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1.
CTP : phosphocholine cytidylyltransferase activity exists in both the microsome and cytosol fractions of adult lung, 36 and 59%, respectively. Although these enzyme activities are stimulated in vitro by added lipid activators (i.e. phosphatidylglycerol), there are significant levels of activity in the absence of added lipid. We have removed endogenous lipid material from microsome and cytosol preparations of rat lung by rapid extraction with isopropyl ether. The extraction procedure did not cause any loss of cytidylyltransferase activity in the cytosol. After the extraction the enzyme was almost completely dependent upon added lipid activator. Isopropyl ether extraction of microsome preparations produced a loss of 40% of the cytidylyltransferase activity, when measured in the presence of added phosphatidylglycerol. Lipid material extracted into isopropyl ether restored the cytidylyltransferase activity in cytosol. The predominant species of enzyme activator in the isopropyl ether extracts was fatty acid. A variety of naturally occurring unsaturated fatty acids stimulated the cytidylyltransferase to the same extent as phosphatidylglycerol. Saturated fatty acids were inactive.  相似文献   

2.
The activity of the low molecular weight form of cytidylyltransferase from fetal lung cytosol and adult liver cytosol was stimulated more by phosphatidylcholine-oleic acid (1:1 molar ratio) vesicles than by phosphatidylglycerol vesicles. Phosphatidylcholine alone did not stimulate the activity, while oleic acid alone produced only slight stimulation. Vesicles prepared from phosphatidylinositol, phosphatidylglycerol-cholesterol (2:1) and phosphatidylglycerol-phosphatidylcholine (1:1) all stimulated the activity to the same extent. Phosphatidylcholine-oleic acid vesicles (molar ratio 2:1) produced less stimulation than 1:1 vesicles. Phosphatidylcholine-palmitic acid vesicles (2:1) were about 50% as active as the corresponding phosphatidylcholine-oleic acid vesicles. All vesicles were in the size range of small unilamellar vesicles as judged by Sephacryl S-1000 chromatography. Stimulation also occurred when phosphatidylcholine vesicles and oleic acid were added separately to the assay. The stimulation by phospholipid vesicles was correlated with the ability of the vesicles to bind cytidylyltransferase, determined by sucrose density centrifugation of the enzyme-vesicles mixtures. We conclude that the stimulation of soluble cytidylyltransferase occurs through binding of the enzyme to anionic membrane surfaces. Suitable anionic membranes can be prepared either from anionic phospholipids, or by the addition of anionic lipids (unesterified fatty acids or phosphatidylglycerol) to phosphatidylcholine.  相似文献   

3.
Addition of oleate, oleyl alcohol, or palmitate to HeLa cell medium resulted in a rapid stimulation of PC synthesis and activation of CTP: phosphocholine cytidylyltransferase. Stimulation was optimal with 0.35 mM oleate, 0.3 mM oleyl alcohol and 5 mM palmitate, or 1 mM palmitate if EGTA were added to the medium. The cytidylyltransferase was activated by translocation of the inactive cytosolic form to membranes. In untreated cells approx. 30% of the total cytidylyltransferase was membrane bound, while in treated cells, 80-90% was membrane associated. Addition of bovine serum albumin (10 mg/ml) to cells previously treated with oleate (0.35 mM) rapidly removed cellular fatty acid, and the membrane-bound cytidylyltransferase activity returned to approx. 30%. Similar results were obtained by extraction of membranes with albumin in vitro. Although 95% of the free fatty acid was extracted, 30-40% of the membrane cytidylyltransferase remained bound. Translocation of cytidylyltransferase between isolated cytosol and microsomal fractions was promoted by addition of oleate, palmitate, oleyl alcohol, and monoolein. Addition of diacylglycerol, lysophosphatidylcholine, lysophosphatidylethanolamine, calcium palmitate, and detergents such as Triton X-100, cholate or Zwittergent did not stimulate translocation of the enzyme. Addition of oleoyl-CoA promoited translocation, however, 40% of it was hydrolyzed releasing free oleic acid. Cytosolic cytidylyltransferase bound to microsomes pre-treated with phospholipase C, which had 7-fold elevated diacylglycerol content. Fatty acid-promoted translocation was blocked by Triton X-100, but not by 1 M KCl. These results suggest that a variety of compounds with differing head group size and charge, and number of hydrocarbon chains can function as translocators, and that hydrophobic rather than ionic interactions mediate the binding of cytidylyltransferase to membranes.  相似文献   

