Stimulation of rat liver mitochondrial <Emphasis Type="Italic">sn</Emphasis>-glycerol-3-phosphate acyltrasferase by polymyxin B <Emphasis Type="Italic">via</Emphasis> enhanced extraction of lysophosphatidic acid

The purpose of this investigation was to determine how polymyxin B stimulates the activity of mitochondrial glycerophosphate acyltransferase. Polymyxin B did not change the integrity of the mitochondrial outer membrane as judged by testing the latency (>80%) of cytochrome oxidase activity. The stimulation totally disappeared when polymyxin B-treated mitochondria were washed. The FA side chain in polymyxin B was unnecessary for stimulation, as the nonapeptide was as effective as the whole antibiotic. The stimulation by polymyxin B or the nonapeptide was observed only in the presence of BSA. Cytochrome c, when added to the incubation medium instead of albumin, did not stimulate the mitochondrial enzyme, but did produce a stimulatory effect of polymyxin B on the mitochondrial acyltransferase. As reported earlier for the bacterial and microsomal acyltransferase, other polycationic compounds such as spermine and spermidine stimulated mitochondrial glycerophosphate acyltransferase. The stimulation of the mitochondrial acyltransferase by spermine and spermidine also occurred only in the presence of BSA. The analysis of the products of esterification demonstrated the presence of more lysophosphatidic acid (LPA) in the polymyxin B-and polyamine-stimulated assays in comparison to their respective control. Furthermore, in comparison to the albumin-treated control, there was 60% more LPA present in the assay supernatant fractions of polymyxin B-treated samples. Our results suggest that polymyxin B stimulates the mitochondrial glycerophosphate acyltransferase activity by enhancing the extraction of more LPA from the mitochondria to the supernatant fraction.     

Effects of streptozotocin-induced diabetes on phosphoglyceride metabolism of the rat liver

We have studied the effect of streptozotocin (SZ)-induced diabetes on fatty acyltransferase and phospholipase enzyme activities involved in the synthesis and degradation of rat liver phosphoglycerides. Neither mitochondrial nor microsomal acyl-CoA: glycerol 3-phosphate acyltransferase (GPAT) activity was altered, although insulin treatment stimulated mitochondrial GPAT activity. However, microsomal acyl-CoA: 1-acylglycerol 3-phosphate acyltransferase (1-acyl-GPAT) activity increased (24–33 per cent, p<0.01) in the diabetic animals using 3 different acyl-CoA donors: palmitoyl-CoA, oleoyl-CoA and linoleoyl-CoA. SZ-induced diabetes also increased acyl-CoA:1-acylglycerol 3-phosphorylcholine acyltransferase (GPCAT) activity (38–45 per cent, p<0.01) with 3 different acyl-CoA donors: oleoyl-CoA, linoleoyl-CoA and arachidonoyl-CoA. 1-acyl-GPAT and GPCAT activity returned to normal with insulin treatment. In contrast to the increased activity of the microsomal fatty acyltransferases 1-acyl-GPAT and GPCAT, SZ-induced diabetes decreased mitochondrial phospholipase A₂ activity and lysophospholipase activity (49–70 per cent, p<0.01). Insulin treatment of the diabetic rats corrected the decreased lysophospholipase and stimulated phospholipase A₂ activity 35 per cent higher than controls. Since microsomal 1-acyl-GPAT and GPCAT are known to have higher activity toward unsaturated fatty acyl-CoA donors, the increased GPCAT activity coupled with the decreased lysophospholipase activity and the increased 1-acyl-GPAT activity in diabetes would tend to increase the formation of newly synthesized phospholipids containing unsaturated fatty acids. This mechanism plus the decreased fatty acid desaturase (4) may be the factors which alter the fatty acid composition of phosphoglycerides in diabetic rat liver microsomes.     

Effect of acylation stimulating protein on the triacylglycerol synthetic pathway of human adipose tissue

Acylation stimulating protein (ASP) is a 14 kDa plasma protein which causesin vitro triacylglycerol synthesis in human adipocytes and fibroblasts to increase substantially. ASP was found to stimulate human adipose tissue microsomal glycerophosphate acyltransferase and diacylglycerol acyltransferase activities by 23% and 90%, respectively. However, phosphatidate phosphohydrolase activity showed no increase in activity, nor did microsomal acyl-CoA synthetase activity. Moreover, ASP did not decrease the apparent K_m of diacylglycerol acyltransferase (DGAT), but rather increased its apparent V_max suggesting direct interaction of ASP with DGAT.     

