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.     

Reaction properties of hydrophobic enzymes and their immobilization on phospholipid composite membranes

Reaction rates of hydrophobic enzymes, aminopeptidase, and alkaline phosphatase, in microsomes prepared from the porcine brush border membrane and in vesicles pre pared from microsomes and phospholipids were measured at various temperatures. Interactions between the hydrophobic enzymes and the phospholipid layers are discussed as well as the effects of fluidity change of phospholipid layers on enzyme activity. Further, reaction properties and stabilities of the immobilized vesicles containing microsomal enzymes were studied.     

Studies in Tetrahymena membranes substrates for desaturation of fatty acyl chains in Tetrahymena pyriformis microsomes

[1-14-C]Palmitoyl-Co A was incubated with Tetrahymena microsomes containing the complete enzyme system for desaturation during various time periods. The level of [1-14C]palmitoleoyl-CoA increased to a maximum during the 1--3 min incubation time, while [1-14C]palmitoleic acid in the phospholipid reached a maximum level during 6--7 min incubation time. The radioactivity of [1-14C]palmitoleic acid in free fatty acid and the triglyceride fraction was not significantly observed upon 3 min incubation. Incubation of [1-14C]palmitoyl-CoA with microsomes in the absence of NADH produced [1-14C]palmitoyl lipid without desaturation. Radioactive palmitic acids in the microsomal lipids were not converted to palmitoleic acids after addition of NADH by the complete enzyme system. When microsomes prepared from cells labeled with [1-14C]palmitic acid or [1-14C]stearic acid were incubated alone in the presence of O2 and NADH, no significant increase in [1-14C]palmitoleic acid in the phospholipid was observed, wherease an increase in [1-14C]linoleic acid and gamma-[1-14C]linolenic acid did occur at the expense of [1-14C]oleic acid in the phospholipid. From these results it can be concluded that the enzyme involving desaturation of palmitic acid to palmitoleic acid requires palmitoyl-CoA as the substrate. However, the possibility of oleoyl and linoleoyl phospholipids being substrates in the desaturation of Tetrahymena microsomes was suggested.     

Effect of docosahexaenoic acid and ascorbate on peroxidation of retinal membranes of ODS rats

Mutant male osteogenic disorder Shionogi (ODS) rats, unable to synthesize ascorbic acid, were fed diets containing a high content of docosahexaenoic acid (DHA) and different amounts of ascorbic acid, to study the effect of DHA on peroxidative susceptibility of the retina and possible antioxidant action of ascorbic acid. ODS rats were fed from 7 weeks of age with diets containing high DHA (6.4% of total energy). A control group received a diet high in linoleic acid. The diets also contained varying amounts of ascorbic acid. Fatty acid compositions and phospholipid hydroperoxides in rod outer segment (ROS) membranes, and retinal ascorbic acid were analyzed. DHA in ROS membranes was significantly increased in rats fed high DHA, compared with the linoleic acid diet. Levels of phospholipid hydroperoxides in the DHA-fed rats were significantly higher than the linoleic acid-fed rats. Ascorbic acid supplementation did not suppress the phospholipid hydroperoxide levels after a high DHA diet, even when the supplement increased the content of retinal ascorbic acid. In conclusion, high DHA feeding induced a marked increase of phospholipid hydroperoxides in ROS membranes of ODS rats. Supplementation of ascorbic acid did not reverse this increase.     

Hepatic microsomal glucuronidation of bilirubin is modulated by the lipid microenvironment of membrane-bound substrate

Hepatocyte intracellular membranes may facilitate the directed movement of bilirubin and other hydrophobic substrates to the active site of UDP-glucuronyltransferase in the endoplasmic reticulum. We postulated that the lipid composition and physical properties of membranes that transport substrate may modulate bilirubin glucuronidation. To examine this hypothesis, we incorporated [14C]bilirubin substrate into the membrane bilayer of small unilamellar liposomes composed of native phospholipid purified from rat hepatic microsomes. The initial velocity of bilirubin glucuronide formation in rat liver microsomes, measured by radiochemical assay, was considerably more rapid than for bilirubin in liposomes of egg phosphatidylcholine (p less than 0.001). Moreover, the ratio of bilirubin diglucuronide to monoglucuronides synthesized was markedly increased (p less than 0.01), approaching that observed in normal rat bile. Although the rates of bilirubin glucuronidation did not correlate with fluidity of the liposomal membrane core region, specific phospholipid head groups were associated with an increase, and cholesterol a decrease, in rates of glucuronidation. Movement of [3H]bilirubin from dual-labeled liposomes to microsomes occurred without concomitant [14C]phospholipid transfer. Thus, the lipid composition of membranes incorporating bilirubin appears to modulate the rate of glucuronidation and the relative rates of bilirubin mono- and diglucuronide formation. Phospholipid head groups on the surface of the bilayer, not the hydrocarbon core regions, may be implicated in the rapid process of membrane transport, which is likely to involve membrane-membrane collisions or diffusion of free substrate rather than membrane fusion.     

