LECITHIN SYNTHESIS, INTRACELLULAR TRANSPORT, AND SECRETION IN RAT LIVER : IV. A Radioautographic and Biochemical Study of Choline-Deficient Rats Injected with Choline-3H

Injection of choline-³H into choline-deficient rats resulted in an enhanced incorporation of the label into liver lecithin, as compared to the incorporation of label into liver lecithin of normal rats. The results obtained with the use of different lecithin precursors indicate that in the intact liver cell, both in vivo and in vitro, exchange of choline with phosphatidyl-choline is not significant. The synthesis and secretion of lecithins by the choline-deficient liver compare favorably with the liver of choline-supplemented rats, when both are presented with labeled choline or lysolecithin as lecithin precursors. Radioautography of the choline-deficient liver shows that 5 min after injection of choline-³H the newly synthesized lecithin is found in the endoplasmic reticulum (62%), mitochondria (13%), and at the "cell boundary" (20%). The ratio of the specific activity of microsomal and mitochondrial lecithin, labeled with choline, glycerol, or linoleate, was 1.53 at 5 min after injection, but the ratio of the specific activity of phosphatidyl ethanolamine (PE), labeled with ethanolamine, was 5.3. These results indicate that lecithin and PE are synthesized mainly in the endoplasmic reticulum, and are transferred into mitochondria at different rates. The site of a precursor pool of bile lecithin was studied in the intact rat and in the perfused liver. Following labeling with choline-³H, microsomal lecithin isolated from perfused liver had a specific activity lower than that of bile lecithin, but the specific activity of microsomal linoleyl lecithin was comparable to that of bile lecithin between 30 and 90 min of perfusion. It is proposed that the site of the bile lecithin pool is located in the endoplasmic reticulum and that the pool consists mostly of linoleyl lecithin.     

Substrate specificity of plasma lysolecithin acyltransferase and the molecular species of lecithin formed by the reaction

Human plasma lecithin-cholesterol acyltransferase also converts lysolecithin to lecithin in the presence of low density lipoproteins. To understand the physiological importance of this lysolecithin acyltransferase reaction, we investigated the molecular species of lysolecithin available for acylation in normal plasma and the lecithins which are formed by the acylation of each of these lysolecithins. Palmitate- and stearate-containing lysolecithins were formed by the lecithin-cholesterol acyltransferase reaction, whereas oleate- and linoleate-containing lysolecithins were formed by the action of post-heparin lipase(s). All the natural lysolecithins were esterified at comparable rates by the isolated enzyme. Lyso platelet-activating factor was esterified about 70% as efficiently as the lysolecithins, while lysophosphatidylethanolamine was esterified at about 30% the rate observed with lysolecithin. The 2-acyl isomers of lysolecithin were acylated to the same extent as the 1-acyl isomers, although considerable isomerization of the former took place during the incubation. There were no net changes in the concentrations of lecithin and lysolecithin after 6 h of incubation with the enzyme, although over 10% of the labeled lysolecithin was converted to lecithin, indicating that the endogenous lecithin serves as the acyl donor in the reaction. When the molecular species of lecithin formed were analyzed by high performance liquid chromatography, the same pattern of fatty acid incorporation was observed with all the lysolecithins used. The bulk of the radioactivity was incorporated into molecular species formed by the acylation with linoleic, oleic, and palmitic acids, in decreasing order. However, in each case, the lecithins formed by acylation with palmitic acid had the highest specific radioactivity, followed by those acylated with linoleic and oleic acids. From these results it is postulated that the enzyme alters the molecular species composition of lecithin in plasma without increasing the net amount of total lecithins.     

ACYLATION OF LYSOPHOSPHATIDES BY PLASMA MEMBRANE FRACTIONS OF RAT LIVER

The plasma membrane fraction of rat liver was isolated and incubated with labeled lysophosphatides in the presence of cofactors; the acylation of lysolecithin to lecithin by the fraction was compared to that of the rough and smooth microsomes. The purity of the isolated fractions was ascertained by enzyme markers and electron microscopy, and the maximal contamination of the plasma membrane fraction by microsomes did not exceed 20%. Under conditions at which the reaction was proportional to the amount of enzyme used, the plasma membrane had a specific activity similar to that of the smooth and rough microsomes. With doubly labeled lysolecithin (containing palmitic acid-¹⁴C and choline-³H) it was shown that the lecithin formed retained the same ratio of the two labels, which indicated that lysolecithin was converted to lecithin through an acylation reaction. The newly formed lecithin was shown to be bound to the plasma membrane fraction; this suggested that it is incorporated into the structure of the membrane itself.     

