共查询到20条相似文献,搜索用时 15 毫秒
1.
Kathleen M. Botham 《Lipids》1991,26(11):901-906
An acid cholesteryl ester hydrolase activity associated with a fraction containing mitochondria and lysosomes from rat lactating
mammary glands was found to have a pH optimum of 5.0. Its sedimentation pattern was closely related to that of the lysosomal
enzyme markers acid phosphatase and β-glucuronidase, suggesting that the activity is associated with the lysosomes. The enzyme
was strongly inhibited by Cu2+, but was inhibited little by other divalent metal ions. Acid cholesteryl ester hydrolase activity was almost completely abolished
byp-hydroxymercuribenzoate, but this effect was reversed in the presence of an equimolar concentration of reduced glutathione
(GSH), indicating that the enzyme requires free sulfhydryl groups for activity. These properties are similar to those of acid,
lysosomal cholesteryl ester hydrolases found in other tissues. Acid cholesteryl ester hydrolase activity was 8–14 fold higher
in mammary tissue from lactating as compared to virgin rats. Neutral cholesteryl ester hydrolase activities associated with
the microsomal and cytosolic subcellular fractions were also increased in lactating glands, but to a lesser extent. In addition,
a 2-fold increase in the activities of both the acid and microsomal neutral enzymes was seen during the first few days of
lactation, while the cytosolic neutral activity remained constant. These results suggest that mammary gland cholesteryl ester
hydrolases have a role in the regulation of cholesterol metabolism in mammary cells, and in the provision of cholesterol for
secretion into milk. 相似文献
2.
Neutral cholesteryl ester hydrolase activity (EC 3.1.1.13) present in microsomes isolated from lactating rat mammary glands
was found to be inhibited by a factor (or factors) occurring in the cytosolic fraction of male rat liver. The inhibitor was
heat-labile, non-dialyzable, destroyed by proteolysis, and was stable following preparation of an acetone/diethyl ether powder
of the cytosolic fraction. The protein also inhibited the activity of hormone-sensitive lipase (HSL) (from bovine adipose
tissue) and esterase fromCandida cylindracea, but seemed to be more active against the neutral hydrolase found in rat liver microsomes. For the mammary gland microsomal
cholesteryl ester hydrolase, the extent of the inhibitory effect was dependent on the concentration of the cytosolic protein,
50% inhibition being achieved by about 100 μg of cytosolic protein, and on the method of initiating the enzyme assay. Kinetic
analysis indicated that, under circumstances where the reaction was initiated by the addition of substrate, the inhibition
was characterized as “uncompetitive”. When an inhibitor/substrate complex was allowed to form in the absence of enzyme, an
element of “competitive” inhibition was introduced into the reaction. Food withdrawal reducted the activity of the inhibitor
in live by 56%, but activity was fully restored by short-term re-feeding. In contrast, feeding a diet high in fat led to a
34% increase in activity. The present findings suggest that the inhibitory factor(s) may be involved in the regulation of
the hydrolysis of cholesteryl esters in the liver and also in other cell types. 相似文献
3.
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. 相似文献
4.
A cytosolic protein, that is inhibitory to neutral cholesteryl ester hydrolase, has been investigated in the livers of female
rats using microsomes isolated from the mammary gland of lactating rats as an enzyme source. To facilitate comparisons, inhibitory
activity is expressed in terms of the amount (μg) of cytosolic protein required to reduce esterase activity by 50% and is
compared to the hepatic content of both cholesterol and cholesteryl esters. The experiments revealed a sexual difference in
the level of inhibitory activity, with the livers of both suckling and mature male animals containing less of the material
than the corresponding females. Alterations in the physiological status of the females, such as pregnancy and lactation, led
to a decrease in the activity of the protein. This was reversed by blocking lactation with a combination of an antiserum to
rat growth hormone and the anti-prolactin drug, bromcoriptine, but not by premature weaning of the animals. Food withdrawal
for 24 hr also had the effect of increasing inhibitory activity. In general the cholesteryl ester content of the livers correlated
with the level of inhibitory activity. Thus the activity of the cytosolic inhibitor of neutral cholesteryl ester hydrolase
responded to changes in both the hormonal and the nutritional status of the female animal. It is suggested that the presence
of the greater cholesteryl ester hydrolase inhibitory activity in the female liver may help to explain the lower risk of coronary
heart disease in premenopausal females by facilitating increased hepatic storage of the sterol in the form of the ester. 相似文献
5.
