A novel assay for measuring recombinant human lysophosphatidylcholine acyltransferase 3 activity.

Lysophosphatidylcholine acyltransferase 3 (LPCAT3) is an important enzyme in phospholipid remodeling, a process that influences the biophysical properties of cell membranes and thus cell function. Multiple lines of evidence suggest that LPCAT3 is involved in several diseases, including atherosclerosis, non-alcoholic steatohepatitis, and carcinoma. Thus, LPCAT3 may have potential as a therapeutic target for these diseases. In the present study, we devised an assay based on reversed-phase HPLC to measure LPCAT3 activity, which may facilitate the identification of LPCAT3 inhibitors and activators. We found that optimal pH and temperature of recombinant human LPCAT3 are 6.0 and 30 °C, respectively. The enzyme Km values for substrates NBD-labelled lysophosphatidylcholine and arachidonoyl CoA were 266.84 ± 3.65 and 11.03 ± 0.51 µmol·L-1 , respectively, and the Vmax was 39.76 ± 1.86 pmol·min-1 ·U-1 . Moreover, we used our new method to determine the IC50 of a known LPCAT inhibitor, TSI-10. In conclusion, this novel assay can be used to measure the effects of compounds on LPCAT3 activity.

Alkyne lipids as substrates for click chemistry-based in vitro enzymatic assays.

Click chemistry is evolving as a powerful tool in biological applications because it allows the sensitive and specific detection of compounds with alkyne or azido groups. Here we describe the use of alkyne lipids as substrates for in vitro enzymatic assays of lipid modifying enzymes. The small alkyne moiety is introduced synthetically at the terminus of the hydrocarbon chain of various substrate lipids. After the assay, the label is click-reacted with the azide-bearing fluorogenic dye 3-azido-7-hydroxycoumarin, followed by the separation of the lipid mix by thin-layer chromatography and fluorescence detection, resulting in high sensitivity and wide-range linearity. Kinetic analyses using alkyne-labeled substrates for lysophosphatidic acid acyltransferases, lysophosphatidylcholine acyltransferases, and ceramide synthases resulted in Michaelis-Menten constants similar to those for radiolabeled or natural substrates. We tested additional alkyne substrates for several hydrolases and acyltransferases in lipid metabolism. In this pilot study we establish alkyne lipids as a new class of convenient substrates for in vitro enzymatic assays.

Identification of two different lysophosphatidylcholine:acyl-CoA acyltransferases (LAT) in pig spleen with putative distinct topological localization.

The lysophosphatidylcholine:acyl-CoA acyltransferase (LAT, EC 2.3.1.23) is an integral membrane protein participating in the membrane turnover and the T-cell activation process. Here, we present data that crude membranes of pig spleen contain two different LAT enzyme activities based on topological localization studies and the enzyme specificities towards various acyl-CoAs. When crude membranes are washed with solutions of high ionic strength the supernatant contains a distinct LAT activity that we refer to as peripheral LAT (pLAT). The majority of LAT activity is found in the membrane pellet also after treatment with CHAPS. The CHAPS-insoluble LAT activity is named integral LAT (iLAT) accordingly. While pLAT prefers arachidonoyl-CoA rather than oleoyl-CoA, iLAT shows no specificity towards both unsaturated acyl-CoAs. Further investigations reveal that the CHAPS-insoluble LAT activity in the membranes can be solubilized by n-octyl glucoside and restored to original activity by reconstitution with artificial membranes. The reconstituted iLAT prefers arachidonoyl-CoA rather than oleoyl-CoA. Despite a great deal of effort by several groups little progress has been made so far in LAT purification because of the enzyme instability. We establish experimental conditions that enhance the stability of both enzyme activities and, therefore, allow further protein purification.

Gestational age dependence of 11 beta-hydroxysteroid dehydrogenase and its relationship to the enzymes of phosphatidylcholine synthesis in lung and liver of fetal rat.

