Acidic residues emulate a phosphorylation switch to enhance the activity of rat hepatic neutral cytosolic cholesterol esterase.

Site-directed mutagenesis of rat hepatic neutral cytosolic cholesteryl ester hydrolase (rhncCEH) was used to substitute acidic, basic or neutral amino acid residues for Ser506, required for activation by protein kinase A. The substitution of acidic Asp506 resulted in esterase activities with cholesteryl oleate, p-nitrophenylcaprylate (PNPC) and p-nitrophenylacetate (PNPA) equivalent to those of native rhncCEH with Ser506. The substitution of 2 acidic residues (Asp505/506), emulating the 2 negative charges of phosphoserine, resulted in a 10-fold greater cholesterol esterase activity than that of native rhncCEH, similar to the activity of rhncCEH treated with protein kinase A. In contrast to mutants with Ser506, protein kinase A did not increase the specific activities of mutants with Asp505/506. The substitution of basic (Lys506) or neutral (Asn506) residues abolished activity with cholesteryl oleate but not PNPC or PNPA. The substitution of neutral Gln for basic residues Lys496/Arg503 also abolished cholesterol esterase activity but not PNPC- and PNPA-esterase activities. These structure-activity relationships are modeled by homology with a recently reported crystal structure for the homologous human triacylglycerol hydrolase. The results suggest that the cholesterol esterase activity of carboxylesterases is enhanced by interactions between one or more basic residues on helix alpha16 (residues 485-503) and acidic groups at residues 505-506 in the adjacent surface loop.

Over-expression of hepatic neutral cytosolic cholesteryl ester hydrolase in mice increases free cholesterol and reduces expression of HMG-CoAR, CYP27, and CYP7A1.

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 beta-galactosidase (AdbetaGAL). 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 7alpha-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. Coinciding 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.

Mutation of residues 423 (Met/Ile), 444 (Thr/Met), and 506 (Asn/Ser) confer cholesteryl esterase activity on rat lung carboxylesterase. Ser-506 is required for activation by cAMP-dependent protein kinase.

Site-directed mutagenesis is used to identify amino acid residues that dictate reported differences in substrate specificity between rat hepatic neutral cytosolic cholesteryl ester hydrolase (hncCEH) and rat lung carboxylesterase (LCE), proteins differing by only 4 residues in their primary sequences. Beginning with LCE, the substitution Met(423) --> Ile(423) alone or in combination with other mutations increased activity with p-nitrophenylcaprylate (PNPC) relative to more hydrophilic p-nitrophenylacetate (PNPA), typical of hncCEH. The substitution Thr(444) --> Met(444) was necessary but not sufficient for expression of cholesteryl esterase activity in COS-7 cells. The substitution Asn(506) --> Ser(506), creating a potential phosphorylation site, uniformly increased activity with both PNPA and PNPC, was necessary but not sufficient for expression of cholesteryl esterase activity and conferred susceptibility to activation by cAMP-dependent protein kinase, a property of hncCEH. The 3 mutations in combination were necessary and sufficient for expression of cholesteryl esterase activity by the mutated LCE. The substitution Gln(186) --> Arg(186) selectively reduced esterase activity with PNPA and PNPC but was not required for cholesteryl esterase activity. Homology modeling from x-ray structures of acetylcholinesterases is used to propose three-dimensional models for hncCEH and LCE that provide insight into the effects of these mutations on substrate specificity.

Regulation of the rat neutral cytosolic cholesteryl ester hydrolase promoter by hormones and sterols: a role for nuclear factor-Y in the sterol-mediated response.