Factors enhancing diacylglycerol acyltransferase activity in microsomes from cell-suspension cultures of oilseed rape

Several factors, including an unidentified endogenous component, were found to stimulate microsomal diacylglycerol acyltransferase (DGAT, EC 2.3.1.20) from a microspore-derived cell-suspension culture of oilseed rape (Brassica napus L. cv. Jet Neuf). At a concentration of 25mM, MgSO₄ and MgCl₂ stimulated microsomal DGAT 25- and 10-fold, respectively. AIP and CoA at concentrations of 2 and 1 mM stimulated the enzyme 2.4- and 12-fold, respectively, although the effects were lessened in the presence of higher Mg²⁺ concentrations. Although microsomal DGAT activity was increased only slightly by the addition of exogenous sn-1,2-diacylglycerol to the reaction mixture, it was increased substantially by the addition of exogenous phosphatidate. sn-Glycerol-3-phosphate and other phospholipids tested did not have this stimulatory effect. DGAT activity did not decrease when microsomes were incubated with ATP in the presence of the cytosolic fraction. This fraction, however, contained a small organic compound(s) that stimulated microsomal DGAT activity.     

Influence of hypo- and hyperthyroidism on rat liver glycerophospholipid metabolism

The effects of hyper- and hypothyroidism on enzyme activities involved in phospholipid metabolism in the rat liver were studied. Hyperthyroidism significantly decreases activities of both microsomal acyl-CoA:glycero-3-phosphate acyltransferase (GPAT) (34%, p<0.01) and microsomal acyl-CoA:1-acylglycero-3-phosphocholine acyltransferase (GPCAT) (28–33%, p<0.01). This may contribute to the decreased proportions of certain unsaturated fatty acids found in microsomal phosphoglycerides in hyperthyroidism. Mitochondrial GPAT, phospholipase A₂ and cytosol lysophospholipase are unaffected by hyperthyroidism. In contrast, hypothyroidism stimulates mitochondrial GPAT (38%, p<0.01) and microsomal GPCAT (14–19%) activities but decreases both mitochondrial phospholipase A₂ (36%, p<0.01) and cytosol lysophospholipase (56%, p<0.01) activities. The increased GPCAT activity may contribute to the increased proportions of certain unsaturated fatty acids found in microsomal phosphoglycerides in hypothyroidism. Triiodothyronine (T₃) treatment of the hypothyroid rat (25 μg/100 g body weight/day for four days) corrected phospholipase A₂ and lysophospholipase activities to the level of the control rat, but failed to correct the increased mitochondrial GPAT activity and not only corrected but lowered GPCAT activity to the level of the hyperthyroid rat.     

The hypotriglyceridemic effect of eicosapentaenoic acid in rats is reflected in increased mitochondrial fatty acid oxidation followed by diminished lipogenesis

The effect of eicosapentaenoic acid (EPA) on fatty acid oxidation and on key enzymes of triglyceride metabolism and lipogenesis was investigated in the liver of rats. Repeated administration of EPA to normolipidemic rats resulted in a time-dependent decrease in plasma triglycerides, phospholipids and cholesterol. The triglyceride-lowering effect was observed after one day of feeding whereas lowering of plasma cholesterol and phospholipids was observed after five days of treatment. The triglyceride content of liver was reduced after two-day treatment. At that time, increased mitochondrial fatty acid oxidation occurred whereas mitochondrial and microsomal glycerophosphate acyltransferase was inhibited. The phosphatidate phosphohydrolase activity was unchanged. Adenosine triphosphate:citrate lyase, acetyl-CoA carboxylase, fatty acid synthetase and glucose-6-phosphate dehydrogenase were inhibited during the 15 d of EPA treatment whereas peroxisomal β-oxidation was increased. At one day of feeding, however, when the hypotriglyceridemic effect was established, the lipogenic enzyme activities were reduced to the same extent in palmitic acid-treated animals as in EPA-treated rats. In cultured rat hepatocytes, the oxidation of [¹⁴C]palmitic acid to carbon dioxide and acid-soluble products was stimulated in the presence of EPA. These results suggest that the instant hypolipidemia in rats given EPA could be explained at least in part by a sudden increase in mitochondrial fatty acid oxidation, thereby reducing the availability of fatty acids for lipid synthesis in the liver for export,e.g., in the form of very low density lipoproteins, even before EPA induced peroxisomal fatty acid oxidation, reduced triglyceride biosynthesis and diminished lipogenesis.     

