial fragments decay at a similar rate to that of the acid-soluble phosphate pool. 6. The possibility of phospholipid exchange processes occurring in the liver cell in vivo is discussed, and it is suggested that only a small but highly labelled part of the endoplasmic-reticulum lipoprotein pool is involved in the transfer.     

Sterol carrier and lipid transfer proteins

The discovery of the sterol carrier and lipid transfer proteins was largely a result of the findings that cells contained cytosolic factors which were required either for the microsomal synthesis of cholesterol or which could accelerate the transfer or exchange of phospholipids between membrane preparations. There are two sterol carrier proteins present in rat liver cytosol. Sterol carrier protein 1 (SCP1) (Mr 47 000) participates in the microsomal conversion of squalene to lanosterol, and sterol carrier protein 2 (SCP2) (Mr 13 500) participates in the microsomal conversion of lanosterol to cholesterol. In addition SCP2 also markedly stimulates the esterification of cholesterol by rat liver microsomes, as well as the conversion of cholesterol to 7 alpha-hydroxycholesterol - the major regulatory step in bile acid formation. Also, SCP2 is required for the intracellular transfer of cholesterol from adrenal cytoplasmic lipid inclusion droplets to mitochondria for steroid hormone production, as well as cholesterol transfer from the outer to the inner mitochondrial membrane. SCP2 is identical to the non-specific phospholipid exchange protein. While SCP2 is capable of phospholipid exchange between artificial donors/acceptors, e.g. liposomes and microsomes, it does not enhance the release of lipids other than unesterified cholesterol from natural donors/acceptors, e.g. adrenal lipid inclusion droplets, and will not enhance exchange of labeled phosphatidylcholine between lipid droplets and mitochondria. Careful comparison of SCP2 and fatty acid binding protein (FABP) using six different assay procedures demonstrates separate and distinct physiological functions for each protein, with SCP2 participating in reactions involving sterols and FABP participating in reactions involving fatty acid binding and/or transport. Furthermore, there is no overlap in substrate specificities, i.e. FABP does not possess sterol carrier protein activity and SCP2 does not specifically bind or transport fatty acid. The results described in the present review support the concept that intracellular lipid transfer is a highly specific process, far more substrate-specific than suggested by the earlier studies conducted using liposomal techniques.     

A phosphatidyl choline exchange protein isolated from germinated castor bean endosperms

A phospholipid exchange protein (PLEP) functioning between theendoplasmic reticulum and the mitochondrion was purified fromthe cytosolic fraction of germinated castor bean endosperms.In the protein fraction eluted from Sephadex G-100 column, theexchange rate reached 7.3µg phospholipids exchanged/mgprotein/15 min, which was 60-fold that of pota to tuber PLEP.The lipid transfer by this protein was specific for phosphatidylcholine and the transfer rate from microsomes to mitochondriawas as high as that from mitochondria to microsomes. Castorbean PLEP transferred phospholipid from castor bean microsomesto mitochondria from other sources such as potato tubers, cauliflowerinflorescences, pumpkin hypocotyls and rat livers, and to liposomes,but not to Avena etioplasts. In addition, it transferred phospholipidfrom potato microsomes to potato mitochondria. (Received November 17, 1978; )     

Lipid metabolism and structure-activity features of the endoplasmic reticulum membrane in regenerating rat liver]

The relationship between the neutral lipid and phospholipid metabolism and some structure-function peculiarities of regenerating rat liver endoplasmic reticulum membranes (13 hours after surgery, i.e., corresponding to the G1-period of the cell cycle) was studied. There was an increase in the degree of the endoplasmic reticulum membrane development and the nonesterified fatty acid (NFA) and triglyceride (TG) content in regenerating rat liver microsomes. The relative specific radioactivity of neutral lipid and phospholipid fractions in regenerating rat liver microsomes was lower than in control animals, presumably due to the high rate of the microsomal lipid exchange in the regenerating liver with other cell organelles. The changes in the lipid content and rate of their metabolism in the regenerating rat liver were associated with the increase in the membrane microviscosity and the decrease in the activity of the membrane-bound enzyme (glucose-6-phosphatase). The differences in the time-dependent changes in the synthesis and metabolism of lipids in the NFA and TG fractions may be regarded as an endogenous factor determining the structure-function peculiarities of endoplasmic reticulum membranes.     