Expression of the rat liver neutral cytosolic cholesteryl ester hydrolase (CEH) gene is regulated by glucocorticoids, thyroxine, and agents that perturb cholesterol metabolism. The present studies identify the putative hormone response elements in the CEH promoter. They also define the roles of two previously identified sterol regulatory elements (SRE-92 and SRE-160) and a putative nuclear factor-Y (NF-Y) binding site with a consensus ATTGG (inverted CCAAT) motif (Natarajan, R., S. Ghosh, and W. M. Grogan. 1998. Biochem. Biophys. Res. Commun. 243: 349;-355). CEH promoter-reporter gene constructs were transiently transfected into HepG2 cells to evaluate promoter activity. Results indicated that the CEH gene has two complex glucocorticoid response units in distal portions of the promoter corresponding to consensus glucocorticoid regulatory sequences as well as putative thyroid hormone response elements. CEH promoter-reporter constructs with the proximal 189 bp of the wild-type or mutated sequences were also transfected into HepG2 cells. Activity of the wild-type construct increased when incubated in sterol depleted media or when co-expressed with a mature sterol regulatory element binding protein (SREBP-2). These responses were suppressed by mutations in SRE-92, SRE-160, or NF-Y, indicating that these cis elements are sufficient for sterol-mediated regulation of the CEH promoter. Gel mobility shift assays further demonstrated that NF-Y binds to the inverted CCAAT box motif and is required for the sterol-mediated regulation. These results indicate that multiple cis-elements regulate transcription of the cholesteryl ester hydrolase (CEH) gene, consistent with the reported regulation of CEH expression.-Natarajan, R., S. Ghosh, and W. M. Grogan. Regulation of the rat neutral cytosolic cholesteryl ester hydrolase promoter by hormones and sterols: a role for nuclear factor-Y in the sterol-mediated response.

Regulation of hepatic neutral cholesteryl ester hydrolase by hormones and changes in cholesterol flux.

To understand molecular events in regulation of hepatic neutral cholesteryl ester hydrolase (EC3.1.1.13; CEH), catalytic activity, protein mass, and mRNA levels were measured in rats with various perturbations of hepatic cholesterol metabolism. Cholesterol feeding decreased activity (56 +/- 2%), mass (44 +/- 2%), and mRNA (14 +/- 3%). The cholesterol precursor mevalonate also decreased activity (42 +/- 6%), mass (76 +/- 3%), and mRNA (23 +/- 16%). Inhibition of cholesterol biosynthesis by lovastatin increased activity (65 +/- 12%) and mRNA (31 +/- 24%). Stimulation of cholesterol efflux by chronic biliary diversion increased activity (138 +/- 34%), mass (29 +/- 7%), and mRNA (146 +/- 28%). Chenodeoxycholate feeding decreased activity (46 +/- 6%) and mRNA (26 +/- 12%). These data suggest rational regulation of CEH in response to changes in cholesterol flux through the liver. In primary hepatocytes, steady-state mRNA markedly decreased during 72-h cultures and addition of L-thyroxine and dexamethasone synergistically maintained mRNA levels near control values. Lovastatin increased mRNA levels by 103 +/- 15%. Taurocholate and phorbol 12-myristate 13-acetate suppressed mRNA (61 +/- 4% and 49 +/- 13%, respectively), suggesting that protein kinase C mediated effects of bile acids on CEH mRNA levels. These data suggest regulation of CEH by hormones and signal transduction in addition to changes in cholesterol flux.

Molecular cloning of the promoter for rat hepatic neutral cholesterol ester hydrolase: evidence for transcriptional regulation by sterols.

Neutral cholesterol ester hydrolase is a key enzyme in regulating hepatic free cholesterol. Using the CEH specific cDNA sequence in the 5'-untranslated region as a primer, we amplified and cloned 1.3 Kb of promoter sequence upstream of the ATG initiation codon. Analysis of the sequence revealed the presence of a consensus GC-box, which can bind the positive transcription factor Sp1, 35 bases upstream from the transcription start site. Transcriptional regulation by agents perturbing cholesterol metabolism was studied in HepG2 cells by transient transfection assays of the promoter activity in deletion constructs linked to the luciferase reporter gene. Three functional sterol response sequences were identified at positions-92, -160, and -280 of the CEH promoter. The sterol response sequence at position -92 was shown to bind SREBP-2. Therefore, the CEH gene is similar to other genes involved in regulation of cholesterol homeostasis, in that it appears to be transcriptionally regulated by sterols.

