Acyltransferase System Research Articles

Enzymatic basis for the formation of pulmonary surfactant lipids by acyltransferase systems

An enzymatic basis for the formation of pulmonary surfactant lipids in rat has been presented. The free fatty acid pools in lung and liver consisted mainly of palmitic, stearic, oleic, and arachidonic acids with relatively less polyunsaturated fatty acids in lung than in liver. The acyl chain specificities of the acyl-CoA synthetase systems in lung and liver microsomes were similar in that most of fatty acids found in the free fatty acid pools were effectively activated by both systems. The acyl-CoA pools had compositions significantly different from those of the free fatty acid pools in lung and liver with relatively more stearate and less polyunsaturated fatty acids. The lung acyl-CoA pool contained mainly palmitate (29%), stearate (31%), and oleate (22%) with very little polyunsaturated acyl-CoAs to compete for esterification. The use of an equimolar mixture of palmitoyl-CoA and arachidonoyl-CoA to acylate the endogenous monoacyl-glycerophosphocholine isomers in the lung microsomes yielded both the 2-palmitate and 2-arachidonate diacyl forms, whereas the major products formed by liver microsomes were the 2-arachidonate and 1-palmitate forms. These results indicate that the 1-acyl isomer is the major monoacyl-glycerophosphocholine species serving as substrate in lung microsomes, whereas both 1-acyl and 2-acyl isomers are present in liver microsomes. Thus, the enrichment of saturated and oligoenoic acids in the acyl-CoA pool combined with the predominance of the 1-acyl isomer in the acyl acceptor pool and the relatively higher selectivity for palmitoyl-CoA by the 1-acyl-GPC acyltransferase activity of lung constitute an important basis for attributing some of the formation of pulmonary surfactant lipids in rats to acyltransferase action.

Specificities and selectivities of glycerol-3-phosphate acyltransferase and monoacylglycerol-3-phosphate acyltransferase from pea and spinach chloroplasts.

In addition to acyl‐CoA, purified glycerol‐3‐phosphate acyltransferases from pea and spinach chloroplasts can also use acyl‐(acyl‐carrier protein), acyl‐ACP, as a substrate for glycerol 3‐phosphate acylation. The enzyme fractions showed absolute specificity for glycerol 3‐phosphate as acyl acceptor. Dihydroxyacetone phosphate was ineffective. Glycerol 3‐phosphate was almost exclusively acylated at the C‐1 position. If mixtures of palmitoyl‐ACP, stearoyl‐ACP and oleoyl‐ACP were offered, the oleoyl group was preferred. These data fully agree with previous experiments on these enzymes carried out with various acyl‐CoA thioesters. Kinetic data determined with different acyl‐ACPs as substrates are consistent with the observed fatty acid selectivity for the oleoyl group. Double labelling experiments with mixtures of oleoyl‐ACP and oleoyl‐CoA demonstrated a preference for ACP‐thioesters.Monoacylglycerol‐3‐phosphate acyltransferase, localized in the envelope of chloroplasts, can also utilize acyl‐ACP as substrate. Envelope fractions of spinach as well as of pea showed a high specificity for the palmitoyl group when ACP‐thioesters or CoA‐thioesters were offered and directed this acyl group to the C‐2 position of the glycerol backbone. Results from competition experiments with [14C]palmitoyl‐ACP and [3H]palmitoyl‐CoA indicate that the membrane‐bound acyltransferase preferably uses ACP‐thioesters for the acylation of 1‐acylglycerol 3‐phosphate. According to fatty acid selectivities and specificities the main product of the recombined acyltransferase systems in chloroplasts of 16:3 plants as well as 18:3 plants is a phosphatidic acid with the oleoyl group at C‐1 and the palmitoyl group at C‐2 whereas molecules with the oleoyl group at both positions are not synthesized.Under appropriate conditions the phosphatidic acid formed by the soluble acyltransferase and spinach envelope was rapidly converted to monogalactosyl diacylglycerol in the presence of UDP‐galactose. In analogous assays with acyltransferase and envelope from pea only a low proportion of labelled phosphatidic acid was converted via diacylglycerol to monogalactosyl diacylglycerol. In both systems monogalactosyl diacylglycerol synthesized in the presence of C16:0‐thioesters and C18:1‐thioesters carried C18:1 at C‐1 and C16:0 at C‐2 in agreement with the fatty acid selectivity of the acyltransferase systems.