Cholesterol esterification by acyl-CoA:cholesterol acyltransferase (ACAT; EC 2.3.1.26) has been studied in microsomes isolated
from the mammary glands of rats in late pregnancy, in early and mid-lactation, and following premature weaning. The mammary
glands were freeze-clamped and the microsomes prepared in the presence of phosphatase inhibitors to preserve the phosphorylation
status of the enzyme. Optimal conditions were established for the assay of enzyme activity in the presence of endogenous cholesterol.
Supplementation of the microsomes with exogenous cholesterol as a dispersion in Triton WR-1339 was shown to lead to an increase
in enzyme activity. Incubation of microsomes with MgATP led to an increase in ACAT activity which could be reversed by treatment
of the microsomes with a phosphoprotein phosphatase preparation from rat liver. The results suggested that ACAT activity in
the mammary gland was activated by phosphorylation in a similar way to that observed for the hepatic enzyme. The mammary glands
from pregnant animals contained a higher level of ACAT activity than did the glands of the lactating animals and this correlated
with the higher cholesteryl ester content of the pregnant glands. The highest level of ACAT activity was found in the weaned
animals but the cholesteryl ester content of the microsomes was lower than expected. The influence of progesterone levels
and changes in feeding patterns during gestation were considered as factors in these variations in ACAT activity. 相似文献
6.
Short term regulation of hepatic cholesterol ester hydrolase by reversible phosphorylation is described. Two different kinase
systems seem to be involved in this regulation. The addition of ATP, cyclic AMP and Mg2+ to rat liver 104,000× g supernatant (S104) produced a 100–140% increase in cholesterol ester hydrolase activity. This stimulation
was abolished when protein kinase inhibitor was added prior to the addition of ATP, cyclic AMP and Mg2+. Cholesterol ester hydrolase activity was also stimulated when calcium ions, phosphatidylserine, and diolein were added to
S104 along with ATP and Mg2+. Diolein in this reaction could be substituted by phorbol 12-myristate 13-acetate. Preincubation of S104 with alkaline phosphatase
resulted in a deactivation of cholesterol ester hydrolase. The addition of increasing concentrations of Mg2+ to S104 produced increasing inhibition of cholesterol ester hydrolase activity, and this effect was blocked by NaF.
It is suggested that rat liver cholesterol ester hydrolase is activated by cyclic AMP dependent protein kinase and protein
kinase C. Deactivation is accomplished by dephosphorylation catalyzed by a phosphoprotein phosphatase, dependent on Mg2+.
This work was presented at the Twenty-Third Southeastern Regional Lipid Conference, held October 26–28, in Cashiers, North
Carolina. 相似文献
7.
The effects of 5 μg/ml of 25-hydroxycholesterol; cholestane-3β, 5α,6β-triol; and cholesterol on acyl CoA cholesterol acyltransferase,
acid cholesteryl ester hydrolase and neutral cholesteryl ester hydrolase was studied in cultured rabbit aortic smooth muscle
cells. After 1 hour incubation, 25-hydroxycholesterol resulted in a fourfold stimulation of acyl CoA cholesterol acyltrans-ferase
activity. No stimulation by 25-hydroxycholesterol was noted before 15 minutes or after 5 hours of incubation. Neither cholestane-3β,5α,6β-triol
nor cholesterol influenced acyl CoA cholesterol acyltransferase activity at any time interval. No significant effects of any
of the sterols were noted on acid cholesteryl ester hydrolase or neutral cholesteryl ester hydrolase activity. The imbalance
between acyl CoA cholesterol acyl trans-ferase and hydrolase activities induced by 25-hydroxycholesterol could result in cholesteryl
ester accumulation by arterial smooth muscle cells, which may be associated with atherosclerosis. 相似文献
8.