4.
The effects of stearic, oleic, and arachidonic acids on phosphatidylcholine biosynthesis in the hamster heart were investigated. When hamster hearts were perfused with labelled choline in the presence of fatty acids, biosynthesis of phosphatidylcholine was stimulated only by stearic acid. Stearic acid was found to accumulate in unesterified (free) form in the hamster heart after perfusion. The stimulation by stearic acid was mediated in vivo by an enhancement of CTP:phosphocholine cytidylyltransferase activity in the microsomal fraction of the hamster heart and the enzyme activity in the cytosolic fraction was not affected. In contrast with the observations in rat hepatocytes, cytidylyltransferase from the hamster heart was not stimulated directly by stearic acid. The selective activation of the microsomal enzyme when the heart was perfused with stearic acid suggests that activation of the enzyme was mediated via the modification of the membrane by stearic acid.  相似文献   

5.
Glycerolphosphate acyltransferase activity in microsomes from rat adipose tissue is shown to decrease with time upon incubation with adipose tissue cytosolic fraction. The inactivation can be prevented with serum albumin and seems to be caused by an increase in endogenous free fatty acid as a consequence of the action of cytosolic lipase(s) on the membrane lipids. Similar inactivation can be observed after short incubation of microsomes with oleic acid at micromolar concentrations. Diacylglycerol acyltransferase is also inhibited by oleic acid, although to a lesser degree. In contrast, glucose-6-phosphatase and NADPH-cytochrome reductase activities are not changed. The oleic acid effect appears to occur upon binding to the microsomal membranes and can be prevented by bovine serum albumin at protein/fatty acid molar ratios above one. These results suggest that free fatty acids may be involved in the modulation of triacylglycerol synthetic enzymes.  相似文献   

6.
We examined the effect of fatty acids on phosphatidylcholine synthesis and cytidylyltransferase activity in Hep G2 cells. Treatment of Hep G2 cells with oleic acid caused an increase in the incorporation of [methyl-14C]choline into phosphatidylcholine and a corresponding decrease in radioactivity in choline phosphate using a pulse-chase procedure. This result is consistent with a fatty acid-induced increase in the cytidylyl-transferase step in the choline pathway. We measured cytidylyltransferase activity in membrane fractions and in cytosol (100,000 x g supernatant or soluble enzyme released by digitonin). The activity increased in both membrane and cytosol. Thus, an increase in total activity occurred. Cytidylyltransferase protein determined by Western blot immunoassay increased after oleic acid treatment. Immunotitration of cytidylyltransferase protein also indicated that an increase in enzyme protein resulted from oleic acid treatment. Cycloheximide did not prevent the oleic acid-induced increase in cytidylyltransferase activity. The increase in enzyme activity was apparent when we measured the activity in the presence or absence of lipid activators. Separation of cytosolic cytidylyltransferase into H- and L-forms showed that the increase in cytosolic activity was due to an increase in H-form. The amount of L-form did not change. We interpret these results to suggest that fatty acid treatment of Hep G2 cells promoted the formation of active cytidylyltransferase (H-form) from a preexisting inactive form. The increased activity was distributed between membranes and the lipoprotein form in cytosol (H-form).  相似文献   

7.
In the studies described here rat liver microsomes containing labeled palmitic, stearic, oleic or linoleic acids were incubated with fatty acid binding protein (FABP) and the rate of removal of14C-labeled fatty acids from the membrane by the soluble protein was measured using a model system. More unsaturated than saturated fatty acids were removed from native liver microsomes incubated with similar amounts of FABP. Thein vitro peroxidation of microsomal membranes mediated by ascorbate-Fe++, modified its fatty acid composition with a considerable decrease of the peroxidizability index. These changes in the microsomes facilitated the removal of oleic and linoeic acids by FABP, but the removal of palmitic and stearic acids was not modified. This effect is proposed to result from a perturbation of membrane structure following peroxidation with release of free fatty acids from susceptible domains.Abbreviations BSA bovine serum albumin - FABP fatty acid binding protein  相似文献   