Age-related changes in catalytic activity, enzyme mass, mRNA, and subcellular distribution of hepatic neutral cholesterol ester hydrolase in female rats.

Activity and protein mass of hepatic neutral cholesteryl ester hydrolase (CEH) were measured in liver cytosol and washed microsomes of female Sprague-Dawley rats aged 3, 4, 7, 9, 13, and 16 wk. CEH mRNA was also measured. The microsomal component varied with age and contributed a greater fraction of total activity in females than previously reported in males. Nevertheless, the cytosolic component accounted for 62-80% of activity and 77-94% of immunoreactive protein in postmitochondrial fractions. Cytosolic and microsomal CEH specific activities, relative to total protein, decreased 94 and 83%, respectively, from 3 to 4 wk, prior to onset of puberty at 5 wk, and increased 360 and 137%, respectively, from 12 to 16 wk. These results contrast with an earlier study, in which cytosolic CEH activity of males increased with puberty and declined after 12 wk. Although cytosolic CEH was activated by protein kinase A and inhibited by alkaline phosphatase treatment at all ages, protein kinase activation peaked at 4 wk, coinciding with the initial decrease in specific activity. Specific activity in cytosol and microsomes correlated with CEH mass at all ages, suggesting that this CEH accounts for most variation in cellular activity. In contrast, CEH mRNA varied little from 3-16 wk, indicating that transcriptional regulation does not make a major contribution to the variation in CEH activity and mass in females, although it may make an important contribution to male-female differences in CEH expression. Specific activities of cytosolic and microsomal CEH, relative to immunoreactive CEH protein mass, exhibited changes consistent with posttranslational regulation. These results indicate gender-specific multivalent regulation of hepatic CEH by posttranslational mechanisms during development of female rats.

Purification and characterization of a 28 kDa cytosolic inhibitor of cholesteryl ester hydrolases in rat testis.

A 28 kDa inhibitory protein was purified from rat testis cytosol by sequential 40-65% ammonium sulfate precipitation, cation exchange chromatography, anion exchange chromatography, and preparative SDS-polyacrylamide gel electrophoresis. The heat-stable, trypsin-labile protein exhibited nonenzymatic, concentration-dependent inhibition of testicular and pancreatic cholesteryl ester hydrolases at all stages of purification. Copurifying at each stage was a 26.5 kDa protein which comprised 25% of the mass of the two proteins. Polyclonal antibodies raised to either or both 28 kDa and 26.5 kDa proteins by direct injection of excised electrophoretic bands cross-reacted with both proteins on western blots, immunoprecipitated both proteins, and neutralized inhibitory activity. Amino acid compositions of the individual proteins electroeluted from SDS-polyacrylamide gels were different from those of other surface-active proteins of similar molecular weights. Both proteins exhibited identical pl of 4.8 on chromatofocusing columns and two-dimensional gel electrophoresis. Although the subcellular distribution of the 28 kDa protein is unknown, its testicular cytosolic concentration, calculated from the purified protein mass, was 8 X 10(-9) mols/L, which probably underestimates the actual concentration by an order of magnitude. This is greater than the minimum concentration required for in vitro inhibition (10(-9) mols/L), consistent with a physiological role for this protein.

Age-related changes in mRNA, protein and catalytic activity of hepatic neutral cholesterol ester hydrolase in male rats: evidence for transcriptional regulation.

