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1.
Ehrlich cells grown in mice fed coconut oil diets (highly saturated) contain about twice as much cholesteryl ester as those grown in mice fed sunflower oil diets (highly polyunsaturated). Acylcoenzyme A: cholesterol acyltransferase (ACAT) activity was 30-100% higher in microsomes prepared from the cells grown on coconut oil (M(c)) than in those prepared from the cells grown on sunflower oil (M(s)). Increased ACAT activity was noted in M(c) with either [1-(14)C]palmitoyl CoA or [1,2-(3)H]cholesterol as the labeled substrate. This occurred at all acyl CoA concentrations tested and, in the [1,2-(3)H]cholesterol assay, with palmitoyl, oleoyl, or linoleoyl CoA as the substrate. The pH optimum for ACAT activity was the same with M(c) and M(s), pH 7.0. ACAT activity obeyed Michaelis-Menten kinetics at palmitoyl CoA concentrations between 1 and 10 micro M. Substrate inhibition occurred at higher concentrations. Kinetic analysis with [1-(14)C]palmitoyl CoA as the substrate indicated that the apparent K(m) for M(c) was 33% smaller than for M(s). There was no difference, however, in apparent V(max) values. The cholesterol and phospholipid contents of M(c) and M(s) were similar, but their fatty acid compositions differed considerably. M(c) contained 2.7 times more monoenoic fatty acid and only half as much polyenoic fatty acid as M(s). Our results indicate that dietary modification of the microsomal fatty acid composition is associated with alterations in the activity of ACAT, an enzyme that is tightly bound to the microsomes. These changes in ACAT activity may be partly responsible for the differences in cholesteryl ester contents of Ehrlich cells grown in mice fed the coconut and sunflower oil diets. 相似文献
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Phospholipid fatty acid modification of rat liver microsomes affects acylcoenzyme A:cholesterol acyltransferase activity 总被引:1,自引:0,他引:1
The effect of phospholipid fatty acyl composition on the activity of acylcoenzyme A:cholesterol acyltransferase was investigated in rat liver microsomes. Specific phosphatidylcholine replacements were produced by incubating the microsomes with liposomes and bovine liver phospholipid-exchange protein. Although the fatty acid composition of the microsomes was modified appreciably, there was no change in the microsomal phospholipid or cholesterol content. As compared to microsomes enriched for 2 h with dioleoylphosphatidylcholine, those enriched with dipalmitoylphosphatidylcholine exhibited 30-45% less acyl-CoA:cholesterol acyltransferase activity. Enrichment with 1-palmitoyl-2-linoleoylphosphatidylcholine increased acyl-CoA:cholesterol acyltransferase activity by 20%. By contrast, dilinoleoylphosphatidylcholine abolished microsomal acyl-CoA:cholesterol acyltransferase activity almost completely. Addition of cofactors that stimulated microsomal lipid peroxidation inhibited acyl-CoA:cholesterol acyltransferase activity by only 10%, however, and did not increase the inhibition produced by submaximal amounts of dilinoleoylphosphatidylcholine. Certain of the phosphatidylcholine replacements produced changes in palmitoyl-CoA hydrolase, NADPH-dependent lipid peroxidase, glucose-6-phosphatase and UDPglucuronyl transferase activities, but they did not closely correlate with the alterations in acyl-CoA:cholesterol acyltransferase activity. Electron spin resonance measurements with the 5-nitroxystearate probe indicated that microsomal lipid ordering was reduced to a roughly similar extent by dioleoyl- or by dilinoleoylphosphatidylcholine enrichment. Since these enrichments produce widely different effects on acyl-CoA:cholesterol acyltransferase activity, changes in bulk membrane lipid fluidity cannot be the only factor responsible for phospholipid fatty acid compositional effect on acyl-CoA:cholesterol acyltransferase. The present results are more consistent with a modulation resulting from either changes in the lipid microenvironment of acyl-CoA:cholesterol acyltransferase or a direct interaction between specific phosphatidylcholine fatty acyl groups and acyl-CoA:cholesterol acyltransferase. 相似文献
