Phospholipid content, composition and biosynthesis during fetal lung development in the rabbit.

The phospholipid content and composition of lung wash and lung tissue as well as the activities of the enzymes involved in the synthesis of phosphatidylcholine and phosphatidylglycerol (the major surface active components of pulmonary surfactant) were studied in the rabbit during fetal lung development. In lung wash the amount of phospholipid increased four-fold during the period 27-31 day's gestation. There was a further ten-fold increase following the onset breathing. During the same period the amount of phosphatidylcholine in lung wash increased from 29% of the total phospholipid to 80% while the amount of sphingomyelin decreased from 38% to 2%. The amount of phosphatidylcholine in lung tissue also increased during development but to a much lesser extent. During fetal lung development the activities of choline kinase and cholinephosphate cytidyltransferase changed little, cholinephosphotranserase decreased while lysophosphatidic acid acyltransferase and lysolecithin acyltransferase increased. There was a postnatal increase in the activities of cholinephosphate cytidyltransferase, cholinephosphotransferase and both acyltransferases. The amount of phosphatidylglycerol, as a percentage of the total phospholipid, in lung wash and lung tissue as well as the activity of pulmonary glycerolphosphate phosphatidyltransferase did not change appreciably during development.     

Activity of cholinephosphotransferase, lysolecithin: lysolecithin acyltransferase and lysolecithin acyltransferase in the developing mouse lung.

1. The present study presents the activity profiles of cholinephosphotransferase, lysolecithin:lysolecithin acyltransferase and lysolecithin acyltransferase at different stages of development of the mouse lung. 2. The specific activity of cholinephosphotransferase, a key enzyme in the de novo synthesis of phosphatidylcholine, increases during the later stages of fetal development until it reaches a maximal value at a gestational age of 17 days, i.e. 2 days before term. Thereafter, the activity of the enzyme declines again until around term. 2. The specific activity of lysolecithin:lysolecithin acyltransferase which catalyzes the transesterification between two molecules of 1-acyl-sn-glycero-3-phosphocholine, appears to be much lower than that of cholinephosphotransferase at gestational ages below 18 days. However, around day 18, the specific activity of lysolecithin:lysolecithin acyltransferase increases dramatically until it almost equals the maximal activity of cholinephosphotransferase measured on day 17. 4. The specific activity of lysolecithin acyltransferase, which catalyzes the direct acylation of 1-acyl-sn-glycero-3-phosphocholine, does not change significantly during the prenatal development and is lower than that of either lysolecithin:lysolecithin acyltransferase or cholinephosphotransferase at all stages of development. 5. These results are discussed in view of the possible role of these enzymes in the biosynthesis of pulmonary 1,2-dipalmitoyl-sn-glycero-3-phosphocholine.     

Studies on pulmonary surfactant. Effects of cortisol administration to fetal rabbits on lung phospholipid content, composition and biosynthesis.

Corticosteroids are known to accelerate maturation of the fetal lung and production of surfactant. We examined the effect of cortisol administration to fetal rabbits on the phospholipid content and composition of lung lavage and lung tissue, as well as on the activities of enzymes involved in the synthesis of phosphatidylcholine and phosphatidylglycerol, the major surface-active components of surfactant. Cortisol was administered by intrauterine injection at 25 days' gestation and the fetuses were delivered at 27 days (full term, 31 days). Saline-injected fetuses, littermates of the cortisol-treated as well as non-littermates, were used as controls. The amount of phospholipid in lung lavage from the hormone-treated fetuses was almost double that of the saline-injected controls and was similar to that of an untreated fetus of more than 30 days' gestation. Similarly, the phospholipid composition of lung lavage from the hormone-treated fetuses was similar to that of an untreated fetus at a greater gestational age. These data, therefore, suggest that cortisol acts by accelerating physiological development. Cortisol administratration stimulated the activity of cholinephosphate cytidylyltransferase and lysolecithin acyltransferase to a small, but statistically significant extent. This is also consistent with an acceleration of normal development. The stimulation of lysolecithin acyltransferase is of interest, since this enzyme is believed to be involved in the synthesis of dipalmitoylglycerophosphocholine, the major surface-active species of phosphatidylcholine. Cortisol administration had no effect on the activities of pulmonary choline kinase, cholinephosphotransferase, lysophosphatidic acid acyltransferase and glycerolphosphate phosphatidyltranferase, although we have previously shown the latter enzyme to be stimulated following a longer period of exposure to the hormone. Saline injection produced some maturational effects presumably as a result of stress, which may be mediated by corticosteroids or other hormones.     