Messenger RNA, protein mass and catalytic activity of hepatic neutral cholesteryl ester hydrolase (CEH) were measured in male Sprague-Dawley rats, aged 6, 8, 9.5, 12 and 24 weeks (wks). CEH mRNA increased 101% from 6 to 9.5 wks, corresponding to onset of puberty, and declined by 52% from 12 to 24 wks. CEH mass was highly correlated with mRNA levels at all ages, increasing 170% from 6 to 9.5 wks and declining 61% from 12 to 24 wks. CEH activity was highly correlated with mass and mRNA from 8-24 wks, but was greater at 6 wks than the activity predicted by the measured mass. In all age groups, activity was consistently increased by activation of endogenous protein kinase A and consistently inhibited by alkaline phosphatase, suggesting that age-related differences in catalytic activity were not due to differences in the level of enzyme phosphorylation. These data suggest transcriptional regulation and indicate an important role for CEH in cholesterol homeostasis in the developing rat.

Molecular cloning and expression of rat hepatic neutral cholesteryl ester hydrolase.

The 1923 bp cDNA for rat hepatic cholesteryl ester hydrolase (CEH) was cloned by screening a lambda gt11 expression library with an oligonucleotide containing the consensus active site sequence for cholesteryl esterases. Expression of a fusion protein, cross-reacting with antibody to the purified liver CEH, was demonstrated by Western blot analysis. The cDNA was sequenced and found to have only 44% homology with pancreatic CEH. Although unique, the cDNA sequence exhibited much greater overall homology with liver carboxylesterases, in both coding and 5'/3' non-coding regions. In Northern blot analysis, the cDNA hybridized with a single band from liver mRNA but not with pancreatic mRNA. The 1.7 kb coding sequence, predicting a 62 kDa protein, was cloned into an Escherichia coli expression system with an inducible promoter and into COS-7 cells. Both expression systems produced a protein which comigrated with liver CEH (66 kDa) on SDS-PAGE and immunoreacted with antibodies to liver CEH on Western blots. Whereas the prokaryotic system produced an inactive protein, expression in COS-7 cells was accompanied by a 5-fold increase in CEH activity and a corresponding increase in immunoreactive protein.

Renal and hepatic family 3A cytochromes P450 (CYP3A) in spontaneously hypertensive rats.

Troleandomycin (TAO), a selective family 3A cytochromes P450 (CYP3A) inhibitor, decreases enhanced in vivo corticosterone 6 beta-hydroxylation and blood pressure in spontaneously hypertensive rats (SHR). Corticosterone 6 beta-hydroxylation was measured in liver and kidney microsomes, to determine ontogeny and the effect of TAO on CYP3A activity at the organ level. SHR kidney CYP3A activity increased from 4 to 8 weeks, stabilized at 11 and 16 weeks, and was much higher than in control (Wistar-Kyoto, WKY) rats at all ages. Hepatic activity showed less consistency in strain difference. TAO produced a relatively large decrease in renal CYP3A activity compared with liver. Although renal CYP3A mRNA was not present in sufficient quantity for detection by northern blot analysis of total RNA, its presence was demonstrated in SHR by reverse transcriptase-polymerase chain reaction amplification. Correlations between renal CYP3A activity and systolic blood pressure in SHR and WKY rats with variations in age, strain and drug treatment are consistent with the role of the enzyme in the pathogenesis of blood pressure elevation in SHR.

Renal corticosterone 6 beta-hydroxylase in the spontaneously hypertensive rat.

Excess 6 beta-OH-corticosterone production by family 3A cytochromes P-450 may play a role in genesis of hypertension in the spontaneously hypertensive rat (SHR), by producing a renal defect in Na+ excretion. Renal cytochromes P-450 may be a causal factor in this genetic model. Since family 3A P-450 is present in rat kidney (collecting duct), the renal family 3A catalytic (6 beta-OHase) and immunoreactive activities were compared in SHR and normotensive control (Wistar-Kyoto; WKY) rats. Corticosterone 6 beta-hydroxylation is markedly higher in SHR than in WKY renal microsomal preparations. Western blot analysis with antibodies to rat and rabbit liver family 3A isoforms demonstrated related proteins. Densitometry revealed greater relative intensity of staining in SHR compared to WKY with both antibodies. Both antibodies inhibited corticosterone 6 beta-hydroxylation by SHR renal microsomes. Increased renal 6 beta-OH-corticosterone production by increased renal family 3A cytochromes P-450 may play a role in the blood pressure elevation in SHR.