Peanut alkaline lipase, (glycerol ester hydrolase EC 3.1.1.3), pH optimum 8.5, was isolated from acetone powders prepared from developing and germinated peanut seed (arachis hypogaea L. var. NC-2). Enzyme activity/seed increased in successive developmental stages. The course of the hydrolytic reaction was linear with regard to enzyme concentration and all times tested up to periods exceeding 60 min. Km for the reaction was determined to be 2.6 times 10-4M. Molecular weight of peanut lipase, as estimated by Sephadex gel filtration and sodium dodecyl sulfate gel electrophoresis, was ca. 55,000. 相似文献
9.
The uptake of lipid from the yolk by the yolk sac membrane of the chick embryo is accompanied by the rapid esterification
of a large proportion of the yolk cholesterol. This could arise from enhanced acyl-CoA:cholesterol acyltransferase (ACAT)
activity and/or inhibition of cholesteryl ester hydrolase (CEH) activity. The activity of ACAT was therefore measured in microsomes
obtained from yolk sac membranes at various stages of development. A high level of activity (up to 929 pmol of cholesteryl
oleate formed per min per mg protein) was found during the second half of this period. Supplementation with exogenous cholesterol
stimulated ACAT activity in microsomes obtained from the tissue at the earlier, but not at the later, stages of development
suggesting that the enzyme became saturated with microsomal cholesterol as development proceeded. Correlating with this, the
concentration of cholesterol in the microsomes increased 4-fold between 9 and 20 d of development. The activity of CEH was
very low in the microsomes and could not be detected in the cytosolic fraction. The activity of a protein, which has been
shown to function as an inhibitor of CEH, was found to be present at all stages of development. The high activity of ACAT,
together with the low activity of CEH and an active CEH inhibitor protein is a combination well suited to promote an essentially
unidirectional conversion of cholesterol to cholesteryl ester. This process may be a major determinant of the rate of lipid
transfer from the yolk to the embryo. 相似文献
10.
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. 相似文献
11.
Fluorescent esters containing pyrenedecanoic acid (P10) or pyrenebutanoic (P4) acid (P4cholesterol, P10cholesterol, P4- and P10-containing triacylglycerols) were synthesized and used as substrates for human pancreatic carboxylic ester hydrolase and
bile salt-stimulated lipase from human milk. Both enzymes were purified by immunoaffinity chromatography. All fluorescent
pyrene derivatives were hydrolyzed by pancreatic carboxylic ester hydrolase and bile salt-stimulated lipase, but at different
rates. The hydrolytic rates of the “short” acyl esters (P4-containing esters) were higher than those of the “long” ones (P10-containing esters). Conditions were optimized for sensitivity of the assay using fluorescent cholesteryl esters. The pH optimum
was 7.5–8.0. Sodium cholate exhibited a stronger activating effect than taurocholate or taurodeoxycholate (maximal activation
was achieved with 5 mmol/L cholate and with a molar ratio cholesteryl ester/cholate around 1∶10). Both pancreatic carboxylic
ester hydrolase and bile salt-stimulated lipase from milk were strongly inhibited by the other amphiphiles tested, namely
phosphatidylcholine and Triton X-100, and were inactivated by low concentrations (10 μmol/L) of the serine-reactive diethyl-paranitrophenyl
phosphate (E600). Both enzymes were strongly inhibited by relatively low concentrations of plasma low density lipoproteins. These studies
indicate that the fluorescent esters containing pyrene fatty acids can be used as substrates for assaying and investigating
the properties of pancreatic carboxylic ester hydrolase as well as bile salt-stimulated lipase from milk. 相似文献
12.