Selective incorporation of polyunsaturated fatty acids into phosphatidylcholine by rat liver microsomes.

The various polyunsaturated fatty acids found in cellular lipids are transferred from their coenzyme A thiol esters to phospholipid acceptors with greatly different maximal velocities. The very low apparent Km values for the thiol esters in acylating 1-acylglycerol-3-phosphocholine could not be directly measured, but their values could be estimated relative to that for arachidonate. The competitive effectiveness of various polyunsaturated acyl-CoAs was estimated by measuring the equivalent concentrations which allow incorporations equal to arachidonyl-CoA. These values help predict the way in which various polyunsaturated acyl-CoAs may be selectively esterified to membrane lipids by the 1-acylglyceol 3-phosphocholine (1-acyl-GPC) acyltransferase system of liver microsomes. The acyl-CoA esters of saturated acids, as well as those for 22:1, 22:2, and 22:3, had negligible ability to compete for the active sites of the 1-acyl-GPC acyltransferase system. The acyl-CoA esters of arachidonate (20:4n-6), eicosatrienoate (20:3n-6), eicosapentaenoate (20:5n-3), and both isomers of linolenate (18:3n-6 and n-3) were handled preferentially by the 1-acyl-GPC acyltransferases. The system from liver has a high selectivity for unsaturated acids but does not appear to discriminate among the polyunsaturated acids of the n-6 and n-3 series that serve as precursors of prostaglandins and leukotrienes.

Lipogenesis in response to an oral glucose load in fed and starved rats

Lipogenesis in livers of fed but not of starved rats is increased after intragastric feeding with glucose. In contrast, lipogenesis in brown adipose tissue increases in both fed and starved animals. These observations suggest that lipogenesis in brown adipose tissue is regulated by mechanisms in addition to, or other than, those operating in liver. The fate of newly synthesized lipid in brown adipose tissue is not known. However, the formation of palmitoyl-carnitine from palmitoyl-CoA and carnitine by mitochondria from brown fat was inhibited by malonyl-CoA. Although inhibition was not 100%, it is implied that mitochondrial uptake of the newly synthesized fat by the carnitine acyltransferase system is restricted under conditions of increased lipogenesis.

Modulation of diacylglycerol acyltransferase by lysophosphatidylcholine and related monochain phospholipids

Acyl-CoA: 1,2-diacylglycerol O-acyltransferase (EC 2.3.1.20) activity of rat liver microsomes was found to be stimulated by 1-acyl- sn-glycero-3-phosphocholine at low concentrations, but was inhibited above 0.2 mM. Diacylglycerol acylation was optimal at 75 μM lysophosphatidylcholine, resulting in a more than 2-fold activation of the enzyme. Acyltransferase activity disappeared above 0.5 mM lysophosphatidylcholine levels. 0.05% sodium taurocholate supplementation reduced diacylglycerol acyltransferase activity by approx. 2 3 over the entire range of lysophosphatidylcholine concentrations. 1- O-Hexadecylpropanediol-3-phosphocholine was shown to mimic lysophosphatidylcholine at stimulatory and at inhibitory concentrations in the absence and in the presence of sodium taurocholate, thus ruling out acyl-CoA depletion due to lysophos-phatidylcholine acylation as a cause of depressed triacylglycerol synthesis at higher lysophosphatidylcholine levels. 1-Acyl- sn-glycero-3-phosphoethanolamine stimulated diacylglycerol acyltransferase to a lesser extent, without showing inhibition at higher concentrations. The data point towards a direct effect of the lysophospholipids on the acyltransferase system.

