Effect of atherosclerosis on lysosomal cholesterol esterase activity in rabbit aorta.

Radiolabeled cholesteryl oleate, when incorporated into phospholipid vesicles, was hydrolyzed at acid pH by an enzyme present in rabbit aortic homogenates. In contrast, cholesteryl oleate presented as an acetone dispersion was not effectively hydrolyzed at acid pH under identical conditions. Using the vesicle preparation as substrate, a sensitive assay system for the acid hydrolase was developed in which hydrolysis was proportional to protein concentration and incubation time, and was independent of substrate concentration. The physical state of the vesicles was apparently not altered by the assay conditions, and no hydrolysis of the vesicle-associated phospholipid was detected. Acid cholesterol esterase activity in atherosclerotic aortic tissue was 2.5-fold greater than that of control tissue, and even greater increases were observed in the activities of other lysosomal enzymes (N-acetyl-beta-d-glucosaminidase and beta-glucuronidase). Glucose-6-phosphatase activity was also increased in aortas from cholesterol-fed animals while 5' nucleotidase activity remained unchanged. Labeled triolein also was incorporated into phospholipid vesicles and was hydrolyzed by an acid lipase in aortic tissue. Similarities between triolein and cholesteryl oleate hydrolysis existed with respect to pH optimum and the effect of cholesterol feeding on activity, suggesting that a single enzyme may hydrolyze both lipids.     

Cholesteryl ester hydrolysis in rat liver lysosomes: different response to female sex hormones

The regulation of the hydrolysis of cholesteryl oleate by female sex hormones was studied in the lysosomal fraction of rat liver. Cholesterol ester hydrolase activity was determined at pH 5.0 with an acetone-dissolved cholesteryl [1-14C]oleate substrate preparation. The administration of a single dose of progesterone decreased the enzyme activity during a 3- to 24-hr period following hormone injection. This effect was not correlated to changes in the lysosomal protein synthesis rate. The lysosomal hydrolysis of cholesteryl esters was also inhibited in a noncompetitive manner by the addition of progesterone at concentrations higher than 100 microM. The esterase failed to respond to the estradiol in vivo as well as in vitro. The findings of the present paper suggest that the lysosomal breakdown of cholesteryl esters in rat liver may be under selective hormonal regulation and that the inhibitory effect of progesterone on the enzyme activity might be, at least in part, responsible for the liver cholesterol ester accumulus produced by the administration of the hormone.     

Characteristics of acid lipase and acid cholesteryl esterase activity in parenchymal and non-parenchymal rat liver cells

(1) Parenchymal and non-parenchymal cells were isolated from rat liver. The characteristics of acid lipase activity with 4-methylumbelliferyl oleate as substrate and acid cholesteryl esterase activity with cholesteryl[1-14C]oleate as substrate were investigated. The substrates were incorporated in egg yolk lecithin vesicles and assays for total cell homogenates were developed, which were linear with the amount of protein and time. With 4-methylumbelliferyl oleate as substrate, both parenchymal and non-parechymal cells show maximal activities at acid pH and the maximal activity for non-parenchymal cells is 2.5 times higher than for parenchymal cells. It is concluded that 4-methylumbelliferyl oleate hydrolysis is catalyzed by similar enzyme(s) in both cell types. (2) With cholesteryl[1-14C]oleate as substrate both parenchymal and non-parenchymal cells show maximal activities at acid pH and the maximal activity for non-parenchymal cells is 11.4 times higher than for parenchymal cells. It is further shown that the cholesteryl ester hydrolysis in both cell types show different properties. (3) The high activity and high affinity of acid cholesteryl esterase from non-parenchymal cells for cholesterol oleate hydrolysis as compared to parenchymal cells indicate a relative specialization of non-parenchymal cells in cholesterol ester hydrolysis. It is concluded that non-parenchymal liver cells in cholesterol ester hydrolysis. It is concluded that non-parenchymal liver cells possess the enzymic equipment to hydrolyze very efficiently internalized cholesterol esters, which supports the suggestion that these cell types are an important site for lipoprotein catabolism in liver.     