Stimualtion of glycerolphosphate phosphatidyltransferase activity in fetal rabbit lung by cortisol administration.

Phosphatidylglycerol is an important component of pulmonary surfactant. Previous studies have shown that direct administration of corticosteroids of thyroxine to the fetus during the latter part of gestation results in accelerated lung maturation with increased surfactant production. We have shown that administration of cortisol to fetal rabbits at 24 days' gestation results 3 days later in a significant increase in the activity of pulmonary glycerolphosphate phosphatidyltransferase, an enzyme involved in the synthesis of phosphatidylglycerol. The activity of the liver enzyme was not affected. Choline phosphotransferase, CDPdiglyceride-inositol phosphatidyltransferase, lysophosphatidic acid acyltransferase and lysolecithin acyltransferase activities were not altered significantly by cortisol treatment. Thyroxine treatment had no effect on any of the enzymes of phospholipid or fatty acid biosynthesis studied.     

Human plasma lecithin-cholesterol acyltransferase. Inhibition of the phospholipase A2-like activity by sn-2-difluoroketone phosphatidylcholine analogues

Lecithin-cholesterol acyltransferase (LCAT) is a plasma enzyme which catalyzes the transacylation of the sn-2-fatty acid of lecithin to cholesterol, forming lysolecithin and cholesteryl ester. We have recently proposed a covalent catalytic mechanism for LCAT in which lecithin cleavage proceeds via the formation of a transition state tetrahedral adduct between the oxygen atom of the catalytic serine residue and the sn-2-carbonyl carbon atom of the substrate (Jauhiainen, M., Ridgway, N.D., and Dolphin, P.J. (1987) Biochim. Biophys. Acta 918, 175-188). This proposal is evaluated here by use of nonhydrolyzable sn-2-difluoroketone phosphatidylcholine analogues, known to inhibit calcium-dependent phospholipase A2. These compounds inhibited the calcium-independent phospholipase A2 activity of LCAT in a time and concentration dependent manner. The most potent analogues had a 100-fold higher affinity for the enzyme than the substrate, lecithin, when present within lecithin/apoA-I proteoliposomes. The inhibition was dependent upon the presence of a difluoromethylene group alpha to the sn-2-carbonyl carbon of the analogues. The inhibition is attributed to the formation of a tetrahedral adduct between the catalytic serine residue of LCAT and the sn-2-carbonyl carbon atom of the analogues which is stabilized by the electronegative fluorine atoms present upon the carbon atom alpha to the carbonyl carbon. This adduct mimics that proposed by us to occur during lecithin cleavage by LCAT, and the data substantiate the existence of this transition state adduct prior to the release of lysolecithin and formation of a fatty acylserine oxyester of the enzyme.     

Mechanisms by which elevated intracellular calcium induces S49 cell membranes to become susceptible to the action of secretory phospholipase A2

Exposure of S49 lymphoma cells to exogenous group IIA or V secretory phospholipase A2 (sPLA2) caused an initial release of fatty acid followed by resistance to further hydrolysis by the enzyme. This refractoriness was overcome by exposing cells to palmitoyl lysolecithin. This effect was specific in terms of lysophospholipid structure. Induction of membrane susceptibility by lysolecithin involved an increase in cytosolic calcium and was duplicated by incubating the cells with calcium ionophores such as ionomycin. Lysolecithin also activated cytosolic phospholipase A2 (cPLA2). Inhibition of this enzyme attenuated the ability of lysolecithin (but not ionomycin) to induce susceptibility to sPLA2. Lysolecithin or ionomycin caused concurrent hydrolysis of both phosphatidylethanolamine and phosphatidylcholine implying that transbilayer movement of phosphatidylethanolamine occurred upon exposure to these agents but that susceptibility is not simply due to exposure of a preferred substrate (i.e. phosphatidylethanolamine) to the enzyme. Microvesicles were apparently released from the cells upon addition of lysolecithin or ionomycin. Both these vesicles and the remnant cell membranes were susceptible to sPLA2. Together these data suggest that lysolecithin induces susceptibility through both cPLA2-dependent and -independent pathways. Whereas elevated cytosolic calcium was required for both pathways, it was sufficient only for the cPLA2-independent pathway. This cPLA2-independent pathway involved changes in cell membrane structure associated with transbilayer phospholipid migration and microvesicle release.     

