Characterization of the isoenzymes of pig-liver esterase. 1. Chemical Studies.

Three different subunits of highly purified pig liver esterase (EC 3.1.1.1) can be separated by analytical dodecyl sulfate electrophoresis, though their relative mobilities are very similar. The same subunit bands are obtained with microsomes, in which the esterases have been labeled with the specific active-site-directed inhibitor bis(4-nitro-[14C]phenyl)phosphate. The heterogeneity of the native trimeric enzyme is much more complex, as is demonstrated by isoelectric focussing and polyacrylamide gel electrophoresis. Fractions of esterase which were partially separated by preparative isoelectric focussing show differences in their subunit composition, their amino acid analyses, their tryptic peptide maps, and their C-terminal amino acids. From these experiments various features of the differing esterase subunits can be deduced. Based on the chemical results and on various experiments which did not indicate any secondary modification of the protein side-chains, the molecular basis of the esterase heterogeneity is discussed. We conclude that the native trimeric esterase is a mixture of numerous hybrids of at least three protein subunits with differing but closely related primary sequences. A comparison of the relative specificity of various preparations of pig liver microsomes indicates that genetic differences concerning the composition of liver esterase exist between individuals.     

Purification and molecular properties of rabbit liver esterase ES-1A

1. The isolation of esterase ES-1A from rabbit liver microsomes/lysosomes is reported. The purification as measured by methylbutyrate-hydrolysing activity, was about 27-fold with a recovery of 2.4%. 2. The resulting product is apparently homogeneous by polyacrylamide (gradient) gel electrophoresis and sodium dodecyl sulphate/polyacrylamide gradient gel electrophoresis after protein staining. The enzyme exhibits heterogeneity after staining for esterase activity and in isoelectric focusing. 3. The molecular mass of the native protein was found to be about 183 kDa (determined by gel filtration and polyacrylamide gel electrophoresis) with a subunit mass of about 63 kDa, indicating a trimeric structure of the enzyme, with subunits of equal size. 4. ES-1A is a glycoprotein and is classified as a carboxylesterase (EC 3.1.1.1). 5. The high degree of similarity of the properties of rabbit ES-1A with those of mouse ES-6A and rat ES-10 suggests that these three esterases may have a common evolutionary origin.     

Thermostable esterase from a thermoacidophilic archaeon: purification and characterization for enzymatic resolution of a chiral compound

Homolog to lipolytic enzymes having the consensus sequence Gly-X-Ser-X-Gly, from the Sulfolobus solfataricus P2 genome, were identified by multiple sequence alignments. Among three potential candidate sequences, one (Est3), which displayed higher activity than the other enzymes on the indicate plates, was characterized. The gene (est 3) was expressed in Escherichia coli, and the recombinant protein (Est3) was purified by chromatographic separation. The enzyme is a trimeric protein and has a molecular weight of 32 kDa in monomer form in its native structure. The optimal pH and temperature of the esterase were 7.4 and 80 degrees C respectively. The enzyme showed broad substrate specificities toward various p-nitrophenyl esters ranging from C2 to C16. The catalytic activity of the Est3 esterase was strongly inhibited by phenylmethylsulfonyl fluoride (PMSF) and diethyl p-nitrophenyl phosphate. Based on substrate specificity and the action of inhibitors, the Est3 enzyme was estimated to be a carboxylesterase (EC 3.1.1.1). The enzyme with methyl (+/-)-2-(3-benzoylphenyl)propionate-hydrolyzing activity to (-)-2-(3-benzoylphenyl)propionic acid displayed a moderate degree of enantioselectivity. The product, (-)-2-(3-benzoylphenyl)propionic acid, rather than its methyl ester, was obtained in 80% enantiomeric excess (e.e.(p)) at 20% conversion at 60 degrees C after a 32-h reaction. This result indicates that S. solfataricus esterase can be used for application in the synthesis of chiral compounds.     

