Isolation of a galactose-free 20-kDa fragment exhibiting butyrylcholine esterase and aryl acylamidase activity from human serum butyrylcholine esterase by limited alpha-chymotrypsin digestion

Purified human serum butyrylcholine esterase (approximately 90-kDa subunit), which also exhibits aryl acylamidase activity, was subjected to limited alpha-chymotrypsin digestion. Three major protein fragments of approximately 50 kDa, approximately 21 kDa and approximately 20 kDa were found to be produced, as observed by SDS-gel electrophoresis of the chymotryptic digest. The purified butyrylcholine esterase could fully bind to a Ricinus-communis-agglutinin-Sepharose column but after chymotryptic digestion about 15-20% of the enzyme activity remained unbound and was recovered in the run-through fractions. Sephadex G-75 chromatography of the chymotryptic digest showed an enzymatically active fragment eluted at an approximate molecular mass of 20 kDa, apart from the undigested butyrylcholine esterase eluted at the void volume. The butyrylcholine esterase fragment that did not bind to Ricinus communis agglutinin also was eluted at an approximate molecular mass of 20 kDa from a Sephadex G-75 column. This enzymatically active low-molecular-mass fragment from Sephadex G-75 chromatography showed a single protein band of approximately 20 kDa on SDS-gel electrophoresis. Neutral sugar analysis of the approximately 20 kDa fragment showed the presence of mannose only, whereas the undigested butyrylcholine esterase showed the presence of both mannose and galactose. Amino-terminal-sequence analysis of the approximately 20 kDa fragment showed the sequence Arg-Val-Gly-Ala-Leu, which agrees with amino acid residues 147-151 reported for human serum butyrylcholine esterase [Lockridge et al. (1987) J. Biol. Chem. 262, 549-557]. Both cholinesterase and aryl acylamidase activities were co-eluted in all chromatographic procedures. The results suggested that limited alpha-chymotrypsin digestion of human serum butyrylcholine esterase resulted in the formation of a approximately 20-kDa enzymatically active fragment with Arg147 as its N-terminal residue and which was devoid of galactose.     

Purification and some properties of an esterase from yeast

An esterase was isolated and purified from baker's yeast by ammonium sulfate precipitation and column chromatographies on Sephacryl S-200, DEAE-Sephacel, chromatofocusing, and DEAE-Sephacel again. The molecular weight of the enzyme was approximately 84,000 on Sephadex G-100 and 40,000 by sodium dodecyl sulfate-poly-acrylamide gel electrophoresis, suggesting a dimer for the activity. This enzyme hydrolyzed short-chain naphthyl esters and p-nitrophenyl esters, and its activity was strongly inhibited by mercuric compounds. The esterase appeared to be an arylesterase (EC 3.1.1.2) and its optimum pH was 8.0 at 30°C.     

A peptidase activity exhibited by human serum pseudocholinesterase

The identity of a peptidase activity with human serum pseudocholinesterase (PsChE) purified to apparent homogeneity was demonstrated by co-elution of both peptidase and PsChE activities from procainamide-Sepharose and concanavalin-A--Sepharose affinity chromatographic columns; comigration on polyacrylamide gel electrophoresis; co-elution on Sephadex G-200 gel filtration and coprecipitation at different dilutions of an antibody raised against purified PsChE. The purified enzyme showed a single protein band on gel electrophoresis under non-denaturing conditions. SDS gel electrophoresis under reducing conditions, followed by silver staining, also gave a single protein band (Mr approximately equal to 90,000). Peptidase activity using different peptides showed the release of C-terminal amino acids. Blocking the carboxy terminal by an amide or ester group did not prevent the hydrolysis of peptides. There was no evidence for release of N-terminal amino acids. Potent anionic or esterase site inhibitors of PsChE, such as eserine sulphate, neostigmine, procainamide, ethopropazine, imipramine, diisopropylfluorophosphate, tetra-isopropylpyrophosphoramide and phenyl boronic acid, did not inhibit the peptidase activity. An anionic site inhibitor (neostigmine or eserine) in combination with an esterase site inhibitor (diisopropylfluorophosphate) also did not inhibit the peptidase. However, the choline esters (acetylcholine, butyrylcholine, propionylcholine, benzoylcholine and succinylcholine) markedly inhibited the peptidase activity in parallel to PsChE. Choline alone or in combination with acetate, butyrate, propionate, benzoate or succinate did not significantly inhibit the peptidase activity. It appeared that inhibitor compounds which bind to both the anionic and esteratic sites simultaneously (like the substrate analogues choline esters) could inhibit the peptidase activity possibly through conformational changes affecting a peptidase domain.     