Non-enzymatic lipid peroxidation of microsomes and mitochondria isolated from liver and heart of pigeon and rat

Studies were carried out to determine the level of ascorbate-Fe2+ dependent lipid peroxidation of mitochondria and microsomes isolated from liver and heart of rat and pigeon. Measurements of chemiluminescence indicate that the lipid peroxidation process was more effective in mitochondria and microsomes from rat liver than in the same organelles obtained from pigeon. In both mitochondria and microsomes from liver of both species a significant decrease of arachidonic acid was observed during peroxidation. The rate C18:2 n6/C20:4 n6 was 4.5 times higher in pigeon than in rat liver. This observation can explain the differences noted when light emission and unsaturation index of both species were analysed. A significant decrease of C18:2 n6 and C20:4 n6 in pigeon liver mitochondria was observed when compared with native organelles whereas in pigeon liver microsomes only C20:4 n6 diminished. In rat liver mitochondria only arachidonic acid C20:4 n6 showed a significant decrease whereas in rat liver microsomes C20:4 n6 and C22:6 n3 decreased significantly. However changes were not observed in the fatty acid profile of mitochondria and microsomes isolated from pigeon heart. In the heart under our peroxidation conditions the fatty acid profile does not appear to be responsible for the different susceptibility to the lipid peroxidation process. The lack of a relationship between fatty acid unsaturation and sensitivity to peroxidation observed in heart suggest that other factor/s may be involved in the protection to lipid peroxidation in microsomes and mitochondria isolated from heart.     

In vitro incorporation of ( 3 H) methyl choline into phosphatidyl choline of rat liver subcellular fractions

Incubation of a rat liver total homogenate with radioactive choline and subsequent isolation of subcellular fractions, at different times, showed similar patterns of labeling. Incubation of microsomes, mitochondria and purified nuclei isolated from rat liver, showed that all fractions were able to incorporate the precursor into phosphatidyl choline. The specific activity was higher in mitochondria and increased in all cases with added supernatant. The addition of microsomes to mitochondria diminished the incorporation of label. Contamination of mitochondria by microsomes, was negligible as shown by undetectable amounts of cytochrome P₄₅₀, while NADPH₂ cytochrome c reductase showed a 10% contamination. A certain amount of radioactivity was incorporated in the absence of ATP and oxidizable substrates due to the presence of substrates and cofactors in the fraction and/or the supernatant. Labeled fractions reincubated with unlabeled choline, showed no loss of radioactivity, proving that incorporation was not due to simple exchange processes. It is concluded that although rat liver mitochondria can acquire part of their own provision of phosphatidyl choline by transference from microsomes, all organelles and specially mitochondria, can independently synthesize this phospholipid.     

Phospholipid composition of mitochondria and microsomes from some transplantable rat tumors

The phospholipid compositions of mitochondria and microsomes from rat liver, kidney, nephroma RA and sarcoma 45 were investigated. The "lipid dedifferentiation" of hepatoma membranes found earlier was shown to extend also to other tumours. However, this phenomenon may concern some, but not all phospholipids.     

Effect of HDL1 infusion on biliary secretion in perfused rat liver

The effects of HDL1 lipoprotein infusion on biliary lipid secretion were studied in thein vitro model of rat perfused liver. A strong increase in bile flow was observed during and after lipoprotein infusion. This caused a significant rise in cholesterol, phospholipid and bile salt secretions. However, only the percentage of cholesterol increased with respect to the other bile lipids. The changes observed in the cholesterol/phospholipid molar ratio values of liver membrane subfractions (i.e., liver plasma membrane, mitochondria plus lysosomes and microsomes) isolated from the perfused rat liver after HDL1 administration were not significant.     

Cholesterol exchange between microsomal, mitochondrial and erythrocyte membranes and its enhancement by cytosol.

1. Cholesterol exchanges between isolated rat liver microsomes and mitochondria and between erythrocytes and microsomes or mitochondria during incubation in vitro. The exchange process is temperature dependent and is no accompanied by a net movement of sterol. 2. cholesterol exchange between the membranes was enhanced by the addition of 105 000 x g supernatant fraction (S105) from rat liver. The degree to which sterol exchange was enhanced was dependent on the amount of this supernatant fraction present in the incubation. 3. enhancement of sterol exchange was not observed with heated S105 fraction, but activity was retained after dialysis or aging at 10 degrees C; these results suggest the presence of a cholesterol-exchange protein in the cytosol from rat liver.     

Lipid dependence of the pyrophosphatase activity of microsomes from rat liver and hepatoma

The lipid dependence of the pyrophosphatase activity of microsomes from rat liver and hepatoma was studied. Two methods were used for modification of the lipid composition of the microsomes: delipidation with organic solvents followed by relipidation with phospholipid vesicles and transformation of the microsomal lipid composition by lipid exchange proteins. In contrast to glucose 6-phosphatase, microsomal pyrophosphatase activity was found to be insensitive to modification of the membrane lipid composition by the above method. Possible causes of the different lipid dependence of various activities of microsomal glucose 6-phosphatase are discussed.     

