Identification of microsomal rat liver carboxylesterases and their activity with retinyl palmitate Sonal P. Sanghani, Wilhelmina I. Davis, Natividad G. Dumaual, Alan Mahrenholz and William F. Bosron Department of Biochemistry and Molecular Biology and of Medicine, Indiana University School of Medicine, Indianapolis, USA Retinyl esters are a major endogenous storage source of vitamin A in vertebrates and their hydrolysis to retinol is a key step in the regulation of the supply of retinoids to all tissues. Some members of nonspecific carboxylesterase family (EC 3.1.1.1) have been shown to hydrolyze retinyl esters. However, the number of different isoenzymes that are expressed in the liver and their retinyl palmitate hydrolase activity is not known. Six different carboxylesterases were identified and purified from rat liver microsomal extracts. Each isoenzyme was identified by mass spectrometry of its tryptic peptides. In addition to previously characterized rat liver carboxylesterases ES10, ES4, ES3, the protein products for two cloned genes, AB010635 and D50580 (GenBank accession numbers), were also identified. The sixth isoen- zyme was a novel carboxylesterase and its complete cDNA was cloned and sequenced (AY034877). Three isoenzymes, ES10, ES4 and ES3, account for more than 95% of rat liver microsomal carboxylesterase activity. They obey Michaelis– Menten kinetics for hydrolysis of retinyl palmitate with K m values of about 1 l M and specific activities between 3 and 8 nmolÆmin )1 Æmg )1 protein. D50580 and AY034877 also hydrolyzed retinyl palmitate. Gene-specific oligonucleotide probing of multiple-tissue Northern blot indicates differen- tial expression in various tissues. Multiple genes are highly expressed in liver and small intestine, important tissues for retinoid metabolism. The level of expression of any one of the six different carboxylesterase isoenzymes will regulate the metabolism of retinyl palmitate in specific rat cells and tis- sues. Keywords: retinyl palmitate hydrolase, carboxylesterase, mass spectrometry, rat, retinol, vitamin A. Vitamin A metabolism [1,2] is a significant area of research because of its diverse role in the regulation of gene expression through retinoic acid receptors. Dietary intake of vitamin A from animal food products is mainly in the form of retinyl esters and retinol, and from plant food products such as provitamin A or b-carotenes. Retinyl esters are converted to retinol in the intestine. After dietary uptake, retinol is converted to retinyl esters in intestinal mucosa and packaged into chylomicrons. These are partially processed during circulation to chylomicron remnants, which contain retinyl esters. Chylomicron rem- nants are rapidly cleared from circulation by liver hepato- cytes where the retinyl esters are hydrolyzed by retinyl ester hydrolases to retinol. The retinol product can either undergo oxidation to retinoic acid for signaling or be secreted into circulation as a complex with retinol binding protein. After meeting the tissue needs, excess retinol is stored in hepatic stellate cells by conversion to retinyl esters (mostly as retinyl palmitate). The stored retinyl esters are the primary vitamin A reservoir in the body and can be mobilized by hydrolysis to retinol by retinyl ester hydro- lases. Hence, retinyl ester hydrolases play very important roles in a variety of cells and tissues to regulate the storage and mobilization of vitamin A [3]. Rats are the most common laboratory model for investigating vitamin A metabolism [3,4]. The rodent hepatic retinyl ester hydrolases are broadly classified into two groups: bile salt-dependent and bile salt-independent. Carboxyl ester lipase is considered as the major bile salt- dependent retinyl ester hydrolase. Recent carboxyl ester lipase knockout studies [5,6] show that both retinoid metabolism and distribution are normal in knockout mice. This suggests that carboxyl ester lipase does not play a significant role in retinoid metabolism and that there may be many different retinyl ester hydrolases in vivo. Another lipase that could play a role in retinoid metabolism is lipoprotein lipase [7]. Two types of bile salt- independent retinyl ester hydrolases have been described in liver microsomes based on their pH optimum as neutral or acid. Both types potentially hydrolyze retinyl palmitate [8]. To date, the specific enzymes and genes responsible Correspondence to W. F. Bosron, Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, MS 207, 635, Barnhill Drive, Indianapolis, IN 46202, USA. Fax: + 317 2744686, Tel.: + 317 2743441, E-mail: wbosron@iupui.edu Abbreviations: CES, carboxylesterase subfamily; SPE, solid phase extraction; LC/ESI-MS, liquid chromatography electrospray ionization mass spectrometry; UPGMA, unweighted pair group method with arithmetic mean. Enzymes and proteins: SWISS-PROT number for ES10 carboxyl- esterase is P16303, that for ES4 carboxylesterase is Q64573 and the number for ES3 carboxylesterase is Q63108. The proteins described in this study as D50580 and AB010635 are the respective products of the genes with GenBank accession numbers D50580 and AB010635. A new carboxylesterase gene was cloned during this study. This cDNA sequence has been submitted to the GenBank and the GI number is AY034877. The protein product of this gene is also called AY034877 in this study. Note: a website is available at http://www.biochemistry.iupui.edu/ (Received 20 February 2002, revised 24 May 2002, accepted 24 May 2002) Eur. J. Biochem. 269, 4387–4398 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03121.x for bile salt-independent acid retinyl ester hydrolase activity have not been characterized [4]. Mentlein and Heymann [9] investigated the retinyl ester hydrolase activity of purified nonspecific carboxylesterases from rat liver microsomes and concluded that only ES4 (also known as hydrolase B [10] or pI 6.2/6.4 esterase [11]) had significant activity with retinyl esters. In the same study, ES10 was reported to have very little activity and ES3 or pI 5.6 esterase [12] had no retinyl palmitate hydrolase activity. Sun et al. [13] purified two neutral bile salt- independent retinyl palmitate hydrolases from rat liver microsomes and identified them as ES2 (serum carboxyl- esterase) and ES10, also known as hydrolase A [14] or pI 6.0 esterase [15]. In a recent review [3], Harrison concluded that there are only three carboxylesterases (ES4, ES10 and ES2) that function as retinyl ester hydrolases in liver. Satoh and Hosokawa [16] proposed a classification scheme for carboxylesterases, CES1 and CES2. ES4, ES10 and ES2 belong to class CES1. In this study, we developed a new procedure for identification of six carboxylesterases utilizing