1-Acyl-lysolecithin acyltransferase and synthesis of biliary lecithins in rat liver

The role of lysolecithin acyltransferase activities in biliary lecithin formation was investigated, using livers perfused in the presence of labeled palmitoyl-lysolecithin and albumin, overloaded or not with linoleic acid. At the end of liver perfusion, the lecithins extracted from microsomes, mitochondria and plasma membranes displayed the same specific activity. Double-labeled lysolecithin was used to prove that labeled lecithins were synthesized by lysolecithin acylation. In the absence or presence of a linoleic acid overload, the level of lysolecithin incorporation into linoleyl and arachidonyl containing lecithin was identical. Hence fatty acids did not influence phosphatidylcholine synthesis by the acylation pathway. In vitro the rate of linoleyl lecithin synthesis was the same in plasma membranes, mitochondria and microsomes provided the linoleyl-CoA concentration was lower than 30 microM. Taurocholate was essential to the excretion of lecithin synthesized from lysolecithin and stimulated its synthesis. The specific activities of the two lecithin molecular species excreted in bile (linoleyl and arachidonyl) were not significantly different. These results enabled us to evaluate the contribution of the lysolecithin pathway to the synthesis of lecithin in liver and bile: this contribution in bile was less than 2% under the perfusion conditions used.     

Acyltransferase-catalyzed cleavage of arachidonic acid from phospholipids and transfer to lysophosphatides in lymphocytes and macrophages

The cleavage of fatty acyl moieties from phospholipids was compared in intact cells and homogenates of mouse lymphocytes (thymocytes, spleen cells) and macrophages. Liberation of free arachidonic acid during incubations of intact cells was only detectable in the presence of albumin. Homogenization of prelabeled thymocytes and further incubation of these homogenates at 37 degrees C resulted in a pronounced decrease of phospholipid degradation and cleavage of arachidonoyl residues, while further incubation of homogenates from prelabeled macrophages produced a greatly increased phospholipid degradation. Homogenates of macrophages but not those of thymocytes contain substantial activities of phospholipase A2 detectable using exogenous radiolabeled substrates. These findings indicate that in thymocytes cleavage of arachidonic acid from phosphatidylcholine is an active process that is not catalyzed by phospholipase A2. Addition of CoA and lysophosphatidylethanolamine to prelabeled thymocyte homogenates induced a fast breakdown of phosphatidylcholine and transfer of arachidonic acid to phosphatidylethanolamine, as in seen during incubations of intact thymocytes or macrophages. The transfer is restricted to arachidonic acid and does not require addition of ATP. Sodium cholate, a known inhibitor of the acyl-CoA:lysophosphatide acyltransferase, completely inhibited this transfer reaction. These results suggest that the CoA-mediated, ATP-independent breakdown of phosphatidylcholine and transfer of arachidonic acid is catalyzed by the acyl-CoA:lysophosphatide acyltransferase operating in reverse.     

Human plasma lecithin-cholesterol acyltransferase. An elucidation of the catalytic mechanism

Human plasma lecithin-cholesterol acyltransferase (LCAT) transacylates the sn-2 fatty acid of lecithin to cholesterol forming cholesteryl ester and lysolecithin. Measurement of the phospholipase A2 and transacylase activities of the enzyme using proteoliposome substrates and following selective chemical modification of serine, histidine, and cysteine residues of pure homogeneous LCAT indicated the following catalytic mechanism: HS-Cys-E-Ser-OH + lecithin in equilibrium HS-Cys-E-Ser-O-FA + lysolecithin, HS-Cys-E-Ser-O-FA in equilibrium FA-S-Cys-E-Ser-OH, FA-S-Cys-E-Ser-OH + cholesterol-OH in equilibrium HS-Cys-E-Ser-OH + cholesterol-O-FA, where FA denotes fatty acid. Modification of 2 LCAT cysteine residues with 5,5'-dithiobis-(2-nitrobenzoic acid) or treatment with ferricyanide inactivated the transacylase but not the phospholipase A2 activity. Modification of 1 serine residue with phenylmethanesulfonyl fluoride or 1 histidine residue with diethyl pyrocarbonate inhibited cholesteryl ester formation and phospholipase A2 activity. Proteoliposome substrates protected both activities against chemical inactivation. Lecithin alone protected the phospholipase A2 activity against phenylmethanesulfonyl fluoride inactivation but not the transacylase against 5,5'-dithiobis-(2-nitrobenzoic acid) inactivation. Incubation of native LCAT with arachidonyl-CoA or the lecithin-apo-A-I proteoliposome resulted in acylation of three enzyme sites, only one of which was stable to neutral hydroxylamine after denaturation. Fatty acylenzyme oxy- and thioesters were demonstrable in both cases. No transfer of arachidonic acid from iodoacetamide-modified LCAT to cholesterol occurred, indicating that the fatty-acylated serine residue cannot directly esterify cholesterol. Cholesterol arachidonate was formed upon incubation of phenylmethanesulfonyl fluoride-modified LCAT with arachidonyl-CoA.     

