Changes in the triacylglycerol content of mitochondrial membranes during development

The lipid content of mitochondria and mitochondrial membranes isolated from foetal, suckling, and adult rat liver mitochondria were compared. In foetal liver mitochondria triacylglycerol made up 26% of the lipids, while in adult rat liver mitochondria the triacylglycerol content was 7%. Esterified fatty acids originating from non-phospholipid sources amounted to 34% in mitochondrial membranes, and this amount decreased in mitochondrial membranes of adult rat liver to 22%. The concentration of phospholipids in the mitochondrial membranes did not change significantly during development.     

BIOGENESIS OF ENDOPLASMIC RETICULUM MEMBRANES : II. Synthesis of Constitutive Microsomal Enzymes in Developing Rat Hepatocyte

The constitutive enzymes of microsomal membranes were investigated during a period of rapid ER development (from 3 days before to 8 days after birth) in rat hepatocytes. The activities studied (electron transport enzymes and phosphatases) appear at different times and increase at different rates. The increase in the enzyme activities tested was inhibited by Actinomycin D and puromycin. G-6-Pase and NADPH-cytochrome c reductase activities appeared first in the rough microsomes, and subsequently in smooth microsomes, eventually reaching a uniform concentration as in adult liver. The evidence suggests that the enzymes are synthesized in the rough part, then transferred to the smooth part, of the ER. Changes in the fat supplement of the maternal diet brought about changes in the fatty acid composition of microsomal phospholipids but did not influence the enzymic pattern of the suckling. Microsomes from 8-day-old and adult rats lose 95% of PLP and 80% of NADH-cytochrome c reductase activity after acetone-H₂O (10:1) extraction. However, one-half the original activity could be regained by adding back phospholipid micelles prepared from purified phospholipid, or from lipid extracts of heart mitochondria, or of liver microsomes of 8-day or adult rats, thus demonstrating an activation of the enzyme by nonspecific phospholipid. The results suggest that during development the enzymic pattern is not influenced by the fatty acid or phospholipid composition of ER membranes.     

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.     

Effect of different levels of thyroid hormones on the fatty acid composition of lipids from rat liver mitochondria

The abundance or deficiency of thyroid hormones in rat organism influence the unsaturation and desaturation indices of total lipid fatty acids and phospholipids in liver mitochondria. The most conspicuous changes were observed in the fatty acid composition of the phospholipid fraction. The changes in the structure and function of rat liver mitochondria are considered to be due to alterations in the fatty acid composition of mitochondrial phospholipids.     

Fatty acid composition of phospholipids in mitochondria and microsomes during diethylnitrosamine carcinogenesis in rat liver

Changes in lipid composition and function of subcellular organelles have been described in transplanted and primary tumours. We examine here the fatty acid composition of individual phospholipids (PL) in hyperplastic nodules and primary hepatoma induced by diethylnitrosamine (DEN), compared to that of normal liver and of transplantable Yoshida AH-130 hepatoma. Phosphatidylcholine and phosphatidylethanolamine fatty acid composition in mitochondria and microsomes from primary hepatoma were markedly different from normal liver; C18:0/C18:1 ratio was lower and the ratio between monosaturated and polyunsaturated fatty acids was higher. Linoleic acid content of mitochondrial cardiolipin, usually very high in normal rat liver, was notably lower in primary hepatoma. Cholesterol/phospholipid ratio in both microsomes and mitochondria from DEN-induced hepatoma was higher than in normal liver. Hyperplastic nodules showed no changes in cholesterol content whereas modifications in fatty acid composition were already observable. These modifications of membrane structure may be related to the functional changes found in nodular cells. Changes in fatty acid composition of membrane phospholipids, occurring in both primary hepatoma and preneoplastic nodules, might be one of the causes for decreased rate of lipid peroxidation peculiar to these tissues.     

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.     

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.     