Cholesteryl ester and triglyceride hydrolysis by an acid lipase from rabbit aorta.

The properties of the triglyceride- and cholesteryl ester-hydrolyzing activity by an acid lipase from rabbit aortic tissue were compared under different experimental conditions. Radiolabeled cholesteryl oleate or triolein was incorporated into phospholipid vesicles by sonication and the resulting preparations were used for in vitro studies. No distinction was observed between triglyceride lipase and cholesterol esterase activity in the aortic cytosol fraction following either thermal inactivation, inhibition by a mercurial, fractionation by ammonium sulfate or acid precipitation, or DEAE-cellulose chromatography. Addition of rabbit lipoproteins to the assay system resulted in inhibition of both cholesterol esterase and triglyceride lipase activity. Parallel changes in the hydrolysis of both substrates also were observed when exogenously added lipids were added to the incubation system in various physical states. Specificities of the enzyme system towards different cholesteryl esters were examined. No differences in the rate of hydrolysis were observed between cholesteryl oleate, palmitate and linoleate. The data suggest that a single acid lipase, presumably of lysosomal origin, has broad specificity towards triglycerides and cholesteryl esters, and may play a role in the hydrolysis of these lipids during intralysosomal degradation of lipoproteins.     

Cholesteryl ester hydrolytic acitivity of rat liver plasma membrane.

Cholesteryl ester hydrolyzing activity of rat liver plasma membranes was studied using acetone-dispersed [4-14-C] cholesteryl oleate as substrate. In contrast to whole liver homogenates which displayed ample activity at both acid (4.5) and neutral (6.2-7.4) pH, purified plasma membrane fractions contained little activity at neutral pH as compared to acid pH. Moreover, rate-zonal sucrose density-gradient centrifugation patterns of plasma membrane rich fractions suggested a specific association with plasma membrane only in the case of the acid activity. These findings suggest that in vivo hepatic cell surface membranes contain little or no cholesteryl ester hydrolytic activity at extracellular pH. They support the possibility that plasma lipoprotein cholesteryl esters enter hepatic parenchymal cells prior to hydrolysis.     

Inhibitors of sterol synthesis. 15-oxygenated steryl ester hydrolase activity of rat liver at neutral and acid pH

5 alpha-Cholest-8(14)-en-3 beta-ol-15-one (15 ketosterol) is a potent inhibitor of cholesterol biosynthesis with significant hypocholesterolemic activity. The results of a recent study (Schroepfer, G.J., Jr., Christophe, A., Chu, A.J., Izumi, A., Kisic, A. and Sherrill, B.C. (1988) Chem. Phys. Lipids 48, 29-58) have indicated that, after intragastric administration of the 15-ketosterol in triolein to rats, most of the compound in intestinal lymph occurs in the form of the oleate ester, which is associated with chylomicrons. Moreover, after intravenous administration of chylomicrons containing the oleate ester of 15-[2,4-3H]ketosterol, rapid and selective uptake of 3H by liver was observed, which was associated with the rapid and substantial appearance of labeled free 15-ketosterol in liver. The present study concerns the capabilities of rat liver fractions to catalyze the hydrolysis of 15-ketosteryl oleate. Efficient hydrolysis was observed at acid pH with a digitonin-solubilized extract of rat liver, with a rate similar to that for the hydrolysis of cholesteryl oleate. The distribution of acid 15-ketosteryl oleate hydrolase of whole liver homogenate on a metrizamide isopycnic density gradient was similar to that of acid cholesteryl oleate hydrolase and acid phosphatase, suggesting that the lysosomal acid lipase is the enzyme responsible for the hydrolysis of the 15-ketosteryl oleate at acid pH. At neutral pH, 15-ketosteryl oleate and cholesteryl oleate was hydrolyzed at similar rates by the microsomal fraction of liver homogenate, whereas the 15-ketosteryl oleate was hydrolyzed at a much lower rate than cholesteryl oleate by the cytosolic fraction. The distribution of neutral 15-ketosteryl oleate hydrolase activity of whole liver homogenate on a metrizamide isopycnic density gradient was most correlated to a microsomal esterase, whereas cholesteryl oleate hydrolase activity was most correlated to a cytosolic enzyme. Both 15-ketosteryl oleate and cholesteryl oleate hydrolase activities were correlated to a mitochondrial marker enzyme.     