Phospholipid metabolism in the rat renal inner medulla

In view of the importance of phospholipids as a source of precursor fatty acids for the high prostaglandin synthesis in the renal inner medulla, we studied pathways of phospholipid esterification and degradation in the rat inner medulla. De novo acylation of [14C]arachidonate occurred predominantly in position 2 of phosphatidylcholine in the microsomal fraction. This newly esterified [14C]arachidonate was accessible to deacylation by a microsomal phospholipase A2 (EC 3.1.1.4) with alkaline optimum which was Ca2+-dependent and resistant to 0.1% deoxycholate. No phospholipase A1 (EC 3.1.1.32) activity against endogenous labeled phosphatidylcholine could be demonstrated in the microsomal fraction. When exogenous phosphatidylcholine labeled at position 2 was deacylated by renomedullary homogenates, labeled free fatty acid but no labeled lysophosphatidylcholine was recovered in the reaction products. This could be attributed to further degradation of generated lysophosphatidylcholine by a cytosolic lysophospholipase (EC 3.1.1.5). Sodium deoxycholate at a concentration of 0.1% or higher inhibited the lysophospholipase and allowed the demonstration of both A2 and A1 alkaline phospholipase activities in the homogenate. The major in vitro pathway of lysophosphatidylcholine disposition is further degradation by a cytosolic lysophospholipase, while reutilization for phosphatidylcholine synthesis through the action of a predominantly microsomal acyltransferase appears to be a minor pathway. In the presence of several acyl-CoAs, reutilization of lysophosphatidylcholine is significantly increased by an acyl-CoA:lysophosphatidylcholine acyltransferase (EC 2.3.1.23) but there is no preferential transfer of arachidonyl-CoA compared to other acyl-CoAs.     

Effects of experimental acute pancreatitis in dogs on metabolism of lung surfactant phosphatidylcholine

Acute haemorrhagic pancreatitis was produced in the dogs by transduodenal injection of autologous bile into the main pancreatic duct. There was no significant change in the activity of three regulatory enzymes of phosphatidylcholine biosynthesis (glycerophosphate acyltransferase, cytidyltransferase and cholinephosphotransferase) in lung; however, there was a 42% decrease in the amount of dipalmitoyl phosphatidylcholine (surfactant) in lung lavage due to acute pancreatitis. The decrease in lavage phospholipid content was associated with 5-fold increase in phospholipase A2 activity of lung lavage, and massive accumulation of osmiophilic spheroid structures in the alveolar space.     

Is a role of phospholipase A(2) in cholesterol gallstone formation phospholipid species-dependent?

Phospholipase A(2) plays a role in cholesterol gallstone formation by hydrolyzing bile phospholipids into lysolecithin and free fatty acids. This study investigated its effects on cholesterol crystallization in model bile systems. Supersaturated model bile solutions with different cholesterol saturation indexes (1.2, 1.4, and 1.6) were prepared using cholesterol, taurocholate, and egg yolk phosphatidylcholine, soybean phosphatidylcholine, palmitoyl-oleoyl phosphatidylcholine, or palmitoyl-linoleoyl phosphatidylcholine. Then the effect of digestion of phosphatidylcholine by phospholipase A(2) on bile metastability was assessed by spectrophotometry and video-enhanced differential contrast microscopy. Addition of phospholipase A(2) caused the release of free fatty acids in a time-dependent manner. Cholesterol crystallization was enhanced by an increased crystal growth rate in model bile containing hydrophilic species such as soybean or palmitoyl-linoleoyl phosphatidylcholine, consisting predominantly of polyunsaturated fatty acids. Because phospholipase A(2) enhanced cholesterol crystallization in bile containing hydrophilic phosphatidylcholine species, but not hydrophobic phosphatidylcholine species, release of polyunsaturated fatty acids by hydrolysis may be responsible for such enhancement. Therefore, the role of phospholipase A(2) in cholesterol gallstone formation depends on the phospholipid species present in bile, so that phospholipid species selection during hepatic excretion is, in part, crucial to the cholesterol stone formation.     