Testicular temperature-labile cholesteryl ester hydrolase. Relationship to isoenzymes from other tissues, correlation with spermatogenesis, and inhibition by physiological concentrations of divalent cations.

Temperature-labile cholesterol ester hydrolase (TLCEH) was purified 2,000-fold from rat testis cytosol using sequential ammonium sulfate precipitation, cation exchange chromatography, and isoelectric focusing chromatography. the purified enzyme, which exhibited a single silver-stained band (66 kDa) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was inhibited 89% by the elevation of the temperature from 32 to 37 degrees C and 65% by treatment with alkaline phosphatase. Its amino acid composition and amino-terminal sequence differed markedly from those of isoenzymes from other tissues, although 6 of 20 residues were conserved. Polyclonal antibodies raised to TLCEH exhibited no cross-immunoreactivity with cytosolic proteins from other rat tissues and inhibited 70% of testis cytosolic CEH. Western blot analysis demonstrated a high correlation between immunoreactive protein and catalytic activity in the testis during maturation of the rat, with a marked increase at the onset of spermatogenesis. TLCEH was inhibited by physiological levels of Cu2+ (I50 = 0.60 microM) and Zn2+ (I50 = 0.75 microM) and by Cd2+ (I50 = 0.15 microM) but not by 0.5-5 mM Mn2+.

Immunological characterization of neutral cholesteryl ester hydrolase from rat liver cytosol.

Rabbit polyclonal antibodies were raised against rat liver bile salt-independent neutral cholesteryl ester hydrolase (CEH) and used for subcellular localization and immunological comparison with isoforms from other tissues. Antibodies exhibited a high degree of specificity for the liver CEH through all stages of purification and neutralized 70-80% of the activity of liver cytosolic CEH. They exhibited various levels of cross-reactivity with cytosolic proteins from other tissues, but reacted weakly with pancreatic and intestinal proteins and did not inhibit pancreatic CEH. Cytosol contained 78% of total cellular CEH activity and 75% of CEH immunoreactive protein. Washed microsomes contained 3% of CEH activity and 5% of CEH protein.

Corticosterone 6 beta-hydroxylation correlates with blood pressure in spontaneously hypertensive rats.

Evidence for increased glucocorticoid 6 beta-hydroxylation (enhanced family 3A cytochrome P-450 activity) is found in certain reversible forms of human hypertension. This association was investigated in the spontaneously hypertensive rat (SHR). The proportion of injected [3H]corticosterone excreted in urine as 6 beta-[3H]OH-corticosterone was four- to fivefold higher in SHR than in control Wistar-Kyoto rats, before and after development of overt hypertension. Both hypertension and 6 beta-hydroxylation were inhibited by troleandomycin (a selective inhibitor of family 3A cytochromes P-450), consistent with a role for increased steroid 6 beta-hydroxylation in the genesis of hypertension in the SHR.

Expression of cytochrome P450 3A in amphibian, rat, and human kidney.