3-thia fatty acid treatment, in contrast to eicosapentaenoic acid and starvation, induces gene expression of carnitine palmitoyltransferase-II in rat liver

The aim of the present study was to investigate the hepatic regulation and β-oxidation of long-chain fatty acids in peroxisomes and mitochondria, after 3-thia- tetradecylthioacetic acid (C₁₄-S-acetic acid) treatment. When palmitoyl-CoA and palmitoyl-l-carnitine were used as substrates, hepatic formation of acid-soluble products was significantly increased in C₁₄-S-acetic acid treated rats. Administration of C₁₄-S-acetic acid resulted in increased enzyme activity and mRNA levels of hepatic mitochondrial carnitine palmitoyltransferase (CPT)-II. CPT-II activity correlated with both palmitoyl-CoA and palmitoyl-l-carnitine oxidation in rats treated with different chain-length 3-thia fatty acids. CPT-I activity and mRNA levels were, however, marginally affected. The hepatic CPT-II activity was mainly localized in the mitochondrial fraction, whereas the CPT-I activity was enriched in the mitochondrial, peroxisomal, and microsomal fractions. In C₁₄-S-acetic acid-treated rats, the specific activity of peroxisomal and microsomal CPT-I increased, whereas the mitochondrial activity tended to decrease. C₁₄-S-Acetyl-CoA inhibited CPT-I activity in vitro. The sensitivity of CPT-I to malonyl-CoA was unchanged, and the hepatic malonyl-CoA concentration increased after C₁₄-S-acetic acid treatment. The mRNA levels of acetyl-CoA carboxylase increased. In hepatocytes cultured from palmitic acid- and C₁₄-S-acetic acid-treated rats, the CPT-I inhibitor etomoxir inhibited the formation of acid-soluble products 91 and 21%, respectively. In contrast to 3-thia fatty acid treatment, eicosapentaenoic acid treatment and starvation increased the mitochondrial CPT-I activity and reduced its malonyl-CoA sensitivity. Palmitoyl-l-carnitine oxidation and CPT-II activity were, however, unchanged after either EPA treatment or starvation. The results from this study open the possibility that the rate control of mitochondrial β-oxidation under mitochondrion and peroxisome proliferation is distributed between an enzyme or enzymes of the pathway beyond the CPT-I site after 3-thia fatty acid treatment. It is suggested that fatty acids are partly oxidized in the peroxisomes before entering the mitochondria as acylcarnitines for further oxidation.     

Application of 2-hydroxypropyl-β-cyclodextrin in the assay of acyl-CoA: cholesterol acyltransferase and neutral and acid cholesterol ester hydrolases

The utility of 2-hydroxypropyl-β-cyclodextrin for increasing the sensitivity of assays for the microsomal acyl-CoA:cholesterol acyltransferase, and the acid lysosomal and the neutral microsomal and cytosolic cholesterol ester hydrolase activity was studied in rat hepatocytes. Enzyme assays, at optimal concentrations of cyclodextrin, were validated by assessing: (i) linearity of product formation with incubation time and protein amount, and saturation with substrate, and (ii) the effect of treatments of cells or of subcellular fractions on enzyme activities. Delivery of cholesterol dissolved in 2-hydroxypropyl-β-cyclodextrin to the acyl-CoA:cholesterol acyltransferase assay mixture raised the enzyme activity more than 8-fold and was twice that measured when cholesterol was added in Triton WR-1339. 2-Hydroxypropyl-β-cyclodextrin itself was partially effective, apparently by making endogenous cholesterol more accesible to the enzyme. Inclusion of 2-hydroxypropyl-β-cyclodextrin in cholesterol ester hydrolase assays using standard micellar substrates doubled the activity estimated in lysosome and microsome preparations and enhanced the cytosolic cholesterol esterase activity by about 50%. Differences in the catalytic activity of acyl-CoA:cholesterol acyltransferase and cholesterol ester hydrolases caused by treatment of hepatocytes with compound 58-035 or 25-hydroxycholesterol, or of subcellular fractions with NaF, were maintained when enzymes were assayed with cyclodextrin. The results indicate that 2-hydroxypropyl-β-cyclodextrin is a suitable vehicle for delivering cholesterol to acyl-CoA:cholesterol acyltransferase and enhances the sensitivity of standard assays of the enzymes governing the intrahepatic hydrolysis of cholesteryl esters.     

