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At least 55 records · Page 3Linked to original sources
PubMed2004

Furcatin hydrolase from Viburnum furcatum Blume is a novel disaccharide-specific acuminosidase in glycosyl hydrolase family 1.

Furcatin hydrolase (FH) is a unique disaccharide-specific acuminosidase, which hydrolyzes furcatin (p-allylphenyl 6-O-beta-D-apiofuranosyl-beta-D-glucopyranoside (acuminoside)) into p-allylphenol and the disaccharide acuminose. We have isolated a cDNA coding for FH from Viburnum furcatum leaves. The open reading frame in the cDNA encoded a 538-amino acid polypeptide including a putative chloroplast transit peptide. The deduced protein showed 64% identity with tea leaf beta-primeverosidase, which is another disaccharide glycosidase specific to beta-primeverosides (6-O-beta-D-xylopyranosyl-beta-D-glucopyranosides). The deduced FH also shared greater than 50% identity with various plant beta-glucosidases in glycosyl hydrolase family 1. The recombinant FH expressed in Escherichia coli exhibited the highest level of activity toward furcatin with a Km value of 2.2 mm and specifically hydrolyzed the beta-glycosidic bond between p-allylphenol and acuminose, confirming FH as a disaccharide glycosidase. The FH also hydrolyzed beta-primeverosides and beta-vicianoside (6-O-alpha-L-arabinopyranosyl-beta-D-glucopyranoside) but poorly hydrolyzed beta-gentiobiosides (6-O-beta-D-glucopyranosyl-beta-d-glucopyranosides), indicating high substrate specificity for the disaccharide glycone moiety. The FH exhibited activity toward p-allylphenyl beta-D-glucopyranoside containing the same aglycone as furcatin but little activity toward the other beta-D-glucopyranosides. Stereochemical analysis using 1H NMR spectroscopy revealed that FH is a retaining glycosidase. The subcellular localization of FH was analyzed using green fluorescent protein fused with the putative N-terminal signal peptide, indicating that FH is localized to the chloroplast. Phylogenetic analysis of plant beta-glucosidases revealed that FH clusters with beta-primeverosidase, and this suggests that the disaccharide glycosidases will form a new subfamily in glycosyl hydrolase family 1.

Amino Acid Sequence↗
PubMed2006

A novel glycoside hydrolase family 105: the structure of family 105 unsaturated rhamnogalacturonyl hydrolase complexed with a disaccharide in comparison with family 88 enzyme complexed with the disaccharide.

YteR, a hypothetical protein with unknown functions, is derived from Bacillus subtilis strain 168 and has an overall structure similar to that of bacterial unsaturated glucuronyl hydrolase (UGL), although it exhibits little amino acid sequence identity with UGL. UGL releases unsaturated glucuronic acid from glycosaminoglycan treated with glycosaminoglycan lyases. The amino acid sequence of YteR shows a significant homology (26% identity) with the hypothetical protein YesR also from B. subtilis strain 168. To clarify the intrinsic functions of YteR and YesR, both proteins were overexpressed in Escherichia coli, purified, and characterized. Based on their gene arrangements in genome and enzyme properties, YteR and YesR were found to constitute a novel enzyme activity, "unsaturated rhamnogalacturonyl hydrolase," classified as new glycoside hydrolase family 105. This enzyme acts specifically on unsaturated rhamnogalacturonan (RG) obtained from RG type-I treated with RG lyases and releases an unsaturated galacturonic acid. The crystal structure of YteR complexed with unsaturated chondroitin disaccharide (UGL substrate) was obtained and compared to the structure of UGL complexed with the same disaccharide. The UGL substrate is sterically hindered with the active pocket of YteR. The protruding loop of YteR prevents the UGL substrate from being bound effectively. The most likely candidate catalytic residues for general acid/base are Asp143 in YteR and Asp135 in YesR. This is supported by three-dimensional structural and site-directed mutagenesis studies. These findings provide molecular insights into novel enzyme catalysis and sequential reaction mechanisms involved in RG-I depolymerization by bacteria.

Acetylglucosamine↗
PubMedDate not supplied

Site-directed mutagenesis of leukotriene A4 hydrolase: distinction of leukotriene A4 hydrolase and aminopeptidase activities.