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We measured the interactive effects of dietary cholesterol and fat on the regulation of hepatic acyl-CoA:cholesterol acyltransferase (ACAT) activity and its relationship to hepatic microsomal lipid composition in guinea pigs fed 15 g/100 g (w/w) fat diets (corn oil, olive oil, or lard) with 0.01, 0.08, 0.17, or 0.33 g/100 g (w/w) added cholesterol. Guinea pigs exhibited a dose dependent increase in hepatic microsomal ACAT activity, with increasing levels of cholesterol intake (P < 0.001) in all dietary fat groups. Animals fed monounsaturated olive oil had the highest hepatic ACAT activity with the exception of the 0.33 g/100 g cholesterol diet (P < 0.001). There were no differences in ACAT activity with intake of polyunsaturated corn oil or saturated lard. Dietary cholesterol resulted in increased microsomal free cholesterol (FC) concentrations in a dose dependent manner but had no effects on microsomal phosphatidylcholine (PC) concentrations. Guinea pigs fed olive oil generally had the highest microsomal FC/PC molar ratios, and hepatic ACAT activities correlated significantly with this parameter. After modification of the lipid compositions of the microsomes from guinea pigs fed the 12 test diets with FC/PC liposome treatment, microsomal ACAT activities remained significantly related to the microsomal FC/PC molar ratios, and dietary fat type did not affect this correlation. Our findings do not support the hypothesis that the stimulation of hepatic ACAT activity with cholesterol intake is enhanced by polyunsaturated fat intake. The data demonstrate that although dietary fat type and cholesterol amount have differential effects on hepatic ACAT activity, substrate availability, expressed as microsomal FC/PC molar ratio, is a major regulator of hepatic microsomal ACAT activity. 相似文献
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Acylcoenzyme A:cholesterol acyltransferase (ACAT) was solubilized from Ehrlich ascites cell microsomes with Triton X-100. After removal of the detergent, ACAT activity per mg protein was reduced by 50 to 65% as compared with untreated microsomes. When this microsomal extract was combined with liposomes composed of cholesterol and egg phosphatidylcholine, the ACAT activity increased 5.4- to 6.7-fold. Under these conditions sucrose density gradient centrifugation indicated that more than 50% of the added lipid was incorporated into vesicles having the same density as the ACAT activity, suggesting the formation of a complex. ACAT activity increased 2.9-fold when the phosphatidylcholine content of the liposomes was raised from 0.5 to 5.0 mumol/mg microsomal protein. By contrast, the ACAT activity increased only 42% when the cholesterol content of the liposomes was raised from 0.17 to 0.57 mumol/mg microsomal protein. Addition of phosphatidylethanolamine to the liposomes produced little change in ACAT activity, whereas the activity was reduced by 25 and 50%, respectively, when sphingomyelin or phosphatidylserine was added. ACAT activity was five times higher when the liposomes were prepared from dioleoylphosphatidylcholine than from saturated phosphatidylcholines, including hydrogenated egg yolk, dimyristoyl or dipalmitoyl phosphatidylcholine. Likewise, the ACAT activity with liposomes made from soybean or egg yolk phosphatidylcholine was almost 3.5-fold greater than with those prepared from the saturated phosphatidylcholines. These results are consistent with the view that the activity of ACAT can be modified by changes in the composition of the membrane lipids with which the enzyme is associated. 相似文献
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Studies on acyl-coenzyme A: cholesterol acyltransferase activity in human liver microsomes 总被引:2,自引:0,他引:2
K Einarsson L Benthin S Ewerth G Hellers D St?hlberg B Angelin 《Journal of lipid research》1989,30(5):739-746
The aim of the present study was to characterize the acyl-coenzyme A: cholesterol acyltransferase (ACAT) activity in human liver microsomes. Liver biopsies were obtained from patients undergoing elective cholecystectomy under highly standardized conditions. In 34 patients the enzyme activity of the microsomal fraction averaged 6.6 +/- 0.7 (mean +/- SEM) pmol.min-1.mg protein-1 in the absence of exogenous cholesterol. Freezing of the liver biopsy in liquid nitrogen increased the enzyme activity five- to sixfold. Similarly, freezing of the microsomal fraction prepared from unfrozen liver tissue increased the enzyme activity about twofold. These results may help to explain previous disparate results reported in the literature. The enhanced ACAT activity obtained by freezing was at least partly explained by a transfer of unesterified cholesterol to the microsomal fraction and possibly also by making the substrate(s) more available to the enzyme. Preincubation of the microsomal