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.     

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.     

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.     

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.     

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.     

Lecithin-cholesterol acyltransferase (LCAT) catalyzes transacylation of intact cholesteryl esters. Evidence for the partial reversal of the forward LCAT reaction

Lecithin-cholesterol acyltransferase (LCAT) catalyzes the intravascular synthesis of lipoprotein cholesteryl esters by converting cholesterol and lecithin to cholesteryl ester and lysolecithin. LCAT is unique in that it catalyzes sequential reactions within a single polypeptide sequence, a phospholipase A2 reaction followed by a transacylation reaction. In this report we find that LCAT mediates a partial reverse reaction, the transacylation of lipoprotein cholesteryl oleate, in whole plasma and in a purified, reconstituted system. As a result of the reverse transacylation reaction, a linear accumulation of [3H]cholesterol occurred during incubations of plasma containing high density lipoprotein labeled with [3H]cholesteryl oleate. When high density lipoprotein labeled with cholesteryl [14C]oleate was also included in the incubation the labeled fatty acyl moiety remained in the cholesteryl [14C]oleate pool showing that the formation of labeled cholesterol did not result from hydrolysis of the doubly labeled cholesteryl esters. The rate of release of [3H]cholesterol was only about 10% of the forward rate of esterification of cholesterol using partially purified human LCAT and was approximately 7% in whole monkey plasma. Therefore, net production of cholesterol via the reverse LCAT reaction would not occur. [3H]Cholesterol production from [3H]cholesteryl oleate was almost completely inhibited by a final concentration of 1.4 mM 5,5'-dithiobis(nitrobenzoic acid) during incubation with either purified LCAT or whole plasma. Addition of excess lysolecithin to the incubation system did not result in the formation of [14C]oleate-labeled lecithin, showing that the reverse reaction found here for LCAT was limited to the last step of the reaction. To explain these results we hypothesize that LCAT forms a [14C]oleate enzyme thioester intermediate after its attack on the cholesteryl oleate molecule. Formation of this intermediate allows [3H]cholesterol to be liberated from the enzyme by exchange with unlabeled cholesterol of plasma lipoproteins. The liberated [3H]cholesterol thereby becomes available for reesterification by LCAT as indicated by its appearance as newly synthesized cholesteryl linoleate.     

Acylation of lysolecithin in the intestinal mucosa of rats

1. The presence of an active acyl-CoA-lysolecithin (1-acylglycerophosphorylcholine) acyltransferase was demonstrated in rat intestinal mucosa. 2. ATP and CoA were necessary for the incorporation of free [1-(14)C]oleic acid into lecithin (phosphatidylcholine). 3. The reaction was about 20 times as fast with [1-(14)C]oleoyl-CoA as with free oleic acid, CoA and ATP. 4. With 1-acylglycerophosphorylcholine as the acceptor, both oleic acid and palmitic acid were incorporated into the beta-position of lecithin; the incorporation of palmitic acid was 60% of that of oleic acid. 5. Of the various analogues of lysolecithin tested as acyl acceptors from [1-(14)C]oleoyl CoA, a lysolecithin with a long-chain fatty acid at the 1-position was most efficient. 6. The enzyme was mostly present in the brush-border-free particulate fraction of the intestinal mucosa. 7. Of the various tissues of rats tested for the activity, intestinal mucosa was found to be the most active, with testes, liver, kidneys and spleen following it in decreasing order.     

Phospholipid removal during degradation of rat plasma very low density lipoprotein in vitro.

The hydrolysis of glycerophospholipids in very low density lipoprotein by enzyme(s) released into circulation after the injection of heparin to rats was studied. [32P]Lysolecithin was formed rapidly from [32P]lecithin when very low density lipoprotein, labeled biosynthetically with 32P, was incubated with postheparin plasma. The [32P]lysolecithin was associated with the plasma protein fraction of density greater than 1.21 g/ml, whereas [32P]lecithin exchanged between very low and high density lipoproteins. Inhibition of the plasma lecithin: cholesterol acyl transferase activity did not change the excess [32P]lysolecithin formation in postheparin plasma, and only a negligible amount of radioactivity was associated with blood cells when the incubation was repeated in whole blood. Analysis of the results has demonstrated that phospholipids are removed from VLDL by two pathways: hydrolysis of glycerophospholipids by the heparin-releasable phospholipase activity (greater than50%) and transfer to high density lipoproteins (less than50%). The tissue origin of the postheparin phospholipase was studied in plasma obtained from intact rats and supradiaphragmatic rats using specific inhibitors of the extrahepatic lipase system (protamine sulfate and 0.5 M NaCl). The phospholipase activity could be ascribed to both the hepatic and extrahepatic lipase systems. It is concluded that hydrolysis of glycerophospholipids is the major mechanism responsible for the removal of phospholipids from very low density lipoprotein during the degradation of the lipoprotein. It is suggested that phospholipid hydrolysis occurs concomitantly with triglyceride hydrolysis, predominantly in extrahepatic tissues.     