Carboxylic ester hydrolases of rat pancreatic juice

An attempt was made to establish the number and characteristics of the enzymes in pancreatic juice that hydrolyze nitrogen- and phosphorus-free esters of fatty acids. For this purpose model compounds were hydrolyzed by lyophilized rat pancreatic juice under conditions that accelerated or inhibited the reactions. Although it is not established with certainty, it is suggested that three enzymes are responsible for the hydrolysis of fatty acid esters. The first enzyme is glycerol-ester hydrolase (EC 3.1.1.3) or lipase. This enzyme hydrolyzes water-insoluble esters of primary alcohols. The reaction occurs at an oil/water interface and is inhibited by bile salts at pH 8. The enzyme is relatively stable at pH 9, but unstable at pH 4. It has a broad pH optimum between 7.5 and 9.5. The second enzyme hydrolyzes esters of secondary alcohols and of other alcohols as well. It has an absolute requirement for bile salts and has a pH optimum at about 8. The enzyme is unstable in pancreatic juice when maintained at pH 9, probably due to the action of trypsin. It may be identical with sterol-ester hydrolase (EC 3.1.1.13). The third enzyme hydrolyzes water-soluble esters. It too has an absolute requirement for bile salts, although a smaller amount is necessary for maximum activity. This enzyme also is unstable at pH 9, but can be differentiated from the preceding enzyme by its stability at pH 4 and its pH optimum of 9.0. Carboxylic-ester hydrolase (EC 3.1.1.1) is not found in pancreatic juice, although it is present in pancreatic tissue.     

Microcalorimetric study of the inhibition of butyrylcholinesterase by carbamates

The inhibition of horse serum butyrylcholinesterase (EC 3.1.1.8) by three carbamates (eserine, neostigmine, and rivastigmine) was studied by flow microcalorimetry at 37 degrees C in Tris buffer (pH 7.5). The kinetics of carbamylation was studied in the absence or presence of the substrate, butyrylcholine, using an extension of the model described by Stojan and coworkers (FEBS Lett. 440 (1998) 85-88). The model was fitted to the data by a nonlinear regression procedure using simulated annealing followed by Marquardt's method. The affinity of the carbamates for the free enzyme increased in the order neostigmine相似文献   

Specificity of alpha-chymotrypsin with exposed carboxyl groups blocked.

The 15 exposed carboxyl groups of alpha-chymotrypsin were modified with glycine ethyl ester at low pH using barbodiimide reagent. The specificity of the modified enzyme (Chy-15) was studied over the pH range of 4 to 9 with both N-acylated and non-N-acylated amino acid esters. The modified enzyme had lower reactivity toward N-acylated esters than non-N-acylated esters compared to the native enzyme. Typical substances such as acetyl- and benzoyl-L-tyrosine ethyl esters retained 4 and 9% activity, whereas phenylalanine ethyl ester was slightly more reactive with the modified than with the native enzyme. The pH-rate profiles of acetyl-L-phenylalanine ethyl ester and tryptophan ethyl and benzyl esters were investigated in detail. Analysis of these profiles revealed three pKa values of approximately 5, 7, and 9 related to a functional carboxyl, imidazoyl, and an amino group, respectively. Since similar pKa values occur for the native enzyme, modification did not block the carboxyl corresponding to pKa 5. A mechanism is proposed for catalysis which includes both the protonated and unprotonated form of the imidazoyl (His-57) and utilizes water rather than a carboxyl (Asp-102) as the proton sink.     