8.
In isolated rat hepatocytes flavaspidic acid, a competitor with free fatty acids for the fatty-acid-binding-protein, decreased the uptake of oleic acid and triglyceride synthesis but stimulated the formation of CO2 and ketone bodies from oleic acid. Flavaspidic acid had no effect on the utilization of octanoic acid. Stimulation of the microsomal fatty-acid-activating enzyme by the fatty-acid-binding protein was reversed by flavaspidic acid. In contrast, the binding protein inhibited the mitochondrial fatty-acid-activating enzyme. Flavaspidic acid not only prevented this inhibition but actually stimulated the enzyme activity. The results indicate that the cytosol fatty-acid-binding protein directs the metabolism of long chain fatty acids toward esterification as well as enhancing their cellular uptake.  相似文献   

9.
Addition of oleic acid to Krebs-II cells stimulated by 9-fold [3H]choline incorporation into choline glycerophospholipids without affecting the selective incorporation of the precursor into diacyl subclass (90% of total [3H]choline glycerophospholipids). The total activity of cytidylyltransferase (E.C. 2.7.7.15), the regulatory enzyme of choline glycerophospholipid synthesis, was increased in the particulate fraction at the expense of cytosol. Free [3H]oleic acid was also associated with the particulate fraction. Subcellular fractionation of membranes on Percoll gradient, indicated that the endoplasmic reticulum, which contained 90% of total cell free oleic acid, was the unique target for the translocation of cytidylyltransferase. [3H]oleic acid was incorporated almost exclusively into phosphatidylcholine and corresponded to a synthesis of 9 nmol/h per 10(6) cells. Based on [3H]choline incorporation a net synthesis of 22 nmol/h per 10(6) cells was determined. However, oleic acid treatment did not change the total amount of phosphatidylcholine (45 nmol/10(6) cells) and other phospholipids; also no modification in the subcellular distribution of phospholipids was observed. It is concluded that the stimulation of the de novo synthesis of phosphatidylcholine which involves translocation of cytidylyltransferase onto the endoplasmic reticulum, is accompanied by a renewal of their polar head group. Also exogenous oleic acid induces an enhanced fatty acid turnover, highly specific for phosphatidylcholine. Therefore, Krebs-II cells exhibited a high degree of regulation of their phosphatidylcholine content, suggesting a parallel stimulation of both synthesis and degradation.  相似文献   

10.
Fetal lung fatty-acid synthase and choline-phosphate cytidylyltransferase activities are increased by glucocorticoids. There is evidence that the hormone increases synthesis of fatty-acid synthase but only increases the catalytic activity of the cytidylyltransferase. Free fatty acids and a number of phospholipids have been reported to stimulate cytidylyltransferase activity in several organs, including the lung. We have addressed the question of whether glucocorticoid induction of fatty-acid synthase mediates the stimulatory effect of the hormone on choline-phosphate cytidylyltransferase activity. Explants of 18-day fetal rat lung were cultured for 48 h with dexamethasone and inhibitors of de novo fatty acid biosynthesis (agaric acid and hydroxycitric acid) being included in the medium for the final 20 h. Dexamethasone increased the activities of fatty acid synthase and choline-phosphate cytidylyltransferase by 84% and 60%, respectively. Agaric acid and hydroxycitric acid completely abolished the stimulatory effect of the hormone on cytidylyltransferase but not on fatty-acid synthase. The inhibitors had no effect on cytidylyltransferase activity in control cultures. Fetal lung choline-phosphate cytidylyltransferase can be maximally stimulated by inclusion of phosphatidylglycerol in the assay mixture and under this condition, cytidylyltransferase activity in control and dexamethasone-treated cultures in the presence and absence of the inhibitors were all increased to the same level. Therefore, the inhibitors did not diminish the capacity of cytidylyltransferase to be fully activated. We suggest that the glucocorticoid induction of fatty-acid synthase in fetal lung results in increased synthesis of fatty acids which in turn, either as free acids or after incorporation into phospholipids, activate choline-phosphate cytidylyltransferase.  相似文献   