Acute effects of L-carnitine on FFA and β-oh-butyrate in man

L, Carnitine (1000 mg) was given intravenously to nine subjects by perfusion lasting four hours. Its effects on plasma levels of Cholesterol, Triglycerides, FFA and B-OH-B were recorded and compared with the effects in the same subjects of a saline solution infusion. Following L, Carnitine a significant drop of FFA was observed 60 minutes after starting infusion while a maximum was reached four hours later. B-OH-B rose significantly at 60′ reaching a plateau which remained unmodified up to the 4th hour. No variations were observed in cholesterol and triglycerides plasma levels. The present data stress the view that Carnitine activates the carnitine acyltransferase system inducing an increased FFA oxidation and a consequential decrease of FFA plasma levels. At the same time, there is an increased production of ketone bodies and of their plasma levels. The shift of energy yielded by FFA oxidation from triglyceride synthesis to ketone body production may be a mechanism of a claimed lipid lowering effect of Carnitine.

1718 CULTURE OF TYPE 2 CELLS FROM FETAL RABBIT LUNGS

Type 2 alveolar epithelial cells synthesize a surfactant which is important for maintaining lung stability. To isolate these cells, fetal rabbit lungs (27-29 days gestation) were digested enzymatically, and differential adhesion to plastic substrate was used to obtain a population enriched in epithelial cells. Cells were cultured using selective medium (with D-valine instead of L-valine) to suppress growth of contaminating fibroblasts; after 4-5 days, the cultures were confluent. Cells were examined at electron microscope level; in a typical culture from 29 day fetuses, approx. 10-40% were identified as “definite” type 2 (cuboidal or elliptical, surface microvilli, cytoplasmic lamellar bodies), and 50-60% were identified as “probable” type 2 (same criteria as for “definite”, except no lamellar bodies). The cells incorporated (14C)-choline into disaturated phosphatidylcholine (dspc), a major component of lung surfactant. Specific activities of the acyl transferase system were compared using palmitoyl-CoA and oleyl-CoA as donor substrates, and 1-sat-2-lyso-pc as receptor substrate. At 27 days gestation, the ratio (palmitoyl-CoA/oleyl-CoA) was 0.39, while at 29 days the ratio was 3.5. These data suggest that changes in acyl transferase donor specificity are important in regulating synthesis of dspc. We conclude that this culture system is a useful model for studying metabolism of lung surfactant.

Selective utilization of palmitoyl lysophosphatidylcholine in the synthesis of disaturated phosphatidylcholine in rat lung: A combined in vitro and in vivo approach

1. 1. The acyl-CoA: lysophosphatidylcholine acyltransferase system in rat lung microsomes was found to utilize selectively 1-[1- 14C]palmitoyl- sn-glycero-3-phosphocholine when compared with 1-[9,10- 3H 2]stearoyl- sn-glycero-3-phosphocholine. This result was found with either palmitoyl-CoA, linoleoyl-CoA or an equimolar mixture of these acyl donors and confirms recent data reported by Holub, Piekarski and Possmayer (Can. J. Biochem. 58 (1980) 434–439). 2. 2. The selective utilization of palmitoyl lysophosphatidylcholine from a mixture of lysophosphatidylcholine species may cause an increased isotopic ratio in phosphatidylcholine when compared with that of total lysophosphatidylcholine. Thus, when rats were injected with a single doubly labelled species i.e. 1-[9,10- 3H 2]palmitoyl- sn-glycero-3-phospho[ methyl- 14C]choline, the isotopic ratio in both total and disaturated phosphatidylcholine from lung was nearly identical to that of the injected substrate. This suggested a direct acylation by lung acyl-CoA: lysophosphatidylcholine acyltransferases. By contrast, when a mixture of 1-[9,10- 3H 2]palmitoyl- sn-glycero-3-phospho [ methyl- 14C]choline and 1-stearoyl- sn-glycero-3-phospho[ methyl- 14C]choline was injected, the 3H 14C ratio in disaturated lung phosphatidylcholine increased to about 1.4-fold that of the injected substrate. 3. 3. These data indicate that increased isotopic ratios in disaturated phosphatidylcholine of lung tissue, after intravenous injection of lysophosphatidylcholine, do not necessarily point to the involvement of lysophosphatidylcholine: lysophosphatidylcholine transacylase in disaturated phosphatidylcholine formation.