Purification and characterization of hemoglobin-hydrolyzing acidic thiol protease induced by leupeptin in rat liver

Intraperitoneal administration of leupeptin to rats induced a hemoglobin-hydrolyzing protease which was most active at pH 3.5 and was insensitive to pepstatin in various tissues such as the liver, kidney, and muscle, as observed previously in adult rat hepatocytes in primary culture (Tanaka, K., Ikegaki, N., and Ichihara, A. (1979) Biochem. Biophys. Res. Commun. 91, 102-107). The induced acidic protease was purified about 600-fold in 30% yield from rat liver by conventional chromatographic techniques. The purified enzyme appeared homogeneous by polyacrylamide gel electrophoresis in the presence or absence of sodium dodecyl sulfate and was a monomeric protein of Mr = 20,000. The enzyme appeared to be a glycoprotein because its induction was blocked by the addition of tunicamycin to cultures of hepatocytes and because the induced protease was absorbed on concanavalin A-Sepharose and eluted with methylglucoside. It seemed to be present in lysosomes and was fairly stable at various pH values and temperatures. It showed endopeptidase activity on various protein substrates, but scarcely hydrolyzed N-substituted derivatives of arginine. It did not hydrolyze esters, showed no aminopeptidase or carboxypeptidase activity, and did not inactivate glucose-6-phosphate dehydrogenase or aldolase. The enzyme appeared to be a thiol protease, since it was strongly inhibited by sulfhydryl-reactive compounds and N-( [N-(1-3-trans-carboxyoxiran-2-carbonyl)-L-leucyl]-agmatine and was not inhibited by reagents specific for carboxyl-, serine-, or metalloproteases. This induced protease could be separated from cathepsins B, D, and H by chromatography. The enzyme was similar to cathepsin L in chromatographic behavior, Mr and pI, but differed from the latter in stability and in its inability to inactivate some enzymes. These results suggest that it differs from any known proteases found previously in rat liver.     

Human lysosomal acid lipase/cholesteryl ester hydrolase. Purification and properties of the form secreted by fibroblasts in microcarrier culture

Lysosomal acid lipase was purified to near homogeneity in a yield of 25-30% from secretions of human fibroblasts grown on microcarriers in spinner culture. Ammonium chloride was added to the serum-free medium to stimulate production of extracellular enzyme and minimize modifications, including proteolytic processing and destruction of the mannose 6-phosphate recognition marker, that have been associated with packaging and maturation of acid hydrolases in lysosomes. Chromatography of secretions by decyl-agarose, hydroxylapatite, phenylboronate-agarose, and gel filtration resulted in greater than 1500-fold purification of the lipase, representing a 10,000-fold increase above the specific activity of intracellular enzyme. The apparent molecular weight of approximately 49,000, estimated for the lipase by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, was similar to that determined for the native enzyme by gel filtration (Mr approximately 47,000). By contrast, a smaller molecular weight (Mr approximately 41,000) was estimated for the intracellular enzyme. The purified enzyme was susceptible to hydrolysis by endo-beta-N-acetylglucosaminidase H, which resulted in at least two new forms, reduced in apparent molecular weight by approximately 4,000-6,000. Treatment with the endoglycosidase did not alter the catalytic activity or heat stability of the acid lipase. However, the treated enzyme was no longer internalized by fibroblasts via the mannose 6-phosphate receptor and thereby had lost the capacity to correct cholesteryl ester accumulation in cultured lipase-deficient cells. Acid fatty acyl hydrolase activity for cholesteryl oleate, triolein, and methylumbelliferyl oleate co-purified. All three esters were hydrolyzed optimally at pH 4.0, but the pH profile was altered by addition of salts or albumin to the phospholipid-bile salt substrate mixtures. In a series of saturated fatty acyl esters of 4-methylumbelliferone, a derivative with an intermediate chain length (9 carbons) was the best substrate and was hydrolyzed at a rate comparable to that of the oleate ester at pH 4. The optimal pH for hydrolysis of the intermediate and shorter chain length esters was higher by about 2 pH units than that for the longer chain esters (pH approximately 4). The activity of the purified lipase was stimulated by several different proteins. The relationship of this effect to the possible requirement for a natural activator substance has not been determined.(ABSTRACT TRUNCATED AT 400 WORDS)     