Stimulation of galactosyltransferase in liver microsomes by lysolecithin

Lysolecithin markedly stimulated membrane-bound UDP-galactose:glycoprotein galactosyltransferase. The parent molecule lecithin, phosphatidylethanolamine, lysophosphatidylethanolamine, phosphatidic acid, lysophosphatidic acid or glycerophosphorylcholine did not activate the enzyme suggesting that both fatty acyl- and phosphorylcholine groups are required for the enzyme activation. The dose-effect of lysolecithin showed sigmoidal kinetics and the Vmax of the enzyme was increased several-fold by lysolecithin. Saturating amounts of Triton masked the effect of lysolecithin. Pre-incubation with phospholipase A also activated the enzyme. A possible role of membrane lysolecithin is indicated in regulating the enzymes of glycoprotein synthesis.     

The kinetic mechanism of acyl-CoA:lysolecithin acyltransferase from rabbit lung

Acyl-CoA:lysolecithin acyltransferase is a key enzyme in the deacylation-reacylation pathway of biosynthesis of molecular species of lecithin. However, the mechanism of the reaction has been little studied. In this paper, the kinetic mechanism of acyl-CoA:lysolecithin acyltransferase, partially purified from rabbit lung, is studied. The double-reciprocal plots of initial velocity vs substrate concentration gave two sets of parallel lines which fitted to a ping-pong equation with the following parameters: Km (palmitoyl-CoA) = 8.5 +/- 2 microM, Km (lysolecithin) = 61 +/- 16 microM, and V = 18 +/- 4 nmol/min/mg protein. Inhibition studies by substrates, alternate substrates, and products supported the ping-pong mechanism, although some nonclassical behavior was observed. Palmitoyl-CoA did not inhibit even at concentrations of 100 Km. In contrast, lysolecithin was a dead-end inhibitor with a dissociation constant of Ki = 930 +/- 40 microM. Alternate substrates and CoA showed alternate pathways for the reaction due to the formation of ternary complexes. Dipalmitoylphosphatidylcholine inhibition pointed to an isomerization of the free enzyme prior to the start of the reaction. From these results, an iso-ping-pong kinetic mechanism for lysolecithin acyltransferase is proposed. The kinetic steps of the reaction are correlated with previous chemical studies of the enzyme.     

Effect of detergents,trypsin, and bivalent metal ions on interfacial activation and functioning of phospholipase D

The effects of detergents, trypsin, and bivalent metal ions on production of phosphatidic and lysophosphatidic acids by the action of phospholipase D (PLD) on lecithin and lysolecithin were studied. It was found that these reaction products and dodecyl sulfate ions activate PLD, whereas other anionic detergents are less effective. A protective effect of the functioning enzyme against its hydrolytic inactivation by trypsin was found. Bivalent metal ions can be arranged in the following sequence by their ability to activate PLD in the hydrolysis of lecithin and lysolecithin: Ca²⁺ > Sr²⁺ > Ba²⁺ > Mg²⁺. These results are considered in relation to a proposed mechanism of activation and functioning of PLD with the participation of clusters of phosphatidates and lysophosphatidates. Such Me²⁺-induced formation of rafts or microdomains from the products of hydrolysis of phospholipids can rationalize not only PLD activation and self-regulation, but also the action of this mechanism on other components and properties of biomembranes. PLD and other lipolytic enzymes can be classified as lateral vector enzymes.     

A rapid assay of acyl-coenzyme A:lysolecithin acyltransferase activity

A simple and rapid procedure for the assay of acyl-coenzyme A:1-acyl-sn-glycero-3-phosphocholine acyltransferase (lysolecithin acyltransferase, LLAT [EC 2.3.1.23]) activity in crude enzyme preparations is described. The incubation system utilizes lysolecithin and [1-14C]-oleoyl-coenzyme A as substrates. Labeled fatty acid released due to accompanying acyl-coenzyme A hydrolase [EC 3.1.2.2]activity is first removed by di-isopropyl ether extraction. The labeled lecithin produced due to LLAT action is then quantitatively recovered by partition of the incubation medium with di-isopropyl ether-n-butanol 60:40 (v/v). Selective extraction of the labeled lecithin formed and avoidance of customary thin-layer chromatographic isolation procedures permits assay of LLAT activity with excellent accuracy at a substantial saving of time. The entire assay can be completed in less than 30 min as compared to 2-3 hrs when following conventional procedures.     