Increase in fetal surfactant synthesis and lung maturity is caused by the glucocorticoidal induction of enzymes required for phosphatidylcholine (PC) synthesis towards the end of gestation. The regulation of gestational age-dependent induction of PC synthesis by glucocorticoids is still unclear. Since 11-beta-hydroxysteroid dehydrogenase (11 beta-HSD) activity and its metabolising capacity for glucocorticoids have been suggested to play a central role in this regulation, we measured the gestational age-dependent changes in 11 beta-HSD and PC synthesizing enzymes in lung and liver of fetal rat. The activity of cholinephosphate cytidyltransferase (CCT; key enzyme in PC synthesis), choline phosphotransferase (CPT) and lysolecithin acyltransferase (LAT) were found to increase gradually in the lung towards the end of gestation, reached peak values at term followed by a decrease of activity reaching finally adult levels. Only CK activity exhibited constant levels until term followed by a slight increase after the birth. In comparison with the lung, the liver enzymes followed a similar pattern, but at a higher rate of activity except for CCT which was higher in the lung. The activity of 11 beta-HSD in fetal lung microsomes was detectable from day 20 and increased towards the end of gestation in the lung and liver of the rat. Oxidase activity was always found to exceed the reductase activity. The activity of 11 beta-HSD continued to increase after delivery and reached peak levels in adult animals in both organs. In order to test the hypothesis, whether 11 beta-HSD activity and PC synthesis are induced by increasing endogenous glucocorticoidal levels, we examined on day 19 of gestation the effect of dexamethasone (DEXA) on enzymatic activities (11 beta-HSD, CCT) and on [14C]choline incorporation in phosphatidylcholine in fetal lung organoid cultures. Additionally, changes in CCT activity in fetal lungs after maternal administration of DEXA were measured. DEXA accelerated 11 beta-HSD and CCT activities as well as [14C]choline incorporation. We conclude, that endogenous glucocorticoids induce PC synthesis as well as 11 beta-HSD activity in lung and liver of the fetal rat. Fetal PC synthesis is not altered by increasing 11 beta-HSD levels, because the increase of free serum corticosterone levels apparently exceeds the metabolising capacity of 11 beta-HSD towards term.

Use of a silicic acid microcolumn to assay acyl-CoA: lysophosphatidylcholine acyltransferase.

A simple and rapid method for assaying acyl-CoA:lysophosphatidylcholine acyltransferase is described. This method is based on silicic acid microcolumn chromatography using labelled lysophosphatidylcholine (lysoPC) as substrate. The reaction was stopped by conventional Folch extraction. The chloroform extract (2 ml) was deposited on the silica gel and pushed through with air, and then elution was performed with methanol/water (50:50, v/v). Under these conditions, only the labelled phosphatidylcholine (PC) synthesized was retained on the gel, and this was then removed from the column and counted immediately. This method gave enzyme activities comparable to those obtained with the TLC method, and has proved to be reproducible. The new method, however, is both faster and safer than the classical TLC method.

Phosphatidylcholine synthesis in isolated type II pneumocytes from ozone-exposed rats.

Phosphatidylcholine (PC) synthesis by alveolar type II cells, as an indicator for the production of pulmonary surfactant, was studied after a 4-h exposure of rats to 4 mg ozone/m3 (2 ppm). Lung ravage fluid analysis after exposure revealed significant increases in proteins, which is indicative for pulmonary injury. When type II cells were isolated immediately and thereafter cultured for 20 h, the rate of PC synthesis in cells derived from ozone-exposed rats was not significantly different from that in cells from unexposed controls. Yet, a decreased rate of PC synthesis was observed when these cells were subsequently exposed to ozone in vitro. The activity of the enzyme glycerolphosphate acyltransferase (GPAT) was slightly enhanced in cultured type II cells isolated from ozone-exposed rats, while the lysophosphatidylcholine acyltransferase (LPCAT) activity was unchanged. However, ozone exposure of rats did result in a significant decrease of PC synthesis when measured in freshly prepared type II cell suspensions, although both GPAT and LPCAT activities were not affected. It is concluded that a decrease in pulmonary surfactant related PC synthesis after ozone exposure of rats can be demonstrated in freshly isolated type II pneumocytes. Cultured type II cells from exposed rats lack this effect and are therefore less useful to study changes in phospholipid biosynthesis after in vivo ozone exposure. The data on in vitro ozone exposure of cultured type II cells, however, support the view that ozone may impair pulmonary surfactant production.

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.

Response of the pulmonary surfactant system to phosgene.

Rats were exposed to 240 ppm X min phosgene (1.0 ppm for 4 hrs) in a Rochester-type chamber. At intervals thereafter over a 4 day period, lungs were removed for determination of wet weight; total, microsomal and surfactant protein concentrations; surfactant phospholipid concentrations; and 1-acyl-2-lyso-phosphatidylcholine: palmitoyl-CoA acyl transferase activity. Immediately upon termination of the phosgene exposure, microsomal protein and acyl transferase activity were reduced below, and lung wet weight was elevated above, control levels. From Day 1 through Day 3 after the exposure, all measured parameters, except for the phosphatidylinositol constituent of the surfactant fraction, were increased above the control values. In general, maximum levels were observed on Day 2; however, the acyl transferase activity and surfactant concentration continued to increase on Day 3. The results suggest components of the pulmonary surfactant system may be involved in maintenance of pulmonary fluid balance and the presence of excess water in the lungs as a result of phosgene exposure may represent a signal for increased synthesis of anti-edematogenic materials in order to promote removal of the inappropriate fluid.

[Activities of phospholipase A1, A2, lysophospholipase and acylCoA: lysophospholipid acyltransferase in ischemic brain of the dog].