fraction, prepared from unfrozen liver tissue, with unlabeled cholesterol increased the enzyme activity about fivefold. This finding indicates that hepatic ACAT in humans can also utilize exogenous cholesterol as substrate. Addition of cholesterol to frozen microsomes prepared from unfrozen liver tissue increased the ACAT activity two- to threefold, whereas addition of cholesterol to microsomes prepared from frozen liver tissue did not further increase the enzyme activity. No evidence supporting the concept that ACAT is activated-inactivated by phosphorylation-dephosphorylation could be obtained by assaying the enzyme under conditions similar to those during which the human HMG-CoA reductase is inactivated-activated. 相似文献
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Acyl-CoA:cholesterol acyltransferase (ACCAT) activity of rat liver microsomes was stimulated by phosphatidylcholine. The stimulatory effect varied with the composition of the phosphatide: dimyristyl-, dipalmityl-, distearyl- and dioleylphosphatidylcholine were stimulatory, whereas dicaproyl- and dilinoleylphosphatidylcholine were not. The results suggest that increased fluidity of the membrane induced by phosphatide is probably not involved in the stimulation of cholesterol esterification. Phosphatide exerted its effect directly on the microsomes and did not extract cholesterol or ACCAT from the microsomes to an appreciable extent.Hydrolysis of microsomal phosphatide suppressed ACCAT activity. Enztme activity was restored with the addition of phosphatidylcholine. The results suggest that phosphatide may be required for cholesterol esterification. 相似文献
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Inhibition of acylcoenzyme A:cholesterol acyltransferase activity in CaCo-2 cells results in intracellular triglyceride accumulation 总被引:1,自引:0,他引:1
The activity of acylcoenzyme A:cholesterol acyltransferase (ACAT) in CaCo-2 cells was inhibited by the ACAT inhibitor, 58-035. The inhibitory effect of this acylamide was specific for cholesterol esterification catalyzed by ACAT; the rates of triglyceride, phospholipid, and cholesterol synthesis were not inhibited by this agent. Cholesteryl esters were depleted in CaCo-2 cells 24 hr after inhibition of ACAT activity, whereas the unesterified cholesterol content increased by 56% after 96 hr. Moreover, inhibiting ACAT activity with 58-035 resulted in a time-dependent 2.5-fold increase in intracellular triglycerides. This accumulation of triglycerides in CaCo-2 cells was associated with a 37% increase in triglyceride synthesis by 96 hr in the presence of 58-035. Triglyceride-rich lipoprotein secretion (d less than 1.006 g/ml) was not affected by inhibiting ACAT activity for up to 6 hr. However, triglyceride-rich lipoprotein secretion was significantly decreased in CaCo-2 cells that were preincubated with 58-035 for 24 to 96 hr. Lipoproteins of density less than 1.006 g/ml that were isolated from CaCo-2 cells incubated with the ACAT inhibitor were deficient in cholesteryl esters and triglycerides compared to lipoproteins isolated from control cells. The data suggest that triglycerides accumulate in CaCo-2 cells in which ACAT activity has been inhibited by 58-035. This accumulation of triglycerides is associated with a modest increase in triglyceride synthesis and a decrease in triglyceride secretion. Altering intracellular cholesterol pools by regulating ACAT activity in the gut could result in the decrease of triglyceride transport and/or the secretion of triglyceride-rich lipoprotein particles of abnormal composition. 相似文献
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beta-sitosterol: esterification by intestinal acylcoenzyme A: cholesterol acyltransferase (ACAT) and its effect on cholesterol esterification 总被引:1,自引:0,他引:1
Rabbits were fed either 10% coconut oil, 10% coconut oil and 1% beta-sitosterol, 10% coconut oil and 1% cholesterol, or 10% coconut oil and 1% beta-sitosterol plus 1% cholesterol for 4 weeks. Microsomal membranes from intestines of animals fed the 1% beta-sitosterol diet had 48% less cholesterol and were enriched twofold in beta-sitosterol compared to membranes from animals fed the coconut oil diet alone. Acylcoenzyme A:cholesterol acyltransferase (ACAT) activity in jejunum and ileum was decreased significantly in animals fed the plant sterol alone. In membranes from animals fed 1% beta-sitosterol and 1% cholesterol, beta-sitosterol content increased 50% whereas cholesterol was modestly decreased compared to their controls fed only