11.
A new model system for the study of phosphatidylcholine biosynthesis is presented. Young rats were fed a diet that contained 5% cholesterol and 2% cholate. After 6 days there was a 2-fold increase in the concentration of plasma phospholipid (243 mg/dl compared to 132 mg/dl for control animals) and a 3-fold increase in the concentration of plasma phosphatidylcholine. The rate of phosphatidylcholine biosynthesis was measured after injection of [Me-3H]choline into the portal veins. The incorporation of tritium into choline, phosphocholine and betaine by liver was similar for experimental and control animals, whereas there was a 3-fold increased incorporation into phosphatidylcholine of the cholesterol/cholate-fed rats. The activities of the enzymes of phosphatidylcholine biosynthesis in cytosol and microsomes were assayed. The only change detected was in the cytosolic and microsomal activities of CTP: phosphocholine cytidylyltransferase which were increased more than 2-fold in specific activity. When total cytidylyltransferase activity per liver was determined, a dramatic translocation of the enzyme to microsomes was observed. The control livers had 24% of the cytidylyltransferase activity associated with microsomes, whereas this value was 61% in the livers from cholesterol/cholate-fed rats. When the cytosolic cytidylyltransferase was assayed in the presence of phospholipid, the enzyme was stimulated several-fold and the difference in specific activity between control and cholesterol/cholate-fed rats was abolished. The increased activity in cytosol appears to be the result of a 2-fold increase in the amount of phospholipid in the cytosol from cholesterol/cholate-fed rats. The data strongly support the hypothesis that the special diet stimulates phosphatidylcholine biosynthesis by causing a translocation of the cytidylyltransferase from cytosol to microsomes where it is activated.  相似文献   

12.
Bovine serum albumin or fatty-acid-binding protein rapidly lose oleic acid when incubated in the presence of dimyristoyl lecithin liposomes. The phenomenon is dependent on vesicle concentration and no measurable quantities of protein are found associated with liposomes. Upon gel filtration on Sepharose CL-2B of incubated mixtures of microsomes containing [1-14C] oleic acid and albumin or fatty-acid-binding protein, association of fatty acid with the soluble proteins could be demonstrated. Both albumin and fatty-acid-binding protein stimulated the transfer of oleic acid from rat liver microsomes to egg lecithin liposomes. These results indicate that albumin is more effective in the binding of oleic acid than fatty-acid-binding protein, which allows a selective oleic acid dissociation during its interaction with membranes.  相似文献   

13.
Supernatant protein factor (SPF) and anionic phospholipids such as phosphatidylglycerol (PG) stimulate squalene epoxidase activity in rat liver microsomes by promoting [3H]squalene uptake as well as substrate translocation (Chin, J., and K. Bloch. 1984. J. Biol. Chem. 259: 11735-11738). This process is postulated to be membrane-mediated and not carrier-mediated. Here we show that treatment of PG with phospholipase A2 in the presence of bovine serum albumin abolishes the stimulatory effect of SPF on epoxidase activity. Disaturated fatty acyl-PGs are not as effective as egg yolk lecithin PG in the SPF effect. These findings suggest an important role for the unsaturated fatty acid moiety of PG. We also show that at submicellar concentrations, cis-unsaturated fatty acids stimulate microsomal epoxidase activity whereas saturated fatty acids do not. This effect is due to an increase in substrate uptake which in turn may facilitate substrate availability to the enzyme.  相似文献   

14.
The purpose of these studies was to determine the properties of the membrane-bound cytidylyltransferase in adult lung and to assess the relationship between the microsomal enzyme and the two forms of cytidylyltransferase in cytosol. Microsomes, isolated by glycerol density centrifugation, contained significantly less cytidylyltransferase than microsomes isolated by differential centrifugation (11.6 +/- 3.2 vs. 30 +/- 11 nmol/min per g lung). The released activity was recovered as H-form cytidylyltransferase. Cytidylyltransferase activity was not removed from microsomes by washing of the microsomal pellet with homogenizing buffer. Triton X 100 extracted all of the cytidylyltransferase from microsomes. The extracted activity was similar to H-form. Chlorpromazine dissociated microsomal enzyme to L-form. Chlorpromazine has been shown previously to dissociate H-form to L-form. These results suggested that microsomal cytidylyltransferase existed in a form similar if not identical to cytosolic H-form. In vitro translocation experiments demonstrated that the L-form of cytidylyltransferase was the species which binds to microsomal membranes. Triton X 100 extraction of microsomes from translocations experiments removed the bound enzyme activity. Glycerol density fractionation indicated that the activity in the Triton extract was H-form cytidylyltransferase. We concluded that the active lipoprotein form of cytidylyltransferase (H-form) is the membrane-associated form of cytidylyltransferase in adult lung; that it is formed after the L-form binds to microsomal membranes and that cytosolic H-form is released from the membrane.  相似文献   