preparative nondenaturing PAGE followed by liquid chromatography electrospray ionization mass spectrometry (LC/ESI-MS). Five of the six protein bands with carboxylesterase activity were identified as protein products of three reported CES1 genes, ES10, ES4, ES3 and two CES2 genes D50580 and AB010635. Amino acid sequencing (Edman method) of the sixth band identified a unique peptide. A new CES2 gene was cloned from a rat liver cDNA library and sequenced (AY034877). The catalytic efficiency of purified carboxylesterases ES10, ES4, ES3, D50580 and AY034877 in hydrolysis of retinyl palmitate was evaluated by a quantitative HPLC assay for retinol production. In contrast to an earlier report [9], we found that all the five carboxylesterases efficiently hydrolyze retinyl palmitate with specific activities of 0.8–8 nmolÆmin )1 Æmg )1 protein. Oligonucleotide probing of multiple-tissue Northern blot showed that all six genes are expressed in liver, but are differentially expressed in small intestine, kidney and skin, known sites that are important for retinoid metabolism [17]. We conclude that differential tissue expression of these retinyl ester hydrolases will be most important for the regulation of vitamin A storage and mobilization in the whole animal. EXPERIMENTAL PROCEDURES Carboxylesterase assays Nonspecific carboxylesterase activity was monitored using 4-methylumbelliferyl acetate as substrate as described by Brzezinski et al. [18]. Briefly, enzyme was incubated with 0.5 m M 4-methylumbelliferyl acetate in 90 m M KH 2 PO 4 , 40 m M KCl, pH 7.3, 37 °C and the product, 4-methyl- umbelliferone, was detected spectrophotometrically at 350 nm. One unit of activity is defined as the amount of enzyme catalyzing the formation of 1 lmol of product, 4-methylumbelliferone, in 1 min. Protein was measured with the Bio-Rad protein assay reagent based on Bradford dye-binding method with BSA as standard [19]. The reaction catalyzed by carboxylesterase to generate retinol from retinyl palmitate was followed by HPLC. All studies involving use of retinoids were carried out under yellow light to prevent photochemical degradation of retinoids. Commercially available retinyl palmitate has many impurities that interfere with the retinyl palmitate hydrolase assay. The presence of retinol in the commercial substrate could produce product inhibition and compromises the sensitivity of the HPLC assay. The photosensitivity of retinoids makes a lengthy purification procedure ineffective. Hence, a short and efficient procedure for purification of retinyl palmitate was developed using solid phase extraction (SPE) Bond Elute C-8 columns (Varian Inc., Palo Alto, CA, USA). Briefly, the extraction cartridges were equilibrated with 3 mL of methanol followed by 3 mL of acetonitrile/ water (60 : 40, v/v). Eleven micromoles of retinyl palmitate in acetonitrile were loaded on the SPE C-8 cartridge and sequentially washed with 6 mL of acetonitrile/water (60 : 40, v/v), 3 mL of ethanol/water (60 : 40, v/v) and 10 mL of methanol/water (90 : 10, v/v). Retinyl palmitate was eluted with 2 mL of methanol. Purity of the sample was checked by HPLC (Agilent 1100 system) using a 5-lm, C-8 Luna column (4.5 · 150 mm, Phenomenex) with isocratic elution with methanol/water (90 : 10, v/v). Retinol elution was monitored at 350 nm. The purified retinyl palmitate stock was stored at )20 °C. Retinyl palmitate hydrolase reactions were performed at 37 °C in 1 mL of 0.05 M Tris-maleate buffer, 20 m M sodium cholate at pH 8. Sodium cholate was added to improve the solubility of retinyl palmitate. Purified rat microsomal carboxylesterases were incubated for 10–60 min with 0.3–20 l M of retinyl palmitate. The reac- tions were stopped by addition of 3 mL of acetonitrile. Ten microliters of 10 l M all-trans-retinal in dimethylsulfoxide were added to each sample as internal standard. We analyzed the retinol standard solution by HPLC and did not detect any all-trans-retinal contamination. A standard curve for retinol was constructed for each experiment and we did not detect any spontaneous oxidation of retinol to retinal. Retinol and all-trans-retinal were extracted with solid phase C-18 columns (Varian Inc.). The columns were washed with 3 mL of methanol and equilibrated with 3 mL of acetonitrile/water (40 : 60, v/v) prior to loading the sample. After loading the samples, columns were washed with 3 mL of acetonitrile/water (40 : 60, v/v) followed by 3 mL of ethanol/water (60 : 40, v/v). Retinol was eluted from the cartridges with 1.8 mL of acetonitrile/methanol (95 : 5, v/v) and dried under nitrogen. The samples were reconstituted in 100 lL of mobile phase constituting of acetonitrile/0.5 M ammonium acetate buffer pH 7.0/tetrahydrofuran (168 : 70 : 10, v/v/v) and 40 lL was injected onto the column. Retinol elution was monitored with a UV detector at 350 nm. Retinol and all-trans-retinal eluted at 10.5 min and 13.0 min, respectively (1 mL min )1 )fromthe5lm, 4.5 · 150 mm Luna C-8 column (Phenomenex). Retinol concentration in experimental samples was calculated from the fit of data to a standard curve by linear regression analysis. The K m and k cat kinetic constants were derived from the fit of data to the Michaelis–Menten equation using GRAFIT 4.0 software (Erithracus Software). Purification of rat liver microsomal carboxylesterases Liver microsomes were prepared from male Sprague– Dawley rats by the method of Pedersen et al.[20].Eighty 4388 S. P. Sanghani et al. (Eur. J. Biochem. 269) Ó FEBS 2002 grams of rat livers were homogenized in 0.22 M mannitol, 70 m M sucrose in 2 m M Hepes, pH 7.4 using a Potter– Elvehjem homogenizer. The microsomes obtained by differential centrifugation were resuspended in 0.1 M Tris, pH 8.5 and frozen at )80 °C until further use. Microsomes were solubilized for 30 min in 0.25% Lubrol at a protein concentration of 5 mgÆmL )1 . Centrifugation at 100 000 g for 30 min gave a supernatant that was subjected to ammonium sulfate fractionation as suggested by Hosokawa et al. [21]. The protein pellet from 30–70% ammonium sulfate saturation was solubilized in 10 m M Tris pH 7.4 containing 1 m M each of Ca 2+ ,Mn 2+ and Mg 2+ chloride salts. This solution was directly applied to a 2.5 · 7cm concanavalin A affinity column (Sigma-Aldrich, St. Louis, MO, USA) in the same buffer. The column was washed with the buffer without divalent cations until the A 280 of the eluant was minimal. The glycosylated proteins were eluted with 10 m M Tris buffer, pH 7.4, 0.2 M sodium chloride and 0.5 M methyl-a- D -mannopyranoside. The fractions contain- ing esterase activity were pooled, concentrated and equili- brated in 20 m M Tris buffer, pH 7.4 (buffer A), using an Amicon 8200 concentrator