Rat liver retinyl palmitate hydrolase activity. Relationship to cholesteryl oleate and triolein hydrolase activities

Studies were conducted to explore relationships in rat liver between retinyl palmitate hydrolase activity and the hydrolytic activities against cholesteryl oleate and triolein. Previous studies have shown positive correlations between these three lipid ester hydrolase activities. In order to extend this work, the hydrolase activities were further purified and characterized. The activities against cholesteryl oleate and triolein resembled retinyl palmitate hydrolase activity in showing great variability from rat to rat as assayed in vitro. The relative levels of the three activities were highly correlated with each other over a 50-fold range of activity in a series of 66 liver homogenates. Partial purification (approx. 200-fold) in the absence of detergents was achieved by sequential chromatography of an acetone powder extract of liver on columns of phenyl-Sepharose, DEAE-Sepharose and heparin-Sepharose. The three hydrolase activities copurified during each of these chromatographic steps. The properties of the three copurifying activities were similar with regard to stimulation of activity by trihydroxy bile salts, pH optimum (near 8.0), and observance of Michaelis-Menten-type saturation kinetics. The three activities were different in their sensitivity towards the serine esterase inhibitors diisopropylfluorophosphate and phenylmethanesulfonyl fluoride, and in their solubility properties in 10 mM sodium acetate, pH 5.0. Thus, triolein hydrolase activity was much less sensitive than the other two activities to the two inhibitors. In addition, the activity against cholesteryl oleate could be separated from the other two activities by extraction of an acetone powder with acetate buffer, pH 5.0. These results indicate that the three lipid hydrolase activities are due to at least three different catalytically active centers, and at least two distinct and separable enzymes. It is likely that three separate but similar enzymes, that appear to be coordinately regulated, are involved.     

Cholate effects on all-trans-retinyl palmitate hydrolysis in tissue homogenates: solubilization of multiple kidney membrane hydrolases

Retinyl ester hydrolysis was observed in the absence of cholate in homogenates of rat lung, liver, kidney, intestine, and testes. Eighty-four percent of the activity in kidney was membrane-associated. The kidney microsomal fraction contained 19% of the total activity and was the only subcellular fraction that had increased specific activity relative to the homogenate (about 1.5-fold). In contrast, the cytosol was the only fraction that was decreased in specific activity (about 3-fold). Cholate (18 mM), reportedly required to observe hydrolysis of all-trans-retinyl esters by rat liver preparations, was not obligatory for activity in kidney homogenates or microsomes. The microsomal activity was solubilized efficiently and with a twofold increase in specific activity by the synthetic detergent 1-S-octyl-beta-D-thioglucopyranoside. Gel-permeation chromatography of the solubilizate suggested that at least two pools of activity existed, with molecular weights in the ranges 70-95 and 30-40 kDa. Neither hydrolyzed cholesteryl oleate. Both were more active in hydrolyzing retinyl palmitate than trioleoylglycerol. The higher mass pool had decreased trioleoylglycerol hydrolase activity relative to the solubilizate. Anion-exchange chromatography separated the lower mass pool into two major peaks. A major peak, distinct from the two peaks observed with the lower mass pool, was observed upon anion-exchange chromatography of the higher mass pool. These data demonstrate that multiple retinyl ester hydrolases, more efficient at hydrolyzing retinyl esters than cholesteryl esters and triacylglycerol, occur in a retinoid target tissue.     

Effect of substrate properties on the activity of lysosomal cholesteryl ester hydrolase.