cholesterol. Intestinal ACAT was unchanged in the animals fed both sterols when compared to their controls. beta-Sitosterol esterification was determined by incubating intestinal microsomal membranes with either [(14)C]beta-sitosterol-albumin emulsion or [(14)C]beta-sitosterol:dipalmitoyl phosphatidylcholine (DPPC) liposomes to radiolabel the endogenous sterol pool. Oleoyl-CoA was then added. The CoA-dependent esterification rate of beta-sitosterol was very slow compared to that of cholesterol using both techniques. An increased amount of endogenous microsomal beta-sitosterol, which occurs in animals fed 1% beta-sitosterol, did not interfere with the stimulation of ACAT activity secondary to cholesterol enrichment of the membranes. Enriching microsomal membranes three- to five-fold with beta-sitosterol did not affect ACAT activity. Freshly isolated intestinal cells were incubated for 1 hour with [(3)H]oleic acid and beta-sitosterol:DPPC or 25-hydroxycholesterol:DPPC. Incorporation of oleic acid into cholesteryl esters did not change in the presence of beta-sitosterol but increased fourfold after the addition of 25-hydroxycholesterol. We conclude that the CoA-dependent esterification rate of cholesterol is at least 60 times greater than that of beta-sitosterol. Membrane beta-sitosterol does not interfere with nor compete with cholesterol esterification. Inadequate esterification of this plant sterol may play a role in the poor absorption of beta-sitosterol by the gut.-Field, F. J., and S. N. Mathur. beta-Sitosterol: esterification by intestinal acylcoenzyme A:cholesterol acyltransferase (ACAT) and its effect on cholesterol esterification. 相似文献
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We analyzed the effects of dietary cholesterol, type of dietary fat, sex and sire progeny family on lecithin-cholesterol acyltransferase activity in 80 adult baboons. The animals were the progeny of 80 dams and 6 sires and were randomly assigned at birth to breast feeding or to one of three formulas containing 0.02, 0.30 or 0.60 mg cholesterol/ml. After weaning at 4 months of age the animals were fed one of four diets that were either high or low in cholesterol with 40% of the calories from either saturated or unsaturated fat. The fractional and molar rates of lecithin-cholesterol acyltransferase activity were measured at 7-8 years of age by an HPLC method. Infant diet (breast vs. formula feeding or level of cholesterol in formula had no effect on enzyme activity later in life. The adult diets that were high in cholesterol decreased the fractional lecithin-cholesterol acyltransferase rate by 20% / compared to diets low in cholesterol (7.89 vs. 9.84%/h, P less than 0.002), but dietary cholesterol did not affect the molar activity. Animals fed the high cholesterol diets had higher unesterified cholesterol concentrations compared to those fed the low cholesterol diets (38.1 mg/dl vs. 31.6 mg/dl, P less than 0.0001). The molar lecithin-cholesterol acyltransferase rate was increased 13% by saturated compared to unsaturated fat (83.3 vs. 73.6 nmol/h per ml plasma, P less than 0.07), but no effect of dietary fat was observed on the fractional enzyme activity. Females compared to males had significantly higher fractional (10.9 vs. 7.14%/h, P less than 0.0001) and molar lecithin-cholesterol acyltransferase activities (99.3 vs. 61.7 nmol/h per ml plasma, P less than 0.0001). After adjustment for the effects of diet and sex we observed differences in the fractional activity (range, 7.2-10.8%/h, P less than 0.04) and in the molar rate (range, 63.6-99.8 nmol/h per ml plasma, P less than 0.07) among the six sire progeny groups. The differences among sire progeny groups are evidence for genetic differences in lecithin-cholesterol acyltransferase activities among the baboon families. 相似文献
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The medium-chain carnitine acyltransferase activity associated with rat liver microsomes is malonyl-CoA sensitive 总被引:2,自引:0,他引:2
K Lilly G E Bugaisky P K Umeda L L Bieber 《Archives of biochemistry and biophysics》1990,280(1):167-174