Formation of phospholipids from sn-glycerol-3-phosphate and free fatty acids or their derivatives by homogenates of soybean cotyledons

Cell-free homogenates of soybean cotyledons contain a sn-glycerol-3-phosphate acyltransferase system which incorporated [U- 14C]- sn-glycerol-3-phosphate into 5 labelled lipids when incubated with palmitic acid in the presence of ATP and CoA. In decreasing order of incorporation of label, the lipids were: lysophosphatidic acid, monoacylglycerol, phosphatidic acid, diacylglycerol and triacylglycerol. The substrate specificity of the acyltransferase system was investigated with the fatty acids shown in order of decreasing rates of reaction; palmitate > stearate > oleate > linoleate > linolenate > laurate. Making these acids more soluble as triethanolamine salts or as polyoxyethylene sorbitan esters did not greatly enhance these rates of reaction. Activity was found in a 10000 g pellet containing plastids, mitochondria and glyoxysomes and also in the lipid layer; the activity in these particulate fractions was enhanced by the addition of cytosol which itself had little activity when gentle methods of cell disruption were used. During cotyledon development the total acyltransferase activity increased, although its specific activity slowly declined due to more rapid synthesis of other proteins. During germination total activity decreased but there was a transient increase in specific activity due to more rapid degradation of other proteins.

Possible involvement of acyltransferase systems in the formation of pulmonary surfactant lipid in rat

Dithiobis (2-nitrobenzoic acid)-resistant and -sensitive glycerophosphate acyltransferase systems were present in rat lung as in liver. The former was specific for palmitate while the latter could incorporate saturated and unsaturated acyl-CoAs comparably. The former has higher affinity for palmitate than the latter indicating that the 1-position of glycerophosphate can be acylated selectively with palmitate under certain conditions. The specificities of 1-acylglycerophosphate and 1-acylglycerophosphocholine acyltransferase systems were similar in lung and liver; both systems showed higher specificities for unsaturated acyl-CoAs. However, the selectivities observed at lower concentrations of phospholipid acceptors in the presence of equimolar mixtures of saturated and unsaturated acyl-CoAs were much different; the lung systems showed relatively higher selectivities for palmitate than the liver systems in the formation of both diacylglycerophosphate and phosphatidylcholine. On the other hand, palmitate was excluded almost completely from the 2-position in the 1-acylglycerophosphoethanolamine acyltransferase systems in lung and liver. These observations provide an enzymatic basis for describing the formation of pulmonary surfactant lipids in rat via acyltransferase systems.

Specificity and selectivity of diacylglycerolphosphate synthesis in Escherichia coli

The glycerolphosphate and 1-acylglycerolphosphate acyltransferase systems in Escherichia coli membranes show relatively low specificities for acylcoenzymes A when maximal velocities for the respective acyl-coenzymes A are compared. However, the selectivities for palmitate and oleate in the acylations of the 1- and 2-positions of glycerolphosphate moiety, respectively, are higher at lower concentrations of acceptors in the presence of an equimolar mixture of palmitoyl-CoA and oleoyl-CoA. More l-palmitoyl-2-oleoyl-glycerolphosphate species and less other species were synthesized at lower concentrations of glycerolphosphate. The fatty acyl moiety at the 1-position of 1-acylglycerolphosphate did not influence significantly the specificity for acyl-coenzymes A of the 1-acylglycerolphosphate acyltransferase system. Thus, the acceptor concentrations being kept low in vivo and in vitro are important for the highly selective incorporations of saturated and unsaturated fatty acids into the 1- and 2-positions of diacylglycerolphosphate, respectively, in the presence of mixtures of saturated and unsaturated acyl-coenzymes A while these acyltransferase systems exhibit relatively low specificies for acyl-coenzymes A when the respective maximal velocities are compared.

Selectivity of diacylglycerophosphate synthesis in subcellular fractions of rat liver