Studies on the phospholipases of rat intestinal mucosa

1. Subcellular distribution and characteristics of different phospholipases of rat intestinal mucosa were studied. 2. The presence of free fatty acid was necessary for the maximal hydrolysis of lecithin (phosphatidylcholine), but there was no accumulation of lysolecithin (1 or 2-acylglycerophosphorylcholine);lysolecithin accumulated when the reaction was carried out in the presence of sodium deoxycholate and at or above pH8.0. 3. The fatty acid-activated phospholipase B as well as lysolecithinase showed optimum activity at pH6.5, whereas for the phospholipase A it was about pH8.6. 4. The bulk of the phospholipase A was present in the microsomal fraction, whereas the phospholipase B and lysolecithinase activities were distributed between the microsomal and soluble fractions of the mucosal homogenate. 5. Phospholipase A was equally distributed between the brush border and brush-border-free particulate fraction, with the brush border having highest specific activity, whereas the other two activities were distributed between the brush-border-free particulate and soluble fractions. 6. Various treatments showed marked differences between the phospholipase A and phospholipase B activities, but not between phospholipase B and lysolecithinase activities. 7. By using (beta[1-(14)C]-oleoyl) lecithin it was shown that the mucosal phospholipase A was specific for the beta-ester linkage of the lecithin molecule.     

Control of prostanoid synthesis: role of reincorporation of released precursor fatty acids

Prostanoid synthesis is limited by the availability of free arachidonic acid. This polyunsaturated fatty acid is liberated by phospholipases and usually is an intermediate of the deacylation-reacylation cycle of membrane phospholipids. In rat peritoneal macrophages, ethylmercurisalicylate (merthiolate) or N-ethylmaleimide (NEM) dose dependently inhibited the incorporation of arachidonic acid into cellular phospholipids, at lower concentrations specifically into phosphatidylcholine. Furthermore, merthiolate could be shown to be a rather selective inhibitor of lysophosphatidylcholine acyltransferase. In contrast, phospholipase A2 activity was not affected over a wide dose range. Consequently, macrophages showed a large increase in prostanoid synthesis (prostaglandin E, prostacyclin and thromboxane) in the presence of both lysophosphatide acyltransferase inhibiting agents. Similar results were obtained with human platelets, in which merthiolate increased the release of thromboxane. Addition of free arachidonic acid also enhanced prostanoid synthesis in macrophages. At optimal concentrations, merthiolate had no further augmenting effect. It is concluded that the rate of prostanoid synthesis is not only controlled by phospholipase A2 activity, but rather by the activity of the reacylating enzymes, mainly lysophosphatide acyltransferase.     

Phospholipid remodeling in human neutrophils. Parallel activation of a deacylation/reacylation cycle and platelet-activating factor synthesis

A23187 stimulated two enzymatic activities of human neutrophils (polymorphonuclear leukocytes), phospholipase A2 and fatty acyl-CoA acyltransferase, which resulted in a stimulated deacylation/reacylation cycle. The incorporation of fatty acids, other than arachidonic or eicosapentaenoic acid, into diacyl and alkylacyl species of choline phosphoglycerides was stimulated by 10-fold by A23187. These fatty acids were exclusively incorporated into the sn-2 position, and [3H]glycerol labeling showed there was no stimulation of de novo synthesis. A23187 also stimulated fatty acid incorporation into other phospholipids, but de novo synthesis accounted for a portion of this uptake. Inhibitors of protein kinase C prevented the stimulated recycling of phosphatidylcholine, and the simultaneous induction of platelet-activating factor synthesis, by inhibiting phospholipase A2 activation. They inhibited [3H]arachidonate release from prelabeled polymorphonuclear leukocytes, but had no effect on in vitro fatty acyl-CoA acyltransferase or acetyl-CoA acetyltransferase activity. Extracts from A23187-treated cells contained a fatty acyl-CoA acyltransferase, which did not utilize arachidonoyl-CoA, that was 2.3-fold more active than that of control extracts. Phosphatase treatment decreased this stimulated activity by 66%. Thus, A23187 stimulated a phospholipase A2 activity that generated both 1-alkyl and 1-acyl lysophosphatidylcholines. A stimulated acetyltransferase used a portion of the alkyl species for platelet-activating factor synthesis, while the acyl species and residual alkyl species were rapidly reacylated to phosphatidylcholine by a stimulated acyl-transferase. Arachidonate, an eicosanoid precursor, was spared by this process.     