Hydrolysis of Endogenous Phospholipids by Rat Brain Microsomes

Phosphatidylcholine of rat brain microsomes was labeled in vivo by intracerebral injection of either [3H]oleic acid or [methyl-3H]choline chloride. These labeled microsomes served both as the enzyme source as well as a source of endogenously labeled substrate. Phospholipase D (PLD) activity was detected with these particles only in the presence of exogenous oleate, its activator. Ca2+ and the ionophore A 23187 inhibit PLD activity of oleate-labeled microsomes. In oleate-labeled particles, besides phosphatidic acid the product of PLD action radioactivity was also detected in diglyceride as a result of resident phosphatidate phosphohydrolase, which hydrolyzed the phosphatidic acid. The phosphatidate phosphohydrolase could not be completely inhibited by KF and propranolol. The release of endogenous fatty acids from labeled phospholipid by a mellitin-stimulated phospholipase A2 also present in these particulates produced minimal stimulation of endogenous PLD. Phosphatidylcholine (PC) and phosphatidylethanolamine (PE) are hydrolyzed by 50% in the presence of mellitin and 90% of the radioactivity was found in the lyso-compounds. Mellitin and oleate together reduced the radioactivity found in lyso-PC and increased that in lyso-PE.     

Measurement and prediction of the rates of spontaneous transfer of phospholipids between plasma lipoproteins

The purpose of this report is to develop a correlation between the hydrophobicity of a phospholipid as measured by reversed-phase high-performance liquid chromatography and its rate of spontaneous transfer and to use this correlation to predict the rate of transfer of any homologous lipid from any lipoprotein. We have studied the mechanism of transfer of a series of fluorescent or radiolabeled phospholipids among natural and reassembled serum lipoproteins. Fluorescent phosphatidylcholines included those with 9-(1-pyrenyl)nonanoic acid in the sn-2 position and lauric, myristic, palmitic, stearic, oleic or linoleic acid at sn-1. The radioactive phosphatidylcholines contained [3H]oleic acid in the sn-2 position and lauric, myristic, or palmitic acid at sn-1. The kinetics of transfer of the pyrene-labeled lipid were followed by changes in the excimer fluorescence, and that of the radioactive lipids by separation of the donor (lipid-apolipoprotein recombinant) from the acceptor (single bilayer vesicles) on a column of Sephacryl S-200. The retention time of each lipid was measured by high-performance hydrophobic chromatography through a Waters radially compressed C18 column eluted with 75% isopropanol and 25% triethylammonium phosphate (0.15 M). A linear relationship was observed between the rate-constant of transfer and the retention time which suggest that the rate of desorption of phosphatidylcholines from lipoproteins and vesicles is controlled predominately by the hydrophobic effect. For a homologous series of lipids, the rate of transfer can be predicted from retention times obtained from hydrophobic chromatography. The kinetics of transfer of 1-lauroyl-2-[9-(1-pyrenyl)nonanoyl] phosphatidylcholine between isolated human serum lipoproteins exhibits a linear correlation between the transfer half-time and the size of the donor lipoproteins. As a consequence, transfer from very-low-density lipoprotein is 10-times slower than that observed from high-density lipoproteins. The observed correlations between phospholipid transfer rates and both the Stokes radius of the donor and the retention time of the phospholipid on a hydrophobic column permit one to calculate the rate of transfer of homologous molecules between lipid-protein complexes. The results predict that the spontaneous transfer of phospholipids between plasma lipoproteins would be too slow to be a physiologically important phenomena.     