Leukotriene (LT) A4 hydrolase catalyzes enzymatic hydration of LTA4 to biologically active substance, LTB4. Biochemical and immuno-histochemical studies have shown that this enzyme is ubiquitously distributed in various cells and tissues. A sequence domain of LTA4 hydrolase was found to be homologous to those of several zinc metalloproteases. Both native and recombinant enzymes were shown to possess equimolar zinc ion and aminopeptidase activity. To examine the molecular mechanism of this enzyme reaction, site-directed mutagenesis experiments were carried out. Single amino acid substitutions at Glu-297 revealed a distinction of two enzyme activities, and suggest that the glutamic acid residue at 297 is essential for aminopeptidase, while the side chain of Glu or Gln is required for LTA4 hydrolase activity. The loss of two enzyme activities in a mutant E319K confirmed the proposal that the presence of a zinc ion in the enzyme is required for both enzyme activities.

Amino Acid Sequence↗
PubMed2004

Small molecule mitochondrial F1F0 ATPase hydrolase inhibitors as cardioprotective agents. Identification of 4-(N-arylimidazole)-substituted benzopyran derivatives as selective hydrolase inhibitors.

In this paper we show that 4-aryl-CH2-imidazole-substituted benzopyran compounds with 3S,4R-stereochemistry are cardioprotective by inhibiting the F1F0 mitochondrial ATP hydrolase. Compounds (e.g., 13) with 3R,4S-stereochemistry act as mitochondrial KATP openers. This resulted from an inversion of stereochemistry for the F1F0 mitochondrial ATP hydrolase vs mitochondrial KATP. Structure-activity relationships for the inhibition of mitochondrial ATP hydrolase are also delineated. It is not clear how 13 (3R,4S) can selectively inhibit the hydrolytic activity of the F1F0 mitochondrial enzyme without interfering with the synthase activity.

Adenosine Triphosphate↗
PubMed2004

Partial amino acid sequence and mRNA analysis of cytosolic pyridoxine-beta-D-glucoside hydrolase from porcine intestinal mucosa: proposed derivation from the lactase-phlorizin hydrolase gene.

We have previously identified and purified a novel beta-glucosidase, designated PNGH (pyridoxine-5'-beta-D-glucoside hydrolase), from the cytosolic fraction of pig intestinal mucosal. PNGH catalyses the hydrolysis of PNG (pyridoxine-5'-beta-D-glucoside), a plant derivative of vitamin B6 that exhibits partial nutritional bioavailability in humans and animals. Preliminary amino acid sequence analysis indicated regions of close similarity of PNGH to the precursor form of LPH (lactase-phlorizin hydrolase), the beta-glucosidase localized to the brush-border membrane. We report in the present study amino acid sequence data for PNGH and results of Northern blot analyses, upon which we propose a common genomic origin of PNGH and LPH. Internal Edman sequencing of the PNGH band isolated by SDS/PAGE yielded data for 16 peptides, averaging 10.8 amino acids in length. These peptides from PNGH (approx. 140 kDa) were highly similar to sequences existing over most of the length of the >200 kDa precursor of rabbit LPH; however, we found no PNGH sequences that corresponded to approx. 350 amino acids between positions 463 and 812 of the LPH precursor, a region encoded by exon 7 of the LPH precursor gene (amino acids 568-784), and no sequences that corresponded to regions near the N-terminus. MS analysis of tryptic peptides yielded 25 peptides, averaging 15 amino acids, with masses that matched segments of the rabbit LPH precursor. Northern blot analysis of pig and human small intestinal polyadenylated mRNA using a non-specific LPH cDNA probe showed an expected approx. 6 kb transcript of the LPH precursor, but also an approx. 4 kb transcript that was consistent with the size predicted from the PNGH protein mass. Using a probe specific to the region encoded by exon 7, hybridization occurred only with the 6 kb transcript. Based on these observations, we propose that both PNGH and LPH enzymes have the same genomic origin, but differ in transcriptional and, possibly, post-translational processing.

Amino Acid Sequence↗
PubMed2003

Identification of a human valacyclovirase: biphenyl hydrolase-like protein as valacyclovir hydrolase.