The effects of the substrate properties on the catalytic activity of lysosomal cholesteryl ester hydrolase from rat liver have been examined with three standard substrate types: vesicle, micelle and emulsion. The pH optimum of the enzyme coincided to 4.5--5.0 with the substrate types employed. The apparent Km values were 15.3, 14.3 and 7.3 microM for vesicle, micelle and emulsion substrates, respectively. In the systems used in this study reaction products, cholesterol and oleic acid, and the nonionic surfactant Tween 80 and Triton X-100 Had an inhibitory effect. The emulsifier phosphatidylcholine and the charged phospholipid phosphatidic acid stimulated the activity. The mixed micelle of sodium taurocholate and phosphatidylcholine was the most potent substrate vehicle. With dipalmitoyl phosphatidylcholine vesicles the enzyme showed maximal activity at the gel-liquid-crystalline transition temperature of the phospholipid. The possible physiological significance of the lysosomal cholesteryl ester hydrolase is discussed with special reference to the form of the substrate.     

Purification and properties of a cholesteryl ester hydrolase from rat liver microsomes

This report describes a purification procedure for a cholesteryl ester hydrolase (CEH) from female rat liver microsomes, and some structural, immunological, kinetic, and regulatory properties of the enzyme that distinguish the microsomal CEH from other hepatic cholesteryl ester-splitting enzymes. CEH was purified 12.4-fold from reisolated microsomes using sequential solubilization by sonication, polyethylene glycol precipitation, fractionation with hydroxyapatite, anion exchange chromatography, and chromatography on hydroxyapatite, with an overall yield of 3.2%. CEH activity was purified 141-fold over nonspecific esterase activity and 56-fold over triacylglycerol lipase activity. In sharp contrast with most esterases and lipases, CEH did not bind to concanavalin A-Sepharose and heparin-Sepharose. After polyacrylamide gel electrophoresis, the purified enzyme exhibited two silver-stained bands, but only the protein electroeluted from the low mobility band had CEH activity. Affinity-purified polyclonal antibodies raised to electroeluted CEH inhibited 90% of the activity of liver microsomal CEH and reacted with a 106 kDa protein band on Western blot analysis. This 106 kDa CEH contains a unique N-terminal amino acid sequence. The purified enzyme had optimal activity at pH 6 and no taurocholate requirements, and was inhibited by the serine active site inhibitor phenylmethylsulfonyl fluoride and by free sulfhydryl specific reagents. It hydrolyzed cholesteryl oleate much more efficiently than trioleine, and hydrolytic activity with p-nitrophenyl acetate was higher than with p-nitrophenyl butyrate. These results indicate that rat liver microsomes contain a bile salt-independent catalytic protein that is relatively specific for cholesteryl ester hydrolysis.     

Lecithin-cholesterol acyltransferase (LCAT) catalyzes transacylation of intact cholesteryl esters. Evidence for the partial reversal of the forward LCAT reaction

Lecithin-cholesterol acyltransferase (LCAT) catalyzes the intravascular synthesis of lipoprotein cholesteryl esters by converting cholesterol and lecithin to cholesteryl ester and lysolecithin. LCAT is unique in that it catalyzes sequential reactions within a single polypeptide sequence, a phospholipase A2 reaction followed by a transacylation reaction. In this report we find that LCAT mediates a partial reverse reaction, the transacylation of lipoprotein cholesteryl oleate, in whole plasma and in a purified, reconstituted system. As a result of the reverse transacylation reaction, a linear accumulation of [3H]cholesterol occurred during incubations of plasma containing high density lipoprotein labeled with [3H]cholesteryl oleate. When high density lipoprotein labeled with cholesteryl [14C]oleate was also included in the incubation the labeled fatty acyl moiety remained in the cholesteryl [14C]oleate pool showing that the formation of labeled cholesterol did not result from hydrolysis of the doubly labeled cholesteryl esters. The rate of release of [3H]cholesterol was only about 10% of the forward rate of esterification of cholesterol using partially purified human LCAT and was approximately 7% in whole monkey plasma. Therefore, net production of cholesterol via the reverse LCAT reaction would not occur. [3H]Cholesterol production from [3H]cholesteryl oleate was almost completely inhibited by a final concentration of 1.4 mM 5,5'-dithiobis(nitrobenzoic acid) during incubation with either purified LCAT or whole plasma. Addition of excess lysolecithin to the incubation system did not result in the formation of [14C]oleate-labeled lecithin, showing that the reverse reaction found here for LCAT was limited to the last step of the reaction. To explain these results we hypothesize that LCAT forms a [14C]oleate enzyme thioester intermediate after its attack on the cholesteryl oleate molecule. Formation of this intermediate allows [3H]cholesterol to be liberated from the enzyme by exchange with unlabeled cholesterol of plasma lipoproteins. The liberated [3H]cholesterol thereby becomes available for reesterification by LCAT as indicated by its appearance as newly synthesized cholesteryl linoleate.     