The relationship of protein and lipid synthesis during the biogenesis of mitochondrial membranes

Summary Rat liver mitochondria were fractionated into inner and outer membrane components at various times after the intravenous injection of¹⁴C-leucine or¹⁴C-glycerol. The time curves of protein and lecithin labeling were similar in the intact mitochondria, the outer membrane fraction, and the inner membrane fraction. In rat liver slices also, the kinetics of³H-phenylalanine incorporation into mitochondrial KCl-insoluble proteins was identical to that of¹⁴C-glycerol incorporation into mitochondrial lecithin. These results suggest a simultaneous assembly of protein and lecithin during membrane biogenesisThe proteins and lecithin of the outer membrane were maximally labeledin vivo within 5 min after injection of the radioactive precursors, whereas the insoluble proteins and lecithin of the inner membrane reached a maximum specific acitivity 10 min after injection.Phospholipid incorporation into mitochondria of rat liver slices was not affected when protein synthesis was blocked by cycloheximide, puromycin, or actinomycin D. The injection of cycloheximide 3 to 30 min prior to¹⁴C-choline did not affect thein vivo incorporation of lecithin into the mitochondrial inner or outer membranes; however treatment with the drug for 60 min prior to¹⁴C-choline resulted in a decrease in lecithin labeling. These results suggest that phospholipid incorporation into membranes may be regulated by the amount of newly synthesized protein available.When mitochondria and microsomes containing labeled phospholipids were incubated with the opposite unlabeled fractionin vitro, a rapid exchange of phospholipid between the microsomes and the outer membrane occurred. A slight exchange with the inner membrane was observed.     

COMPOSITION OF CELLULAR MEMBRANES IN THE PANCREAS OF THE GUINEA PIG : II. Lipids

The lipid composition of rough and smooth microsomal membranes, zymogen granule membranes, and a plasmalemmal fraction from the guinea pig pancreatic exocrine cell has been determined. As a group, membranes of the smooth variety (i.e., smooth microsomes, zymogen granule membranes, and the plasmalemma) were similar in their content of phospholipids, cholesterol and neutral lipids, and in the ratio of total lipids to membrane proteins. In contrast, rough microsomal membranes contained much less sphingomyelin and cholesterol and possessed a smaller lipid/protein ratio. All membrane fractions were unusually high in their content of lysolecithin (up to ~20% of the total phospholipids) and of neutral lipids, especially fatty acids. The lysolecithin content was shown to be due to the hydrolysis of membrane lecithin by pancreatic lipase; the fatty acids, liberated by the action of lipase on endogenous triglyceride stores, are apparently scavenged by the membranes from the suspending media. Similar artifactually high levels of lysolecithin and fatty acids were noted in hepatic microsomes incubated with pancreatic postmicrosomal supernatant. E 600, an inhibitor of lipase, largely prevented the appearance of lysolecithin and fatty acids in pancreatic microsomes and in liver microsomes treated with pancreatic supernatant.     

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.     

Lysolecithin as regulator of de novo lecithin synthesis in rat liver microsomes.

1-Acyl-sn-glycero-3-phosphocholine (lysolecithin) was found to affect 1,2-diacyl-sn-glycerol:CDPcholine cholinephosphotransferase (CPT; EC 2.7.8.2) activity of rat liver microsomes in a concentration dependent, characteristic manner. Cholinephosphate transfer was activated at lysolecithin concentrations below 0.5 mM with a maximum stimulation occurring at 75–100 μM lysolecithin levels. At concentrations above 0.5 mM, CPT activity was inhibited by lysolecithin. It was shown that CPT inhibition by lysolecithin is competitive (Ki ≈ 0.6 mM) with respect to CDPcholine. The possible role of lysolecithin as regulator of de novo lecithin synthesis in vivo is outlined.     