Intracellular distribution of lipolytic activity in the female gametophyte of germinating Douglas fir seeds 总被引:3,自引:0,他引:3
Acid (pH 5.2) and neutral (pH 7.1) lipase activity was studied in order to localize the sites of lipolysis in cellular fractions
of catabolic organ of Douglas fir seed. Cellular particles were separated by differential centrifugation of the tissue homogenate
and identified by electron microscopy. Emulsified native neutral lipids were provided as a substrate to protein body, mitochondrial,
microsomal and soluble fractions, and endogenous lipids were used as a substrate for light and heavy fat body fractions. Little
difference was observed in average specific activity of the two enzyme systems in dry seeds, but acid lipase activity increased
sevenfold and neutral lipase activity fourfold during germination. Highest specific activity of both enzyme systems was found
to be associated with the heavy fat bodies and the soluble fraction. Heavy fat bodies contained an ample quantity of endogenous
substrate while the soluble fraction consisted of little substrate. Experimental data indicated that the soluble fraction
was the source of lipases, and the heavy fat bodies were the site of in situ lipolysis. 相似文献
13.
Fibric acid derivatives are used to treat hyperlipidemia and have wide ranging effects on lipid metabolism. The action of
these compounds on cholesterol esterification, catalyzed by acyl-coenzyme A:cholesterol acyltransferase (ACAT), has been quite
widely studied, but their effect on cholesteryl ester hydrolysis and the enzyme neutral cholesteryl ester hydrolase (nCEH)
has been largely ignored. Male rats were therefore fed for 10 d on a standard chow diet supplemented with either clofibrate
or bezafibrate, to study their effects on plasma lipid levels and hepatic cholesterol metabolism. Plasma triacyglycerols were
not significantly altered by these diets, but bezafibrate significantly lowered plasma cholesterol levels (29.7%,P<0.01). When expressed per unit weight of DNA, both fibrates reduced the hepatic content of triacylglycerol, cholesterol and
cholesteryl esters (40, 18.7, 16.5 and 66.7, 28.6, 34.2% for clofibrate and bezafibrate, respectively). ACAT activity was
significantly reduced by both drugs, but clofibrate (65% inhibition) was more effective than bezafibrate (35% inhibition).
The most dramatic effect of the diets was a marked increase in the activity of both the microsomal and the cytosolic nCEH.
When expressed on a whole liver basis, the effect of bezafibrate on the cytosolic enzyme (13.6-fold increase in activity)
was much greater than that of clofibrate (4.8-fold increase). Increases in the activity of a cytosolic protein that inhibits
the activity of nCEH were also noted, but these changes were relatively small. The results suggest that the activation of
nCEH, in combination with the inhibition in ACAT activity, contributes to a decrease in the cholesteryl ester content of the
liver which may influence the secretion of very low density lipoprotein. 相似文献
14.
Hans Stam Sietske Broekhoven-Schokker Kees Schoonderwoerd Willem C. Hülsmann 《Lipids》1987,22(2):108-115
Cholesteryl esterase activities were determined in homogenates of rat heart (ventricles), isolated, calcium-tolerant, cardiac
myocytes and aortic tissue and were compared with acid and neutral triglyceride lipase activities in these fractions.
Using cholesteryl oleate/phosphatidylcholine/taurocholate emulsions and digitonin pretreatment of the enzyme fractions, acid
and neutral cholesteryl esterase activities were measured in all tissue preparations. In contrast to the acid and neutral
triglyceridase and acid cholesteryl esterase activity, the neutral cholesteryl esterase activity was subject to substrate
inhibition. Upon isolation of cardiac myocytes, and in contrast with the recovery of neutral triglyceride lipase activity,
only a small portion of the neutral cholesteryl esterase (6%) was recovered, suggesting that nonmyocyte neutral cholesteryl
esterase activity markedly contributes to the relatively high activity detectable in whole ventricular homogenates. The recovery
of large amounts of neutral cholesteryl esterase activity in the supernatant of collagenase-digested heart tissue, obtained
during the isolation of myocytes, which is also markedly enriched in activities of two endothelial marker enzymes (5′-nucleotidase
and angiotesine-converting enzyme) may indicate the predominant contribution of neutral cholesteryl esterase activity from
coronary endothelial cells to this activity detectable in ventricular homogenates. Relative to the activity in ventricular
and myocyte homogenates, aorta homogenates possessed the highest specific neutral cholesteryl esterase activity. We propose
that in addition to coronary endothelium, smooth muscle cells also contribute to the neutral cholesteryl esterase activity
in ventricular homogenates. Pretreatment of rats with carrageenan an agent toxic to macrophages, lymphocytes and fibroblasts,
induced a significant drop in myocardial neutral cholesteryl esterase and triglyceride lipase activity, suggesting that interstitially
trapped macrophages may also contribute to lipolytic activities present in whole ventricular homogenates. Our data indicate
that caution has to be taken upon extrapolation of experimental findings in heart homogenates to myocardial muscle cells. 相似文献
15.