Inactivation of Enzymes by Linoleic Acid Hydroperoxides and Linoleic Acid

The inactivation of the enzymes by linoleic acid hydroperoxides (LAHPO) was tested in connection with the toxicity of oxidized fat. At the same time, the inhibition of enzyme activities by linoleic acid was also tested. Ribonuclease (RNase), trypsin, chymotrypsin and pepsin which are considered to be simple proteins and not to be SH-enzymes were chosen as the enzymes. RNase was largely inhibited by LAHPO, but the other enzymes were inhibited by linoleic acid as well as LAHPO. The inhibition of each enzyme occurred at different pH. This fact may show that the inhibition occurs by binding of such hydrophobic compounds to the enzyme, and that the surface exposition of hydrophobic region may depend on the pH. Not only the reaction of some specific amino acid residue in the protein molecules with LAHPO, but also the binding of these hydrophobic compounds must be remembered in the mechanism of inhibition.     

Cloning and overexpression of the oah1 gene encoding O-acetyl-L-homoserine sulfhydrylase of Thermus thermophilus HB8 and characterization of the gene product

The oah1 gene of an extremely thermophilic bacterium, Thermus thermophilus HB8, was cloned, sequenced, and overexpressed in Escherichia coli cells. The gene product having a high O-acetyl-L-homoserine sulfhydrylase (EC 4.2.99.10) activity was purified to homogeneity, with a recovery of approximately 40% and a purification ratio of 81-fold, both calculated from the cell-homogenate. The protein showed molecular masses of approximately 163000 (for the native form) and 47000 (for the subunit). The isoelectric point was pH 6.0. The optimum temperature and pH for the activity were approximately 70 degrees C and pH 7.8, respectively. The enzyme was also shown to be very stable at high temperature (90% activity remaining at 90 degrees C for 60 min at pH 7.8) and in a wide range of pH (pH 4-12 at room temperature). The absorption spectrum showed a peak at 425 nm, and hydroxylamine hydrochloride (0.1 mM) inhibited approximately 90% of the activity, suggesting formation of a Schiff base with pyridoxal 5'-phosphate. The enzyme showed an apparent K(m) value of 6.8 mM for O-acetyl-L-homoserine, a V(max) value of 165 micromol/min per mg of protein at a fixed sulfide concentration of 5 mM, and also an apparent K(m) value of approximately 1.3 mM for sulfide (with 25 mM acetylhomoserine). L-Methionine (1 mM) inhibited the enzyme activity by 67%. Based on these findings, it was discussed that this enzyme might be inactive under ordinary conditions but might become active as an alternative homocysteine synthase in T. thermophilus HB8, only under such conditions as deficiency in transsulfuration, bringing about a sufficient amount of sulfide available in the cell.     