Valacyclovir is the 5'-valyl ester prodrug of acyclovir, an effective anti-herpetic drug. Systemic availability of acyclovir in humans is three to five times higher when administered orally as the prodrug. The increased bioavailability of valacyclovir is attributed to carrier-mediated intestinal absorption, via the hPEPT1 peptide transporter, followed by the rapid and complete conversion to acyclovir. The one or more human enzymes responsible for in vivo activation of the prodrug to the active drug and its conversion sites, however, have not been identified. In this report, we describe the purification, identification, and characterization of a human enzyme that activates valacyclovir to acyclovir. A protein with significant hydrolytic activity toward valacyclovir, the 5'-glycyl ester of acyclovir, and the 5'-valyl ester of zidovudine (AZT), was purified from Caco-2 cells derived from human intestine. Using a non-redundant data base search, the N-terminal 19-amino acid sequence of the purified 27-kDa, basic protein revealed a perfect match within the N terminus of a serine hydrolase, Biphenyl hydrolase-like (BPHL, gi:4757862) protein, previously cloned from human breast carcinoma. Recombinant BPHL exhibited significant hydrolytic activity for both valacyclovir and valganciclovir with specificity constants (kcat/Km), 420 and 53.2 mm-1.s-1, respectively. We conclude that BPHL may be an important enzyme activating valacyclovir and valganciclovir in humans and an important new target for prodrug design.

Acyclovir↗
PubMed1990

Separation and differential activation of rat liver cytosolic cholesteryl ester hydrolase, triglyceride lipase and retinyl palmitate hydrolase by cholestyramine and protein kinases.

Cholesteryl ester hydrolase (CEH), triacylglycerol lipase (TGL) and retinyl palmitate hydrolase (RPH) were measured in 104,000 x g supernatants from rat liver under optimal conditions for measurement of cytosolic CEH. Similar levels of hydrolytic activity were seen with oil droplet dispersions of cholesteryl oleate, trioleoylglycerol and retinyl palmitate. No cytosolic TGL activity was seen with substrate presented in the triton-albumin emulsion used for measurement of lipoprotein lipase-like TGL associated with hepatic plasma membrane. Cytosolic CEH, TGL and RPH were differentially partially purified by both ammonium sulfate precipitation and anion exchange fast protein liquid chromatography (FPLC). Of the tree activities, only CEH was stimulated by cholestyramine feeding and by activators of protein kinases A and C. All three activities were inhibited by alkaline phosphatase treatment, although to different degrees. It is concluded that these activities are catalyzed by at least three differentially regulated enzymes with a high degree of specificity for their respective substrates.

Animals↗
PubMed1989

Relative specificities of inhibition of acid cholesteryl ester hydrolase and neutral cholesteryl ester hydrolase in cultured rabbit aortic smooth muscle cells by esterastin and cholesteryl oleyl ether.

Rabbit aortic smooth muscle cells in culture were incubated with 0.04-500 M esterastin. Acid cholesteryl ester hydrolase (ACEH) and neutral cholesteryl ester hydrolase (NCEH) activities were inhibited to a comparable degree, with 50% inhibition occurring in the range of 0.4 M esterastin. Cells incubated with cholesteryl oleyl ether showed 50% inhibition of NCEH at 5.0 M, but no inhibition of ACEH over a concentration range of 0.2-20 M. This relative specificity of cholesteryl oleyl ether for NCEH can be employed to study the relative roles of ACEH vs. NCEH in preventing cellular cholesteryl ester accumulation.

Animals↗
PubMed1986

Hepatic microsomal cholesterol epoxide hydrolase: selective inhibition by detergents and separation from xenobiotic epoxide hydrolase.

Cholesterol epoxide hydrolase (CEHase) activities of mouse liver microsomes were completely inhibited by Emulgen 108 and Lubrol PX and approximately half by cholate, Triton WR-1339, and Tween 80 when preincubated with 0.1% of these detergents, while their xenobiotic epoxide hydrolase (XEHase) activities were markedly enhanced under the same preincubation conditions. The CEHase and XEHase activities were separately eluted from a gel column applied with mouse liver microsomes which were solubilized in a phosphate buffer containing cholate, glycerol, urea, dithiothreitol, and EDTA.

Animals↗
PubMed1990

Leukotriene A4 hydrolase: an epoxide hydrolase with peptidase activity.

Purified leukotriene A4 hydrolase from human leukocytes is shown to exhibit peptidase activity towards the synthetic substrates alanine-4-nitroanilide and leucine-4-nitroanilide. The enzymatic activity is abolished after heat treatment (70 degrees C, 30 min). At 37 degrees C these substrates are hydrolyzed at a rate of 380 and 130 nmol/mg/min, respectively, and there is no enzyme inhibition during catalysis. Apo-leukotriene A4 hydrolase, obtained by removal of the intrinsic zinc atom, exhibits only a low peptidase activity which can be restored by the addition of stoichiometric amounts of zinc. Reconstitution of the apoenzyme with cobalt results in a peptidase activity which exceeds that of enzyme reactivated with zinc. Preincubation of the native enzyme with leukotriene A4 reduces the peptidase activity. Semipurified preparations of bovine intestinal aminopeptidase and porcine kidney aminopeptidase do not hydrolyze leukotriene A4 into leukotriene B4.