The direct activation of sterol ester hydrolase (E.C. 3.1.1.13) in homogenates of bovine corpus luteum by N6O2′·dibutyryl cyclic adenosine 3′∶5′-phosphate, (dibutyryl cAMP), adenosine triphosphate (ATP), and Mg2+ has been demonstrated. Variability in the extent of activation by the additions was minimized by homogenization of the tissue
in 5 mM Mg2+. Baseline sterol ester hydrolase activity was primarily associated with the 105,000 × g soluble fraction, and significant
activation of the enzyme preparation preincubated with dibutyryl cAMP, ATP and Mg2+ occurred within the first 15 min, prior to addition of substrate. A requirement for protein kinase in the system was demonstrated
by blocking the cofactor-dependent enzyme activation with commercial protein kinase inhibitor. 相似文献
16.
The fatty acid composition and metabolic activity of cholesterol esters in milk and mammary tissue (cow, sow and goat) were
investigated. Cholesterol esters of freshly secreted milks incubated at 40 C for 2 hr showed no change in fatty acid composition
and no incorporation of 1-14C-palmitate. The fatty acid composition of cholesterol esters in the milk of all three species exhibited elevated levels of
a unique group of fatty acids when compared to other milk ester lipid classes. This group was comprised of monounsaturated
acids and acids with odd numbers of carbons. Tissue cholesterol esters contained lower levels of these acids. Evidence from
experiments in which an odd carbon acid (C15) was infused into the lactating mammary gland indicated that the group of unique acids is preferentially retained in the
cholesterol ester fraction which is secreted with milk. These infusion experiments also provided evidence that cholesterol
esters accumulate in developing milk fat globules in a manner paralleling triglyceride accumulation, and that acyl moieties
of cholesterol esters may be desaturated in the form of the intact ester. Our results are compatible with the hypothesis that
acyl moieties of cholesterol esters in lactating mammary tissue are turning over rapidly.
Paper No. 3496 in the journal series of the Pennsylvania Agricultural Experiment Station. 相似文献
17.
An extracellular lipase (EC 3.1.1.3) from Geotrichum marinum was purified 76-fold with 46% recovery using Octyl Sepharose 4 Fast Flow and Bio-Gel A 1.5 m chromatography. The purified enzyme showed a prominent band on SDS-PAGE and a single band on native PAGE based on the activity staining. The molecular mass of the lipase was estimated to be 62 kDa using SDS-PAGE and Bio-Gel A chromatography, indicating that the lipase likely functions as a monomer. The pl of the lipase was determined to be 4.54. The apparent V(max) and Km were 1000 micromol/min/mg protein and 11.5 mM, respectively, using olive oil emulsified with taurocholic acid as substrate. The lipase demonstrated a pH optimum at pH 8.0 and a temperature optimum at 40 degrees C. At 6 mM, Na+, K+, Ca2+, and Mg2+ stimulated activity, but Na+ and K+ at 500 mM and Fe2+ and Mn2+ at 6 mM reduced lipase activity. The anionic surfactant, taurocholic acid, and the zwitterionic surfactant, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, enhanced the activity at 0.1 mM. Other anionic surfactants such as SDS and sodium dioctyl sulfosuccinate, the cationic surfactants methylbenzethonium bromide and cetyltriethylammonium bromide, and the nonionic surfactants Tween-20 and Triton X-100 inhibited the lipase activity to different extents. The lipase was found to have a preference for TG containing cis double bonds in their FA side chains, and the reaction rate increased with an increasing number of double bonds in the side chain. The lipase had a preference for ester bonds at the sn-1 and sn-3 positions over the ester bond at the sn-2 position. 相似文献
18.