Family 3A mammalian liver cytochromes P450 (3A1, rat; 3A3/4, human) catalyze the 6 beta-hydroxylation of endogenous steroids and are steroid inducible. Our recent finding that A6 cells (a toad kidney epithelial cell line) contain corticosterone 6 beta-hydroxylase activity as a steroid-inducible microsomal cytochrome P450 raised the possibility that corticosterone 6 beta-hydroxylase activity in the A6 cells is catalyzed by a member of the 3A family. We found that incubation of A6 cell microsomes from dexamethasone-induced cells with antibodies against family 3A proteins specifically inhibited corticosterone 6 beta-hydroxylase activity. Microsomes from A6 cells analyzed on immunoblots developed with family 3A specific antibodies revealed immunoreactive proteins and treatment of A6 with corticosterone or dexamethasone increased the amounts of 3A immunoreactive protein(s). Furthermore, A6 RNA hybridized with 3A cDNAs on Northern blots and genomic DNA from A6 cells hybridized with a 3A cDNA on a Southern blot. Thus, toad kidney A6 cells express a family 3A P450 that is immunochemically, functionally, and genetically related to the mammalian liver 3A proteins. Prompted by these findings in amphibian kidney, we examined mammalian kidney for evidence of family 3A proteins. Immunocytochemical studies of frozen cryostat sections of normal adult rat kidney incubated with 3A1 antibody showed immunoreactivity only with collecting duct. Immunoblot analysis of human kidney microsomes found three protein bands representing 3A3/4, 3A5, and a 53-kDa Mr protein immunoreactive with human 3A antibody. An unexpected finding was the polymorphic expression of 3A3/4 in human kidney with only one of seven (14%) adult human kidneys tested expressing this protein while 3A5, a protein which is polymorphically expressed in adult human livers, was routinely present in the adult human kidney samples tested. Since human fetal liver contains a family 3A P450 we examined human fetal kidney microsomes by immunoblot analysis with human liver 3A antibody and found expression of a protein tentatively identified as 3A7. Thus, like A6 amphibian cells, family 3A P450 proteins and mRNAs are prominent, functional components in the kidney of mammals, including man.

Effects of perturbations in hepatic free and esterified cholesterol pools on bile acid synthesis, cholesterol 7 alpha-hydroxylase, HMG-CoA reductase, acyl-CoA:cholesterol acyltransferase and cytosolic cholesteryl ester hydrolase.

Effects of expansion of the hepatic free cholesterol pool on bile acid and cholesterol metabolism and homeostasis were examined in rats fed cholesterol in high-fat diets or treated with oleyl-p-(n-decyl)-benzenesulfonate (ODS) or progesterone. Cholesterol feeding for 10-16 days, which increased free (33%) and esterified (6-fold) cholesterol, had no effect on cholate synthesis, total bile acid synthesis, or cholate turnover, whereas these activities were increased 60-80% by ODS and progesterone, which produced only small increases (19%) in free cholesterol. Cholesterol feeding reduced beta-hydroxy-beta-methylglutaryl (HMG)-CoA reductase (72%) and cholesteryl ester hydrolase (48%) and increased acyl-CoA:cholesterol acyltransferase (184%), whereas ODS and progesterone reversed these compensatory responses in cholesterol-fed rats. Cholesterol 7 alpha-hydroxylase was changed no more than 22% by any treatment. A bolus of ODS elevated biliary cholesterol output 41% and shifted biliary bile acid synthesis and composition toward 12-deoxy bile acids. These effects were not seen in ODS-fed or progesterone-treated rats, in which cholesteryl ester stores were depleted. It is concluded that effects of free cholesterol on bile acid synthesis and biliary cholesterol are probably mediated by specific precursor or regulatory pools which can be independently regulated and which represent a relatively small fraction of hepatic free cholesterol.

Membrane structural dynamics of plasma membranes of living human prostatic carcinoma cells differing in metastatic potential.