Coenzyme A-mediated transacylation of sn-2 fatty acids from phosphatidylcholine in rat lung microsomes

Evidence was obtained for a CoA-dependent transfer of linoleate from rat lung microsomal phosphatidylcholine to lysophosphatidylethanolamine without the intervention of a Ca2+-requiring phospholipase A2 activity and ATP. To study this CoA-mediated transacylation process, microsomes were prepared in which the endogenous phosphatidylcholine was labeled by protein-catalyzed exchange with phosphatidylcholines containing labeled fatty acids in the sn-2-position. The apparent Km for CoA in the transfer of arachidonate from phosphatidylcholine to 1-acyllysophosphatidylethanolamine was 1.5 microM. At saturating lysophosphatidylethanolamine concentrations, the transacylation was linear with the amount of microsomal protein, i.e., a fixed percentage of the labeled fatty acid was transferred independent of the amount of microsomal protein. A maximal transfer of 12.2% for arachidonate and 2.0% for linoleate from the respective phosphatidylcholines to lysophosphatidylethanolamine was observed in 30 min. With 1-acyl-2-[1-14C]arachidonoylphosphatidylcholine as acyl donor, lysophosphatidylethanolamine was the best acceptor followed by lysophosphatidylglycerol and lysophosphatidylserine. Lysophosphatidate barely functioned as acceptor. These data provide further evidence for the widespread occurrence of CoA-mediated transacylation reactions. The arachidonate transacylation from phosphatidylcholine to other phospholipids in lung tissue may contribute to the low level of arachidonate in pulmonary phosphatidylcholine.     

Histochemical demonstration of phospholipase B (lysolecithinase) activity in rat tissues.

A method has been developed for the histochemical demonstration of phospholipase B (lysolecithinase) of rat tissues. The enzyme attacks lysolecithin with liberation of 1 mole of glycerylphosphorylcholine and 1 mole of fatty acid. The recommended procedure involves use of 6-10 micro frozen sections, fixed in cold calcium-formol and incubated at 37 degrees C in Tris buffered medium at pH 6.6 containing 2.2 X 10(-3) M lysolecithin and 1% cobalt acetate. The fatty acid liberated by enzymatic hydrolysis is trapped as a cobalt precipitate and is then converted to a black-brown precipitate by treatment with dilute ammonium sulfide in cold isotonic saline. Equivalent amounts of fatty acid and glycerylphosphorylcholine are recovered by extraction and analysis of the incubated sections and of the incubation medium, thus proving that lysolecithin hydrolysis occurs under the proposed reaction conditions. Staining is reduced by treating the sections with copper ions, mercury compounds, alcohols, acetone and by heating at 60 degrees C prior to incubation with substrate. Lowering of the pH of the incubation medium has similar effect. These findings are interpreted as evidence of the enzymatic nature of the reaction. Cells exhibiting a positive staining are found in the lamina propria of the intestinal villi and crypts, in the red pulp of the spleen and in the interstitial tissue of lung, liver and thymus. Similar elements are present in bone marrow smears and in leukocyte preparations obtained by peritoneal lavage. The morphologic and staining characteristics of these cells correspond to those of the eosinophilic leukocytes. Physical and chemical agents (x-irradiation, corticosteroids) which sharply decrease the number of eosinophils also reduce the number of cells shown histochemically to hydrolyze lysolecithin. A correspondent diminution of phospholipase B activity of homogenates of the same tissues can be shown in vitro. Differences in tissue distribution and chemical properties distinguish the phospholipase B from less specific esterases and lipases.     