Insights into the physiological role of pig liver esterase: Isoenzymes show differences in the demethylation of prenylated proteins

The possible physiological role of PLE (E.C. 3.1.1.1) located in the endoplasmic reticulum (ER) of pig liver cells in the conversion of endogenous compounds was investigated as it was reported, that PLE acts as prenylated methylated protein methyl esterase (PMPMEase) hydrolysing methylesters of prenylated proteins. Using the specific PMPMEase substrate benzoyl–glycyl–farnesyl–cysteine methyl ester (BzGFCM), six different PLE isoenzymes expressed recombinantly in the yeast Pichia pastoris were found active. Activities ranged from 1.6–15.6 mU per mg protein and it is suggested that Pro285 has a major influence on high activity. In addition, the role of the C-terminal HAEL retention signal for translocation of pig liver esterase (PLE) in the endoplasmic reticulum (ER) of eukaryotic cells was studied using the γ-isoenzyme of PLE expressed in Pichia pastoris. Using truncated versions (HAE, HA, H and without retention signal) of the enzyme it was found that in contrast to earlier reports no influence of the signal peptide on the expression rate of PLE was found. However, higher enzyme activities were obtained in the periplasmatic fraction compared to the supernatant irrespective of the presence or absence of HAEL and the trimeric formation seems to occur in the supernatant of P. pastoris X33 enabling an easier transition of monomeric forms through cell membranes.     

Burkholderia cepacia胆固醇酯酶及其分子伴侣基因的串联表达及酶学性质的研究

为实现胆固醇酯酶的可溶性表达,以Burkholderia cepacia ST-200胆固醇酯酶为研究对象,将酶及其分子伴侣通过人工添加的SD序列在大肠杆菌中进行串联表达。通过SDS-PAGE电泳分析,胆固醇酯酶成功获得可溶性表达,摇瓶水平的总酶量为1 077 U/L。经过镍柱纯化之后可以获得一条相对分子量约为37 kDa的单一条带。利用圆二色谱解析胆固醇酯酶的二级结构,并测定Tm值,从最适温度、最适pH、热稳定性、pH稳定性及有机溶剂耐受性等方面对胆固醇酯酶进行研究,结果表明该酶在pH为7.0,45℃条件下表现出最大活力。     

The esterase from the thermophilic eubacterium Bacillus acidocaldarius: structural-functional relationship and comparison with the esterase from the hyperthermophilic archaeon Archaeoglobus fulgidus

The esterase from the thermophilic eubacterium Bacillus acidocaldarius is a thermophilic and thermostable monomeric protein with a molecular mass of 34 KDa. The enzyme, characterized as a "B-type" carboxylesterase, displays the maximal activity at 65 degrees C. Interestingly, it is also quite active at room temperature, an unusual feature for an enzyme isolated from a thermophilic microorganism. We investigated the effect of temperature on the structural properties of the enzyme, and compared its structural features with those of the esterase from the hyperthermophilic archaeon Archaeoglobus fulgidus. In particular, the secondary structure and the thermal stability of the esterase were studied by FT-IR spectroscopy, while information on the conformational dynamics of the enzyme were obtained by frequency-domain fluorometry and anisotropy decays. Our data pointed out that the Bacillus acidocaldarius enzyme possesses a secondary structure rich in alpha-helices as described for the esterase isolated from Archaeoglobus fulgidus. Moreover, infrared spectra indicated a higher accessibility of the solvent ((2)H(2)O) to Bacillus acidocaldarius esterase than to Archaeoglobus fulgidus enzyme suggesting, in turn, a less compact structure of the former enzyme. The fluorescence studies showed that the intrinsic tryptophanyl fluorescence of the Bacillus acidocaldarius protein was well represented by the three-exponential model, and that the temperature affected the protein conformational dynamics. The data suggested an increase in the protein flexibility on increasing the temperature. Moreover, comparison of Bacillus acidocaldarius esterase with the Archaeoglobus fugidus enzyme fluorescence data indicated a higher flexibility of the former enzyme at all temperatures tested, supporting the infrared data and giving a possible explanation of its unusual relative high activity at low temperatures. Proteins 2000;40:473-481.     