Role of Phospholipid Transfer Protein in the Exchange of Phospholipids between Microsomes and Chloroplasts

When microsomes containing phosphatidylcholine labelled with[1-¹⁴C]-linoleate were incubated with pea or spinach chloroplasts,active transfer of this phospholipid took place in the presenceof phospholipid transfer protein. This transfer also was demonstratedby incubating unlabelled microsomes, chloroplasts and the phospholipidtransfer protein in the presence of [1-¹⁴C]-acetate. The reconstitutedsystems could synthesize fatty acids which were acylated inmicrosomal phosphatidylcholine. The transfer of this phospholipidto chloroplasts is mediated by the transfer protein. Our resultssuggest a role for phospholipid transfer protein in the synthesisof chloroplast lipids. (Received October 25, 1983; Accepted July 18, 1984)     

Some characteristics of a high molecular weight lipid-protein aggregate and its possible role in intracellular fatty acid metabolism

Several physical aspects of a high molecular weight lipid-protein aggregate separated by gel chromatography from chick and rat liver cytosol and its possible role in intracellular fatty acid metabolism were investigated. Electron microscopic examination of the high molecular weight lipid-protein aggregate indicated spherical particles with a diameter range of 200-600 A. This structure is consistent with a microemulsion particle of triglyceride encapsulated by phospholipid and protein. Uptake of fatty acids by microsomes occurred from the same lipid-protein aggregate, and the triglycerides synthesized in microsomes also became associated with these particles in the cytosol. The lipid-protein aggregate prepared by different homogenization methods showed identical ratios of components, but these ratios changed following incubation. These findings lend support to the concept that this aggregate plays a physiological role in intracellular lipid metabolism, and may be identifiable with previously reported subcellular fatty acid and triglyceride pools.     

Early effects of dietary orotic acid upon liver lipid synthesis and bile cholesterol secretion in rats

Dietary orotic acid is known to cause impaired fatty acid synthesis and increased cholesterol synthesis in rats. We found that the impaired fatty acid synthesis occurs during the first day of orotic acid feeding and, in studies with albumin-bound [1-14C]palmitic acid, an associated decrease in the rate of esterification of this fatty acid into triacylglycerol, phospholipid, and cholesteryl ester was observed. These changes may result from the known decreases in liver levels of adenine nucleotides or, as reported here, from decreased liver CoASH levels in orotic acid-fed rats. The increase in hepatic cholesterol synthesis occurred during the second day of orotic acid feeding. It was detected by increased incorporation of [1,2-14C]acetate into cholesterol by liver slices and by a 7-fold increase in HMG-CoA reductase activity. At the same time the biliary output of cholesterol was increased 2-fold and studies using 3H2O revealed that the output of newly synthesized cholesterol in bile was increased 5-fold. The content of cholesteryl ester in hepatic microsomes decreased during orotic acid feeding but free cholesterol was unchanged. The findings are interpreted to suggest that the increased bile cholesterol secretion caused by orotic acid is a result of impaired hepatic cholesterol esterification and that the increase in HMG-CoA reductase activity is a result of diminished negative feedback due to the depleted content of cholesteryl ester in the hepatic microsomes.     

Enhancement of the Stability of Insoluble Calcium Particles Using a Phospholipid Coating

Calcium is one of the most important elements in the human body. Insoluble calcium particles are often used in calcium-fortified food products, such as calcium-fortified milk, dairy beverages or protein powders. However, their suspension may be unstable often leading to precipitation in such products. In this study, three different kinds of insoluble calcium particles, i.e. hydroxyapatite (HA), tricalcium phosphate (TCP) and calcium carbonate (CaCO₃) were coated with amphiphilic phospholipids using a solvent-exchange method. Suspension stability of these insoluble calcium particles was effectively improved with phospholipid coating, especially for HA and TCP, as more phospholipids were coated on the surface of these two calcium particles than CaCO₃. Phospholipid coating increased the electrostatic repulsions between particles, preventing the particles from aggregating and precipitating. In addition, the digestibility of phospholipid-coated insoluble calcium particles was tested in simulated gastric juice, and the dissolution time of these insoluble calcium particles was prolonged through phospholipid coating.     

Covalent cross-linking of photosensitive phospholipids to human serum high density apolipoproteins (apoHDL).

Human serum high density apolipoproteins were reassociated with three different lecithin species substituted with radioactively labelled photosensitive azido fatty acids, bis([3H]-16-azidopalmitoyl)-, bis([3H]12-azidooleoyl)- and bis([3H]18-azidolinoleoyl)glycerophosphocholine. The lipoprotein particles were reconstituted from a mixture of azido-labelled phosphatidylcholine and non-labelled dioleoylglycerophosphocholine (1:9). Excess lipid was separated from the homogeneous particles by Bio-Gel A-5m. The molecular weight, stoichiometry, fluorescence and circular dichroism of the reconstituted particles were determined before and after photoactivation with covalent cross-linking of the phospholipids with the apoproteins. The physical parameters of the reconstituted lipoproteins remained unperturbed by the cross-linking reaction between the generated nitrenes and apolipoprotein A-I and A-II. Thus the hydrophobic interactions of the phospholipid molecules with the apoproteins have been proved for the first time by a chemical method.     