Microcalorimetric study of phospholipid binding to human apo-HDL

The binding of lysolecithin and synthetic short-chain lecithins: di-caproyl, di-lauroyl and di-myristoyl lecithins to a human apo-high density lipoprotein (apo-HDL) was followed by microcalorimetry. Complex formation was checked by ultracentrifugal flotation.The binding reaction was very rapid and strongly exothermal. The apparent binding enthalpy ΔH_B together with the complex composition were computed from the binding curves. Both quantities were of the same order of magnitude for lysolecithin and for the shorter chain lecithins while the binding of di-myristoyl lecithin was characterized by a more highly exothermal reaction.The structure of the lipid phase strongly influences the enthalpy change. In the case of lysolecithin and of the shorter chain lecithins; which form micellar structures in water, the enthalpy change is mainly due to apoprotein-phospholipid complex formation.The disrupture of the myelin figures formed by the di-myristoyl lecithin accounts for the complementary heat effect.The phospholipid composition of the complexes isolated by ultracentrifugal flotation was lower than that determined by microcalorimetry, due to the presence of high salt concentrations in the ultracentrifuge.     

Demonstration of enzymatic conversion of lysolecithin to lecithin in normal human plasma

An enzyme in normal human plasma that converts [1-acyl ¹⁴C] lysolecithin to lecithin is demonstrated. This enzyme is inhibited by heparin and is not derived from platelets or other blood elements. The synthesis of lecithin from labeled lysolecithin was not stimulated by ATP and CoA or by oleyl CoA and there was nearly an equal distribution of labeled fatty acid between the two positions of lecithin indicating that the enzyme may be a lysolecithin: lysolecithin acyl transferase (LLAT). The enzyme is associated with the lipoproteins of the plasma, and may have a physiological role in the formation of saturated cholesterol esters in plasma.     

Reversible activation/inactivation of the deacylation/acylation cycle in rat liver microsomes

Rat liver microsomes incorporate [¹⁴C]palmitoyl CoA into membrane phospholipids via the deacylation/acylation cycle. This activity is reversibly inactivated/activated by treatment of the microsomes with ATP, MgCl₂, and 105,000g supernatant or with 105,000g supernatant alone. These observations suggest that the acylation cycle is controlled by a mechanism involving phosphorylation/dephosphorylation. As the pool of lysolecithin in the membranes is not altered by conditions increasing incorporation of palmitoyl CoA into phospholipid, it is probable that the site of regulation of deacylation/acylation is at the acyltransferase rather than the phospholipase.     

Lipids of the adrenal medulla: Lysolecithin, a characteristic constituent of chromaffin granules

1. The lipid composition of microsomes, mitochondria and chromaffin granules, obtained from homogenates of bovine adrenal medulla, has been investigated. 2. The three types of particle showed characteristic differences of phospholipid and cholesterol content. The lipid composition of microsomes and mitochondria resembled that of corresponding particles from other tissues. The chromaffin granules contained 19% of the cholesterol and 14% of the phospholipids of the low-speed supernatant. 3. Thin-layer chromatography indicated the presence of these phospholipids in extracts from each particle: lecithin, lysolecithin, phosphatidylethanolamine (partly plasmalogen), phosphatidylserine, phosphatidylinositol and sphingomyelin. 4. On quantitative analysis of the phospholipids, chromaffin granules were found to contain a high concentration of lysolecithin (17% of the lipid phosphorus). Mitochondria and microsomes, on the other hand, contained very little lysolecithin (less than 2% of the lipid phosphorus).     

Topographic distribution of enzymes involved in glycerolipid synthesis in rat small, intestinal epithelium.