containing YM 30 membrane. The concentrated protein sample was subjected to preparative nondenaturing gel electrophoresis using the Bio-Rad Prep Cell model 491. The proteins were separated on 1.5 cm of a 4% stacking gel and 4 cm of 6% separating gel poured in a 37-mm diameter tube. The protein eluting from the gel was collected in 0.025 M Tris and 0.192 M glycine buffer at 1 mLÆmin )1 . Carboxylesterase activity was measured in the eluant and the peaks with esterase activity were pooled, concentrated and equilibrated in buffer A. Peaks were subjected to analytical nondenaturing PAGE prior to tryptic digestion and analysis by LC/ESI-MS. Less abundant carboxylesterase peaks were further puri- fied by anion exchange chromatography. A MonoQ HR 5/5 column was equilibrated in buffer A and the concentrated protein samples from preparative nondenaturing gel elec- trophoresis were injected. The column was washed with buffer A at 0.5 mLÆmin )1 for 10 min followed by a linear gradient of 0–50% 20 m M Tris with 1 M NaCl (buffer B) over 60 min, followed by 5 mL of 50% buffer B. Carboxyl- esterases were identified by 4-methylumbelliferyl acetate assay, concentrated and equilibrated in buffer A. These purified enzymes were used for steady state kinetic studies. Non-denaturing polyacrylamide gel electrophoresis (nondenaturing PAGE) Carboxylesterases were separated by analytical discontinu- ous nondenaturing PAGE using the Ornstein–Davis buffer system [22] as described by Dean et al. [23]. Gels were stained for carboxylesterase activity by incubating them for 15minin100m M phosphate buffer, pH 6.5, containing 0.02% 4-methylumbelliferyl acetate. After imaging, the gels were stained with coomassie blue and the protein bands corresponding to the carboxylesterase activity were cut out from the gel for digestion with trypsin and analysis of tryptic peptides by mass spectrometry. Mass spectrometry of tryptic peptides Protein was in-gel digested with 10% (w/w) sequencing- grade trypsin (Worthington) as described by Speicher et al. [24]. Digests and peptide extractions were performed at 37 °C with shaking using a Thermomixer (Eppendorf). Peptide extracts were concentrated (Speed Vac, Savant Instruments, Inc.) prior to analysis. Reversed-phase chro- matography was performed with a fused silica column (300 lmID· 20 cm) packed with a 10-lm, 300 A ˚ Vydac C-18 matrix [25]. Peptide elutions were performed with an Applied Biosystems 140D HPLC system using linear gradient of 0–95% acetonitrile in water containing isopropanol/acetic acid/trifluoroacetic acid (0.2 : 0.1 : 0.001 v/v/v). Column effluents, at 6 lLÆmin )1 , were infused directly into a Finnigan LCQ ion-trap mass spectrometer. Data was acquired using XCALIBUR software (Finnigan) by employing repeating cycles of a full scan, high resolution scan and finally a collisionally induced decomposition (CID) scan of the parent ion selected in the first scan of the cycle. To identify parent peptides and corresponding proteins, the fragmentation data was processed by Sequest software (Thermoquest) and used to query a rat database extracted from the nonredundant protein database obtained from NCBI. Automated amino acid sequencing Proteins were digested with 10% (w/w) trypsin (Promega, sequencing grade) in 40 m M ammonium bicarbonate at 37 °C overnight. Concentrated peptide mixtures were separated on a 500-lmID· 20 cm fused silica capillary column packed with C-18 matrix (Vydac). Linear elution gradients were generated using an ABI 172 HPLC with acetonitrile/water/trifluoroacetic acid buffers and peaks were collected manually based on the UV signal. Individual peptide fractions were sequenced using a Procise cLC (Perkin Elmer Applied Biosystems) employing vendor- supplied reagents for Edman degradation. PCR amplification of a new isoenzyme Reverse transcription and PCR amplifications were per- formed with the Perkin Elmer GeneAmp RNA PCR kit. One microgram of rat liver RNA from Sprague–Dawley rats was reverse-transcribed following the supplier’s proto- col using random hexamer primers. Degenerate primers were designed to the peptide sequences identified by amino acid sequencing and 0.3 l M of each primer, 5¢-GTNCAYCCNACNCCNATGTCYGA-3¢ and 5¢-CTCR TARAARTANACAGGRGC-3¢,wasused.The50lL PCR mixture contained 2 m M Mg 2+ ,0.2m M of each nucleotide and 2.5 U of Amplitaq. The rat liver cDNA was denatured at 95 °C for 2 min and amplified using a DNA Thermal Cycler (Perkin-Elmer) with 35 cycles of 95 °Cfor 1min,58°C for 30 s and 72 °C for 1 min. The 1 kb PCR product was identified, cloned and sequenced. Marathon Ready rat liver cDNA, anchor primers AP1 and AP2 (Clontech, Palo Alto, CA, USA), and gene specific primers 5¢-AGGCCCAGGAACGGGATTCC-3¢ for 5¢ RACE and 5¢-GATAAATCTGAGGTGGTCTACAAG-3¢ for 3¢ RACE were used to amplify the new gene with the PCR reagent concentrations described above. The cDNA was denatured at 95 °C for 2 min and then amplified using following 35 cycles, 95 °C for 30 s and 68 °C for 3 min. The 5¢ RACE product was cloned and sequenced. The 3¢ RACE product was further amplified by nested PCR using AP2 Ó FEBS 2002 Hydrolysis of retinyl palmitate by carboxylesterases (Eur. J. Biochem. 269) 4389 and nested gene specific primer 5¢-GGAATCCCTGTG TTCCTGGGCCT-3¢. Both the 5¢-and3¢-RACE products were cloned and sequenced. The complete cDNA for this carboxylesterase was cloned by amplifying rat liver cDNA with the primers 5¢-CTGAGATTCAACCATGCCTTTGGC-3¢ and 5¢-TTG CCCAGAATGATAACACAGAGG-3¢ that were desig- nedtothemost5¢ and 3¢ regions, respectively, from the sequence information from the race products. Pfu polymer- ase (Stratagene) was used for this PCR reaction at 2.5 U per 50 lL. Reactions were performed as described by Strata- gene and the final primer concentration was 1 l M for each primer. Marathon Ready rat liver cDNA was denatured at 95 °C followed by 35 cycles of 95 °Cfor45 s,65 °Cfor45 s and 72 °C for 3 min and final elongation at 72 °Cfor5 min. The 1.8 kb PCR product was cloned into the Zero Blunt TOPO cloning vector (Invitrogen Corp.) and three clones were picked and sequenced entirely in both direction. Determination of pI Isoelectric focusing was carried out on a LKB Bromma 2117 Multiphor instrument using premade IsoGel agarose IEF Plates (FMC). About 1–5 lgofeachisoenzymein 10 m M Tris buffer and 4 lg of IEF markers (Sigma- Aldrich) pI 3.6–9.3 were loaded on the gel in duplicate. Proteins were focused under constant power of 10 W for 45–60 min. Half of the gel was immediately stained for total protein with coomassie blue and the duplicate half was stained for activity as described for nondenaturing PAGE. The pI for each isoenzyme was calculated from the standard curve and the average of the