The role of cardiolipin as an acyl donor in dog heart N-acylethanolamine phospholipid biosynthesis

N-Acylethanolamine phospholipids were produced from endogenous substrates with dog heart mitochondrial and microsomal preparations. With mitochondria the N-acyl group contained 13.8% linoleate, with microsomes only 3.6%. Cardiolipin comprised 18.5% of mitochondrial and 3.3% of microsomal lipid P and contained 93.7 and 72.4% linoleic acid, respectively. Incubation of dog heart subcellular fractions with [1-14C]linoleoyl cardiolipin in the presence of Ca2+ resulted in the formation of N-acylethanolamine phospholipids labeled primarily in the N-acyl and 1-O-acyl moieties. The data indicate that cardiolipin is the major source of linoleic acid used in the N-acylation of ethanolamine phospholipids by transacylase activity.     

Preparation and use of a photoactivatable glucose-6-phosphate analogue

A benzophenone-containing derivative of glucose-6-phosphate, 6-[(3-([2,3-3H2]-p-benzoyl dihydrocinnamidoylpropyl-1-oxy)phosphoryl]-D-glucopyranose ([3H]BZDC-Glc-6-P) was synthesized and employed to photoaffinity label proteins on intact rat liver microsomes. The use of a non-photoactivatable, UV-transparent desoxy analogue of BZDC, named p-benzyldihydrocinnamoyl (BnDC), is introduced as a general method to achieve competition when hydrophilic ligands are modified with hydrophobic photophores.     

Phospholipid metabolism in roots and microsomes of sunflower seedlings: Inhibition of choline phosphotransferase activity by boron

The action of boron on phospholipid composition and synthesis in roots and microsomes from sunflower seedlings has been studied. The fatty acid composition and relative amounts of individual molecular species of phospholipids in roots and microsomes were very similar. In both the content of phospholipids was decreased and the relative levels of their component fatty acids changed by treatment with 50 ppm of boron. This concentration of boron in the culture medium was found to inhibit the in vivo [1-^14C] acetate incorporation into root lipids and that of [Me-¹⁴C] choline into phosphatidylcholine of root microsomes. Cytidine-5-diphospho (CDP)-[Me-¹⁴C] choline incorporation into phosphatidylcholine of isolated microsomes was also inhibited by 50 ppm of boron when present in the growth medium of seedlings. These results indicate that the decrease in phosphatidylcholine labelling from [¹⁴C] choline observed when root microsomes were treated with boron would be caused by a decrease in CDP-choline phosphotransferase activity.     

Biosynthesis and processing of rat alpha 1-antitrypsin

Various biosynthetic forms of rat alpha 1-antitrypsin (alpha 1AT) have been isolated by immunoprecipitation of in vitro and in vivo synthesized products. Rat alpha 1AT is synthesized in a rabbit reticulocyte system as a 45,000-Da preprotein with a 23-amino acid signal sequence. The majority of the amino acids in the signal sequence have been identified and resemble the signal peptides of other secretory proteins with respect to the abundance and positions of hydrophobic amino acids. Evidence from the translation of rat liver RNA in the presence of dog pancreas microsomes, from the translation of rat liver polysomes, and from tunicamycin-treated rat hepatocytes established that cleavage of the signal peptide of pre-alpha 1AT results in the formation of a 42,000-Da protein, the polypeptide backbone of mature alpha 1AT. A 50,000-Da glycoprotein is immunoprecipitated from translations programmed with rat liver microsomes or with rat liver mRNA and dog pancreas microsomes. Cotranslational glycosylation of alpha 1AT appears to occur in a stepwise fashion since three glycosylated forms of alpha 1AT (approximately 45,000, 47,000, and 50,000 Da) can be detected in polysome translations. These proteins are susceptible to cleavage by endo-beta-N-acetylglucosaminidase H and are digested to the same product, indicating that they have identical polypeptide chains. Two intracellular forms of alpha 1AT were detected in cultured rat hepatocytes, a 50,000- and a 52,000-Da protein; only the larger protein was immunoprecipitated from the medium of these cells. Digestion with endo-beta-N-acetylglucosaminidase H indicated that the 50,000-Da protein is a core glycosylated processing intermediate, whereas the 52,000-Da protein, which comigrated with purified serum alpha 1AT, appears to contain complex carbohydrate sidechains. When glycosylation was inhibited by incubation of hepatocytes with tunicamycin, a nonglycosylated 42,000-Da protein was immunoprecipitated from the cells and the culture medium, indicating that glycosylation of alpha 1AT is not essential for its secretion.     