Fatty acid specificity of the lysosomal acid cholesterol esterase in intact human arterial smooth muscle cells

The fatty-acid specificity of the lysosomal cholesterol esterase was examined in cultured human arterial smooth muscle cells. The lysosomal compartment of cultured cells was enriched with cholesteryl esters by incubation of cells with 0.2 mg/ml low-density lipoprotein and 50 microM chloroquine for 24 h. The hydrolysis of cholesteryl esters was subsequently induced by incubating cells in a medium containing 5% lipoprotein-deficient serum without chloroquine. Cellular cholesteryl ester mass was markedly reduced after 23 h in the lipoprotein-deficient serum. Fatty-acid analysis of cholesteryl esters in cells before and after the 23 h incubation with lipoprotein-deficient serum revealed that polyunsaturated cholesteryl esters (linoleate and arachidonate) were preferentially hydrolyzed compared to cholesteryl oleate or saturated cholesteryl esters. An increase in the ratio of cholesteryl oleate to cholesteryl linoleate was observed even when the cellular activity of acyl-CoA:cholesterol acyltransferase was inhibited with Sandoz Compound 58-035. We conclude that, in human arterial smooth muscle cells, the lysosomal acid cholesterol esterase preferentially hydrolyzes polyunsaturated cholesteryl esters.     

Reversible accumulation of cholesteryl esters in macrophages incubated with acetylated lipoproteins

Mouse peritoneal macrophages accumulate large amounts of cholesteryl ester when incubated with human low-density lipoprotein that has been modified by chemical acetylation (acetyl-LDL). This accumulation is related to a high-affinity cell surface binding site that mediates the uptake of acetyl-LDL by adsorptive endocytosis and its delivery to lysosomes. The current studies demonstrate that the cholesteryl ester accumulation can be considered in terms of a two-compartment model: (a) the incoming cholesteryl esters of acetyl-LDL are hydrolyzed in lysosomes, and (b) the resultant free cholesterol is re-esterified in the cytosol where the newly formed esters are stored as lipid droplets. The following biochemical and morphologic evidence supports the hydrolysis-re-esterification mechanism: (a) Incubation of macrophages with acetyl-LDL markedly increased the rate of cholesteryl ester synthesis from [14C]oleate, and this was accompanied by an increase in the acyl-CoA:cholesteryl acyltransferase activity of cell-free extracts. (b) When macrophages were incubated with reconstituted acetyl-LDL in which the endogenous cholesterol was replaced with [3H]-cholesteryl linoleate, the [3H]cholesteryl linoleate was hydrolyzed, and at least one-half of the resultant [3H]cholesterol was re-esterified to form [3H]cholesteryl oleate, which accumulated within the cell. The lysosomal enzyme inhibitor chloroquine inhibited the hydrolysis of the [3H]cholesteryl linoleate, thus preventing the formation of [3H]cholesteryl oleate and leading to the accumulation of unhydrolyzed [3H]cholesteryl linoleate within the cells. (c) In the electron microscope, macrophages incubated with acetyl-LDL had numerous cytoplasmic lipid droplets that were not surrounded by a limiting membrane. The time course of droplet accumulation was similar to the time course of cholesteryl ester accumulation as measured biochemically. (d) When acetyl-LDL was removed from the incubation medium, biochemical and morphological studies showed that cytoplasmic cholesteryl esters were rapidly hydrolyzed and that the resultant free cholesterol was excreted from the cell.     