Schistosoma mansoni: a comparative study of plasma and erythrocyte lipid alterations in the experimentally infected mouse and in selected human patients

Alterations of plasma and erythrocyte lipids associated with hepatosplenic schistosomiasis mansoni were studied in the mouse and in human patients. Qualitative and quantitative differences were observed between the two species which indicated that the experimentally infected mouse should not be used as a model for altered lipid metabolism associated with Schistosoma mansoni infections in man. Also blood lipid values should not be used as prophylactic indicators for experimental therapeutical studies in the infected mouse, although lipid determinations could have clinical value in studies of human patients. In infected mice plasma cholesterol and phospholipid were significantly reduced (40 and 25%, respectively), but proportions of individual plasma phospholipids were unchanged. In contrast, only plasma cholesterol was reduced in human patients with compensated or decompensated hepatosplenic schistosomiasis (16 and 29%, respectively); of the individual phospholipids, lecithin was significantly increased and lysolecithin was decreased. The percentage of plasma total cholesterol was reduced in infected mice and patients suggesting that hypocholesterolemia is due mainly to decreased cholesteryl ester. Lipid changes also occurred in erythrocytes. Those of infected mice had significantly elevated membrane phospholipid content and no changes in cholesterol or in the proportions of the individual phospholipid fractions. In marked contrast, the erythrocytes of two groups of human patients had significantly higher levels of cholesterol without a raised total phospholipid concentration. Moreover, decreased proportions of lysolecithin and increased proportions of lecithin were apparent although only the increased membrane lecithin associated with compensated patients was statistically significant.     

Biosynthesis of retinal phospholipids: incorporation of radioactivity from labeled phosphorylcholine and cytidine diphosphate choline

Phosphorylcholine-1,2-(14)C and choline-1,2-(14)C-labeled cytidine diphosphate choline are incorporated into lecithin by whole homogenates and particulate fractions of rat retina with optimal incorporation of label by the microsomal fraction. The soluble fraction contains a factor(s) which stimulates incorporation of label with release of inorganic phosphate. Mg(++) is required for optimal incorporation of intermediates into lecithin in the presence of added diglycerides; without added diglycerides, incorporation of phosphorylcholine or cytidine diphosphate choline was moderately stimulated by preincubating the system in the absence of Mg(++) with added phosphatidic acid and by adding this mixture to fresh enzyme and the complete incubation mixture (including Mg(++)). The results show that the retina is capable of de novo synthesis of phosphatides and suggest that the rod outer segments depend on the pigment epithelium and(or) the inner rod segments for a source of phospholipids. Coenzyme A and ATP added to whole homogenate of retina did not significantly increase the incorporation of CDP-choline-1,2-(14)C into lecithin but slightly increased the radioactivity found in lysolecithin and sphingomyelin. Rats with hereditary retinitis pigmentosa have an abnormally high lipid phosphorus content of the retina, but they do not incorporate labeled CDP-choline into lecithin of retina at a higher rate than do normal animals.     

Distribution of lecithin-cholesterol acyltransferase (LCAT) in human plasma lipoprotein fractions. Evidence for the association of active LCAT with low density lipoproteins

The distribution of lecithin-cholesterol acyltransferase (LCAT) in human plasma was assessed by measuring both LCAT mass and activity in plasma fractions separated by sequential flotation ultracentrifugation, single-spin gradient ultracentrifugation, dextran sulfate-Mg²⁺ precipitation or agarose gel filtration. Although most of the LCAT was found to be associated with the high density lipoprotein fraction, a small amount of active LCAT (approximately 1% of the plasma LCAT mass and activity) was consistently associated with the low density lipoprotein fraction. LCAT was not found in the very low density lipoprotein fraction. The LDL-associated LCAT may play an important role in the acylation of lysolecithin by lysolecithin acyltransferase activity of LCAT.     

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.     

Some properties of purified phospholipase D and especially the effect of amphipathic substances

1. The soluble phospholipase D of cabbage was purified by heat treatment, acetone precipitation and electrophoresis on a density gradient of aqueous glycerol. 2. The purified enzyme slowly attacked a lecithin suspension whereas ultrasonically treated lecithin was hydrolysed more rapidly. 3. Diethyl ether stimulated the hydrolysis of both the lecithin suspension and ultrasonically treated lecithin. 4. Ca²⁺ was essential for the hydrolysis (optimum about 0·04m); it could not be replaced by Mg²⁺ or cationic amphipathic substances. 5. The reaction had a sharp pH optimum at pH5·4, irrespective of the physical form of the lecithin substrate or the activator used. 6. Anionic amphipathic substances such as dodecyl sulphate, phosphatidic acid, triphosphoinositide and monocetyl phosphoric acid, were potent activators of the reaction: other acidic lipids were relatively inactive. 7. Cationic amphipathic substances inhibited the hydrolysis; however, they also reversed the inhibition caused by using an excess of anionic amphipathic substance as activator. 8. The activation produced by amphipathic substances could not be correlated with their effect on the ζ-potential or size of the substrate particles. 9. The addition of activating anionic amphipaths to lecithin induces the latter to adsorb enzyme from solution. In the absence of Ca²⁺ the enzyme is denatured on the highly negatively charged surface, but in the presence of Ca²⁺ (or Mg²⁺) it is protected from denaturation. It is suggested that this adsorption is an essential prerequisite for ready enzymic hydrolysis. 10. The hydrolysis of lecithin by the enzyme was strongly inhibited by protamine sulphate (0·1mg./ml.) and by choline and ethanolamine. 11. Ultrasonically treated phosphatidylethanolamine, or mixed particles of phosphatidylethanolamine plus dodecyl sulphate, were slowly attacked by the enzyme provided that Ca²⁺ was present.     