Studies on the characterization of the sodium-potassium transport adenosine triphosphatase. Purification and properties of the enzyme from the electric organ of Electrophorus electricus

An unspecific carboxylesterase was purified 180-fold from acid-precipitated human liver microsomes. The final preparation was homogeneous on disc electrophoresis and polyacrylamide gel electrophoresis in the presence of 6.25 M urea at pH 3.2. A single symmetrical peak was also found on gel filtration and on velocity sedimentation in the analytical ultracentrifuge, whereas slight heterogeneity was observed on isoelectric focusing.The amino acid composition of the purified enzyme is presented. From the results the partial specific volume (0.745 ml × g^?1) and the minimal molecular weight (60,000) could be calculated. Fingerprint maps of tryptic peptides from the carboxymethylated enzyme are shown.The molecular weight as determined by gel filtration, disc electrophoresis, and analytical ultracentrifugation is in the range of 181,000–186,000. For the molecular weight of the subunits a value of 61,500 has been obtained by sodium dodecylsulfate polyacrylamide gel electrophoresis. The equivalent weight of the enzyme has been estimated to be 62,500 from stoichiometry of its reaction with diethyl-p-nitrophenyl-phosphate. Partial cross-linking of the subunits with dimethyl suberimidate and subsequent sodium dodecylsulfate polyacrylamide gel electrophoresis yielded three bands with molecular weights of 60,000, 120,000, and 180,000.From these results it is concluded that human liver esterase is a trimeric protein. It is composed of three subunits of equal size, and there is one active site per subunit.     

Purification and properties of arginine esterases from Bitis arietans (puff adder) venom

An arginine esterase (FT1) was purified from B. arietans venom by gel-filtration and ion-exchange chromatography. The purified enzyme contains 21.6% of carbohydrate, 240 amino acids including 12 half-cystine residues and has a mol. wt of approximately 43,000. The purified enzyme has a high esterolytic activity towards N-alpha-benzoyl-L-arginine ethyl ester but shows no proteolytic activity against Azocoll and no clotting activity with fibrinogen. The N-terminal sequence of the arginine esterase from B. arietans venom shares a significant degree of sequence homology with the arginine esterase of B. nasicornis, the thrombin-like enzyme of C. adamanteus and the kallikrein-like enzymes of C. atrox venoms. It would appear that the arginine esterase from B. arietans venom exists in various multiple forms of the enzyme.     

Oligomeric structure of mammalian purine nucleoside phosphorylase in solution determined by analytical ultracentrifugation

The influence of phosphate, ionic strength, temperature and enzyme concentration on the oligomeric structure of calf spleen purine nucleoside phosphorylase (PNP) in solution was studied by analytical ultracentrifugation methods. Sedimentation equilibrium analysis used to directly determine the enzyme molecular mass revealed a trimeric molecule with Mr = (90.6 +/- 2.1) kDa, regardless the conditions investigated: protein concentration in the range 0.02-1.0 mg/ml, presence of up to 100 mM phosphate and up to 200 mM NaCl, temperature in the range 4-25 degrees C. The sedimentation coefficient (6.04 +/- 0.02) S, together with the diffusion coefficient (6.15 +/- 0.11) 10(-7) cm2/s, both values obtained from the classic sedimentation velocity method at 1.0 mg/ml PNP concentration in 20 mM Hepes, pH 7.0, yielded a molecular mass of (90.2 +/- 1.6) kDa as expected for the trimeric enzyme molecule. Moreover, as shown by active enzyme sedimentation, calf spleen PNP remained trimeric even at low protein concentrations (1 microg/ml). Hence in solution, similar like in the crystalline state, calf spleen PNP is a homotrimer and previous suggestions for dissociation of this enzyme into more active monomers, upon dilution of the enzyme or addition of phosphate, are incorrect.     