1. The enzymes involved in glycerolphosphate and monoacylglycerol acylation of rat small intestine were more active in villi than in crypts. Monoglyceride acyltransferase (EC 2.3.1.22) was found to be absent from crypts. 2. In the villi, the enzymes are mainly localized in microsomes, although low activities of palmitoyl-CoA synthetase (EC 6.2.1.3), glycerolphosphate acyltransferase (EC 2.3.1.15) and cholinephosphotransferase (EC 2.7.8.2) are found in mitochondria. Mitochondria lack monoglyceride acyltransferase and lysolecithin acyltransferase (EC 2.3.1.23), both of which are involved in the reacylation of alimentary partial glycerides. Therefore, this process is confined to microsomes. 3. The monoacylglycerol and lysolecithin acyltransferases, as well as choline-phosphotransferase, are probably localized within the endoplasmic reticulum, since these enzymes are relatively Nagerse resistant (subtilisin; EC 3.4.2.1, compared with palmitoyl-CoA synthetase and glycerolphosphate acyltransferase, which are highly Nagarse-sensitive and therefore probably localized on the outside of the microsomes (and mitochondria). 4. The physical separation of alimentary product reacylation from de novo synthetic processes provides the basis of metabolic compartmentation observed by other workers. 5. The use of sucrose instead of a salt medium for the isolation and homogenization of small intestinal epithelial cells allowed the separation of mitochondria and microsomes by differential centrifugation without mutual contamination. 6. Phospholipids were found to stimulate glycerolphosphate acylation in vitro. 7. The glycerolphosphate and monoacylglycerol acylation pathways are not competitive.     

Phosphatidylglycerol in lung surfactant. II. Subcellular distribution and mechanism of biosynthesis in vitro.

Lamellar inclusion bodies, apparent precursors for alveolar surfactant lining, have remarkably similar phospholipid composition to surfactant from alveolar lavage, but distinctly different from other fractions studied: mitochondria, microsomal fraction containing endoplasmic reticulum membranes, plasma membranes and nuclei. Surfactant contained (as % of total phospholipid phosphate): 75.5-77.0% lecithin, 11.0-11.2% phosphatidylglycerol, 4.2-4.6% phosphatidylethanolamine, 3.0-3.2% phosphatidylinositol, 1.5-1.7% bis-(monoacylglycerol) phosphate, 1.2-1.9% phosphatidylserine, and 0.7-1.5% sphingomyelin. Fatty acids of phosphatidylglycerol from lamellar bodies were similar to those from microsomes but different from those in mitochondria. Lung homogenate in continuous sucrose density gradient displayed two major activity peaks of phosphatidylglycerol synthesis: the heavier from mitochondria; the lighter from endoplasmic reticulum. Studies on mechanism of phosphatidylglycerol synthesis in vitro revealed (in these two fractions) CDP-diglyceride and sn-glycerol phosphate precursors to phosphatidylglycerol phosphate, that hydrolysed to phosphatidylglycerol. In microsomes disaturated CDP-diglycerides were 1.6-1.9 times more active substrates than in mitochondria, whereas CDP-diglycerides from egg lecithin were almost equally active. In contrast to lung mitochondria no cardiolipin synthesis was detected in microsomes. The highest specific activities for phosphatidate cytidyltransferase, CDP-diglyceride-inositol phosphatidyltransferase, choline phosphotransferase, and phosphatidylethanolamine methyltransferase were all found in microsomes. The present in vitro studies and additional evidence (M. Hallman and L. Gluck, (1975) Fed. Proc. 34, 274) support the hypothesis that de novo synthesis of surfactant lecithin phosphatidylinositol and phosphatidylglycerol takes place in the endoplasmic reticulum of alveolar cells.     

Comparative study of lipid transfer between cell organelles using lipid-transfer proteins in vitro

The transfer of de novo synthesized lipids from microsomes to lipid non-synthesizing membranes was studied in vivo and in vitro from the ratios of specific radioactivities of [14C]cholesterol, [14C] and [32P]phosphatidylcholine and [32P]phosphatidylethanolamine in the nuclei and mitochondria to that in microsomes. The radioactivity of lipids transferred from microsomes to mitochondria and nuclei was identical both in vitro and in vivo and when the lipid-exchange protein of the 105 000 g supernatant was used. Acceleration of lipid metabolism in the liver of gamma-irradiated rats was concomitant with the increase in the rate of labeled cholesterol transfer cation to liver cell nuclei and mitochondria, but remained unchanged in in vitro studies involving lipid-exchange protein. The reduction of phosphatidylethanolamine transfer to the nuclei in vitro and in vivo diminished in the same way. The existence in the cell of mechanisms of transfer of de novo synthesized cholesterol other than lipid-exchange protein is postulated.     