major bands is reported. Oligonucleotide probing of rat multiple-tissue Northern blot for expression of carboxylesterases Oligonucleotides specific for each isoenzyme were designed from sequence alignments. The oligonucleotides are as follows: for ES10 5¢-ATCAGCTTAGCAATGGGCTTG CTA-3¢;ES45¢- TCGGCAGCACTACATTGTCAAC-3¢; ES3 5¢- GAGTCTCCGTGCAAATCCAGCG-3¢; D50580 5¢-TGTTCTTCAGAACAGCCCGCATG-3¢; AB010635 5¢-CAGCGGGAATCATCTTGAAGACC-3¢ and for AY034877 5¢-AGGCCCAGGAACACAGGGATTCC-3¢. The specificity of oligonucleotides for ES10, ES4, ES3 and D50580 was verified by slot blot analysis. The oligonucleo- tides were labeled with [c- 32 P]dATP (Perkin Elmer) using T 4 polynucleotide kinase (Promega). The labeled oligonucleo- tides were desalted using a G25 spin column (Pharmacia Amersham) and used to probe a 12-tissue rat Northern blot (Origene Technologies). The specific activities of the oligonucleotide probes were between 12 and 17 · 10 8 cpmÆlg )1 and b-actin probe was 2.7 · 10 8 cpmÆlg )1 . Oligonucleotide probes were heated at 80 °Cfor3min with 100 lL of sonicated salmon sperm DNA (Stratagene) prior to hybridization. The blot was prehybridized for 30 min in Quikhyb solution (Stratagene), hybridized for 16–20 h at 55 °C and washed with 2 · NaCl/Cit containing 0.1% SDS, two washings for 15 min at room temperature followed by one 30 min wash at 55 °C. The radioactive blot was developed by exposure to a phosphoimager screen for 24–48 h and analyzed in a Bio-Rad Phosphoimager. RESULTS Separation and isolation of rat carboxylesterases by preparative nondenaturing PAGE In this study, we developed a simple procedure for purification of rat liver microsomal carboxylesterases resulting in separation of six different isoenzymes. Conca- navalin A affinity chromatography, as shown in Tables 1 and 2, is an effective step in this procedure because it enriched glycosylated carboxylesterases activity by 5.6-fold with 69% recovery. Five glycosylated carboxylesterase activities were separated on preparative nondenaturing PAGE as shown in Fig. 1. All activities were concentrated and the enzymes were readily identified on nondenaturing PAGEasshowninlanes1–5inFig.2.Morethan90%of carboxylesterase activity loaded onto preparative nondena- turing PAGE was recovered in the five peaks. As seen in Table 1, peak 1 was purified 6.3-fold on PAGE and exhibits a single protein band (lane 1, Fig. 2B) with specific activity of 42 UÆmg )1 . The specific activities for peaks 2–5 are lower Table 1. Purification of rat liver microsomal carboxylesterases. Carb- oxylesterases were purified from microsomes prepared from livers of male Sprague–Dawley rats. The glycosylated and nonglycosylated carboxylesterases were separated during concanavalin A affinity chromatography. Peaks 1–5 on preparative PAGE represent five gly- cosylated carboxylesterase activities that bound to concanavalin A affinity column and separated on nondenaturing PAGE. The purifi- cation of nonglycosylated carboxylesterases that did not bind to con- canavalin A column is not shown. One unit of activity is defined as the amount of enzyme catalyzing formation of 1 lmol of product, 4-methylumbelliferone, in 1 min. Specific activity is defined as micro- moles of 4-methylumbelliferone product formed per minute per mg of protein under the conditions described in Experimental procedures. Purification step and enzyme form Total protein (mg) Total activity (U) Specific activity (UÆmg )1 ) Lubrol high speed supernatant 1879 1450 0.77 Ammonium sulfate fractionation 827 974 1.18 Concanavalin A 101 675 6.7 Preparative PAGE (210 U loaded) Peak 1 4.45 186.5 41.9 Peak 2 1.23 20.7 16.7 Peak 3 0.71 7.3 10.2 Peak 4 0.74 4.7 6.3 Peak 5 0.38 1.3 3.6 Table 2. Peak 3, 4 and 5 activities from three separate preparations were combined and subjected to MonoQ chromatography. Before MonoQ After MonoQ Total protein (mg) Total activity (U) Specific activity (UÆmg )1 ) Total protein (mg) Total activity (U) Specific activity (UÆmg )1 ) Peak 3 3.7 15.8 4.3 0.3 9 30.1 Peak 4 3.94 8.4 2.1 0.69 3.8 5.5 Peak 5 3.0 5.4 1.8 0.09 2.6 29.7 4390 S. P. Sanghani et al. (Eur. J. Biochem. 269) Ó FEBS 2002 (Tables 1 and 2). While each peak is pure with respect to cross contamination by the other isoenzymes (lanes 2–5, Fig. 2A), peaks 2–5 exhibitmultiple protein bands(lanes 2–5, Fig. 2B). The purity of peak 2 was optimized by pooling fewer fractions and peaks 3–5 were purified to homogeneity by ion exchange chromatography using a MonoQ column (Table 2). The purified carboxylesterases were used to study their retinyl palmitate hydrolase activity. Separation of nonglycosylated carboxylesterases The flow-through from the concanavalin A column had only 2% or 19 units of carboxylesterase activity with a specific activity of 0.04 UÆmg )1 of protein. Three faint activity bands were detected on nondenaturing PAGE (not shown). Partial purification of these three activities allowed their separation on nondenaturing PAGE (not shown) and their identification by LC/ESI-MS. Identification of carboxylesterases by mass spectrometry Each carboxylesterase protein band that was separated by preparative and analytical nondenaturing PAGE was characterized by the LC/ESI-MS methods. Bands were cut out of the analytical nondenaturing gel, digested with trypsin and the mixture of peptides was separated by capillary HPLC. Sequence analysis was performed by electrospray tandem mass spectrometry. Each protein was analyzed from at least two different preparations. An example of a total ion chromatogram of tryptic peptides from peak 4 is shown in Fig. 3. The results of the MS sequencing experiments for the five purified carboxylester- ase isoenzymes are summarized in Fig. 4. As shown in Fig. 1, peak 1 (Fig. 2, lane 1) is the most abundant carboxylesterase from rat liver accounting for 85% of the total activity recovered from preparative nondenaturing PAGE. This protein was positively identified as ES10 by MS analysis with as high as 26% protein sequence coverage (Fig. 4). It is known to be a homotrimer [26] and exhibits the most cathodic mobility of all carboxylesterase activities on nondenaturing PAGE. Peak 2 (Fig. 2, lane 2) accounted for 9.4% of the total activity recovered from preparative PAGE (Table 1) and it appears as a doublet as shown in Fig. 4. The relative abundance of these bands varies from preparation to preparation. In one preparation where the bands were equally present, both of them were individually analyzed by mass spectrometry of their tryptic peptides. Table 3 lists the peptides identified for each band as an example and the peptides shown in bold were identified in both the bands. As seen in Table 3, both the bands were identified to be products of the ES4 gene [27] with as high as Fig. 1. Elution profile of glycosylated carboxylesterases from prepara- tive nondenaturing PAGE. Fractions were assayed for carboxylesterase activity with 4-methylumbelliferyl acetate as substrate as described in materials and methods. Activity in UÆmL )1 is plotted as a function of fraction number, identifying the peaks with carboxylesterase activity. The peaks are labeled 1–5 based on their cathodic to anodic mobility on nondenaturing PAGE shown in Fig. 2A, lane G. Fig. 2. Non-denaturing polyacrylamide gel of rat liver microsomal carboxylesterases stained for activity and protein. The glycoprotein fraction of microsomal extract and the five activity peaks seen in Fig. 1 were individually concentrated and 15 lg of protein from each peak was separated by nondenaturing PAGE. Panel A is stained for carb- oxylesterase activity and panel B is the same gel stained with coomassie blue. Arrows mark the positions of the protein bands with carboxy- lesterase activity. Lane 1 is peak 1; lane 2 is peak 2; lane 3 is peak 3; lane 4 is peak 4; lane 5 is peak 5 as seen in Fig. 2 and lane G is glycosylated proteins that bind to concanavalin A resin that was purified by pre- parative nondenaturing PAGE (Fig. 1). Fig. 3. Total ion chromatogram of carboxylesterase peak 4. The protein band with carboxylesterase activity in peak 4 (Fig. 1) was cut out from the nondenaturing polyacrylamide gel, marked by an arrow in lane 4 of Fig. 2. The protein was in-gel digested with trypsin and the mixture of tryptic peptides was injected into capillary HPLC and analyzed by ESI-MS. The mass-to-charge ratios of the ion with the largest response in the major peaks are indicated in the chromatogram. Ó FEBS 2002 Hydrolysis of retinyl palmitate by carboxylesterases (Eur. J. Biochem. 269) 4391 45% sequence coverage. There is a faint band below the doublet of peak 2 (Fig. 4) and in one preparation we pooled the fractions corresponding to this band and identified it to be ES4 by LC/ESI-MS with 32% coverage. Peak 3 (Fig. 2, lane 3) accounts for only 3.3% of activity recovered from preparative PAGE (Table 1). It was sequenced from three Table 3. Peptides identified by mass spectrometry for the two bands in peak 2 (Fig. 4). The two bands for peak 2 were individually sequenced and the peptides identified for both the bands are listed below. The peptides that were common to both the bands are shown in bold type in the list for band a. Boxed amino acids are unique to ES4 (13) in comparison to hydrolase B (12). Underlined amino acids are unique to ES4 in comparison to hydrolase C (10). Observed (M + H) + using monoisotopic m/z ion in high resolution segment. Peptide number Predicted (M + H) + ion Observed (M + H) + ion Identified fragment ions/total fragment ions Peptide position in ES4 (GI:2494386) Peptide sequence ES4 band a 1 1451.9 1451.9 16/26 51–64 LGVPFAKPP L GSLR 2 1600.8 1601.4 16/26 65–78 FAPPQPAEPWSFVK 3 976.5 976.5 12/14 97–104 MNDLLTNR 4 686.4 686.5 7/8 238–242 NL FHR 5 1630.9 1631.4 19/30 243–258 AISESGVV FL P GLLTK 6 1618.9 1618.7 17/24 287–299 QKTEEELLEIM KK 7 1947.0 1947.5 24/32 313–329 ESYHFLSTVVDNVVLPK 8 702.4 702.3 9/10 333–338 EILAEK 9 918.5 918.8 12/14 375–382 MAI TLLEK 10 1593.8 1593.8 11/28 419–433 IGDV SF SI PSVMVSR 11 1573.7 1573.3 13/26 461–474 HVVGDHADDLYSVF 12 626.4 626.4 9/10 475–480 GAPILR 13 977.4 977.4 12/16 481–489 DGASEEEIK 14 911.5 911.6 11/12 497–503 FWANFAR 15 1368.7 1368.2 12/20 511–521 GLPHWPQYDQK 16 1747 1747.0 24/28 537–551 LKAEEVAFWTQLLAK 17 1255.6 1255.7 10/18 552–561 R Q PQPHHNEL ES4 band b 1 1498.8 1498.5 10/26 37–50 Y VSLEGVTQSVAVF 2 2375.2 2375.7 16/44 133–155 LPVMVWIHGGGMTLGGASTYDGR 3 2644.4 2644.3 19/88 310–332 DNKESYHFLSTVVDNVVLPKDPK 4 1478.8 1478.8 14/24 339–351 NFNTVPYIVGINK 5 1975.08 1975.0 23/34 383–400 FAS LYGIPEDIIPVAIEK 6 904.5 904.4 11/14 411–418 IRDGILAF 6 1584.8 1584.2 12/24 448–460 QYYPSFSSPQRPK 7 1305.7 1305.7 12/22 481–492 DGASEEEIKLSK 8 1751.9 1751.9 1728 522–536 EEYLQIGATTQQSQR Fig. 4. Summary of mass spectrometry results of glycosylated carboxylesterases. A nondenaturing gel of glycosylated fraction of rat liver microsomal proteins that were loaded onto preparative nondenaturing PAGE is shown at the left. The gel is stained for activity and the activity bands for corresponding peaks are labeled with arrows. In this gel, peak 2 appeared as a doublet (band a and b). Each peak was further separated on nondenaturing PAGE and the bands with activity were cut out and in-gel digested with trypsin. The peptide mixture for each band was analyzed by LC/ESI-MS. The percentage of amino acid sequence identified for each isoenzyme in two or three different experiments is reported along with the carboxylesterase isoenzyme identified from nr database as the primary match for each activity. 4392 S. P. Sanghani et al. (Eur. J. Biochem. 269) Ó FEBS 2002 different experiments and was identified as ES3, also called the pI 5.6 isoenzyme, with 23–42% sequence coverage. Peak 4 (Fig. 2, lane 4) accounts for 2% of total activity recovered from preparative PAGE. It was sequenced six times and a unique match was not found in the database. In all six experiments, only 7–9% of the protein sequence was identical with D50580 or AB010635 (shown in bold in Fig. 5). This band was subjected to amino acid sequencing and identified as described below. Peak 5 (lane 5, Fig. 2 and Table 1) accounted for only 0.6% of the activity recovered from preparative PAGE (Table 1). This protein was identified as the product of the gene D50580, also called CE21p [28], with 18–30% sequence coverage in three separate experiments. The three minor activities that accounted for about 2% of the microsomal activity and did not bind to concanavalin A column were also analyzed by LC/ESI-MS (not shown). One peak was identified as ES4 with 16% coverage. A second peak was a doublet and both the bands could not be assigned to any known carboxylesterase. The third peak was a doublet on nondenaturing PAGE. Both bands were individually sequenced and identified as protein products of the gene AB010635 with 27% sequence coverage. Identification of a new rat liver carboxylesterase isoenzyme (AY034877) Initial attempts to identify the carboxylesterase activity in peak 4 (Figs 1 and 4) identified a few peptides that are present in D50580 and AB010635 (shown in bold type in Fig. 5). However, the majority of peptides from peak 4 could not be positively identified in the protein database. The analysis of the mass spectrometry data of the tryptic peptides relies on the presence of carboxylesterase sequences in the protein database and consequently it cannot identify a new carboxylesterase sequence. Hence, tryptic peptides were purified and sequenced by automated