Factors influencing juvenile hormone esterase activity in the wax moth, Galleria mellonella

The effects of juvenile hormone, antiallatotropins, selected surgical procedures and starvation on the juvenile hormone esterase levels in Galleria larvae and pupae were investigated. JH reduced JH esterase activity in larvae but induced the enzyme in 1-day-old pupae. In vitro studies confirmed that the peak of synthesis and/or release of JH esterase from the fat body of last instar larvae occurred 4 days after ecdysis. These studies also showed that fat body from JH-treated larvae released much less enzyme than controls. Antiallatotropins, precocene 2 and ZR 2646 also reduced JH esterase levels in larvae, but ZR 2646 induced JH esterase in pupae. In starved larvae, JH esterase did not increase during the first five days. A minimum of 36 hr of feeding was necessary for the larval esterase activity to increase on schedule on day 4 of the last larval stadium. When day-l larvae were ligated behind the head or the prothorax, they had lower JH esterase levels and yet showed a slight increase in the enzyme when the larvae reached the age of 4 days. The significance of these results is discussed in relation to the possible control of esterase activity during metamorphosis.     

Purification and characterization of rat urinary esterase A1

An enzyme, esterase A1, which hydrolyzes tosyl-arginine methyl ester (Tos-Arg-OMe) was separated from esterase A2 and kallikrein of male rat urine and purified by a procedure involving ammonium sulfate fractionation, ion exchange chromatography, hydrophobic chromatography and gel filtration. The resulting preparation was apparently homogeneous, as assessed by polyacrylamide gel electrophoresis. The molecular weight of the preparation was estimated to be 27,000 by SDS-polyacrylamide gel electrophoresis and 30,000 by gel filtration. The enzyme was more specific for arginine methyl esters than for lysine methyl esters. The optimum pH determined with Tos-Arg-OMe as a substrate was 8.0 and the Km was 11.8 mM. The Tos-Arg-OMe esterolytic activity of esterase A1 was inhibited by soybean trypsin inhibitor, but not by aprotinin. In immunodiffusion analysis, the antiserum to esterase A1 formed immunoprecipitin arcs with this enzyme and the urine collected from rat bladder, but not with esterase A2, kallikrein, plasma and the urine collected from ureters. These results indicate that rat urinary esterase A1 differs from esterase A2 and kallikrein. The esterase A1 appears to be produced by accessory sex glands and excreted via the spermiduct into the urine.     

Identity of a cytosolic neutral cholesterol esterase in rat liver with the bile salt stimulated cholesterol esterase in pancreas

A neutral cholesterol esterase has been purified to homogeneity from the cytosolic fraction of rat liver. The 105,000 x g supernatant fraction of rat liver was applied to a DEAE-cellulose column to isolate a partially purified fraction of hepatic cholesterol esterase. Immunoblot analysis of the partially purified liver fraction with the anti-porcine pancreatic cholesterol esterase IgG demonstrated a single band with a molecular weight of 67,000. The hepatic protein was then isolated by immunoaffinity chromatography technique using a column constructed with antibodies prepared against the pancreatic cholesterol esterase. Characterization of the hepatic cholesterol esterase revealed that the hepatic enzyme shared antigenic epitopes with the pancreatic cholesterol esterase and was similarly activated by addition of bile salt such as taurocholate. Moreover, amino-terminal sequencing analysis of the hepatic cholesterol esterase showed an identical sequence with the pancreatic enzyme. Taken together, these results showed that the cholesterol esterases in the liver and the pancreas are very similar and possibly identical proteins.     

Characterization of a thermostable esterase activity from the moderate thermophile Bacillus licheniformis

A new esterase activity from Bacillus licheniformis was characterized from an Escherichia coli recombinant strain. The protein was a single polypeptide chain with a molecular mass of 81 kDa. The optimum pH for esterase activity was 8-8.5 and it was stable in the range 7-8.5. The optimum temperature for activity was 45 degrees C and the half-life was 1 h at 64 degrees C. Maximum activity was observed on p-nitrophenyl caproate with little activity toward long-chain fatty acid esters. The enzyme had a KM of 0.52 mM for p-nitrophenyl caproate hydrolysis at pH 8 and 37 degrees C. The enzyme activity was not affected by either metal ions or sulfydryl reagents. Surprisingly, the enzyme was only slightly inhibited by PMSF. These characteristics classified the new enzyme as a thermostable esterase that shared similarities with lipases. The esterase might be useful for biotechnological applications such as ester synthesis.     