Effect of dietary fat saturation on acylcoenzyme A:cholesterol acyltransferase activity of rat liver microsomes

The saturation of the fat contained in the diet has been observed to affect the acylcoenzyme A:cholesterol acyltransferase (ACAT) activity of rat liver microsomes. ACAT activity in microsomes (Mp) prepared from livers of rats fed a polyunsaturated fat-enriched diet containing 14% sunflower seed oil was 70-90% higher than in microsomes (Ms) prepared from livers of rats fed a saturated fat-enriched diet containing 14% coconut oil. This difference was observed within 20 days after the diets were begun, the earliest time tested, and persisted throughout the 70-day experimental period. The difference was noted at all [1-14C]palmitoyl CoA concentrations tested, 2.5-33 micronM, and at temperatures between 18 and 40 degrees C. Arrhenius plots revealed a single transition in enzyme activity, occurring at 29 degrees C in both microsomal preparations. Likewise, the activation energy above this transition was the same in Mp and Ms, 12.5 KCal/mol. Addition of albumin to the incubation medium increased the ACAT activity of both microsome preparations, but the difference between Mp and Ms persisted. Mp was enriched in polyenoic fatty acids, primarily 18:2 and 20:4, while Ms was enriched in monoenoic acids. Although the 20:4 increase in Mp occurred in all phosphoglycerides, it was especially pronounced in the serine and inositol phosphoglyceride fraction. There were no differences in the phospholipid or cholesterol content, phospholipid head group composition, or protein composition of the two microsomal preparations. The possibility is discussed that the changes in ACAT activity result from the differences in fatty acid composition of the microsomes. Other microsomal enzymes exhibited varying responses to these dietary fatty acid modifications. Palmitoyl CoA hydrolase and NADPH cytochrome c reductase activities were unchanged. UDP glucuronyl transferase activity was 50% higher in Mp, but glucose-6-phosphatase and NADH cytochrome b5 reductase activities were 25% higher in Ms. Therefore, dietary fat modifications do not produce a uniform effect on the activity of microsomal enzymes.     

Preparation of S-( )-ketoprofen by coupling the enantioselective hydrolysis of ketoprofen methyl ester with the photo-oxidation of methanol

A novel method for preparation of S-(+)-ketoprofen is presented involving coupling enantioselective hydrolysis of ketoprofen methyl ester catalyzed by a surfactant-coated-lipase with the photo-oxidation of methanol in a water-saturated organic solvent. The effect of photocatalytic conversion of methanol into water and carbon dioxide on the hydrolysis of ketoprofen methyl ester and the stability of the enzyme was investigated. The photo-oxidation of methanol shifted the equilibrium of the hydrolysis toward the formation of ketoprofen, increasing the equilibrium conversion ratio and improving the enantioselectivity. Because the surfactant-coated lipase and ketoprofen methyl ester dissolved in the organic solvent and ketoprofen was absorbed on the TiO₂ photocatalyst particles, the separation procedures could be simplified and the stability of the enzyme was increased.     

Preparation of S-(+)-ketoprofen by coupling the enantioselective hydrolysis of ketoprofen methyl ester with the photo-oxidation of methanol

A novel method for preparation of S-(+)-ketoprofen is presented involving coupling enantioselective hydrolysis of ketoprofen methyl ester catalyzed by a surfactant-coated-lipase with the photo-oxidation of methanol in a water-saturated organic solvent. The effect of photocatalytic conversion of methanol into water and carbon dioxide on the hydrolysis of ketoprofen methyl ester and the stability of the enzyme was investigated. The photo-oxidation of methanol shifted the equilibrium of the hydrolysis toward the formation of ketoprofen, increasing the equilibrium conversion ratio and improving the enantioselectivity. Because the surfactant-coated lipase and ketoprofen methyl ester dissolved in the organic solvent and ketoprofen was absorbed on the TiO₂ photocatalyst particles, the separation procedures could be simplified and the stability of the enzyme was increased.