A method for direct incorporation of radiolabeled cholesteryl ester into human plasma low-density-lipoproteins: preparation of tracer substrate for cholesteryl ester transfer reaction between lipoproteins

[14C]Cholesteryl ester was directly incorporated into human plasma low-density lipoproteins (LDL) for the purpose of preparing a tracer substrate for investigation of the cholesteryl ester transfer reaction between plasma lipoproteins. The radiolabeled cholesteryl oleate was sonicated with egg phosphatidylcholine to form cholesteryl ester-containing liposomes. The liposomes were incubated with plasma fraction of density greater than 1.006 at 37 degrees C in the presence of dithionitrobenzoic acid. When the distribution of the radiolabeled cholesteryl ester was equilibrated among liposomes and lipoprotein fractions, the mixture was applied to an affinity chromatography column of dextran sulfate-cellulose (LA01) (Arteriosclerosis 4, 276-282). LDL was eluted by increasing the NaCl concentration and was finally isolated as a floating fraction by ultracentrifugation at a solvent density of 1.063 (adjusted with NaCl). The chemical composition, electrophoretic mobility and density of the labeled LDL were consistent with those of the native LDL. Radioactivity in this preparation was present exclusively in cholesteryl ester. Apolipoprotein B100 was preserved intact throughout the procedure. When the rate of cholesteryl ester transfer was measured between LDL and high-density lipoproteins by using this labeled LDL, the kinetics was consistent with the equilibrium transfer model, but the apparent rate measured was slightly higher than that measured with the labeled LDL prepared by the method using the intrinsic cholesterol esterification reaction of plasma.     

Characterization of multiple forms of cholesteryl ester hydrolase in the rat testis

Cholesterol ester hydrolase (EC 3.1.1.13) activity from the 104,000 X g supernatant of rat testis was fractionated into 28-kDa, 72-kDa, and 420-kDa molecular mass forms by high performance size exclusion chromatography. The 72-kDa and 420-kDa forms (temperature-labile) were completely inactivated by elevation of temperature from 32 to 37 degrees C. Apparent disaggregation of the 420-kDa form suggested that the 72-kDa and 420-kDa enzymes are monomeric and multimeric forms of the same enzyme. The 28-kDa form was shown to be a different enzyme (temperature-stable) which retained activity at 37 degrees C. In contrast, cholesteryl ester hydrolase activities from 104,000 X g supernatants of liver or adrenal gland were unaffected and increased 4-fold, respectively, by elevation of temperature from 32 to 37 degrees C. Both testicular enzymes exhibited pH optima at about 7.3, and were activated by sodium cholate at concentrations near the critical micellar concentration (0.03-0.07%), but inhibited by higher concentrations. The temperature-labile cholesteryl ester hydrolase exhibited a high specificity for cholesteryl esters of monoenoic fatty acids of 18-24 carbons, especially nervonate (24:1), whereas the temperature-stable cholesteryl ester hydrolase exhibited highest specificity for cholesteryl oleate and arachidonate. Neither enzyme hydrolyzed cholesteryl acetate, myristate, palmitate, linoleate, or docosahexaenoate . Both enzymes reached maximum rates of hydrolysis at 150 microM substrates, with each substrate and at both reaction temperatures. Substrate inhibition was observed at higher concentrations (200 microM). The temperature-labile cholesteryl ester hydrolase was induced 20-fold in hypophysectomized rats by injection of follicle-stimulating hormone (FSH) and was localized in Sertoli cells, the target cells for FSH, but was not induced by luteinizing hormone. The temperature-stable cholesteryl ester hydrolase was induced by both FSH and LH and was found in both Sertoli cells and Leydig cells, the respective target cells for FSH and luteinizing hormone. Neither form of the enzyme was present at detectable levels in the germinal cells. The unique properties, localization, and hormonal regulation of both temperature-labile and temperature-stable cholesterol ester hydrolases suggest important roles for these enzymes in the testis.     