Lipids of dystrophic and normal mouse muscle: whole tissue and particulate fractions

Myofibrillar, mitochondrial, and microsomal fractions were prepared from normal and dystrophic mouse limb muscle by differential centrifugation and analyzed for phospholipids and cholesterol. Fatty acids and aldehydes of neutral lipids and of phospholipids from whole muscle and particulate fractions were also determined. Normal microsomes contained more lecithin and less total ethanolamine phospholipids and cardiolipin than mitochondria. The myofibrils had an intermediate phospholipid composition, but their cholesterol-phospholipid ratio was smaller than that of the other two fractions. Except for an increased percentage of phosphatidalethanolamine in the dystrophic mitochondria, only the composition of the dystrophic microsomes differed from normal by containing less lecithin but more total ethanolamine phospholipid, phosphatidalethanolamine, sphingomyelin, and cholesterol. No significant differences were found in the fatty acid composition of neutral lipid extracts from normal and dystrophic preparations, but there was a significant decrease in the percentage of 22:6 in phospholipids from both dystrophic whole muscle and microsomes (-25% and -37%, respectively), whereas the 20:4 content was unaltered. By contrast, the percentages of 18:0 and total fatty aldehyde increased significantly. Phospholipid extracts from all dystrophic samples showed a significant decrease in 16:0 and an increase in 18:1 as compared with the normal.     

Biosynthesis of mitochondrial phospholipids using endogenously generated diglycerides.

When isolated mitochondria or microsomes from rat liver were treated with phospholipase C, the incorporation of radioactive phospholipid precursors was markedly enhanced, presumably as a result of production of diglycerides by hydrolysis of endogenous phospholipids. Incorporation of CDP[14C]choline into lecithin in rat liver or BHK-21 mitochondria could be attributed to residual contamination from elements of the endoplasmic reticulum, with added diglycerides or with endogenous diglycerides produced by the phospholipase C treatment. A similar stimulation of [gamma32P]ATP incorporation into phospholipids was observed with exogenous or endogenous diglycerides, but the mitochondrial diglyceride kinase in either case was also related to the degree of microsomal contaminants. It was concluded that previous studies showing negligible capacity of mitochondria for lecithin biosynthesis de novo were not explainable on the basis of limited accessibility of added diglycerides, and that formation of phosphatidic acid by diglyceride kinase was not of significance in rat liver mitochondria.     

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.     

Fatty acid synthesis in vivo. Transfer of lipids from microsomes to other subcellular fractions of rat liver

At various times after the intraperitoneal injection of Na acetate-1-C¹⁴ to male Wistar rats, the labelled fatty acids are nonuniformely distributed among the lipids of liver microsomes, mitochondria and cell sap. The changes observed in the specific radioactivity of the neutral and phospholipids support the hypothesis that a transfer of these lipids takes place from the site of synthesis (endoplasmic reticulum) to mitochondria and cell sap. This phenomenon is probably responsible for the decline of microsomal fatty acids in favour of the mitochondrial and soluble fractions. In this connection, the deacylation-reacylation process does not seem to be involved.     

Phospholipid exchange activity in developing rat brain

Phospholipid exchange activity has been determined in the supernatant fraction of rat brain from birth through to maturity by measuring the protein-catalysed transfer of total and individual 32P-labelled phospholipids from microsomal membranes to mitochondria, and the transfer of [14C]phosphatidylcholine from liposomes to mitochondria. Transfer activity has also been compared in brain and liver supernatant. Overall phospholipid exchange activity in the brain increased only slightly with age. The activity at birth was 75% of the adult value. However, the transfer of individual phospholipids showed markedly different trends during postnatal brain development. The transfer of phosphatidylinositol (PI) and ethanolamine phospholipids increased postnatally to a maximum at 9 days of age, with lowest values in adult brain. Phosphatidylcholine (PC) transfer increased from 9 days to reach maximum values in the mature brain. The transfer of sphingomyelin was highest immediately after birth. PI transfer activity was higher in brain than liver, while PC and ethanolamine phospholipid transfer activity was higher in liver. The heterogeneity of phospholipid exchange proteins in central nervous system tissue is reflected in the developmental changes in exchange activity towards individual phospholipids. The various exchange proteins appear to have separate induction mechanisms. The presence of exchange-protein activity from birth in the rat indicates the functional importance of phospholipid transport during cell acquisition and membrane proliferation. Activity is not primarily associated with membrane formation such as the formation of the myelin sheath, and therefore is more likely to be involved in the process of phospholipid turnover.     