Rigidity of the outer hemileaflet of the plasma membrane of two prostatic carcinoma cell lines with different metastatic potential, 1-LN and 1-LN-EMS-10, was assessed by steady-state anisotropy, using a battery of fluorescent probes. The "bulk" membrane rigidity sensed by diphenylhexatriene, trimethylammonio-DPH, 1-palmitoyl-2-[DPH-ethylcarbonyl]-phosphatidylcholine, and 10-pyrenedecanoic acid indicated slightly higher rigidity in the membrane of the highly metastatic line (1-LN). This was accompanied by 26% greater mole fraction of cholesterol and 9% lower phospholipid, resulting in 40% greater cholesterol/phospholipid ratio. Phosphatidylethanolamine was increased 12%, but corresponding decreases in phosphatidylserine and phosphatidylinositol resulted in no significant change in molar ratio of choline/noncholine phospholipids. Whereas unsaturation index was slightly higher in 1-LN, fatty acids of 1-LN plasma membranes contained 15% more 18:1, 43% more 20:4, 26% more 22:4, and 38% less 18:2. Anisotropy gradients were determined for the two cell lines using a series of n-(9-anthroyloxy) fatty acid probes with n = 2, 3, 6, 7, 9, 12, and 16. Gradients differed only in position of anisotropy maxima, which occurred with n = 6, in 1-LN, and n = 7, in 1-LN-EMS-10. Possible relationships between observed anisotropy gradients and differences in membrane cholesterol and fatty acid composition are discussed.

Rapid three-step purification of a hepatic neutral cholesteryl ester hydrolase which is not the pancreatic enzyme.

A rat liver cytosolic cholesteryl ester hydrolase (CEH) was purified 12,600-fold by ammonium sulfate precipitation, cation exchange chromatography and gel permeation high-performance liquid chromatography, with an overall yield of 20%. Its properties are compared to those of pancreatic CEH, with which it has sometimes been identified. Liver CEH exhibited a single silver stained band following SDS-polyacrylamide gel electrophoresis (Mr = 66 kDa), was activated by 0.5-10 mM taurocholate but was strongly inhibited by higher levels of taurocholate, which activate pancreatic CEH. Whereas bile salts are known to induce formation of a hexamer of pancreatic CEH, in the current study, 0.5 mM taurocholate dissociated a multimeric form of liver CEH to monomer. Liver CEH did not coelute with pancreatic CEH from cation exchange and chromatofocusing columns, exhibited no immunoreactivity with anti-rat pancreatic CEH IgG in Western blots, was not inhibited by anti-rat pancreatic CEH IgG and had a different amino acid composition from pancreatic CEH. In contrast to liver CEH, which is known to be activated by protein kinases A and C, pancreatic CEH was unaffected by cofactors for protein kinase A and was inhibited by cofactors for protein kinase C.

Comparisons of steady-state anisotropy of the plasma membrane of living cells with different probes.

We have used an extended Perrin equation which was in agreement with literature data for steady-state anisotropy (rSS) for a wide variety of artificial and isolated biological membranes labeled with various probes (Van der Meer et al. (1986) Biochim. Biophys. Acta 854, 38-44 to obtain the static component (r infinity) for the intact plasma membranes of living cells. We show that lipid structural order parameters can be obtained for DPH and TMA-DPH in the plasma membranes of intact cells. We have examined the relationship between 'fractional limiting hindered anisotropy', r infinity/r0, which is related to the lipid structural order parameter, of DPH, TMA-DPH, DPHpPC, and a series of depth-dependent probes (n-(9-anthroyloxy) fatty acids, with n = 2-16), using data from 19 cell types. There was a linear relationship between r infinity/r0 values of DPH and TMA-DPH, but the relationship between either of these probes was non-linear with respect to DPHpPC or the series of fatty acid probes. The relationship between r infinity/r0 values of DPHpPC and the series of fatty acid probes was linear, suggesting that they not only undergo similar motions in the membrane, but also experience similar types of restriction to motion, a type which is different from that experienced by DPH and TMA-DPH. We show that for the plasma membranes of living cells, 'second degree' order parameters can be estimated for DPH and TMA-DPH, and propose that the parameter r infinity/r0, or the 'fractional limiting hindered anisotropy', analogous to a 'first degree' order parameter, can be estimated for DPHpPC and the depth-dependent fatty acid probes to evaluate the density of membrane packing.