Purification and characterization of cathepsin L-like proteinase from goat brain

Cathepsin L-like proteinase was purified approximately 1708-fold with 40% activity yield to an apparent electrophoretic homogeneity from goat brain by homogenization, acid-autolysis at pH 4.2, 30-80% (NH4)2SO4 fractionation, Sephadex G-100 column chromatography and ion-exchange chromatography on CM-Sephadex C-50 at pH 5.0 and 5.6. The molecular weight of proteinase was found to be approximately 65,000 Da, by gel-filtration chromatography. The pH optima were 5.9 and 4.5 for the hydrolysis of Z-Phe-Arg-4mbetaNA (benzyloxycarbonyl-L-phenylalanine-L-arginine-4-methoxy-beta-naphthylamide) and azocasein, respectively. Of the synthetic chromogenic substrates tested, Z-Phe-Arg-4mbetaNA was hydrolyzed maximally by the enzyme (Km value for hydrolysis was 0.06 mM), followed by Z-Val-Lys-Lys-Arg-4mbetaNA, Z-Phe-Val-Arg-4mbetaNA, Z-Arg-Arg-4mbetaNA and Z-Ala-Arg-Arg-4mbetaNA. The proteinase was activated maximally by glutathione in conjunction with EDTA, followed by cysteine, dithioerythritol, thioglycolic acid, dithiothreitol and beta-mercaptoethanol. It was strongly inhibited by p-hydroxymercuribenzenesulphonic acid, iodoacetic acid, iodoacetamide and microbial peptide inhibitors, leupeptin and antipain. Leupeptin inhibited the enzyme competitively with Ki value 44 x 10(-9) M. The enzyme was strongly inhibited by 4 M urea. Metal ions, Hg(2+), Ca(2+), Cu(2+), Li(2+), K(+), Cd(2+), Ni(2+), Ba(2+), Mn(2+), Co(2+) and Sn(2+) also inhibited the activity of the enzyme. The enzyme was stable between pH 4.0-6.0 and up to 40 degrees C. The optimum temperature for the hydrolysis of Z-Phe-Arg-4mbetaNA was approximately 50-55 degrees C with an activation energy Ea of approximately 6.34 KCal mole(-1).     

Multiple forms of barley root acid phosphatase: purification and some characteristics of the major cytoplasmic isoenzyme

The major acid phosphatase form (orthophosphoric-monoester phosphohydrolase (acid optimum), EC 3.1.3.2) was purified from the soluble extract of barley roots. The enzyme is homogeneous on polyacrylamide gel electrophoresis and moves as a single band of Mr approximately 38,000 in the presence of sodium dodecyl sulphate. The molecular weight of the native enzyme was Mr 77,600 and 79,000 as determined, respectively, by gel filtration on a Sephadex G-100 column and by density gradient ultracentrifugation. The isoelectric point was about 6.28. The enzyme is competitively inhibited by molybdate (Ki = 9 x 10(-7) M). NaF, Ag(+), Hg(2+), Pb(2+) and Zn(2+) are also inhibitors, while other cations showed no effect. The enzyme hydrolyzes a wide variety of natural and synthetic phosphate esters. In particular, the enzyme seems to be active on ATP, o-phosphotyrosine, o-phosphoserine and glucose 1-phosphate. The pH dependence studies between pH 4-8 using p-nitrophenylphosphate as substrate and diethylpyrocarbonate inactivation indicate the presence of essential histidine residue at the active site.     

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.     

Purification and some properties of intracellular esterase from Pseudomonas fluorescens

A novel esterase was found in Pseudomonas fluorescens cells and purified to homogeneity as determined by polyacrylamide gel electrophoresis. The esterase was extracted from the cells by freeze-thawing and hypotonic treatment. Purification was achieved by ammonium sulfate precipitation, followed by successive chromatographies on DEAE-cellulose and benzylamine-agarose and then electrophoresis. The enzyme catalyzed the hydrolysis of methyl esters, such as methyl butyrate, but its hydrolyzing activity decreased with increase in the chain length of the alcohol moiety, and it did not catalyze the hydrolysis of triacylglycerols, such as triacetin. In contrast, the enzyme acted on various acyl residues in a series of methyl esters, such as dimethyl succinate, methyl methacrylate, and dimethyl malate. The optimum pH for activity of this enzyme with methyl butyrate was 7.0-8.5. The enzyme was inhibited by phenylmethylsulfonylfluoride. Its molecular weight was estimated as 48,000 by molecular sieve electrophoresis and gel filtration on Sephadex G-150.     