Lecithin biosynthesis in liver mitochondrial fractions

The pretreatment of rat liver mitochondrial fractions with phospholipase C preparations enhanced the incorporation of cytidine diphospho-[¹⁴C]-choline into phospholipids several-fold. Similar pretreatment of the microsomal fraction produced a similar stimulation. When the extent of microsomal contamination in the mitochondria was determined, and increments of pretreated microsomes were added to the mitochondria, the incorporation values extrapolated to zero for zero microsomal contamination. It was concluded that lecithin biosynthesis from endogenous diglycerides in the mitochondrial fractions could be ascribed to contaminating microsomes.     

Newly made phosphatidylserine and phosphatidylethanolamine are preferentially translocated between rat liver mitochondria and endoplasmic reticulum

The translocation of: (i) phosphatidylserine (PtdSer) from its site of synthesis on microsomal membranes to its site decarboxylation in mitochondrial membranes and (ii) phosphatidylethanolamine (PtdEtn) from the mitochondria to its site of methylation to phosphatidylcholine on microsomal membranes has been reconstituted in cell-free systems consisting of rat liver mitochondria and microsomes. Two types of systems have been reconstituted. In one, the translocation of newly made PtdSer or PtdEtn was examined by incubation of microsomes and mitochondria with [3-3H]serine. In the other, membranes were prelabeled with radioactive PtdSer or PtdEtn, and the transfer of these two lipids between mitochondria and microsomes was monitored. For the transfer of both PtdSer from microsomes to mitochondria and PtdEtn from mitochondria to microsomes, newly made phospholipids were translocated much more readily than pre-existing phospholipids. The data suggest that with respect to their translocation between these two organelles, the pools of newly synthesized PtdSer and PtdEtn were distinct from the pools of "older" phospholipids pre-existing in the membranes. Transfer of neither phospholipid in vitro depended on the presence of cytosolic proteins (i.e. soluble phospholipid transfer proteins) or on the hydrolysis of ATP, although there was some stimulation of PtdSer transfer by ATP and several other nucleoside mono-, di-, and triphosphates. The data are consistent with a collision-based mechanism in which the endoplasmic reticulum and mitochondria come into contact with one another, thereby effecting the transfer of phospholipids. The proposal that there is contact between the endoplasmic reticulum and mitochondria is supported by the recent isolation of a membrane fraction having many, but not all, of the properties of the endoplasmic reticulum, but which was isolated in association with mitochondria (Vance, J. E. (1990) J. Biol. Chem. 265, 7248-7256).     

The adenine nucleotide translocator in foetal, suckling and adult rat liver mitochondria

The adenine nucleotide content of rat liver mitochondria was shown to increase significantly after birth. On the other hand, it was found that the ligand-binding properties of the adenine nucleotide translocator were essentially the same in foetal, suckling and adult rat liver mitochondria. These results are compatible with the proposal that the accumulation of adenine nucleotides which occurs during mitochondrial biogenesis and maturation is effected by a pathway different from the adenine nucleotide translocator.     

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.     

Differentiation of phospholipases A in mitochondria and lysosomes of rat liver

Highly purified mitochondria from rat liver contain a phospholipase A that catalyzes removal of 2-fatty acids, with a pH optimum above pH 8.0. Lysosomal preparations appeared to have two phospholipases A associated with them, one with a pH optimum at about pH 4.0, the second between pH 6.0 and 7.0. Mitochondrial phospholipase A hydrolyzed exogenous phospholipid as fast as or faster than endogenous phospholipid. The difference in specific radioactivity of (14)C-ethanolamine-labeled endogenous mitochondrial phospholipid before and after incubation indicates that a fraction of mitochondrial phosphatidyl ethanolamine is hydrolyzed more rapidly than the mitochondrial phospholipids as a whole. Acyl bond hydrolysis of exogenous and endogenous phospholipid by mitochondria was stimulated by free fatty acid, Ca(++), or in certain cases, monoacyl phospholipids or by treatments that disrupt the mitochondrial membrane. Of various fatty acids tested, lauric, myristic, oleic, and linoleic were most effective. ADP and ATP inhibited mitochondrial phospholipase, probably because they compete for Ca(++). Mg(++) also behaved as a competitive inhibitor; the effect was overcome by relatively little Ca(++).     