Edman degradation (Procise). The sequenced peptides are boxed in Fig. 5. The de novo sequencing approach resulted in identification of one unique peptide LTVHPTPMSED that did not match any known protein in the nonredundant database. A degenerate 5¢ primer designed to this peptide and a 3¢ primer to peptide QAPVYFYE were synthesized (Fig. 4). These degenerate primers amplified a 1-kb DNA product from Sprague–Dawley rat liver cDNA. The product was cloned and the sequence identified it as a new cDNA. Gene-specific primers were designed for 5¢ and 3¢ race reactions to amplify the newly identified sequence. The complete cDNA for the new isoenzyme was obtained by PCR using pfu polymerase (Stratagene) and the sequence is shown in Fig. 6. This sequence was submitted to GenBank and its accession number is AY034877. The protein in the second peak that did not bind to concanavalin A is a doublet. Both protein bands in this peak were also identified as the products of AY034877 gene. There was 17 and 23% coverage for these bands, respectively, by mass spectromet- ric analysis of tryptic peptides. Isoelectric point (pI) analysis The determination of pI values for carboxylesterases by isoelectric focusing is difficult because the isoenzymes exhibit multiple bands. Nevertheless, the isoenzymes have been routinely identified in the literature by their pI values. All of the isolated proteins were equilibrated in the same buffer before isoelectric focusing and the reported pI values are the average of the major protein bands with carboxyl- esterase activity from two to three different experiments (Table 4). The experimentally determined pI values are compared to values calculated from the protein sequence without carbohydrate and the N-terminal signal peptide. Steady state kinetics of retinyl palmitate hydrolysis The purpose of this study was to investigate the role of the five purified carboxylesterases as retinyl palmitate Fig. 5. Amino acid sequence for a new carboxylesterase isoenzyme (AY034877). The translated amino acid sequence of the protein encoded by new carboxylesterase gene (GI:AY034877) is shown. This protein has the predicted signal peptide at N-terminal shown in italics. The position of asparagine in the potential glycosylation site is indicated (*). The active site residues and conserved cysteines were identified by homology with other carboxylesterases, are marked by arrows and (d), respectively. Peptides identified by automated amino acid sequencing are boxed. The peptides identified by mass spectrometry prior to identification of new gene are shown in bold. The peptides identified by mass spectrometry after adding the AY034877 sequence to the database are underlined. Ó FEBS 2002 Hydrolysis of retinyl palmitate by carboxylesterases (Eur. J. Biochem. 269) 4393 hydrolases. Studies on solubility of retinyl palmitate in 50 m M Tris-maleate buffer pH 8.0 with and without 20 m M cholate show that sodium cholate increases the solubility of retinol palmitate in assays to 50 l M .Duetoalimited amount of AY034877, we determined its specific activity with a single concentration of retinyl palmitate (20 l M ). D50580 was unstable so we have reported the range of k cat values determined after 10- and 20-min incubations. Retinol product formation was linear for 60 min with isoenzymes ES10, ES4 and ES3. The kinetics of retinol palmitate hydrolysis was studied for ES10, ES4 and ES3 under steady- state conditions. The HPLC chromatogram in Fig. 7B shows the elution profile of the product retinol and the internal standard all-trans-retinal with AY034877 isoen- zyme. The ratio of peak area for retinol to internal standard was estimated for each sample and quantitated using the standard curve for retinol generated under identical condi- tions for each experiment. The kinetic constants for the five purified carboxylesterases are summarized in Table 5. The K m values for retinyl palmitate for the ES10, ES4 and ES3 isoenzymes were about 1 l M . ES4 was the most efficient enzyme studied with a specific activity of 7.5 nmolÆ min )1 Æmg )1 . AY034877 (Fig. 7B) was the least efficient enzyme with a specific activity of 0.8 nmolÆmin )1 Æmg )1 . Most importantly, all five isoenzymes can function as retinyl palmitate hydrolases. ES10 and ES4 are estimated to contribute 94% of the liver retinyl palmitate activity. Tissue distribution of rat liver carboxylesterase The high sequence homology among these isoenzymes (up to 80%) makes it difficult to study their tissue distribution by Northern blot analysis using full-length cDNAs as probes. Hence, specific oligonucleotide probes were designed for each isoenzyme to identify their specific cDNAs. Slot-blot cross-hybridization experiments for Fig. 6. cDNA sequence for new isoenzyme. The cDNA sequence for new glycosylated rat liver carboxylesterase is reported in GenBank as GI:AY034877. The start and the stop codons are shown in bold. The coding region is shown in upper case and the UTR is shown in lower case. The sequence of last 33 nucleotides in the 3¢-UTR was from the 3¢-RACE clones and the polyadenylation signal is underlined. The translated protein sequence is shown in Fig. 5. 4394 S. P. Sanghani et al. (Eur. J. Biochem. 269) Ó FEBS 2002 ES10, ES3, ES4 and D50580 showed an absolute specificity of each oligonucleotide probe to their respective cDNA (data not shown). A multiple-tissue Northern blot was sequentially probed with an oligonucleotide specific for each isoenzyme. Message for ES10 was 2.2 kb, ES4 and AY037877 were 2.0 kb, ES3 was 2.1 kb, D50580 was 2.4 kb and AB010635 was 2.3 kb, no additional bands were observed in all six Northern blots. In agreement with the protein purification in Tables 1 and 2, mRNAs for all five glycosylated isoenzymes are expressed in the liver (Fig. 8). AB010635 was identified in the fraction that did not bind concanavalin A and in agreement with protein identification its message is detected in the liver. Multiple isoenzymes are also expressed in the kidney, ES4, ES3, AB010635 and AY034877, and in small intestine, ES3 and AY034877. The isoenzymes are differentially expressed in other tissues. For example, only ES10 is seen in lung and testis, both ES10 and ES3 are seen in skin. Stomach expresses AB010635 and AY034877 both isoenzymes belong to CES2 family. No isoenzyme expression was detected in brain, thymus, heart or spleen. The even loading of all samples was confirmed with cDNA probe for b-actin, which was supplied by Origene Technologies. Table 4. Properties of microsomal rat liver carboxylesterases. The experimental isoelectric point (pI) for all rat liver carboxylesterases were determined as described in material and methods the values reported in the table are average