Topographical Distribution of Base Exchange Activities in Rat Brain Subcellular Fractions

Abstract: Enrichment in the base-exchange activities was found in the micro-somal fraction of rat brain, with less activity being associated with nuclei, mitochondria and synaptosomes. The distribution of the choline base exchange in microsomal subfractions differed from that for serine and ethanolamine and these three activities seemed asymmetrically distributed in the microsomes. Choline exchange activity was trypsin-sensitive and presumably was located on the cytoplasmic side of the microsomes, while serine and ethanolamine exchange activities were trypsin-insensitive and were assumed to be located on the luminal side of the microsomes. Treatment of rat brain microsomes with phospholipases A, C and D produced significant losses of membrane-bound base exchange activities. Some activity was restored in phospholipase C-treated microsomes by exogenous phospholipid, but significant restoration was not observed in phospholipase A-treated microsomes by such additions. Exogenous phospholipid stimulated choline and ethanolamine exchange activities, but not serine exchange activity of phospholipase D-treated microsomes. The exchange activities of rat brain microsomes differed in their responses to treatment with phospholipases, choline exchange activity in general being more sensitive than either serine or ethanolamine activities.     

Some properties of phosphatidylcholine exchange protein purified from beef liver

A phospholipid exchange protein has been purified 2680-fold from beef liver. The assay of the exchange activity of the protein was based on the transfer of [¹⁴C]phosphatidylcholine from microsomes labeled with [¹⁴C]phosphatidylcholine to liposomes. The homogeneity of the protein has been established by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoelectrophoresis and isoelectric focusing. The protein has a molecular weight of approximately 22000 and an isoelectric point of 5.8. The amino acid composition has been determined. The protein contains one disulfide bridge and has glutamic acid as the N-terminal amino acid. Phospholipid, tentatively identified as phosphatidylcholine, was found to be present in the protein preparation. The protein stimulated specifically the exchange of phosphatidylcholine between mitochondria and microsomes from rat liver.     

Lipid composition of the Golgi apparatus of rat kidney and liver in comparison with other subcellular organelles.

Golgi apparatus isolated from both rat liver and rat kidney have been characterized with respect to their neutral and phospholipid content and their phosphopipid composition and compared with mitochondria, rough endoplasmic reticulum and plasma membranes. In addition, the distribution of sulfatide in the subcellular fractions of rat kidney was determinich are rich in cholesterol esters and ubiquinone. Removal of about 75% of the cisternal contents of rat liver Golgi reduced its content of cholesterol esters but not of ubiquinone. The Golgi complex of liver most closely resembles endoplasmic reticulum in its phospholipid composition except for a higher content of sphingomyelin. Removal of most of the contents of the Golgi cisternae did not appreciably alter the phospholipid composition of the Golgi apparatus of liver. Goligi apparatus from kidney has a phospholipid composition which resembles liver Golgi much more closely than it does any other cell fraction from kidney. The sulfatide content of kidney Golgi, the cell fraction richest in this glycolipid, is about 14% of the total lipid present in this fraction. Sulfatide was present in plasma membranes, mitochondria and rough microsomes, but at about one-third the level found in Golgi. Sulfatide is the main glycosphingolipid present in all the cell fractions from kidney which were studied.     

Effect of chronic ethanol treatment on the t-butyl hydroperoxide-dependent lipid peroxidation in rat liver

1. The effect of chronic ethanol consumption on the level of the t-butyl hydroperoxide (Bu'OOH)-induced lipid peroxidation in rat liver homogenate and subcellular fractions was measured using chemiluminescence technique and malondialdehyde formation. 2. It was shown that under the action of ethanol the rate of lipid peroxidation was decreased in the whole and "postnuclear" liver homogenates. 3. Ethanol significantly decreased the intensity of lipid peroxidation in microsomes, but did not affect the Bu'OOH-dependent process in mitochondria. 4. The level of lipid peroxidation was reduced after incubation of the total particulate fraction (mitochondria plus microsomes) with the undialysed cytosol from ethanol-treated rat liver. Dialysis of the cytosol prevented depressive effect of ethanol treatment on lipid peroxidation. 5. Reduced glutathione (0.1-1.0 mM) was shown to decrease the rate of lipid peroxidation in rat liver microsomes, but did not affect its level in mitochondria. 6. Pyrazole injections to rats reduced and phenobarbital treatment increased the level of the Bu'OOH-dependent lipid peroxidation in liver microsomes. 7. The data obtained indicate that the Bu'OOH-dependent lipid peroxidation is not an appropriate marker of the ethanol-induced oxidative stress in rat liver cells.     

Extensive exchange of rat liver microsomal phospholipids

Liver microsomal fractions were prepared from rats injected with a single dose of choline [¹⁴C] methylchloride or with single or multiple doses of ³²P_i. Exchangeability of microsomal phospholipids was determined by incubation with an excess of mitochondria and phospholipid exchange proteins derived from beef heart, beef liver or rat liver. Labeled phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine and phosphatidylinositol were found to act as a single pool and were 85–95% exchangeable in 1–2 h. High latencies of mannose-6-phosphate phosphohydrolase activities and impermeability of microsomes to EDTA proved that phospholipid exchange proteins did not have access to the intracisternal space. If microsomal membranes are largely composed of phospholipid bilayers, the experiments suggest that one or more of the phospholipid classes in microsomal membranes undergo rapid translocation between the inner and outer portions of the bilayer.