Lysophosphatidylcholine acyltransferase activity in Saccharomyces cerevisiae: Regulation by a high-affinity Zn2+ binding site

Saccharomyces cerevisiae cells were demonstrated to contain lysophosphatidylcholine (lysoPtdCho) acyltransferase (E.C. 2.3.1.23) activity. The enzyme displayed K _m(app) of 69 μM for lysoPtdCho and 152 μM for oleoyl CoA. Enzyme activity was not affected by the addition of 1 mM Mg²⁺, Mn²⁺, Ca²⁻, or 200 mM EDTA. However, Zn²⁺ inhibited lysoPtdCho acyltransferase activity to 33% control values at 0.1 mM and to 7% at 1.0 mM Zn²⁺. To further explore the possibility that lysoPtdCho acyltransferase may contain a high-affinity Zn²⁺ binding site, we tested the strong Zn²⁺ chelator o-phenanthroline for its ability to inhibit enzyme activity. LysoptdCho acyltransferase activity was inhibited to 18 and 27%, respectively, those of control values in the presence of 2 and 1 mM o-phenanthroline, implying that a high-affinity Zn²⁺ binding site exists in lysoPtdCho acyltransferase or in an accessory protein that is essential for protein stability and/or activity. Saccharomyces cerevisiae lysoPtdCho acyltransferase activity displayed a broad lysoPtdCho fatty acyl chain substrate specificity utilizing lysoPtdCho molecules ranging in length from C₁₀−C₂₀ (the entire range tested). In addition, the enzyme was capable of using the ether-linked analog of lysoPtdCho, 1-O-alkyl-2-hydroxy-sn-3-glycerophosphocholine, as a substrate. The ability of S. cerevisiae to incorporate radiolabeled 1-O-alkyl-2-hydroxy-sn-3-glycerophosphocholine into phosphatidylcholine in vitro was exploited to demonstrate a direct precursor-product relationship between lysoPtdCho molecules and their incorportation into phosphatidylcholine in vivo. Identical labeling results were obtained in S. cerevisiae cells disrupted for their major transacylase activity, PLB1, demonstrating that the incorporation of lysolipid was via acyltransferase, and not transacylase, activity.     

Mechanism of lysophosphatidylcholine accumulation in the ischemic canine heart

The metabolism of lysophosphatidylcholine (LPC) in non-ischemic and ischemic canine heart was investigated byin vitro enzyme analysis. Selected subcellular fractions were assayed for the LPC-producing enzyme phospholipase A and the LPC-eliminating enzymes LPC:acyl-CoA acyltransferase, LPC:LPC transacylase and lysophospholipase. The canine heart was found to contain all enzymes differing, however, in subcellular distribution and specific activity. Phospholipase A activity did not change significantly in any of the fractions prepared from the ischemic tissue of hearts rendered ischemic for 1, 3 or 5 hr when compared to non-ischemic tissue. Changes in the activity of the microsomal LPC:acyl-CoA acyltransferase over the course of 5 hr of ischemia were observed. Significant decreases in the activity of the cytosolic and microsomal lysophospholipases were detected especially after 3 and 5 hr of ischemia. Similarly, a decrease in the activity of the microsomal LPC:LPC transacylase was noted after 3 and 5 hr of ischemia. Our results suggest that impaired catabolism of LPC rather than an enhanced production of LPC is the principal mechanism for the increase in LPC levels in the ischemic canine heart.     