Localization and some properties of lysosomal dipeptidases in rat liver.

1. The rates of hydrolysis of 26 synthetic dipeptides by extracts from highly purified lysosomal fractions from rat liver at pH 5.0 and by whole liver homogenates at pH 7.4 have been determined. Extracts from the lysosomal fractions hydrolysed most peptides at a lower rate per mg protein than the homogenates, and some peptides not at all. 2. Properties of two dipeptidases present in the extracts from the lysosomal fractions, splitting Ile-Glu and Leu-Gly, respectively, were studied in greater detail. The enzyme that hydrolysed Ile-Glu was strongly activated by dithiothreitol, showed optimal activity at pH 4.5 and had a molecular weight of about 120 000. Leu-Gly dipeptidase did apparently not contain an essential thiol group and had a molecular weight of approx. 90 000. It showed maximal activity at pH 6.5. 3. After differential centrifugation of liver homogenates, Ile-Glu and Leu-Gly-splitting activities were determined in the fractions, under the optimal conditions mentioned above. The Ile-Glu-hydrolysing enzyme activity showed about the same distribution as the lysosomal marker enzyme acid phosphatase. Leu-Gly-splitting activity, however, was largely present in the cytosol fraction, with only a small peak in the lysosomal fraction. We obtained evidence that the activities present in the lysosomal fraction and in the cytosol fraction were due to different enzymes, and that one of these enzymes was localized exclusively in lysosomes. 4. It is concluded that some dipeptides originating from intralysosomal proteolysis might be split by lysosomal dipeptidases, whereas others are probably hydrolysed only in the extra-lysosomal compartment of the cell.     

Neutral cholesteryl ester hydrolase in the rat lactating mammary gland: regulation by phosphorylation-dephosphorylation

The characteristics of neutral cholesteryl ester hydrolase activities found in the microsomal and cytosolic subcellular fractions of rat lactating mammary tissue were investigated. The enzymes were assayed using cholesteryl oleate dispersed as a mixed micelle with phosphatidylcholine and sodium taurocholate (molar ratio 1:4:2) as substrate. This method gave activities approx. 20-fold higher than those seen when cholesteryl oleate was added in ethanol. Addition of phosphatidylcholine and sodium taurocholate to the assays using the ethanol-dissolved substrate did not increase the activities observed. When the cholesteryl oleate was dispersed with phosphatidylcholine only (molar ratio, 1:4) the activity of the two neutral cholesteryl ester hydrolases was also decreased considerably compared to that found with mixed micelles. In this case, however, approx. 60% of the cytosolic, but only 10% of the microsomal activity, was restored by separate addition of sodium taurocholate. The activities of both the microsomal and the cytosolic neutral cholesteryl ester hydrolases were inhibited by MgCl2, and this inhibition was almost completely reversed by the addition of an equimolar concentration of ATP. At a fixed concentration of MgCl2 increasing concentrations of ATP increased the enzyme activities in a dose-dependent way. The activity of the microsomal, but not the cytosolic enzyme was enhanced by a cyclic AMP-dependent protein kinase and both activities were inhibited by alkaline phosphatase (bovine milk). These results provide evidence for the regulation of neutral cholesteryl ester hydrolases in the rat lactating mammary gland by mechanisms involving phosphorylation-dephosphorylation and therefore suggest that these enzymes may be under hormonal control.     