Relative contributions of two glycerophosphate acyltransferase systems to the synthesis of molecular species of diacylglycerophosphate were studied. A dithiobis(nitrobenzoate)-resistant system found mainly in mitochondrial outer membranes incorporated palmitate selectively into the 1-position of glycerophosphate, while a dithiobis(nitrobenzoate)-sensitive system in microsomes utilized palmitoyl-, oleoyl-, and linoleoyl-CoAs at comparable rates when the maximal velocities obtained in the presence of saturating amounts of a single acyl-CoA and glycerophosphate were compared. The level of available acyl-CoAs was found to be an important factor controlling the relative contributions of dithiobis(nitrobenzoate)-resistant and dithiobis(nitrobenzoate)-sensitive systems; the former with higher affinity for palmitoyl-CoA produced more 1-palmitoylglycerophosphate than the latter at lower concentrations of an equimolar mixture of palmitoyl-CoA and linoleoyl-CoA. Since the acylation at the 2-position yielding diacylglycerophosphate was highly selective for linoleate under these conditions, 1-palmitoyl-2-linoleoylglycerophosphate was the major product formed at lower concentrations of acyl-CoAs, while both 1-palmitoyl-2-linoleoylglycerophosphate and 1,2-dilinoleoylglycerophosphate species were formed in the presence of higher concentrations of these acyl-CoAs. Thus, the molecular species of diacylglycerophosphate synthesized in rat liver is not confined to 1-saturated acyl-2-unsaturated acylglycerophosphate species, but is variable depending on the relative concentrations of substrates. This mechanism provides an explanation for the apparent discrepancy in the specificities for acyl-CoAs of glycerophosphate acyltransferase systems and the synthesis of various molecular species of glycerophospholipids in rat liver in vivo depending on the nutritional conditions.

14C]palmitate uptake in isolated rat liver mitochondria: effects of fasting, diabetes mellitus, and inhibitors of carnitine acyltransferase.

The rapid association of Na-[16-(14)C]palmitate with isolated rat liver mitochondria was measured by an oil separation method. This association was time and temperature-dependent and was absolutely dependent on the presence of exogenous ATP and CoASH and partially dependent on exogenous carnitine. Carnitine dependence was enhanced at lower concentrations of [(14)C]palmitate. At 6.5 micro M [(14)C]palmitate (molar ratio of palmitate to albumin equal to 0.54), the rate of association was linear for 20 sec and was increased more than 100% in the presence of carnitine. Carnitine-dependent association was inhibited by 2-bromopalmitate, an inhibitor of carnitine acyltransferase I, but not by (+)-octanoylcarnitine, a presumed inhibitor of carnitine acyltransferase II. The association of [(14)C]palmitate with mitochondria was enhanced from 190 to 330% in mitochondria isolated from fasted animals and from 160 to 230% in mitochondria isolated from diabetic, ketotic animals as compared to control animals. The enhanced association with mitochondria from fasted animals was inhibited by 2-bromopalmitate. These studies demonstrate a method of evaluating fatty acid association with mitochondria which, because of its dependence on carnitine and carnitine acyltransferase I activity, most likely represents true uptake into mitochondria. Furthermore, these studies indicate that the carnitine-dependent uptake of fatty acids into mitochondria is enhanced in the two ketotic states evaluated and that the carnitine acyltransferase system may be a regulatory site in ketone body production.

Bile acid conjugation in organ culture of human fetal liver

An organ culture system for prolonged maintenance of human fetal liver has been developed and used to investigate the formation of bile acid conjugates. Multiple liver specimens were obtained from human abortuses and stillbirths ranging from 10 to 29 weeks of gestation. Within 3 hr of hysterotomy, small fragments of liver were established in organ culture. The morphological integrity of the explants was demonstrated by light and electron microscopy. Functional viability was determined by adding radiolabeled primary bile acid to the medium and assaying the taurine and glycine conjugates formed. Primary bile acid was taken up by the tissues, conjugated with taurine and glycine, and secreted into the medium at a constant rate during 10 days in vitro and in constant increments during a selected 24-hr period. The results establish that human fetal liver survives intact for periods up to 10 days in vitro. Taurine conjugates of the primary bile acids predominate throughout gestation and conjugates of cholic acid are synthesized in preference to those of chenodeoxycholic acid. Supplementation of the medium with taurine results in enhanced taurocholate formation with competitive inhibition of glycocholate synthesis, suggesting one acyl transferase system for both taurine and glycine. Finally, in medium supplemented with hydrocortisone there is a reversal of the glycine-taurine ratio seen in fetal liver.

Regulation of the fatty acid composition of alkyl ether phospholipid in Ehrlich ascites tumor cells. The substrate specificities of 1-O-alkylglycerol 3-phosphate and 1-O-alkylglycero-3-phosphocholine acyltransferases.