Immunological comparison of cholesterol esterases.

Rat pancreas cholesterol esterase has been immunologically compared with rat intestinal cholesterol esterase. Monospecific precipitating antisera against purified rat pancreas cholesterol esterase were produced in rabbits. Immune IgG, isolated from the antisera, crossreacted with the cholesterol esterase of intestine in the immunodiffusion assay with a pattern of complete identity. Titration of the pancreatic and intestinal enzyme with immune IgG revealed a maximum precipitation (99 and 98%) and maximum inhibition of enzyme activity (66 and 65%) when the ratio of enzyme activity (units) to immune IgG (mg) was 4.1 and 4.0, respectively. The immunological identity demonstrated in these studies lend support to the concept that intestinal cholesterol esterase is derived from the pancreatic enzyme. In additional studies, the immune IgG was employed in the immunodiffusion assay to test for cross-reaction with cholesterol esterases prepared from rat aorta, adrenal, and liver and with cholesterol esterases prepared from the pancreas of rabbit, dog, cow, and guinea pig. There was no evidence of cross-reaction in any case. Further, cholesterol esterase prepared from the pancreas of rabbit, dog, and cow retained full enzymatic activity when titrated with immune IgG.     

Esterase activity of the mitochondria oligomycin-sensitive ATPase complex]

It was found that mitochondrial oligomycin-sensitive ATPase (OS-ATPase) possesses the esterase activity with respect to some carboxylic acid esters with phenols and arylalcane alcohols. The substrate specificity of the esterase found was studied. The effects of some inhibitors and activators of ATPase on the enzyme activity were demonstrated. It was found that ADP inhibits the enzyme from submitochondrial particles containing factor F1 and does not inhibit the enzyme from the particles devoid of this factor. The data obtained suggest that esterase is localized in the hydrophobic part of the oligomycin-sensitive ATPase complex and are indicative of the functional interrelationship between the esterase and ATPase activities.     

Purification and properties of an acetylxylan esterase from Thermobifida fusca

An acetylxylan esterase from Thermobifida fusca NTU22 was purified 51-fold as measured by specific activity from crude culture filtrate by ultrafiltration concentration, Sepharose CL-6B and DEAE-Sepharose CL-6B column chromatography. The overall yield of the purified enzyme was 14.4%. The purified enzyme gave an apparent single protein band on an SDS-PAGE. The molecular mass of purified enzyme as estimated by SDS-PAGE and by gel filtration on Sepharose CL-6B was found to be 30 and 28kDa, respectively, indicating that the acetylxylan esterase from T. fusca NTU22 is a monomer. The pI value of the purified enzyme was estimated to be 6.55 by isoelectric focusing gel electrophoresis. The N-terminal amino acid sequence of the purified esterase was ANPYERGP. The optimum pH and temperature for the purified enzyme were 8.0 and 80°C, respectively. The Zn(2+), Hg(2+), PMSF and DIPF inhibited the enzyme activity. The K(m) value for p-nitrophenyl acetate and acetylxylan were 1.86μM and 0.15%, respectively. Co-operative enzymatic degradation of oat-spelt xylan by purified acetylxylan esterase and xylanase significantly increased the acetic acid liberation compared to the acetylxylan esterase action alone.     