Acid lipase in rat intestinal mucosa: physiological parameters

Previous studies have shown that up to a half of infused triacylglycerol does not exit the intestine via lymphatics. This suggests the presence of a mucosal lipase which could provide fatty acids for potential transport via the portal vein. The present study describes an acid-active lipase in rat intestinal mucosa. Acid lipase was assayed using a glyceryl tri[14C]oleate emulsion (pH 5.8). Mucosal homogenates were differentially centrifuged to yield cellular organelles and cytosol. Cells were sequentially released from villi using citrate and EDTA. The enzyme was found to be most active in the proximal quarter intestine and in the upper third of villi. Its greatest activity was in the lysosomal fraction. Esophageal diversion demonstrated that lingual lipase was not the precursor of the mucosal acid lipase. Bile salts stimulated activity 3- to 5-fold, but other neutral or anionic detergents were inhibitory. Of the detergents tested, taurocholate at super critical micellar concentrations could restore activity only with SDS. Sepharose 6B chromatography suggested that the enzyme partitioned into an SDS and taurocholate mixed micelle. We conclude that mucosal acid lipase is a distinct, intrinsic enzyme of the intestinal mucosa. It is predominantly lysosomal in origin. The location of its greatest activity in the villus tips of the proximal intestine suggests that it is potentially involved in mucosal triacylglycerol disposal.     

Processing of different liposome markers after in vitro uptake of immunoglobulin-coated liposomes by rat liver macrophages

We compared the metabolic fate of [³H]cholesteryl[¹⁴C]oleate, [³H]cholesteryl hexadecylether, ¹²⁵I-labeled bovine serum albumin and [³H]inulin as constituents of large immunoglobulin-coupled unilamellar lipid vesicles following their internalization by rat liver macrophages (Kupffer cells) in monolayer culture. Under serum-free conditions, the cholesteryl oleate that is taken up is hydrolyzed, for the greater part, within 2 h. This occurs in the lysosomal compartment as judged by the inhibitory effect of the lysosomotropic agents monensin and chloroquin. After hydrolysis, the cholesterol moiety is accommodated in the cellular pool of free cholesterol and the oleate is reutilized for the synthesis mainly of phospholipids and, to a lesser extent of triacylglycerols. During incubation in plasma, however, substantial proportions of both the cholesterol and the oleate are shed from the cells, predominantly in the unesterified form. When the liposomes are labeled with the cholesteryl ester analog [³H]cholesteryl hexadecylether only a very small fraction of the label is released from the cells, even in the presence of plasma. Similar to the label remaining associated with the cells, the released label is identified in that case as unchanged cholesteryl ether. The liposomal aqueous phase marker ¹²⁵I-labeled bovine serum albumin is also readily degraded intralysosomally and the radioactive label is rapidly released from the cells in a trichloroacetic acid-soluble form. Also, as much as 20% of the aqueous phase marker [³H]inulin that becomes cell-associated during a 2-h incubation with inulin-containing liposomes, is released from the cells during a subsequent 4-h incubation period in medium or rat plasma. The usefulness of the various liposomal labels as parameters of liposome uptake and intracellular processing is discussed.     

Enzyme replacement therapy in fibroblasts from a patient with cholesteryl ester storage disease

Enzyme replacement has long been considered only a remote possibility in the treatment of a wide range of genetic disorders, many manifested as lysosomal storage diseases. The complexity of having a particular enzyme gain access to the lysosomal compartment in a specific cell seemed insurmountable. We report here on an attempt to introduce the enzyme cholesteryl esterase into fibroblasts from a patient with cholesteryl ester storage disease (CESD). The enzyme gains access to the lysosomal compartment and the accumulating cholesteryl ester by virtue of being carried into the cell conjugated to a ligand (insulin or apoprotein B [apoB]) that binds to its own specific receptor and is internalized by the well-described process of receptor-mediated endocytosis. Regardless of whether the enzyme enters the cell via the insulin receptor or via the low-density lipoprotein (ApoB) receptor, it can be found associated with a lysosomal fraction and is effective in lowering levels of accumulated substrate, cholesteryl ester. The time course of the substrate degradation and the dependence on the receptor density and receptor density and receptor-ligand interaction indicate that the enzyme is simply being carried to the site of substrate accumulation by virtue of the fact that that is the destination of the ligand (along with its conjugated enzyme) following internalization.