The inhibitory guanine nucleotide-binding protein Gi2α induces and potentiates adipocyte differentiation

The present study further elucidates the involvement of the α-subunit of the GTP-binding protein G_i2 in the differentiation of murine 313-L1 cells. Control and vector-transfected cells attained a fully differentiated adipocyte phenotype showing ample lipid droplets. Cells expressing wild type (WT)-G_i2α or the constitutively active R179E-G_i2α, however, became enlarged, less confluent, and produced large amounts of lipids. Differentiation consistently increased the triglyceride (TAG) content in control cells. In both WT-G_i2α and R179E-G_i2α clones, a marked increase in TAG could be detected even prior to insulin/dexamethasone/isobutyl methylxanthine exposure. The activity of palmitoyl-CoA synthetase (PCS) and glycerophosphate acyltransferase (GPAT) also increased upon differentiation. WT-G_i2α and R179E-G_i2α overexpression also enhanced PCS and GPAT activities even before differentiation medium was added. The total amount of phospholipids (PL) generally increased upon differentiation; however, pre- and postdifferentiation values were insignificantly different in cells expressing WT-G_i2α and R179E-G_i2α. Differentiation altered the PL profile with a relative shift from phosphatidylcholine and phosphatidylethanolamine to phosphatidylinositol (PI) in differentiated cells. Finally, differentiation yielded a general increase in the activity of basal PI-phospholipase-C activity. Again, cells expressing WT-G_i2α and R179E-G_i2α demonstrated elevated enzyme activity and enhanced second messenger accumulation subsequent to differentiation. In summary, cells with the R179E-mutants of G_i2α exhibited stimulated lipid turnover and accumulation in both undifferentiated and differentiated cells.     

Properties of palmityl-CoA: L-α-glycerolphosphate acyl transferase from bovine mammary microsomes

Palmityl-coenzyme A: L-α-glycerolphosphate acyltransferase is the most active acyltransferase of bovine mammary microsomes, with a specific activity ranging from 8–20 nmoles min⁻¹ mg⁻¹ protein. Corresponding acylation rates of 2.2, 1.4, 2.1, and 0.6 nmoles min⁻¹ mg⁻¹ were obtained for myristyl-, stearyl-, oleyl- and linoleyl-coenzyme A, respectively. Optimum pH of palmityltransferase was 7.7, and activity was not affected by buffer molarity in range 25–150 mM. Inhibitory effects of palmityl-coenzyme A (10 μM/0.1 mg microsomal protein) was relieved by bovine serum albumin. Sonication magnesium and ethylenediaminetetraacetic acid enhanced activity. Delipidation of microsomes reduced activity by 84%; restoration of extracted lipids achieved 70% of original activity. Apparent Km and Vmax values of 4.1 and 260 μM and 9.5 and 8.2 nmole min⁻¹ mg⁻¹ were determined for palmityl-coenzyme A and D,L-α-glycerolphosphate, respectively, using untreated microsomes. The enzyme was stable as lyophilized microsomes when stored at −30 C. Phosphatidic acid was the major product and marked quantities of diglycerides were formed, especially when microsomal protein was increased.     

Lysolecithin acyltransferase in hamster intestinal mucosa

1-Acyl-lysolecithin acyltransferase has been demonstrated in the microsomal fraction of hamster intestinal mucosa. The characteristics of the enzyme, with respect to substrate concentration, time of incubation and protein concentration, were studied. Ca⁺⁺ was found to severely inhibit enzymatic activity. More modest inhibitors were found to be Mg⁺⁺ and F⁻; EDTA and albumin had no effect. Enzyme activity was reduced when palmityl CoA was substituted for oleoyl CoA as substrate. The specific activity of intestinal microsomes was modestly greater than liver microsomes.     