of 2–3 different experiments. The calculated pI for all carboxylesterases without the signal peptide and carbohydrate was determined using PROTPARAM software available at the ExPASy website (http:// www.expasy.ch/tools/protparam.html). The potential N-glycosylation sites [36] were predicted using SCANPROSITE software and the location of asparagines for each isoenzyme is tabulated. The positions of conserved cysteines and active site serine, glutamic acid and histidine residues are determined by homology to cholinesterases [37] whose X-ray structure is known. Isoenzyme Experimental pI Calculated pI Positions of predicted residues Glycosylated Asn Conserved Cys Active site triad Ser Glu His ES10 6.05 6.2 79, 489 88, 116, 273, 284 221 353 466 ES4 6.7 6.3 79 87, 116, 273, 284 221 353 466 ES3 5.7 5.6 79, 107, 489 87, 116, 273, 284 221 353 466 AY034877 6.0 6.1 275 95, 122, 279, 290 227 344 456 D50580 5.3 5.6 261 272 550 92, 119, 276, 287 224 341 452 AB010635 a 4.8 5.4 None 97, 125, 282, 293 230 347 459 a AB010635 was expressed in low amount in the fraction of the microsomal extract that did not bind to concanavalin A sepharose (not shown). Fig. 7. HPLC profile for retinyl palmitate hydrolase assay. Panel A shows the HPLC profile of an assay without addition of enzyme and panel B shows the profile for a reaction mixture with AY034877 carboxylesterase. Retinol elutes at 10.5 min and the internal standard, all-trans-retinal, elutes at 13.0 min. The absorbance of retinol is monitored at 350 nm. Table 5. Summary of kinetics of retinyl palmitate hydrolysis by carb- oxylesterases. Steady state kinetics for hydrolysis of retinyl palmitate by carboxylesterase was studied at 37 °Cin1mLof0.05 M Tris- maleate buffer, 20 m M sodium cholate at pH 8. Purified carboxylest- erase isoenzymes were incubated for 10–60 min with 0.3–20 l M of retinyl palmitate. The retinol formed was extracted with SPE method described in methods and separated by HPLC on a 5-lm, 4.5 · 150 mm Luna C-8 column (Phenomenex) and eluant was monitored at 350 nm. All-trans-retinal was used as an internal stand- ard. The amount of retinol in each sample was quantitated from the standard curve for retinol run with each experiment. The relative percentage of retinyl palmitate activity for each isoenzyme eluting from preparative PAGE was calculated from the specific activity of the purified hydrolases and the number of milligrams recovered on pre- parative PAGE (Table 1). ND, not determined. Enzyme K m (l M ) k cat (min )1 ) k cat /K m (l M )1 Æ min )1 ) Relative percentage retinyl palmitate activity ES10 1.16 ± 0.12 0.22 ± 0.04 0.19 60 ES4 1.4 ± 0.5 0.45 ± 0.1 0.32 34 ES3 0.89 ± 0.54 0.19 ± 0.6 0.21 3 AY034877 ND 0.05 a ND 3 D50580 ND 0.17–0.27 a,b ND 0.05 a k cat s were estimated at single concentration of retinyl palmitate, 20 l M . b Due to instability of D50580 enzyme the range of k cat estimates for 10 and 20 min incubations is reported. Ó FEBS 2002 Hydrolysis of retinyl palmitate by carboxylesterases (Eur. J. Biochem. 269) 4395 DISCUSSION Liver is the primary site for the complex regulation vitamin A uptake from chylomicrons, storage of vitamin A esters and mobilization of retinol for transport to target tissues [3]. The primary goal of this study was to identify carboxyles- terases that are expressed in rat liver and investigate their ability to function as retinyl palmitate hydrolases. We identified five glycosylated carboxylesterases in rat liver microsomal extracts that had activity with 4-methylumbel- liferyl acetate as substrate. In agreement with the literature, ES10 and ES4 (Fig. 1, peaks 1 and 2) accounted for > 80% of 4-methylumbelliferyl acetate and retinyl palmitate hydrolase activity (Table 5). Hence they are the most abundant broad substrate specificity carboxylesterases in rat liver [29]. The multiplicity and tissue-specific expression of the less abundant rat liver carboxylesterases are controver- sial. Minor peaks 3–5 (Tables 1 and 2) account for only 6% of 4-methylumbelliferyl hydrolase activity in the liver. This low abundance makes their purification challenging. More- over, the identification of rat liver carboxylesterases based on pI values is difficult because of the complexity of their banding patterns and the variation in the reported values from one laboratory to another [30]. These carboxylesterase isoenzymes have overlapping substrate specificities so it is equally difficult to differentiate them by substrate specificity [28,16]. Hence, we decided to purify and sequence five nonspecific carboxylesterases by LC/ESI-MS prior to evaluating them as retinyl palmitate hydrolases. An efficient purification procedure was developed to separate the carboxylesterase isoenzymes from rat liver microsomes. Concanavalin A chromatography was used to isolate the microsomal glycoproteins and preparative non- denaturing PAGE was used to separate isoenzymes. Five carboxylesterase forms were purified (Fig. 2 and Table 1). Recovery from nondenaturing PAGE was always > 90%. From three separate preparations, the yield of low abun- dance carboxylesterases ES3, AY034877 and D50580 proved very reproducible. Their identity was determined by individually sequencing the tryptic peptides for all five peaks from multiple preparations by LC/ESI-MS. Four of the five peaks were positively identified by LC/ESI-MS (Fig. 4). Peak 1 was confirmed as ES10, also known as hydrolase A, pI 6.0 or RH1. Peak 2 exhibited two major bands on nondenaturing PAGE as shown in Fig. 4. Both bands were positively identified as ES4 (Table 3). There are three closely related genes reported in the database: ES4 [27], hydrolase B [10] and hydrolase C [31]. The amino acid sequence for ES4 and hydrolase B differs by only 10 amino acids; that of ES4 and hydrolase C differs by 38 amino acids. The amino acids that would differentiate between ES4 and hydrolase B (boxed in Table 3) and ES4 and hydrolase C are underlined in Table 3 and were examined from both bands and agree with the ES4 sequence. Peak 3 was identified as ES3 and peak 5 was confirmed to be protein product of gene D50580. Tryptic peptides for peak 4 yielded high quality MS data as shown in Fig. 3 but no known protein could be assigned to this peak from analysis of the nonredundant protein database. Peak 4 appeared to be a new carboxylesterase isoenzyme. From a combination of Edman amino acid sequencing, design of degenerate oligonucleotide primers, PCR, and 5¢-and3¢-RACE, a new isoenzyme, AY034877 was cloned. The previous MS data for peak 4 was reanalyzed with the new sequence and 27% of the protein sequence could be assigned to the newly identified protein, as shown by peptides underlined in Fig. 5. The protein encoded by AB010635 gene does