Aminopeptidases↗
PubMed2000

Identification of oxidized protein hydrolase of human erythrocytes as acylpeptide hydrolase.

Partial amino acid sequence of 80 kDa oxidized protein hydrolase (OPH), a serine protease present in human erythrocyte cytosol (Fujino et al., J. Biochem. 124 (1998) 1077-1085) that is adherent to oxidized erythrocyte membranes and preferentially degrades oxidatively damaged proteins (Beppu et al., Biochim. Biophys. Acta 1196 (1994) 81-87; Fujino et al., Biochim. Biophys. Acta 1374 (1998) 47-55) was determined. The N-terminal amino acid of diisopropyl fluorophosphate (DFP)-labeled OPH was suggested to be masked. Six peptide fragments of OPH obtained by digestion of DFP-labeled OPH with lysyl endopeptidase were isolated by use of reverse-phase high-performance liquid chromatography, and the sequence of more than eight amino acids from the N-terminal position of each peptide was determined. Results of homology search of amino acid sequence of each peptide strongly suggested that the protein was identical with human liver acylpeptide hydrolase (ACPH). OPH showed ACPH activity when N-acetyl-L-alanine p-nitroanilide and N-acetylmethionyl L-alanine were used as substrates. Glutathione S-transferase (GST)-tagged recombinant ACPH (rACPH) was prepared by use of baculovirus expression system as a 107-kDa protein from cDNA of human erythroleukemic cell line K-562. rACPH reacted with anti-OPH antiserum from rabbit. rACPH showed OPH activity when hydrogen peroxide-oxidized or glycated bovine serum albumin was used as substrates. As well as the enzyme activities of OPH, those of rACPH were inhibited by DFP. The results clearly demonstrate that ACPH, whose physiological function has not yet been well characterized, can play an important role as OPH in destroying oxidatively damaged proteins in living cells.

Amino Acid Sequence↗
PubMed2003

A novel member of glycoside hydrolase family 88: overexpression, purification, and characterization of unsaturated beta-glucuronyl hydrolase of Bacillus sp. GL1.

Unsaturated beta-glucuronyl hydrolase of Bacillus sp. GL1 catalyzes the hydrolytic release of unsaturated glucuronic acids from oligosaccharides produced through the reactions of polysaccharide lyases such as gellan, xanthan, hyaluronate, and chondroitin lyases. An overexpression system for the enzyme was constructed in Escherichia coli cells involving regulation of the enzyme gene under the T7 promoter and terminator. The expression level of the enzyme in E. coli cells was 250-fold higher than that in Bacillus sp. GL1 cells. The enzyme expressed in E. coli cells was purified and characterized. The optimal pH and temperature, and substrate specificity of the purified enzyme were similar to those of the native enzyme from Bacillus sp. GL1 cells, although the enzyme expressed in E. coli cells underwent self-assembly into polymeric forms through the formation of intermolecular disulfide bonds. Circular dichroism analysis indicated that the secondary structure of the enzyme was rich in alpha-helices. Genes showing high identity (over 40% identity) with that of the enzyme were found in the genomes of some pathogenic bacteria, such as Streptococcus pyogenes and Streptococcus pneumoniae, which cause serious diseases (e.g., meningitis and pneumonia). Therefore, the enzyme of Bacillus sp. GL1 and the streptococcal proteins form a new glycoside hydrolase family, 88.

Amino Acid Sequence↗
PubMed1999

Schizosaccharomyces pombe Aps1, a diadenosine 5',5' "-P1, P6-hexaphosphate hydrolase that is a member of the nudix (MutT) family of hydrolases: cloning of the gene and characterization of the purified enzyme.