LYSOLECITHIN AND SPHINGOSINEPHOSPHORYL-CHOLINE IN THE METABOLISM OF BRAIN PHOSPHOLIPIDS OF THE RHESUS MONKEY (MACAC A MULATTA): EFFECTS OF DEVELOPMENT

The rates of incorporation of palmitate from palmitoyl CoA into lecithin or sphingomyelin by homogenates of neural tissue of rhesus monkeys were greatly increased by the addition of lysolecithin or sphingosinephosphorylcholine (SPC). Labelled lysolecithin and SPC were also incorporated into lecithin and sphingomyelin. There was a low level of nonenzymic sphingomyelin formation from SPC and palmitoyl CoA. Choline from CDP-choline was rapidly incorporated into lecithin and slowly into SPC and sphingomyelin by homogenates of pons. The activity of lysolecithin acyl hydrolase (EC 3.1.1.5) was high throughout life in homogenates of pons and cerebral cortex. The rate of utilization of palmitoyl CoA and lysolecithin for phospholipid synthesis was higher in neural tissue from fetal and neonatal monkeys than from adults. Lysolecithin was acylated most rapidly with palmitoleic, linoleic and arachidonic acids. All phospholipid metabolic activities were higher in the cerebral cortex than in the pons.     

Antioxidant effect of conjugated linoleic acid and vitamin A during non enzymatic lipid peroxidation of rat liver microsomes and mitochondria

In the study reported here the effect of conjugated linoleic acid (CLA) and vitamin A on the polyunsaturated fatty acid composition, chemiluminescence and peroxidizability index of microsomes and mitochondria isolated from rat liver was analyzed. The effect of CLA on the polyunsaturated fatty acid composition of native microsomes was evidenced by an statistically significant p < 0.007 decrease of linoleic acid C18:2 n6, whereas in mitochondria it was observed a decrease p < 0.0001 of arachidonic acid C20:4 n6 when compared with vitamin A and control groups. Docosahexaenoic acid C22:6 n3 in mitochondria was reduced p < 0.04 in CLA and vitamin A groups when compared with control. After incubation of microsomes or mitochondria in an ascorbate (0.4 mM)-Fe⁺⁺ (2.15 M) system (120 min at 37°C) it was observed that the total cpm/mg protein originated from light emission: chemiluminescence was lower in liver microsomes or mitochondria obtained from CLA group (received orally: 12.5 mg/daily during 10 days) than in the vitamin A group (received intraperitoneal injection: daily 0.195 g/kg during 10 days). CLA reduced significantly maximal induced chemiluminescence in microsomes relative to vitamin A and control groups, whereas in mitochondria the effect was observed relative to control group The polyunsaturated fatty acid composition of liver microsomes or mitochondria changed by CLA and vitamin A treatment. The polyunsaturated fatty acids mainly affected when microsomes native and peroxidized from control group were compared were linoleic, linolenic and arachidonic acids, while in vitamin A group linoleic and arachidonic acid were mainly peroxidized, whereas in CLA group only arachidonic acid was altered. In mitochondria obtained from the three groups arachidonic acid and docosahexaenoic acid showed a significant decrease when native and peroxidized groups were compared. As a consequence the peroxidizability index, a parameter based on the maximal rate of oxidation of fatty acids, show significant changes in the CLA group compare vitamin A and control groups. The simultaneous analysis of peroxidizability index, chemiluminescence and fatty acid composition demonstrated that CLA is more effective than vitamin A protecting microsomes or mitochondria from peroxidative damage.     