Activity for the acylation of 1-O-alkyl-GP was found in the microsomes of Ehrlich ascites tumor cells. The reaction product was shown to be 1-O-alkyl-2-acyl-GP by identifying the acetolysis product as 1-O-alkyl-2-acyl-3-acetylglycerol. The acyl transfer activity to 1-O-alkyl-GP was significantly lower than that to 1-acyl-GP. The substrate specificity of 1-O-alkyl-GP acyltransferase was rather broad as regards thiol esters. Similar specificity was observed with 1-acyl-GP acyltransferase. In contrast to these acyltransferase systems, the 1-acyl- and 1-O-alkyl-GPC acyltransferases were specific for polyunsaturated fatty acyl-CoA's. Since a high percentage of polyunsaturated fatty acid and a little palmitic acid were located at the 2-position of 1-O-alkyl-2-acyl-GPC(E), the observed specificities for acyl-COA's of these acyltransferase systems can be considered in relation to the fatty acid composition at the 2-position of 1-O-alkyl-2-acyl-GPC(E) in the cells.

Isolation and properties of a glycerophosphate acylating fraction in the fat body of Schistocerca gregaria (Forskäl).

An acyl-CoA-L-alpha-glycerophosphate acyltransferase system has been found in the fat body of the locust Schistocerca gregaria (Forskäl). After homogenization and differential centrifugation the enzyme system has been localized in two distinct particulate fractions. In both fractions phosphatidic acid was the main reaction product. The 10 000 g -30 000 g particulate fraction was further studied. The enzyme system is very sensitive to pH and Mg2+ concentration. An apparent Km of 0.3-0.5 mM for glycerophosphate was measured. The substrate concentration curve for palmitoyl-CoA is influenced by the protein concentration in the assay medium. This effect would partly explain the non-lineariy of the acylation reactions with respect to enzyme concentration. These observations are correlated with physiological phenomena.

Acyltransferase systems involved in phospholipid metabolism in Saccharomyces cerevisiae

Membrane preparations from Saccharomyces cerevisiae OC-2 catalyzed the acylation of glycerophosphate, 1-acyl and 2-acyl isomers of monoacylglycerophosphate, and 1-acyl and 2-acyl isomers of monoacylglycerylphosphorylcholine. The acyl-CoA:glycerophosphate acyltransferase system (EC 2.3.1.15) showed a broad specificity for acyl-CoAs when the maximal velocities were compared under optimized conditions. The acyl-CoA:2-acylglycerophosphate acyltransferase activity was much lower than the 1-acyl-glycerophosphate acyltransferase activity. Although the 1-acylglycerophosphate acyltransferase system utilized saturated and unsaturated acyl-CoAs at comparable rates, the acylations at the 1- and 2-positions were relatively more selective for palmitate and oleate, respectively, when assayed in the presence of palmitoyl-CoA, oleoyl-CoA, 1-acylglycerophosphate, and 2-acylglycerophosphate. The acyl-CoA:1-acylglyceryl-phosphorylcholine acyltransferase system (EC 2.3.1.23) was relatively more specific for unsaturated acyl-CoAs, while the acyl-CoA:2-acylglycerylphosphorylcholine acyltransferase system (EC 2.3.1.23) utilized both palmitoyl-CoA and oleoyl-CoA at a comparable rate. Although various acyltransferase systems showed a different degree of specificity for acyl-CoAs, the positional distribution of fatty acids in the phospholipid molecules could not be explained simply by the observed specificities. Zymolyase, β-1,3-glucanase from Arthrobacter luteus, was used successfully for the protoplast formation. Subcellular fractionation of the protoplast revealed that these acyltransferase activities were localized mainly in the microsomal fraction. However, the glycerophosphate and 1-acylglycerophosphate acyltranferase activities in the mitochondrial fraction could not be explained by the contamination of microsomes in this fraction. These observations are apparently inconsistent with a current concept that the mitochondrial fraction is the major site of phospholipid synthesis in yeast.