15.
1. The effects of unsaturated fatty acids on drug-metabolizing enzymes in vitro were measured by using rat and rabbit hepatic 9000g supernatant fractions. 2. Unsaturated fatty acids inhibited the hepatic microsomal metabolism of ;type I' drugs with inhibition increasing with unsaturation: arachidonic acid>linolenic acid>linoleic acid>oleic acid. Inhibition was independent of lipid peroxidation. Linoleic acid competitively inhibited the microsomal O-demethylation of p-nitroanisole and the N-demethylation of (+)-benzphetamine. 3. The hepatic microsomal metabolism of ;type II' substrates, aniline and (-)-amphetamine, was not affected by unsaturated fatty acids. 4. The rate of reduction of p-nitrobenzoic acid and Neoprontosil was accelerated by unsaturated fatty acids. 5. Linoleic acid up to 3.5mm did not decelerate the generation of NADPH by rat liver soluble fraction, nor the activity of NADPH-cytochrome c reductase of rat liver microsomes. Hepatic microsomal NADPH oxidase activity was slightly enhanced by added linoleic acid. 6. No measurable disappearance of exogenously added linoleic acid occurred when this fatty acid was incubated with rat liver microsomes and an NADPH source. 7. The unsaturated fatty acids used in this study produced type I spectra when added to rat liver microsomes, and affected several microsomal enzyme activities in a manner characteristic of type I ligands.  相似文献   

16.
The changes in linoleyl-CoA desaturase activity of rat liver microsomes were studied after a single intraperitoneal injection of 11-deoxycorticosterone or aldosterone at physiological doses. Fatty acid of plasma and different liver fractions, and physical properties of microsomal membranes were also investigated. It was found that the specific activity of delta 6 desaturase decreased 4-fold 24 hr after the injection of aldosterone or deoxycorticosterone. Pretreatment of the rats with aldosterone led to a significant decrease in the percent distribution of palmitic, arachidonic, docosapentaenoic and docosahexenoic acids, with a concomitant increase in palmitoleic, oleic and linoleic acids in plasma and liver homogenates, microsomes and cytosol fractions. A similar pattern was observed after deoxycorticosterone administration. The changes resulted in a decreased unsaturation index of all fractions studied and were well-correlated with the increase observed in fluorescence depolarization of the hydrophobic probe 1,6-diphenylhexatriene in liver microsomal membranes. The interlipid and lipid/protein ratios in microsomes remained constant after hormonal treatment. These results are consistent with the idea that the inhibition of delta 6 desaturase activity and the alterations in fatty acid composition induced by mineralocorticoids, are solely responsible for the measured decrease in liver microsomal membrane fluidity.  相似文献   

17.
Activities of ethanolaminephosphotransferases (EC 2.7.8.1) and choline phosphotransferases (EC 2.7.8.2) in microsomal fractions from brains and livers of mature rats are increased several fold by the addition of 1,2-diacyl-sn-glycerols or 1-alkyl-2-acyl-sn-clycerols. Oleic acid added with diacylglycerols stimulated further the synthesis of lecithins by liver microsomes, confirming the work of Sribney and Lyman (Can J. Biochem. 51: 1479-1486, 1973). With alkylacylglycerols, oleic and stearic acids were inhibitory and linoleic acid was even more inhibitory for the synthesis of both 1-alkyl-1-acyl-sn-glycero-3-phosphorylcholines and the corresponding ethanolamine compounds with microsomes from both tissues. Free fatty acids without added diglycerides had mixed effects. These results are best explained by postulating the presence of two isoenzymes each for ethanolaminephosphotransferase and cholinephosphotransferase of which only one is affected by free fatty acids. Regulation of the phosphotransferases by free fatty acids may determine the proportion of CDP-choline and CDP-ethanolamine used for synthesis of diacyl and alkylacyl types of these phosphoglycerides.  相似文献   