Nonaprenyl-4-hydroxybenzoate transferase, an enzyme involved in ubiquinone biosynthesis, in the endoplasmic reticulum-Golgi system of rat liver

The properties and distribution of nonaprenyl-4-hydroxybenzoate transferase in rat liver were investigated with subcellular fractions, liver perfusion, and in vivo labeling with [3H]solanesyl-PP. In addition to some ubiquinone-9, only one labeled intermediate, i.e. nonaprenyl-4-hydroxybenzoate, was obtained. In the total microsomal fraction, the enzyme had a pH optimum of 7.5 and was completely inhibited by Triton X-100 and deoxycholate, but not by taurodeoxycholate and beta-octyl glucoside. Liver, kidney, and spleen demonstrated the highest activities of nonaprenyl-4-hydroxybenzoate transferase. Upon subcellular fractionation, high specific activities were found in smooth II microsomes and Golgi III vesicles. The enzyme was also found in lysosomes and plasma membranes, but only at low levels in rough and smooth I microsomes and mitochondria and not at all in peroxisomes and cytosol. When the product of the transferase reaction was used as a substrate in vitro and in a perfusion system, the only product obtained was end product ubiquinone-9. Although the transferase reaction was associated with the inner, luminal surface of microsomal vesicles, the terminal reaction(s) for ubiquinone-9 synthesis are found at the outer cytoplasmic surface. The results suggest that the major site for ubiquinone synthesis is the endoplasmic reticulum-Golgi system, which also participates in the distribution of ubiquinone-9 to other cellular membranes.     

Origins of the phospholipids in animal mitochondria

As is the case for the assembly of protein components of the membranes in animal mitochondria, the bilayer phospholipids arise from a complicated interplay of intra- and extra-mitochondrial reactions. Our early studies indicated that the bulk of mitochondrial phospholipids (typified by phosphatidylcholine) had their origin in the endoplasmic reticulum and were transported to the mitochondria as complexes with phospholipid-exchange proteins. The polyglycerophosphatides (typified by diphosphatidylglycerol) were apparently synthesized in situ by intramitochondrial membrane-bound enzymes using CDP-diglycerides as intermediates. The case for the precursors in the latter pathway is less clear, although evidence has been presented for dual localization of enzymes for glycerophosphate acylation and CTP:phosphatidate cytidylyl transfer in both mitochondria and microsomes. Phosphatidylethanolamine also shows evidence for two sites of origin: by translocation from its site of synthesis in the endoplasmic reticulum and by translocation of phosphatidylserine followed by decarboxylation within the mitochondria. In the latter case mitochondrial phosphatidylserine decarboxylase may play an important role in the regulation of phospholipid metabolism throughout the cell.     

Prostaglandin precursor fatty acids in serum and bile as influenced by oophorectomy, contraceptive steroids and pregnancy. An experimental study in the cat

Variations in the occurrence of prostaglandin precursor fatty acids might be of importance for the pathogenesis of gallstones. Pregnancy and use of contraceptive steroids increase the risk of gallstones. The present study reports the relative fatty acid composition in serum and biliary phospholipids studied by gas-liquid chromatography in four groups of female cats, which were on a standard diet: 1) oophorectomized animals, 2) animals on contraceptive steroids, 3) pregnant animals and 4) control animals. It was consistently found that the portions of palmitic and linoleic acid were higher and stearic and arachidonic acid were lower in biliary than in serum lecithin. In biliary lysolecithin, sphingomyelin and cephaline there were only small portions of linoleic and negligible amounts of arachidonic acid. Oophorectomy, contraceptive steroids or pregnancy did not induce any gross changes in the fatty acid pattern of lecithin in serum or bile. In animals treated with contraceptive steroids a reduced portion of linoleic acid was seen in the bile lecithin, and in pregnant animals there was a reduction of omega 3 and omega 6 fatty acids in biliary lecithin.     

Biosynthesis and characterization of a phosphatidic acid analog containing beta-hydroxy fatty acid

A new phospholipid was shown to be biosynthesized in liver mitochondria from labeled 1-alkyl-$\text{sn}$-glycerol-3-phosphate and labeled fatty acid in the presence of ATP and CoA and its structure was shown to be 1-alkyl-2-(3-hydroxy)acyl-$\text{sn}$-glycerol-3-phosphate. Fatty acids in mitochondria were oxidized to the β-hydroxy derivatives which were utilized for the acylation of alkyl glycerophosphate. Free long chain β-hydroxy acids were also utilized by mitochondria and microsomes in the presence of ATP and CoA for the acylation of glycerophosphate derivatives to form the phosphatidate analogs.