The data presented herein show that both rough and smooth endoplasmic reticulum contain a medium-chain/long-chain carnitine acyltransferase, designated as COT, that is strongly inhibited by malonyl-CoA. The average percentage inhibition by 17 microM malonyl-CoA for 25 preparations is 87.4 +/- 11.7, with nine preparations showing 100% inhibition; the concentrations of decanoyl-CoA and L-carnitine were 17 microM and 1.7 mM, respectively. The concentration of malonyl-CoA required for 50% inhibition is 5.3 microM. The microsomal medium-chain/long-chain carnitine acyltransferase is also strongly inhibited by etomoxiryl-CoA, with 0.6 microM etomoxiryl-CoA producing 50% inhibition. Although palmitoyl-CoA is a substrate at low concentrations, the enzyme is strongly inhibited by high concentrations of palmitoyl-CoA; 50% inhibition is produced by 11 microM palmitoyl-CoA. The microsomal medium-chain/long-chain carnitine acyltransferase is stable to freezing at -70 degrees C, but it is labile in Triton X-100 and octylglucoside. The inhibition by palmitoyl-CoA and the approximate 200-fold higher I50 for etomoxiryl-CoA clearly distinguish this enzyme from the outer form of mitochondrial carnitine palmitoyltransferase. The microsomal medium-chain/long-chain carnitine acyltransferase is not inhibited by antibody prepared against mitochondrial carnitine palmitoyltransferase, and it is only slightly inhibited by antibody prepared against peroxisomal carnitine octanoyltransferase. When purified peroxisomal enzyme is mixed with equal amounts of microsomal activity and the mixture is incubated with the antibody prepared against the peroxisomal enzyme, the amount of carnitine octanoyltransferase precipitated is equal to all of the peroxisomal carnitine octanoyltransferase plus a small amount of the microsomal activity. This demonstrates that the microsomal enzyme is antigenically different than either of the other liver carnitine acyltransferases that show medium-chain/long-chain transferase activity. These results indicate that medium-chain and long-chain acyl-CoA conversion to acylcarnitines by microsomes in the cytosolic compartment is also modulated by malonyl-CoA. 相似文献
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The relationships between cholesterol 7 alpha-hydroxylase activity, pool of free microsomal cholesterol, and degree of substrate saturation of the enzyme were studied in untreated (n = 5), cholesterol-fed (n = 4), and cholestyramine-treated (n = 6) gallstone patients undergoing cholecystectomy. Highly accurate methods based on isotope dilution-mass spectrometry were used for assay of the cholesterol 7 alpha-hydroxylase activity and for determination of the concentration of free cholesterol in the microsomes. The cholesterol-enriched diet increased the cholesterol 7 alpha-hydroxylase activity about twofold. Cholestyramine treatment was associated with a five- to sixfold increase of the cholesterol 7 alpha-hydroxylase activity. The concentration of free microsomal cholesterol remained essentially unchanged. The apparent degree of saturation of the enzyme was calculated to be 85% in the untreated patients, 86% in the cholesterol-fed patients, and 67% in those treated with cholestyramine. A significant negative correlation was obtained between enzyme activity and apparent substrate saturation. It is concluded that the apparent substrate saturation of the cholesterol 7 alpha-hydroxylase in human liver microsomes is high but that availability of cholesterol may limit the enzyme activity to some extent a high bile acid synthesis rates. 相似文献
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Buprenorphine is a partial opioid agonist available in France as an alternative to methadone in the treatment of opiate-dependent individuals. Twenty deaths have been reported in patients who have ingested buprenorphine in combination with benzodiazepines. Since buprenorphine and many benzodiazepines are CYP3A substrates, the effect of buprenorphine on CYP3A activity was examined in order to assess the likelihood of a pharmacokinetic interaction. The formation of 6beta-hydroxytestosterone was measured in dexamethasone-induced rat liver microsomes and in human liver microsomes under control conditions and in the presence of buprenorphine. Buprenorphine was found to be a weak inhibitor of CYP3A with a 50% decrease in enzyme activity occurring at a concentration of 118 microM (IC50) in human liver microsomes. IC50 was 0.3 microM for ketoconazole in the same system. Since the IC50 for buprenorphine is roughly 2000 times higher than typical plasma concentrations, this drug is unlikely to cause clinically significant inhibition of CYP3A in patients. Excessive CNS depression due to the combination of buprenorphine and benzodiazepines is most likely due to additive or synergistic pharmacologic effect unrelated to a pharmacokinetic interaction between the drugs. 相似文献
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