Phospholipid composition and fatty acid patterns of isolated phospholipids from rabbit reticulocyte mitochondria

The phospholipid composition and fatty acid patterns of individual phospholipid classes were determined in mitochondria from rabbit reticulocytes. Compared to mitochondria from rat liver reticulocyte, mitochondria exhibit about twice the amount of phospholipids. The phospholipid pattern of reticulocyte mitochondria (phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol and cardiolipin) is comparable with other mitochondrial species. Mitochondrial fractions from reticulocytes are characterized, however, by an additional content of sphingomyelin. This sphingomyelin differs in its fatty acid composition from the sphingomyelin of the plasma membrane. The fatty acid patterns of all other phospholipids essentially correspond to those of mitochondria from other sources and to those of plasma membranes as well.     

Role of lamellar inclusions in surfactant production: studies on phospholipid composition and biosynthesis in rat and rabbit lung subcellular fractions.

Lamellar inclusion bodies in the type II alveolar epithelial cell are believed to be involved in pulmonary surfactant production. However, it is not clear whether their role is that of synthesis, storage, or secretion. We have examined the phospholipid composition and fatty acid content of rabbit lung wash, lamellar bodies, mitochondria, and microsomes. Phosphatidylcholine and phosphatidylglycerol, the surface-active components of pulmonary surfactant, accounted for over 80% of the total phospholipid in lung wash and lamellar bodies but for only about 50% in mitochondria and microsomes. Phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, and sphingomyelin accounted for over 40% of the total in mitochondria and microsomes but for only 6% in lung wash and 15% in lamellar bodies. The fatty acid composition of lamellar body phosphatidylcholine was similar to that of lung wash, but different from that of mitochondria and microsomes, in containing palmitic acid as a major component with little stearic acid and few fatty acids of chain length greater than 18 carbon atoms. The biosynthesis of phosphatidylcholine and phosphatidylglycerol was examined in the mitochondrial, microsomal, and lamellar body fractions from rat lung. Cholinephosphotransferase was largely microsomal. The activity in the lamellar body fraction could be attributed to microsomal contamination. The activity of glycerolphosphate phosphatidyltransferase, however, was high in the lamellar body fraction, although it was highest in the mitochondria and was also active in the microsomes. These data suggest that the lamellar bodies are involved both in the storage of the lipid components of surfactant and in the synthesis of at least one of those components, phosphatidylglycerol.     

Electron-spin resonance studies of lipid-modified microsomes from Friend erythroleukemia cells

The fatty acid composition of cultured Friend erythroleukemia cells was modified by supplementation of the medium with oleic or linoleic acid. There was a 30% reduction in saturated and a 35% reduction in polyunsaturated fatty acids in microsomal phospholipids when the cells were grown in media supplemented with oleic acid, and a 3-fold increase in polyunsaturated fatty acids when the cells were grown in linoleic acid-supplemented media. Electron-spin resonance studies with the 5-nitroxystearate probe demonstrated that there was no appreciable change in microsomal lipid mobility as measured by the order parameters. In contrast, changes in lipid mobility were detected with the spin-label probe when microsomes were first isolated from Friend erythroleukemia cells and subsequently modified by incubation with liposomes composed of either dioleoyl- or dilinoleoylphosphatidylcholine plus bovine liver phospholipid-exchange protein. The fatty acid compositional changes produced in these microsomes were similar to those obtained when the intact cells were grown in media containing supplemental fatty acids. These findings indicate that the lipid mobility of Friend cell microsomes can be altered by phospholipid replacements in vitro, but that this does not occur when similar microsomal fatty acid modifications are produced during culture of the intact cell.