O-Acetylhomoserine sulfhydrylase of the fission yeast Schizosaccharomyces pombe: partial purification, characterization, and its probable role in homocysteine biosynthesis

A crude extract of Schizosaccharomyces pombe cells catalyzed sulfhydrylation of both O-acetyl-L-serine and O-acetyl-L-homoserine with H2S, but did not synthesize cystathionine from O-acetyl-L-homoserine and L-cysteine. The O-acetylhomoserine sulfhydrylase [EC 4.2.99.10] was very unstable; however, it could be stabilized by the addition of 25% (w/w) sucrose or glycerol. The optimal pH for activity was 8.0 and that for stability was 7.0. The enzyme was purified approximately 300-fold from an ammonium sulfate-precipitated fraction. L-Methionine was the most effective inhibitor among the amino acids examined. It inhibited the enzyme competitively with respect to OAH with a Ki value of 2.6 mM. Sulfhydrylase activity was inhibited to various extents by some carbonyl reagents, but sulfhydryl reagents such as p-chloromercuribenzoic acid, 5,5'-dithio-bis(2-nitrobenzoic acid), and monoiodoacetic acid had no inhibitory effect. The enzyme also reacted with O-succinylhomoserine and L-homoserine to synthesize homocysteine directly, but could not utilize cysteine as a co-substrate in place of H2S. In the sulfhydrylation reactions, Km values for the substrates ranged from 10.4-12.5 mM. The enzyme was resolved to the apoenzyme by incubation with phenylhydrazine and reactivated by the addition of pyridoxal 5'-phosphate, whose Km value was 0.083 microM. The molecular weight of the enzyme was estimated to be approximately 186,000 by gel filtration and 170,000 by ultracentrifugation in sucrose density gradients. The isolectric point of the protein was pH 4.1. The characteristics of this enzyme are compared with those of physiologically functional sulfhydrylases reported for other organisms, and the possibility of the enzyme functioning as a homocysteine synthase is discussed.     

Impact of reaction conditions on the laccase-catalyzed conversion of bisphenol A

The oxidative conversion of aqueous BPA catalyzed by laccase from Trametes versicolor was conducted in a closed, temperature-controlled system containing buffer for pH control. The effects of medium pH, buffer concentration, temperature and mediators and the impacts of dissolved wastewater constituents on BPA conversion were investigated. The optimal pH for BPA conversion was approximately 5, with greater than half maximal conversion and good enzyme stability in the range of 4-7. The stability of the enzyme was not impacted by buffer concentration, nor was BPA conversion. Despite the observation that the enzyme tended to be inactivated at elevated temperatures, enhanced conversion of BPA was observed up until a reaction temperature of 45 degrees C. Of the mediators studied, ABTS was most successful at enhancing the conversion of BPA. Dissolved wastewater constituents that were studied included various inorganic salts, organic compounds and heavy metal ions. BPA conversion was inhibited in the presence of anions such as sulfite, thiosulfate, sulfide, nitrite and cyanide. The metal ions Fe(III) and Cu(II) and the halogens chloride and fluoride substantially suppressed BPA conversion, but the presence of selected organic compounds did not significantly reduce the conversion of BPA.     

Purification and characterization of a carboxylesterase from rabbit liver lysosomes

Carboxylesterase [EC 3.1.1.1] was purified from rabbit liver lysosomes by means of detergent solubilization, and by hydroxyapatite, phenyl-Sepharose and chromatofocusing column chromatographies. The purified enzyme appeared to be homogeneous on SDS-polyacrylamide gel electrophoresis and its molecular weight was estimated to be 58,000. This enzyme was eluted at an isoelectric point of approximately 5.8 by chromatofocusing, and exhibited a broad pH optimum of between 6.0 and 9.0. The enzyme hydrolyzed 4-methylumbelliferyl esters of saturated fatty acids (C2-C12), and it also hydrolyzed p-nitrophenylacetate, methyl butyrate, and tributyrin, but not acetanilide. Its activity was completely inhibited by diisopropyl-fluorophosphate (DFP) and phenylmethylsulfonyl fluoride (PMSF) at 10(-4) M, but was not affected by eserine, or by alpha- or beta-naphthyl acetate at 10(-3) M. Various metal ions (Mg2+, Mn2+, Ca2+, Co2+, Cu2+, Zn2+, Ni2+) at 10(-3) M also had no effect on the enzyme activity.     