Regulation of rat liver microsomal cholesterol ester hydrolase by reversible phosphorylation

The regulation of neutral cholesterol ester hydrolase activity by changes in its phosphorylation state was studied in rat liver microsomes. Treatment with cAMP-dependent protein kinase resulted in increased enzyme activity, which was further enhanced by the addition of cAMP and MgATP. Consistent activations were also achieved with MgCl₂ and MgATP, the magnesium effect being abolished by ethylenediaminetetraacetic acid and adenosine triphosphate. Cholesterol ester hydrolase was activated twofold by free calcium and Ca²⁺/calmodulin; this latter effect was blocked by the chelator ethyleneglycol-bis(β-aminoethyl ether)N,N,N′,N′-tetraacetic acid and the calmodulin antagonist trifluoperazine. The phosphatase inhibitors pyrophosphate and glycerophosphate led to marked and dose-dependent increases in esterase activity, whereas okadaic acid elicited no effect. Furthermore, pyrophosphate and okadaic acid did not change the increases in enzyme activity promoted by Ca²⁺, Ca²⁺/calmodulin, Mg²⁺ and MgATP. Cholesterol ester hydrolase was inactivated in a concentration-dependent manner by nonspecific alkaline phosphatases. In cAMP-dependent protein kinase/cAMP- or Ca²⁺/calmodulin-activated microsomes, a time-dependent loss of activation in cholesteryl oleate hydrolysis was caused by alkaline phosphatase. These findings suggest that microsomal cholesterol ester hydrolase is activated through cAMP and Ca²⁺/calmodulin phosphorylation, whereas enzyme deactivation is dependent on phosphatase action.     

Altered Lipid Parameters in Hepatic Subcellular Membrane Fractions Induced by Fumonisin B1

Alteration of lipid constituents of cellular membranes has been proposed as a possible mechanism for cancer promotion by fumonisin B₁ (FB₁). To further investigate this hypothesis a dietary dosage which initiates and promotes liver cancer (250 mg FB₁/kg) was fed to male Fischer rats for 21 days and the lipid composition of plasma, microsomal, mitochondrial and nuclear subcellular fractions determined. The effect of FB₁ on the cholesterol, phosphatidylcholine (PC) and phosphatidylethanolamine (PE), as well as sphingomyelin (SM) and the phospholipids-associated fatty acid (FA) profiles, were unique for each subcellular membrane fraction. PE was significantly increased in the microsomal, mitochondrial and plasma membrane fractions, whereas cholesterol was increased in both the microsomal and nuclear fraction. In addition SM was decreased and increased in the mitochondrial and nuclear fractions, respectively. The decreased PC/PE and polyunsaturated/saturated (P/S) FA ratio in the different membrane fractions suggest a more rigid membrane structure. The decreased levels in polyunsaturated fatty acids in PC together with a pronounced increase in C18:1ω9 and C18:2ω6 were indicative of an impaired delta-6 desaturase. The increased ω6/ω3 ratio and decreased C20:4ω6 PC/PE ratio due to an increase in C20:4ω6 in PE relatively to PC in the different subcellular fractions suggests a shift towards prostanoid synthesis of the E2 series. Changes in the PE and C20:4ω6 parameters in the plasma membrane could alter key growth regulatory and/or other cell receptors in lipid rafts known to be altered by FB₁. An interactive role between C20:4ω6 and ceramide in the mitochondria, is suggested to regulate the balance between proliferation and apoptosis in altered initiated hepatocytes resulting in their selective outgrowth during cancer promotion effected by FB₁.     

A Novel Assay of DGAT Activity Based on High Temperature GC/MS of Triacylglycerol

Diacylglycerol acyltransferase (DGAT) catalyzes the final step in the acyl-CoA-dependent biosynthesis of triacylglycerol (TAG), a high-energy compound composed of three fatty acids esterified to a glycerol backbone. In vitro DGAT assays, which are usually conducted with radiolabeled substrate using microsomal fractions, have been useful in identifying compounds and genetic modifications that affect DGAT activity. Here, we describe a high-temperature gas chromatography (GC)/mass spectrometry (MS)-based method for monitoring molecular species of TAG produced by the catalytic action of microsomal DGAT. This method circumvents the need for radiolabeled or modified substrates, and only requires a simple lipid extraction prior to GC. The utility of the method is demonstrated using a recombinant type-1 Brassica napus DGAT produced in a strain of Saccharomyces cerevisae that is deficient in TAG synthesis. The GC/MS-based assay of DGAT activity was strongly correlated with the typical in vitro assay of the enzyme using [1-¹⁴C] acyl-CoA as an acyl donor. In addition to determining DGAT activity, the method is also useful for determining substrate specificity and selectivity properties of the enzyme.     