The fission yeast Schizosaccharomyces pombe contains a gene on chromosome I that encodes a hypothetical nudix hydrolase, YA9E. The gene, designated aps1, has been cloned and the protein has been purified from Escherichia coli with a yield of 10 mg of Aps1/L of culture. Aps1, composed of 210 amino acids with a calculated molecular mass of 23 724 Da, behaves as a monomer with a sedimentation coefficient of 1.92 S as determined by analytical ultracentrifugation. The effective hydrodynamic radius is about 29 A as determined by both analytical ultracentrifugation and gel-filtration chromatography. Aps1, whose expression was detected in S. pombe by Western blotting, is an enzyme that catalyzes the hydrolysis of dinucleoside oligophosphates, with Ap6A and Ap5A being the preferred substrates. The major reaction products are ADP and p4A from Ap6A and ADP and ATP from Ap5A. Values of Km for Ap6A and Ap5A are 19 microM and 22 microM, respectively, and the corresponding values of kcat are 2.0 s-1 and 1.7 s-1, respectively. The enzyme has limited activity on Ap4A and negligible activity on Ap3A, ADP-ribose, and NADH. Aps1 catalyzes the hydrolysis of mononucleotides with decreasing activity in order from p5A to AMP. Optimal activity with Ap6A as substrate is observed at pH 7.6 and in the presence of 0.1-1 mM MnCl2. Aps1 is the first nudix hydrolase isolated from S. pombe, and it is the first enzyme identified with this specific substrate specificity and reaction products.

Bacterial Proteins↗
PubMed1996

Crystal structure of L-2-haloacid dehalogenase from Pseudomonas sp. YL. An alpha/beta hydrolase structure that is different from the alpha/beta hydrolase fold.

L-2-Haloacid dehalogenase catalyzes the hydrolytic dehalogenation of L-2-haloalkanoic acids to yield the corresponding D-2-hydroxyalkanoic acids. The crystal structure of the homodimeric enzyme from Pseudomonas sp. YL has been determined by a multiple isomorphous replacement method and refined at 2.5 A resolution to a crystallographic R-factor of 19.5%. The subunit consists of two structurally distinct domains: the core domain and the subdomain. The core domain has an alpha/beta structure formed by a six-stranded parallel beta-sheet flanked by five alpha-helices. The subdomain inserted into the core domain has a four helix bundle structure providing the greater part of the interface for dimer formation. There is an active site cavity between the domains. An experimentally identified nucleophilic residue, Asp-10, is located on a loop following the amino-terminal beta-strand in the core domain, and other functional residues, Thr-14, Arg-41, Ser-118, Lys-151, Tyr-157, Ser-175, Asn-177, and Asp-180, detected by a site-directed mutagenesis experiment, are arranged around the nucleophile in the active site. Although the enzyme is an alpha/beta-type hydrolase, it does not belong to the alpha/beta hydrolase fold family, from the viewpoint of the topological feature and the position of the nucleophile.

Amino Acid Sequence↗
PubMed1997

Lysine 129 of CD38 (ADP-ribosyl cyclase/cyclic ADP-ribose hydrolase) participates in the binding of ATP to inhibit the cyclic ADP-ribose hydrolase.

CD38 catalyzes not only the formation of cyclic ADP-ribose (cADPR) from NAD+ but also the hydrolysis of cADPR to ADP-ribose (ADPR), and ATP inhibits the hydrolysis (Takasawa, S., Tohgo, A., Noguchi, N., Koguma, T., Nata, K., Sugimoto, T., Yonekura, H., and Okamoto, H. (1993) J. Biol. Chem. 268, 26052-26054). In the present study, using purified recombinant CD38, we showed that the cADPR hydrolase activity of CD38 was inhibited by ATP in a competitive manner with cADPR. To identify the binding site for ATP and/or cADPR, we labeled the purified CD38 with FSBA. Sequence analysis of the lysylendopeptidase-digested fragment of the labeled CD38 indicated that the FSBA-labeled residue was Lys-129. We introduced site-directed mutations to change the Lys-129 of CD38 to Ala and to Arg. Neither mutant was labeled with FSBA nor catalyzed the hydrolysis of cADPR to ADPR. Furthermore, the mutants did not bind cADPR, whereas they still used NAD+ as a substrate to form cADPR and ADPR. These results indicate that Lys-129 of CD38 participates in cADPR binding and that ATP competes with cADPR for the binding site, resulting in the inhibition of the cADPR hydrolase activity of CD38.

ADP-ribosyl Cyclase↗
PubMed1999

The Saccharomyces cerevisiae YOR163w gene encodes a diadenosine 5', 5"'-P1,P6-hexaphosphate (Ap6A) hydrolase member of the MutT motif (Nudix hydrolase) family.