Isolation and characterization of a murine serum esterase which hydrolyzes a tumor promoter, 12-O-tetradecanoyl phorbol 13-acetate

An enzyme which catalyzes the following esterase reaction was isolated from mouse serum: 12-O-tetradecanoyl phorbol 13-acetate (TPA) + H2O----phorbol 13-acetate + tetradecanoic acid. The recovery was 0.18% of total serum protein and 820-fold purification was achieved. The enzyme is composed of a single polypeptide chain with sugar moiety; its molecular weight was estimated to be 77,000. Its sugar content is 15%, the isoelectric point was 4.3, and the alpha-helix content was 15.3% . The activity is stable between pH 5 and 9 under 40 degrees C; it is insensitive to 2-mercaptoethanol and is not dependent on divalent cations. The optimal pH is around 7.5. The apparent Km for TPA is 6.6 X 10(-7)M. The hydrolysis of [3H]TPA is inhibited by phorbol diesters and phorbol 12-myristate, but not by phorbol and phorbol 13-acetate. The activity is inhibited to some extent by phosphatidylcholine, cholesterol, and lanosterol, but not by free fatty acids, fatty acid esters of glycerol, cholesterol esters, or cholestanol. The enzyme hydrolyzes ester linkages, but not peptide linkages of synthetic substrates. Esterase inhibitors and serine-reactive reagents affect the activity. Although sera from rodents displayed strong activity, such activity was not detected in human serum. Unlike lipoprotein lipase, the serum enzyme activity was not enhanced by treatment of the animal with heparin. These characteristics and the amino acid composition do not agree with any of the reported characteristics of known serum enzymes with esterase activity.     

Purification and properties of a novel extra-cellular thermotolerant metallolipase of Bacillus coagulans MTCC-6375 isolate

A novel extra-cellular lipase from Bacillus coagulans MTCC-6375 was purified 76.4-fold by DEAE anion exchange and Octyl Sepharose chromatography. The purified enzyme was found to be electrophoretically pure by denaturing gel electrophoresis and possessed a molecular mass of approximately 103 kDa. The lipase was optimally active at 45 degrees C and retained approximately 50% of its original activity after 20 min of incubation at 55 degrees C. The enzyme was optimally active at pH 8.5. Mg2+, Cu2+, Ca2+, Hg2+, Al3+, and Fe3+ at 1mM enhanced hydrolytic activity of the lipase. Interestingly, Hg2+ ions resulted in a maximal increase in lipase activity but Zn2+ and Co2+ ions showed an antagonistic effect on this enzyme. EDTA at 150 mM concentration inhibited the activity of lipase but Hg2+ or Al3+ (10mM) restored most of the activity of EDTA-quenched lipase. Phenyl methyl sulfonyl fluoride (PMSF, 15 mM) decreased 98% of original activity of lipase. The lipase was more specific to p-nitrophenyl esters of 8 (pNPC) and 16 (pNPP) carbon chain length esters. The lipase had a Vmax and Km of 0.44 mmol mg(-1)min(-1) and 28 mM for hydrolysis of pNPP, and 0.7 mmol mg(-1)min(-1) and 32 mM for hydrolysis of pNPC, respectively.     

Spontaneous reactivation of phosphinylated human erythrocyte acetylcholinesterase and human serum butyrylcholinesterase

This report documents studies on the spontaneous reactivation of human erythrocyte acetylcholinesterase and human serum butyrylcholinesterase following inhibition by organophosphinate esters. The spontaneous reactivation reactions were carried out at 26.0 degrees C in 0.10 M phosphate buffer of pH 7.6. Based upon results at 24 h, human serum butyrylcholinesterase inhibited with 4-nitrophenyl methyl (4-methoxyphenyl) phosphinate was the most responsive (92.5% recovery) of the nine esters studied. Using the same criteria, the most active compound in the human erythrocyte acetylcholinesterase studies was 4-nitrophenyl methyl(phenyl)phosphinate (74.2% recovery). With seven of the nine compounds examined the response was greater from the serum enzyme than from the erythrocyte enzyme.