PROPERTIES OF MEMBRANE-BOUND DOPAMINES-HYDROXYLASE IN CHROMAFFIN GRANULES FROM BOVINE ADRENAL MEDULLA

Abstract— Properties of membrane-bound and soluble dopamine-β-hydroxylase were studied. Both enzyme forms have identical affinities for tyramine as the substrate. Arrhenius plots of the membrane-bound activity displayed a discontinuity at 29°C, the activation energy changing from 20,500 cal/mol below 29°C to 9500 cal/mol above 29°C. The soluble enzyme, like the purified enzyme, did not show discontinuities in Arrhenius plots, the activation energies being 18,500 cal/mol and 16,500 cal/mol respectively. The membrane-bound enzyme showed a discontinuity in the p K^m for tyramine versus reciprocal temperature plot, with a transition at 29°C, whereas the soluble enzyme failed to show such transition.
The membrane-bound dopamine-β-hydroxylase is solubilized by Triton X-100 as well as by lysolecithin. Of lecithin, lysophosphatidyl ethanolamine and phosphatidyl serine, lysolecithin was the only phospholipid to induce solubilization of membrane-bound dopamines β-hydroxylase. The 29°C-transition was not removed by treatment either with lysolecithin or with Triton X-100 used at concentration of up to 2%. This could indicate a solubilization of dopamine-β-hydroxylase with an associated lipid moiety which cannot be dispersed from the enzyme molecule and which still affects the activation energy and Michaelis constant for tyramine.
Results are discussed in terms of the relationship between lipid organization and enzyme activities; the significance of the solubilization by lysolecithin during exocytosis is considered in terms of the exocytosis process and fate of the chromaffin granule.     

Human plasma lecithin-cholesterol acyltransferase. The vicinal nature of cysteine 31 and cysteine 184 in the catalytic site

Lecithin-cholesterol acyltransferase (LCAT) is a plasma enzyme which catalyzes the transacylation of the fatty acid at the sn-2 position of lecithin to cholesterol forming lysolecithin and cholesteryl ester. The substrates for and products of this reaction are present within the plasma lipoproteins upon which the enzyme acts to form the majority of cholesteryl ester in human plasma. We proposed a covalent catalytic mechanism of action for LCAT (Jauhiainen, M., and Dolphin, P. J. (1986) J. Biol. Chem. 261, 7032-7034) in which serine and histidine residues mediate lecithin cleavage and two cysteine residues cholesterol esterification. With the aid of sulfhydryl reactive trivalent organoarsenical compounds which are specific for vicinal thiols we have probed the geometry of the catalytic site. p-Aminophenylarsendichloride noncompetitively inactivates cholesterol esterification (Ki = 0.23 mM) by LCAT via alkylation of both catalytic cysteine residues. This reagent does not significantly inactivate lecithin cleavage by LCAT. Full enzyme activity is restored by treatment with 2,3-dimercapto-1-propanesulfonic acid. Treatment of LCAT with p-bromoacetylaminophenylarsenoxide blocks the subsequent incorporation of diisopropyl fluorophosphate and iodoacetamide and inactivates both cholesterol esterification and lecithin cleavage. These activities are not restored following 2,3-dimercapto-1-propanesulfonic acid treatment. However, the reduced cysteine thiols are regenerated and can catalyze cholesteryl arachidonate formation from arachidonyl-CoA. The control reagent, bromoacetylaniline, which lacks the sulfhydryl-reactive arsenical moiety, does not inactivate LCAT nor is this reagent incorporated into the LCAT protein. We conclude that the two catalytic cysteine residues of LCAT (Cys31 and Cys184) are vicinal with a calculated distance between their sulfur atoms of 3.50-3.62 A. The additional residue alkylated by the bifunctional reagent is within the catalytic site and may represent a previously identified catalytic serine or histidine residue.     

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.