Specificity and induction of undecyl acetate esterase from Pseudomonas cepacia grown on 2-tridecanone

Undecyl acetate esterase from Pseudomonas cepacia grown on 2-tridecanone was strongly inhibited by organophosphates and other esterase inhibitors. Also, p-chloromercuribenzoate at 1 x 10(-4) M showed a 70% inhibition of esterase activity. The enzyme hydrolyzed both aliphatic and aromatic acetate esters at substrate concentrations of 0.25 M. Under these conditions the highest reaction rate was toward undecyl acetate. No lipase or proteolytic activity was demonstrated. Undecyl acetate esterase was classified as a carboxylesterase (B-esterase). Cell-free activity studies on the production of undecyl acetate esterase grown on different carbon sources plus zymogram studies demonstrated that the enzyme was inducible when 2-tridecanone, 2-tridecanol, undecyl acetate and, to a lesser extent, 1-undecanol were growth substrates. Induction of undecyl acetate esterase during oxidation of 2-tridecanone supports the view that undecyl acetate is an intermediate in the degradation of the methyl ketone.     

Electrophoretic data in designing strategies for purification and identification of a highly polymorphic bacterial esterase

We have purified a bacterial enzyme, designated esterase M, by tailoring an efficient and rapid strategy with information derived from titration curves of proteins in crude extract. The pH-dependent stability of the enzyme activity observed by titration pattern allowed an acidic pH treatment of extract and a cationic exchange chromatography at pH 4.1. These two steps were followed by an anionic exchange chromatography and a preparative electrophoresis. Thus, the enzyme was purified about 2000-fold within two days with a recovery of 13.3%. The electrophoretic variants of esterase M were investigated for their molecular relationship through the specific effect of antibodies on esterase electrophoretic pattern (immunosubtractive electrophoresis) which is applicable to large series of samples. By this process, we have demonstrated the presence of common antigenic determinants among the electromorphs of esterase M produced by the three species of motile Aeromonas.     

Study on the production of acetyl esterase and side-group cleaving glycosidases of ammonia fungi

Acetyl esterase was found to be widely distributed in ammonia fungi in a screen comprising 26 species (71 strains). No great differences appeared in enzyme production for acetyl esterase, beta-xylosidase, alpha-arabinosidase, or beta-glucosidase between different strains of the same species, but differences were detected between different genera. Acetyl esterase of Coprinus phlyctidosporus and Lyophyllum tylicolor may act cooperatively with beta-glucosidase. An increase in urea concentration significantly affected enzyme activity. It was supposed that urea used as 20 mg/g litter may solubilize leaf nutrients. At 20 mg urea added/g litter, a sizable increase in beta-glucosidase activity of C. phlyctidosporus and L. tylicolor was found, whereas a decrease in enzyme production of alpha-arabinosidase and beta-xylosidase was detected in some strains. Acetyl esterase and beta-glucosidase of C. phlyctidosporus, L. tylicolor, C. leucocephala 589, and C. rhombisperma 248 were most active in acidic conditions (pH 5.3-6.3), whereas acetyl esterase of L. nuda 561 and L. tarda 564 was most active in alkaline conditions (pH 8.3).     

In vitro activation of a soluble cholesterol esterase from bovine adrenals by a cAMP-dependent protein kinase.

Properties and partial purification of the bovine adrenal cholesterol esterase from the 100000 X g supernatant fraction were investigated. Variations of the enzyme activity with time-dependent (enzymatic) and time-dependent (non enzymatic) effects have been demonstrated. Mg2 has been proved to inhibit the enzyme activity by a non-enzymatic effect in 50mM Tris/HCl buffer, pH 7.4. A time-dependent inactivation of the cholesterol esterase has been observed in the same buffer. The enzyme could be protected from this enzymatic inactivation by its substrate, cholesterol oleate. cAMP, ATP and Mg2 cuase a time-dependent stimulation of the enzyme in 50mM Tris/HCl buffer, pH 7.4. This result suggests that corticotropin activates the soluble cholesterol esterase from bovine adrenals via cAMP-dependent protein kinase. This view is strengthened by the incorporation of 32P radioactivity from [gamma-32P] ATP into the protein fraction of the 100,000 X g supernatant. The protein-bound 32P radioactivity could be co-purified with the enzyme activity during the partial purification of the soluble cholesterol esterase.