Possible Role of Different Yeast and Plant Lysophospholipid:Acyl-CoA Acyltransferases (LPLATs) in Acyl Remodelling of Phospholipids

Recent results have suggested that plant lysophosphatidylcholine:acyl‐coenzyme A acyltransferases (LPCATs) can operate in reverse in vivo and thereby catalyse an acyl exchange between the acyl‐coenzyme A (CoA) pool and the phosphatidylcholine. We have investigated the abilities of Arabidopsis AtLPCAT2, Arabidopsis lysophosphatidylethanolamine acyltransferase (LPEAT2), S. cerevisiae lysophospholipid acyltransferase (Ale1) and S. cerevisiae lysophosphatidic acid acyltransferase (SLC1) to acylate lysoPtdCho, lysoPtdEtn and lysoPtdOH and act reversibly on the products of the acylation; the PtdCho, PtdEtn and PtdOH. The tested LPLATs were expressed in an S. cervisiaeale1 strain and enzyme activities were assessed in assays using microsomal preparations of the different transformants. The results show that, despite high activity towards lysoPtdCho, lysoPtdEtn and lysoPtdOH by the ALE1, its capacities to operate reversibly on the products of the acylation were very low. Slc1 readily acylated lysoPtdOH, lysoPtdCho and lysoPtdEtn but showed no reversibility towards PtdCho, very little reversibility towards PtdEtn and very high reversibility towards PtdOH. LPEAT2 showed the highest levels of reversibility towards PtdCho and PtdEtn of all LPLATs tested but low ability to operate reversibly on PtdOH. AtLPCAT2 showed good reversible activity towards PtdCho and PtdEtn and very low reversibility towards PtdOH. Thus, it appears that some of the LPLATs have developed properties that, to a much higher degree than other LPLATs, promote the reverse reaction during the same assay conditions and with the same phospholipid. The results also show that the capacity of reversibility can be specific for a particular phospholipid, albeit the lysophospholipid derivatives of other phospholipids serve as good acyl acceptors for the forward reaction of the enzyme.     

Enzymic regulation of arachidonate metabolism in brain membrane phosphoglycerides

The metabolism of arachidonate in brain membrane phosphoglycerides was investigated in vivo by intracerebral injection of labeled arachidonate and by in vitro assay of enzymic systems associated with the metabolism. After intracerebral injection, labeled arachidonate was incorporated rapidly into brain phosphoglycerides with radioactivity distributed mainly in diacyl-sn-glycero-3-phosphoinositols (GPI) and diacyl-sn-glycero-3-phosphocholines (GPC). Some evidence of a metabolic relationship between diacyl-sn-glycerophosphoinositols (diacyl-GPI) and diacylglycerols was observed. Among the phosphoglycerides labeled with [¹⁴C] arachidonoyl groups, diacyl-GPI were most rapidly metabolized in brain microsomal and synaptosomal fractions. The decay of diacyl-GPI in brain synaptosomes may be represented by two pools with half-lives of 5 hr and 5 days. Three types of enzymic systems related to metabolism of the polyunsaturated fatty acids in brain were investigated. The first system involves the cyclic events relating the ATP-dependent activation of polyunsaturated fatty acids (PUFA) to their acylCoA by the acylCoA ligase and subsequent hydrolysis of acylCoA to free fatty acids by the acylCoA hydrolase. It is apparent that fatty acid activation and hydrolysis is under strigent control in order to maitain suitable levels of free fatty acids and acylCoA in the brain tissue for various metabolic use. Factors involved in the regulation may include the level of ATP, divalent cations and the nature of substrates. The second enzymic system pertains to deacylation via phospholipase A₂ and reacylation via the acyltransferase of membrane phosphoglycerides. In brain tissue, activity of the acyl transferase is generally higher than that of the phospholipase A₂. Factors known to affect specificity of the acyltransferase include substrate concentration and the nature of the acyl groups and lysophosphoglycerides. The acyltranferase(s) in brain preferentially transfers arachidonate to 1-acyl-GPI. Activity of the acyltransferase can be inhited by a number of lypophilic compounds including local anesthetics and cell surface agents. Activity of the phospholipase A₂ in brain may depend on the physical form of the substrates, i.e., whether the substrates are in monomeric or micellar form. The third process is associated with the degradation of diacyl-GPI by enzymes present in brain subcellular membranes. Incubation of brain subcellular membranes with 1-acyl-2-[¹⁴C] arachidonoyl-GPI yielded labeled diacylglycerols and arachidonate. The phospholipase C action is specific for hydrolysis of diacyl-GPI. The arachidonate released from incubation of labeled diacyl-GPI may be the result of phospholipase A₂ action which is not specific for diacyl-GPI or the hydrolysis by lipase acting on the diacylglycerols formed from the phospholipase C activity. Enzymic hydrolysis of diacyl-GPI is most active in the microsomal fraction, but uoon disruption of synaptosomes, enzyme in synaptic plasma membranes is also active in degradating this glycerophospholipid. In general, the results of in vitro studies are in good agreement with those observed in vivo and the information yielded has contributed towards understanding the metabolism of polyunsaturated fatty acids in brain subcellular membranes.     