The YOR163w open reading frame on chromosome XV of the Saccharomyces cerevisiae genome encodes a member of the MutT motif (nudix hydrolase) family of enzymes of Mr 21,443. By cloning and expressing this gene in Escherichia coli and S. cerevisiae, we have shown the product to be a (di)adenosine polyphosphate hydrolase with a previously undescribed substrate specificity. Diadenosine 5',5"'-P1, P6-hexaphosphate is the preferred substrate, and hydrolysis in H218O shows that ADP and adenosine 5'-tetraphosphate are produced by attack at Pbeta and AMP and adenosine 5'-pentaphosphate are produced by attack at Palpha with a Km of 56 microM and kcat of 0.4 s-1. Diadenosine 5',5"'-P1,P5-pentaphosphate, adenosine 5'-pentaphosphate, and adenosine 5'-tetraphosphate are also substrates, but not diadenosine 5',5"'-P1,P4-tetraphosphate or other dinucleotides, mononucleotides, nucleotide sugars, or nucleotide alcohols. The enzyme, which was shown to be expressed in log phase yeast cells by immunoblotting, displays optimal activity at pH 6.9, 50 degrees C, and 4-10 mM Mg2+ (or 200 microM Mn2+). It has an absolute requirement for a reducing agent, such as dithiothreitol (1 mM), and is inhibited by Ca2+ with an IC50 of 3.3 mM and F- (noncompetitively) with a Ki of 80 microM. Its function may be to eliminate potentially toxic dinucleoside polyphosphates during sporulation.

Acid Anhydride Hydrolases↗
PubMed1977

Identification of acylcholine acyl-hydrolase with carboxylic ester-hydrolase in human serum.

The relationship between pseudocholine esterase [acylcholine acyl-hydrolase, EC 3.1.1.8] and non-specific esterase [carboxylic ester-hydrolase, EC 3.1.1.1] in human serum was investigated. The purified preparation (purified 500-fold) which had both pseudocholine esterase and non-specific esterase activities, was found to give a single band with faint tailing on polyacrylamide gel electrophoresis. The ratio of the specific activity of pseudocholine esterase to that of non-specific esterase remained essentially the same during the purification procedures. Furthermore, the pseudocholine esterase was demonstrated to be identical with the non-specific esterase by immunochemical studies. All these results suggest that activities of pseudocholine esterase and non-specific esterase in human serum derive from the same enzyme molecule. Observation of Yoshida-cho in Ehime after the application of organophosphorus insecticide supported our results: the activity of pseudocholine esterase was found to be reduced with a concomitant decrease in the activity of non-specific esterase. Based on these results, the physiological significance of the esterase is discussed.

Butyrylcholinesterase↗
PubMed1999

Soluble epoxide hydrolase in rat inflammatory cells is indistinguishable from soluble epoxide hydrolase in rat liver.

Soluble epoxide hydrolase (sEH) is a ubiquitous mammalian enzyme for which liver and kidney are reported to have the highest activity. We have shown that the soluble epoxide hydrolase (sEH) activity present in rat neutrophils and macrophages is kinetically, immunologically, and physically indistinguishable from rat liver cytosolic sEH. Cytosol from rat liver or inflammatory cells and recombinant rat sEH were incubated with trans-diphenylpropene oxide (tDPPO), a selective substrate for sEH. The tDPPO hydration activity we observed in inflammatory cell cytosol was lower than that from liver. The Km for tDPPO hydration observed in rat inflammatory cell cytosol was the same as the Km for rat liver cytosol (10 microM). Recombinant rat sEH and cytosol from rat liver or inflammatory cells were incubated with the sEH inhibitors, chalcone oxide, 4-fluorochalcone oxide, and 4-phenylchalcone oxide. The IC50 values were 40, 8, and 0.4 microM, respectively, in all samples. Furthermore, sEH activity could be completely immunoprecipitated out of the samples, and the amount of antibody required to do so was apparently identical, regardless of the source of enzyme. SDS-polyacrylamide gel electrophoresis followed by Western blot analysis revealed a single sEH band with a molecular weight of 62 kDa. Isoelectric focusing followed by Western blot analysis revealed multiple bands containing tDPPO-hydrating activity. Although the inflammatory cell bands had the same pattern as those from liver cytosol, the recombinant sEH showed a different banding pattern. These multiple bands were not an artifact of the IEF gel selected. Furthermore, in a 2-dimensional IEF gel, the bands re-migrated to the same position. The presence of sEH in inflammatory cells suggests that this enzyme may have an important endogenous function.

Animals↗