Effect of albumin on acyl-CoA: lysolecithin acyltransferase,lysolecithin : lysolecithin acyltransferase and acyl-CoA hydrolase from rabbit lung

Acyl-CoA : lysolecithin and lysolecithin : lysolecithin acyltransferases, as well as acyl-CoA hydrolase are important enzymes in lung lipid metabolism. They use amphiphylic lipids as substrates and differ in subcellular localization. In this sense, lipid-protein interactions can be an essential factor in their activity. We have studied the effect of albumin, as lipid-binding protein model, in the activities of these enzymes. Acyl-CoA hydrolase was inhibited in the presence of albumin, whereas acyl-CoA : lysolecithin acyltransferase showed a complex effect of activation depending on both albumin concentration and palmitoyl-CoA/lysolecithin molar ratio. Lysolecithin : lysolecithin acyltransferase was affected differentially on its two activities. Hydrolysis remained unaffected and transacylation was inhibited by albumin. These results are consequence of the interaction of albumin with both lipidic substrates that changes their critical micellar concentration.Abbreviations TNS 6-(p-toluidino)-2-naphthalene-sulfonic acid - CMC Critical Micellar Concentration - LP Lysolecithin (1-acyl-sn-glycero-3-phosphocholine) - PalmCoA palmitoyl-CoA     

Effect of lipids on activity and conformation of lysolecithin:lysolecithin acyltransferase from rabbit lung

Summary Lysolecithin:lysolecithin acyltranferase is an enzyme which in several previous studies has shown a dual behavior catalyzing two types of reaction, transacylation or hydrolysis, with the same substrate. Both activities have shown to be dependent on several environmental conditions and among them, the presence of lipids.The addition of several classes of lipids activated in all the cases the enzyme, decreasing the hydrolysis/transacylation molar ratio. This effect was higher for PC/PE/Chol mixture than for other lipids assayed. Circular dichroism spectra of the enzyme did not show any change with the addition of lipids, concluding that the effect of lipids was not due to any structural change in the protein. The hypothesis has been made of an influence of lipids on the physical state of the substrate as well as, possibly, on the enzyme-substrate interaction.The significance of these effects on the physiological role of lysolecithin:lysolecithin acyltransferase from soluble fraction of rabbit lung is discussed.Abbreviations Chol cholesterol - CMC critical micellar concentration - DPPC dipalmitoylphosphatidylcholine - FA fatty acid - H/T hydrolysis/transacylation molar ratio - LPC lysophosphatidylcholine - PC phosphatidylcholine - PE phosphatidylethanolamine - TG triglyceride - UV ultraviolet     

Substrate specificity of neutral phospholipase D from rat brain studied by selective labeling of endogenous synaptic membrane phospholipids in vitro.

We have designed a novel approach for studying the specificity of neutral phospholipase D from rat brain synaptic plasma membranes for endogenous phospholipid substrates in native membranes. A procedure was established that provides synaptic membranes labeled in selected phospholipids. This labeling procedure exploits the presence of endogenous acyl-coenzyme A synthetase and acyl-coenzyme A:lysophospholipid acyltransferase in synaptosomes for acylating various lysophospholipid acceptors with radioactive fatty acid. With [3H]arachidonate for acylation and optimal concentrations of the respective lysophospholipids, membranes were labeled in either of the following phospholipids: phosphatidylcholine (93% of total label in phospholipids), 1-O-alkyl-phosphatidylcholine (87%), phosphatidylinositol (90%), phosphatidylethanolamine (85%), phosphatidylethanolamine-plasmalogen (81%) or phosphatidylserine (59%). These membranes were employed to study the substrate specificity of the neutral, oleate-activated rat brain phospholipase D. This phospholipase exhibited almost absolute specificity for the choline-phospholipids phosphatidylcholine and 1-O-alkyl-phosphatidylcholine: 0.34% of the former labeled substrate were transphosphatidylated to phosphatidylpropanol during the assay and 0.28% of the latter. Activity toward other phospholipids was barely detectable and could largely be accounted for by utilization of residual labeled phosphatidylcholine present in those preparations. The phospholipase D exhibited some preference for fatty acids in the C-2 position of phosphatidylcholine in the following order: 2-oleoyl-phosphatidylcholine (0.67% of this labeled phosphatidylcholine were converted to phosphatidylpropanol), 2-myristoyl-phosphatidylcholine (0.60%), 2-palmitoyl-phosphatidylcholine (0.46%) and 2-arachidonoyl-phosphatidylcholine (0.34%). The present approach of labeling membrane phospholipids in vitro could be useful in studies of phospholipase specificity as an alternative to the use of sonicated vesicles or mixed detergent-phospholipid micellar systems.