The existence of a soluble plasmalogenase in guinea pig tissues

The distribution of plasmalogenase for the hydrolysis of 1-alkenyl-2-acyl-sn-glycero-3-phosphoethanolamine (plasmenylethanolamine) in the subcellular fractions of guinea pig tissues was examined. Plasmalogenase activity was found in high abundance in the cytosolic fractions of the brain and the heart. Assessment of microsomal marker enzyme activities in the cytosolic fraction revealed that plasmalogenase activity in the cytosol was not due to microsomal contaminations. The characteristics of the cytosolic plasmalogenase were very similar to the microsomal enzyme with respect to the pH profile of the reaction, the presence of divalent cations and K_m values for plasmenylethanolamine. However, the cytosolic enzyme was slightly less stable at 55°C than the microsomal enzyme. Cytosolic enzyme activity was eluted as a broad peak in Sepharose 6B chromatography with an average molecular weight of 250,000. Our results demonstrate that most of brain plasmalogenase activity is soluble which makes the brain cytosol an excellent source to initiate the purification of this enzyme.     

Characterization of oleoyl-12-hydroxylase in castor microsomes using the putative substrate, 1-acyl-2-oleoyl-sn-glycero-3-phosphocholine

We have characterized the oleoyl-12-hydroxylase in the microsomal fraction of immature castor bean using the putative substrate, 1-acyl-2-oleoyl-sn-glycero-3-phosphocholine (2-oleoyl-PC). Previous characterizations of this enzyme used oleoyl-CoA as substrate and relied on the enzyme transferring oleate from oleoyl-CoA to lysophosphatidylcholine to form 2-oleoyl-PC (acyl-CoA:lysophosphatidylcholine acyltransferase) in addition to oleoyl-12-hydroxylase. The present assay system and characterization use 2-oleoyl-PC as substrate (oleoyl-12-hydroxylase alone). Use of the actual substrate for assay purposes is important for the eventual purification of the oleoyl-12-hydroxylase. Ricinoleate (product of oleoyl-12-hydroxylase) and linoleate (product of oleoyl-12-desaturase) were identified as metabolites of oleate of 2-oleoyl-PC by high-performance liquid chromatography and gas chromatography/mass spectrometry. The activity of oleoyl-12-hydroxylase in the microsomal fraction reached a peak about 44 d after anthesis of castor, while the activity of oleoyl-12-desaturase reached a peak about 23 d after anthesis. The optimal temperature for the oleoyl-12-hydroxylase was about 22.5°C, and the optimal pH was 6.3. Catalase stimulated oleoyl-12-hydroxylase while bovine serum albumin and CoA did not activate oleoyl-12-hydroxylase. The phosphatidylcholine analogue, oleoyloxyethyl phosphocholine, inhibited the activity of oleoyl-12-hydroxylase. These results further support the hypothesis that the actual subtrate of oleoyl-12-hydroxylase is 2-oleoyl-PC.