Short-term activation of microsomal cholesterol ester hydrolase by glucagon, cAMP analogues, and vasopressin in isolated rat
hepatocytes is described. Glucagon led to a dose-and time-dependent activation of cholesteryl oleate hydrolysis, but values
returned to basal levels within 120 min. Exposure of isolated hepatocytes to 0.5 mM concentrations of dibutyryl-cAMP or 8-[4-chlorophenylthio]-cAMP,
or 25 μM forskolin caused persistent activation of cholesterol ester hydrolase activity after a lag period of 30 min. The
three agents resulted in early marked intracellular accumulation of cAMP that declined progressively, and moderate and sustained
reductions in the diacylglycerol content. The actions of glucagon on hepatocytes were inhibited by pretreatment of cells with
10 nM [8-arginine] vasopressin. Vasopressin elicited a consistent and sustained increase in cholesterol ester hydrolase activity
and diacylglycerol without affecting cAMP while reducing the effect of glucagon on cAMP. Furthermore, the effects of glucagon
and vasopressin on the activation of cholesterol ester hydrolase were not additive despite the similarity of their stimulation
of diacylglycerol formation. Blockade of vasopressin-mediated activation of cholesterol ester hydrolase and diacylglycerol
content were induced by excess prazosin. These data suggest that stimulation of microsomal cholesterol ester hydrolase in
isolated liver cells may involve at least two signal transduction systems. 相似文献
19.
The microsomal bound phosphatidic acid phosphohydrolase from lactating rat mammary tissue had a specific activity of six nmoles
per mg protein per minute. The optimum pH was 7.0; magnesium at 1.3 mM was required for maximum activity, and at low substrate
concentrations magnesium lowered the Km of the enzyme for phosphatidic acid. Diglycerides exerted little effect while diglyceride
ether stimulated enzyme activity. Inorganic salts, i.e., potassium phosphate and potassium chloride, enhanced rates of phosphatidic
acid hydrolysis under standard assay conditions. 相似文献
20.
Hepatic neutral cytosolic cholesteryl ester hydrolase (hncCEH) is a key enzyme in the regulation of hepatic free cholesterol
(FC). In examining the effects of over-expression of this enzyme on cholesterol homeostasis, mice were infected with a recombinant
adenovirus construct (AdCEH) of the rat hncCEH cDNA driven by the human cytomegalovirus promoter. Cholesteryl esterase and
p-nitrophenylcaprylate (PNPC) esterase activities were measured in liver postmitochondrial supernatants at 1, 3, 7, and 11
d after infection with AdCEH or a control virus expressing β-galactosidase (AdβGAL). The PNPC esterase activity of AdCEH mice
peaked threefold higher than controls on day 2, declining on subsequent days. In contrast, cholesteryl esterase peaked eightfold
higher than controls on day 3, indicating a shift in substrate selectivity of hncCEH. Hepatic FC peaked at 144% of controls,
7 d postinfection. The mRNAs for cholesterol 7α-hydroxylase, sterol 27-hydroxylase, and HMG-CoA reductase decreased to 47,
46, and 58% of controls, respectively, on day 7, coinciding with peak FC concentrations. Coiniding with increased cholesteryl
esterase activity, hepatic esterified cholesterol dropped precipitously from day 3 onward, to 11% of controls by day 11. Hepatic
TAG levels also declined, consistent with the reported TAG lipase activity of hncCEH. These results demonstrate elevation
of FC and depletion of cholesteryl esters by over-expression of hncCEH, which were resistant to compensatory responses by
other enzymes of cholesterol homeostasis. 相似文献