not contain a consensus glycosylation sequence and consistent with this we only found this isoenzyme in the protein fraction that did not bind concanavalin A (not shown). Hence, analyses by mass spectrometry resulted in identifi- cation of three known carboxylesterases ES10, ES4 and ES3, two protein products of cloned genes AB010635 and D50580 and one new carboxylesterase, AY034877. We were unable to identify ES2 (serum carboxylesterase) or hydro- lase C in our rat liver microsomal extracts. So far we have described at least four proteins arising from four different genes in the hydrolase ÔCÕ region [29] suggesting that the identification of isoenzymes from this region is more complicated than previously appreciated. The six rat carboxylesterases were divided into two groups based on the sequence homology analysis, CES1 and CES2 [16]. There is about 70% identity within a class and about 50% identity between classes. This classification was further supported by phylogenetic analysis (Fig. 9). In this study, a nongapped alignment of all known rat carboxyl- esterase protein sequences was created by ClustalW method using MACVECTOR 7.0 software (Oxford Molecular Ltd) followed by generation of a tree by the neighbor joining method (Fig. 9). The same tree was generated using PHYLIP package (http://evolution.genetics.washington.edu/phylip. html) with PROTDIST and NEIGHBOR software. For the six carboxylesterases identified in this study, ES10, ES4 and ES3 isoenzymes belong to CES1 and D50580; and AB010635 and AY034877 belong to CES2, as predicted by sequence identity analysis. Phylogenetic tree analysis of the carboxylesterase supergene family has been described by Satoh and Hosokawa by UPGMA method and the tree was rooted to human class I (hCE-1) isoenzyme [16]. In agreement with that study, all three rat CES1 isoenzymes Fig. 8. Multiple tissue northern analysis for rat liver carboxylesterase. Gene specific oligonucleotides were designed for each rat liver carb- oxylesterase. The same blot was probed sequentially for each isoen- zyme. Origene Technologies supplied b-actin probe along with the blot. Oligo probes were hybridized at 55 °C in QuikHyb solution and washed at 55 °Cin2· NaCl/Cit with 0.1% SDS. 4396 S. P. Sanghani et al. (Eur. J. Biochem. 269) Ó FEBS 2002 [...]... example, the use of sodium cholate in our assay increases the solubility of retinyl palmitate, purification of retinyl palmitate substrate prior to use, efficient sample preparation by SPE in our assay compared to extraction and the identity of isoenzymes studied We found ES4 to be most efficient in hydrolyzing retinol palmitate with Vmax of 7.5 nmolÆ min)1Æmg)1 in agreement with the reported Vmax of 7.3 nmolÆmin)1Æmg)1... value of 0.2 min)1 (Table 5) for both ES10 and ES3 suggests that they can efficiently hydrolyze retinyl palmitate Based on the analysis of three separate experiments for purification on nondenaturing preparative PAGE ES10 and ES4 account for more than 83% of carboxylesterase protein in rat liver microsomes From their retinyl palmitate catalytic efficiency, we estimate that ES10 and ES4 account for 94% of. .. 2002 Hydrolysis of retinyl palmitate by carboxylesterases (Eur J Biochem 269) 4397 Fig 9 Phylogenetic tree for rat liver carboxylesterases The phylogenetic tree was generated by MACVECTOR 7.0 software Neighbor joining method used a nongapped CLUSTALW alignment of all the carboxylesterase proteins to generate the tree GI accession numbers for their cDNA identifies the carboxylesterases and their trivial... characterization and biosynthesis of pI-6.4 esterase, a carboxylesterase of rat liver microsomal extracts Biochem J 254, 51–57 12 Robbi, M & Beaufay, H (1994) Cloning and sequencing of rat liver carboxylesterase ES-3 (egasyn) Biochem Biophys Res Commun 203, 1404–1411 13 Sun, G., Alexson, S.E & Harrison, E.H (1997) Purification and characterization of a neutral, bile salt-independent retinyl ester hydrolase from rat. .. B., Hullihen, J., Decker, G.L., Soper, J.W & Bustamente, E (1978) Preparation and characterization of mitochondria and submitochondrial particles of rat liver and liver- derived tissues Methods Cell Biol 20, 411–481 21 Hosokawa, M., Maki, T & Satoh, T (1987) Multiplicity and regulation of hepatic microsomal carboxylesterases in rats Mol Pharmacol 31, 579–584 22 Ornstein, L (1964) Disc electrophoresis... metabolism of retinyl esters For example, expression of AY034877 and ES3 in small intestine suggest that they may be involved in dietary vitamin A uptake The pharmacokinetics of retinyl ester metabolism will be determined by the tissue specific expression of carboxylesterase and their catalytic efficiency for ester hydrolysis Such understanding of retinyl ester metabolism will be possible only upon identification. .. Purification and characterization of two rat liver microsomal carboxylesterases (hydrolase A and B) Arch Biochem Biophys 315, 495–512 30 Simon, B., de Looze, S., Ronai, A & von Deimling, O (1985) Identification of rat liver carboxylesterase isozymes (EC 3.1.1.1) using polyacrylamide gel electrophoresis and isoelectric focusing Electrophoresis 6, 575–582 31 Yan, B., Yang, D & Parkinson, A (1995) Cloning and expression... 94% of total carboxylesterase retinyl palmitate hydrolase activity and hence will be most important carboxylesterase isoenzymes in liver retinoid metabolism Of the three CES2 proteins identified in this study the very low abundance of AB010635 prevented us from investigating its role as retinyl palmitate hydrolase Both D50580 and AY034877 were not stable at 37 °C and so their Michaelis–Menten kinetics... signal is a variant of the consensus eukaryotic endoplasmic reticulum retention signal of KDEL [34] All three rat liver CES1 isoenzymes, ES10, ES4 and ES3, followed Michaelis–Menten kinetics for hydrolysis of retinyl palmitate The Km value for all three isoenzymes was about 1 lM, much lower than previously determined Km values for pig retinyl palmitate hydrolase (27.5 lM) [35] and rat serum carboxylesterase... & Krisch, K (1973) Purification and molecular properties of an unspecific carboxylesterase (E1) from rat- liver microsomes Eur J Biochem 36, 120–128 27 Robbi, M., Van Schaftingen, E & Beaufay, H (1996) Cloning and sequencing of rat liver carboxylesterase ES-4 (microsomal palmitoyl-CoA hydrolase) Biochem J 313, 821–826 28 Sone, T & Wang, C.Y (1997) Microsomal amidases and carboxylesterases Comp Toxicol . Identification of microsomal rat liver carboxylesterases and their activity with retinyl palmitate Sonal P. Sanghani, Wilhelmina. polyacrylamide gel of rat liver microsomal carboxylesterases stained for activity and protein. The glycoprotein fraction of microsomal extract and the five activity