In this report, the sequential changes of phospholipase A1, A2, lysophospholipase and acylCoA: lysophospholipid acyltransferase activities in ischemic dog brain were investigated. The purpose of this study was to examine the enzymic changes of deacylation-reacylation cycle of biomembrane phospholipid in ischemia. Hemispheric non-blood supply models were produced by occlusion of main intracranial trunk arteries in dogs according to Suzuki's method. The sample was spooned out and frozen immediately with liquid nitrogen at the predetermined time. The assay of phospholipase A1, A2 and lysophospholipase activities was done by our method and acylCoA: lysophospholipid acyltransferase activity was according to Corbin and Sun's method. The activities of phospholipase A1, A2 and lysophospholipase did not show significant changes within 60 minutes after arterial occlusion. However these activities showed significant high value at 120 minutes and decreased gradually after then. On the other hand, acylCoA: lysophospholipid acyltransferase activity showed gradual decrease. These findings show that enzymic degradiation of acyl group of phospholipid in the brain is highest at about 120 minutes after complete ischemia. The importance of acyl groups of phospholipids for biomembrane structure and the function is well recognized. The turnover of these acyl groups in normal brain biomembrane is also well known. Some types of enzymic system related to this turnover has been investigated. Phospholipase A1, A2, lysophospholipase and acylCoA: lysophospholipid acyltransferase belong to these enzymic systems. There have been some reports that the content of free fatty acids in the ischemic brain increases in early stage.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Familial respiratory distress syndrome in three consecutive full-term infants. Case reports and documentation of lung enzyme activities.

Familial respiratory distress syndrome in full-term newborn infants is a rare occurrence. Our patient delivered three consecutive full-term infants who developed findings consistent with respiratory distress syndrome. All three died from autopsy-proven hyaline membrane disease. Analysis of the activities of four enzymes that play an important role in the biosynthesis of lecithin (choline kinase, choline phosphotransferase, phospholipase A and lysolecithin acyltransferase) failed to disclose an abnormality in lung samples in our patient with familial respiratory distress syndrome.

Subcellular distribution of lysophosphatidylinositol and lysophosphatidylcholine acyltransferases in rat brain.

Lysophosphatidylinositol acyltransferase utilizing arachidonoyl CoA and lysophosphatidylcholine acyltransferase utilizing linoleoyl CoA were measured in subcellular fractions of rat brain. In general, the distribution of activities paralleled that of NADPH--cytochrome c reductase. It is concluded that the endoplasmic reticulum is the major site of these acyltransferase activities in rat brain.

Fractionation of membrane vesicles. II. A method for separation of membrane vesicles bearing different enzymes by free-flow electrophoresis.

Free-flow electrophoresis was used to subfractionate membrane vesicles from calf thymocyte plasma membranes. The fractionation resulted in a separation of vesicle populations bearing four different enzymes: alkaline nitrophenyl-phosphatase (orthophosphoric-monoester phosphohydrolase (alkalin optimum) EC 3.1.3.1), gamma-glutamyltransferase (EC 2.3.2.2), (Mg2+ + Na+ + K+)-ATPase (ATP phosphohydrolase, EC 3.6.1.3) and acyl-CoA:lysophosphatidylcholine acyltransferase (acyl-CoA:1-acylglycero-3-phosphocholine-O-acyltransferase, EC 2.3.1.23). The specific content of cholesterol and total phospholipid coincided with the distribution of membrane-bound protein. However, vesicles migrating towards the cathode had a higher molar ratio of cholesterol to phospholipid (0.75) compared to those migrating to the anode (0.55). Sodium dodecyl sulphate-gel electrophoresis of pooled vesicle fractions also demonstrates distinct differences in their protein pattern. Electron-micrographic thin sections show that the vesicle populations have a similar morphology and size distribution. These results are discussed in terms of heterogeneity of the original thymocytes, contamination with intracellular membranes and a heterogeneous structure of the plasma membrane.

Mitochondrial and microsomal phospholipids of Morris hepatoma 7777.

The phospholipids of both mitochondrial and microsomal membranes from normal liver, host liver, and Morris hepatoma 7777 were isolated, separated, and quantitated. The total as well as the individual fatty acid concentrations and compositions were determined. The total phosphlipids isolated from tumor mitochondria were idly altered, compared with mitochondria from other normal or host liver. The polyenoic acids were decreased, and there was a concomitant increase in the monoenes. When the respiratory control was determined, the tumor mitochondria exhibited a significant decrease in this parameter. The tumor microsomal membrane fraction, on the other hand, contained about 50% less phospholipid than the controls. The fatty acid patterns of the total as well as the individual phospholipids were quite similar to those observed in the mitochondria. The species of phosphatidylcholine from both membrane fractions were separated by argentation chromatography of the intact molecules, and, as predicted by the fatty acid compositions, the major species of the tumor was the monoenoic/dienoic fraction. The acyl coenzyme A:1-acyl glycerophosphorylcholine acyltransferases, which aid in controlling the fatty acid composition of phospholipids, were measured. The very marked increase in activity of these enzymes toward polyenoic as well as monoenic fatty acids suggested that the polyenoic acids were not available for use in the resynthesis of the phosphatidylcholines in the tumor.