18.
The enzymes responsible for the biosynthesis of phosphatidylglycerol, CTP:phosphatidate cytidylyltransferase, CDP-diacylglycerol: glycerophosphate phosphatidyltransferase and phosphatidylglycerophosphate phosphatase demonstrated a coordinate increase in activity in fetal rat lung at term when the demand for pulmonary surfactant increases. The activity of CTP:cholinephosphate cytidylyltransferase, the enzyme responsible for CDP-choline production also increased in the perinatal period. The activity of cholinephosphate cytidylyltransferase in fetal and neonatal cytosol was stimulated by the addition of phosphatidylglycerol but no effect was noted with cytosol from adult lung. These results are consistent with the suggestion that the activity of cholinephosphate cytidylyltransferase, a potential rate-determining enzyme in pulmonary phosphatidylcholine synthesis, may be regulated in the perinatal period both through an activation by phosphatidylglycerol and by an increase in total enzyme units.  相似文献   

19.
The effects of oleic acid on the activities of cytosolic HMG-CoA (3-hydroxy-3-methylglutaryl-CoA) synthase, AcAc-CoA (acetoacetyl-CoA) thiolase and AcAc-CoA synthetase, as well as microsomal HMG-CoA reductase, all enzymes in the pathway of cholesterol biosynthesis, were studied in the isolated perfused rat liver. Oleic acid bound to bovine serum albumin, or albumin alone, was infused for 4 h at a rate sufficient to sustain an average concentration of 0.61 +/- 0.05 mM fatty acid during the perfusion. Hepatic cytosol and microsomal fractions were isolated at the termination of the perfusion. Oleic acid simultaneously increased the activities of the cytosolic cholesterol-biosynthetic enzymes 1.4-2.7-fold in livers from normal fed rats and from animals fasted for 24 h. These effects were accompanied by increased net secretion by the liver of cholesterol and triacylglycerol in the very-low-density lipoprotein (VLDL). We confirmed the observations reported previously from this laboratory of the stimulation by oleic acid of microsomal HMG-CoA reductase. In cytosols from perfused livers, the increase in AcAc-CoA thiolase activity was characterized by an increase in Vmax. without any change in the apparent Km of the enzyme for AcAc-CoA. In contrast, oleic acid decreased the Km of HMG-CoA synthase for Ac-CoA, without alteration of the Vmax. of the enzyme. The Vmax. of AcAc-CoA synthetase was increased by oleic acid, and there was a trend towards a small increase in the Km of the enzyme for acetoacetate. These data allow us to conclude that the enzymes that supply the HMG-CoA required for hepatic cholesterogenesis are stimulated, as is HMG-CoA reductase, by a physiological substrate, fatty acid, that increases rates of hepatic cholesterol synthesis and cholesterol secretion. Furthermore, we suggest that these effects of fatty acid on hepatic cholesterol metabolism result from stimulation of secretion of triacylglycerol in the VLDL by fatty acids, and the absolute requirement of cholesterol as an important structural surface component of the VLDL necessary for transport of triacylglycerol from the liver.  相似文献   

20.
The fatty acid composition of cultured Friend erythroleukemia cells was modified by supplementation of the medium with oleic or linoleic acid. There was a 30% reduction in saturated and a 35% reduction in polyunsaturated fatty acids in microsomal phospholipids when the cells were grown in media supplemented with oleic acid, and a 3-fold increase in polyunsaturated fatty acids when the cells were grown in linoleic acid-supplemented media. Electron-spin resonance studies with the 5-nitroxystearate probe demonstrated that there was no appreciable change in microsomal lipid mobility as measured by the order parameters. In contrast, changes in lipid mobility were detected with the spin-label probe when microsomes were first isolated from Friend erythroleukemia cells and subsequently modified by incubation with liposomes composed of either dioleoyl- or dilinoleoylphosphatidylcholine plus bovine liver phospholipid-exchange protein. The fatty acid compositional changes produced in these microsomes were similar to those obtained when the intact cells were grown in media containing supplemental fatty acids. These findings indicate that the lipid mobility of Friend cell microsomes can be altered by phospholipid replacements in vitro, but that this does not occur when similar microsomal fatty acid modifications are produced during culture of the intact cell.  相似文献   

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