Differential increase of hepatic peroxisomal, mitochondrial and microsomal carnitine acyltransferases in clofibrate-fed rats

Hepatic peroxisomes, mitochondria and microsomes from control and clofibrate-treated animals were separated by isopycnic sucrose gradient centrifugation and the carnitine acyltransferase system studied in each of these organelles. Clofibrate treatment produced a 13-fold increase in the total activity of carnitine acetyltransferase and a 5-fold increase in carnitine octanoyl- and palmitoyl-transferase activities. The specific activities of the transferases in all three subcellular locations increased, but to different extents. Peroxisomal and microsomal carnitine acetyltransferases doubled in specific activity; the mitochondrial enzyme increased 10-fold. Peroxisomal, mitochondrial and microsomal carnitine octanoyltransferases all increased 3-fold in specific activity. Carnitine palmitoyltransferase, which is found only in mitochondria, increased 3-fold in specific activity. These differential increases changed the per cent distribution of total carnitine acetyltransferase from 50 per cent in the mitochondria of control livers to 90 per cent in treated livers. Peroxisomes from clofibrate-treated livers had a consistently greater isopycnic density in sucrose gradients. Total catalase activity increased 2-fold upon treatment and a greater percentage of it was found in the paniculate fractions. The specific activity of peroxisomal catalase and urate oxidase remained the same as in controls. Carnitine acetyl- and octanoyltransferases are the first reported enzymes whose peroxisomal specific activity increases with clofibrate treatment. Preliminary results of treatment with another membrane-inducing drug, phenobarbital, indicated no change in peroxisomal density, catalase distribution and activity, and no effect on the specific activities of the peroxisomal, mitochondrial and microsomal carnitine acyltransferases.

Metabolism of arachidonate and stearate injected simultaneously into the mouse brain.

The metabolism of a polyunsaturated and a saturated fatty acid in brain membrane phosphoglycerides was examined by injecting simultaneously a mixture of 14C-arachidonate and 3H-stearate into the mouse brain and isolating the microsomal and synaptosomal fractions at 1-40 min after injections. Both types of labeled fatty acids were utilized more readily in the microsomal than the synaptosomal fractions in brain. However, labeled arachidonate was incorporated more rapidly into membrane phosphoglycerides than was stearate. In both subcellular fractions, the relative specific radioactivity (3H and 14C) of diacyl-glycerophosphorylinositol (diacyl-GPI) was higher than other types of phosphoglycerides such as diacyl-glycerophosphorylcholine (diacyl-GPC) and diacyl-glycerophorylethanolamine (diacyl-GPE). Furthermore, the apparent rates of incorporation of radioactivity into diacyl-GPI was more rapid for the 14C-arachidonate than for the 3H-stearate. Results of the experiment have demonstrated obvious differences in metabolism between stearate and arachidonate in brain. The more rapid transfer of arachidonate to diacyl-GPI is probably due to the presence of an acyl transferase system specially active for the transfer of arachidonyl groups to diacyl-GPI.

Effects on cell adhesion and membrane fluidity of changes in plasmalemmal lipids in mouse l929 cells

ABSTRACT It has recently been shown that the lipid composition of the plasmalemma may be altered by taking advantage of the acyltransferase system to incorporate selected fatty acids into phosphatidyl lipids of the plasmalemma. Such lipid alterations have been shown to effect changes in cell adhesion in chick neural retina cells. Based on the fluid-mosaic model, it has been suggested that such changee in cell behaviour may be related to the degree of plasmalemmal fluidity. In the present study, when mouse L929 cells are incubated in the presence of stearic or arachidic acid (saturated), cell adhesion levels, as measured by an assay based on cellsubstrate interaction, increase. Similar incubation with linoleic or linolenic acid (unsaturated) decreases cell adhesion. In addition, we have found significant changes in plasmalemmal fluidity, using an assay based on patching of fluorescent antibody. Unsaturated fatty acids have a fluidizing effect on the membrane, while saturated fatty acids exert a solidifying effect, when substituted into the membrane. Although alternative interpretations are possible, we suggest that membrane fluidity changes are induced by alterations in lipid thermal transition temperatures of plasmalemmal phosphatidyl compounds, in which fatty acid substitutions were made. We also suggest that changes in membrane fluidity might affect adhesion by altering the aggregability of any membrane sites that may be involved in adhesion. Although a very fluid membrane may allow rapid lateral migration of sites, it might also aid their rapid dispersal, thereby reducing adhesion.