A high cysteine containing thiol proteinase from the latex of Ervatamia heyneana: purification and comparison with ervatamin B and C from Ervatamia coronaria

A cysteine protease, with a high cysteine content and a high degree of amino terminal sequence homology with ervatamins B and C, has been purified from the latex of Ervatamia heyneana (Family Apocynaceae). The enzyme designated as heynein (M(r) = 23 kDa) has a comparatively high cysteine content (11), high isoelectric point (10.8), and high stability against pH (2.5-11.5), temperature (63 degrees C, 15 min), strong denaturants, and organic solvents. The enzyme has high specific activities for natural substrates such as casein and azoalbumin. The pH and temperature optima are pH 8.0-8.5 and 52 +/- 2 degrees C, respectively. Hydrolysis of synthetic substrates and digestion of bovine serum albumin confirm a distinct specificity of heynein as compared to ervatamins and papain. Also, heynein has distinct immunogenicity as monitored by enzyme-linked immunosorbent assay and Ouchterlony's double immunodiffusion. Strong enzyme activation by reducing agents such as beta-mercaptoethanol, dithiothreitol, and strong enzyme inhibition by thiol proteinase inhibitors such as E-64 and iodoacetic acid have evidenced heynein to be a cysteine protease. High stability, specific activity, and easy purification may make heynein a potential protease for food and biotechnology applications.     

Estimates of the chromium(VI) reducing capacity in human body compartments as a mechanism for attenuating its potential toxicity and carcinogenicity

Cathepsin L was purified from carp hepatopancreas by a method involving ammonium sulfate precipitation and a series of column chromatographies, in which the enzyme had an affinity toward Concanavalin A and Cibacron Blue F3GA. Its homogeneity was established by Native-PAGE, but two protein bands corresponding to molecular masses of 30,000 (single chain) and 24,000 (heavy chain) migrated on SDS-PAGE. The enzyme exhibited a maximum activity for carbobenzoxy-L-phenylalanyl-L-arginyl-4-methylcoumaryl-7-amide (Z-Phe-Arg-MCA) at pH 5.5-6.0 and 50 degrees C and the remarkable stability at pH 5.0-6.5 and below 40 degrees C. All tested cysteine protease inhibitors and TLCK and chymostatin markedly inhibited its activity, whereas the other serine protease inhibitors and a metalloprotease inhibitor negligibly affected it. In addition, several metal compounds reduced either its activity or stability to differing extents. Although EDTA alone caused an only marginal activation of the enzyme, its maximum activation required both 2 mM cysteine and 1 mM EDTA. The enzyme had an ability to hydrolyze three peptidyl-MCA substrates including Z-Phe-Arg-MCA, but all kinetic constants indicate that Z-Phe-Arg-MCA is the optical substrate to the enzyme.     

Purification and properties of dipeptidyl amino-peptidase I from chicken liver (author's transl)

Dipeptidyl aminopeptidase I (E.S. 3.4.14.1) from chicken liver was purified by the following steps: homogenization at pH 5, thermic precipitation, acetone fractionation and Sephadex G-100, DEAE-cellulose and organomercurial-Sepharose column fractionations. The purified enzyme appears to be homogeneous by polyacrylamide gel electrophoresis at both pH 4.5 and 8.3 and has an isoelectric point of 5.7 +/- 0.05. The molecular weight of the enzyme reale 167,000 +/- 17,000 on the Sephadex G-150 column chromatography. The optimum pH for hydrolysis of Gly-Phe-p-nitroanilide (GPNA) and Gly-Phe-B-naphthylamide was 5.8. The value of Km for the hydrolysis of GPNA was estimated at 3.3 mM. The enzyme required halide ions for activity and was activated by thiol reagents (dithiothreitol, cysteine and 2-mercaptoethanol). Accordingly, DAP I was inhibited by thiol-blocking reagents (PCMB, IAA, Hg2+). The enzyme oxidation with oxygen current was fostered by chloride anion (50 nM); nevertheless the activity was recovered when cysteine was present in the incubation mixture; the latter, besides, seems to perform as enzyme protector.     

Cystalysin, a 46-kilodalton cysteine desulfhydrase from Treponema denticola, with hemolytic and hemoxidative activities

A 46-kDa hemolytic protein, referred to as cystalysin, from Treponema denticola ATCC 35404 was overexpressed in Escherichia coli LC-67. Both the native and recombinant 46-kDa proteins were purified to homogeneity. Both proteins expressed identical biological and functional characteristics. In addition to its biological function of lysing erythrocytes and hemoxidizing the hemoglobin to methemoglobin, cystalysin was also capable of removing the sulfhydryl and amino groups from selected S-containing compounds (e.g., cysteine) producing H2S, NH3, and pyruvate. This cysteine desulfhydrase resulted in the following Michaelis-Menten kinetics: Km = 3.6 mM and k(cat) = 12 s(-1). Cystathionine and S-aminoethyl-L-cysteine were also substrates for the protein. Gas chromatography-mass spectrometry and high-performance liquid chromatography analysis of the end products revealed NH3, pyruvate, homocysteine (from cystathionine), and cysteamine (from S-aminoethyl-L-cysteine). The enzyme was active over a broad pH range, with highest activity at pH 7.8 to 8.0. The enzymatic activity was increased by beta-mercaptoethanol. It was not inhibited by the proteinase inhibitor TLCK (N alpha-p-tosyl-L-lysine chloromethyl ketone), pronase, or proteinase K, suggesting that the functional site was physically protected or located in a small fragment of the polypeptide. We hypothesize that cystalysin is a pyridoxal-5-phosphate-containing enzyme, with activity of an alphaC-N and betaC-S lyase (cystathionase) type. Since large amounts of H2S have been reported in deep periodontal pockets, cystalysin may also function in vivo as an important virulence molecule.     

Effect of tetramethyl pyrazine on coronary vasoconstriction induced by endothelin-1 in dogs

Cyclomaltodextrin glucanotranferase (CGTase) catalyzes the degradation of starch to form alpha-, beta- and gamma-cyclodextrin. Based on cyclodextrin formation, an alkalophilic Bacillus sp. ATCC 21783 was used for its high CGTase production ability, using corn and rice flakes as substrates. Maximum enzyme production was achieved after 96 hours at pH 9.0, temperature 37 degrees C, and 1% (w/v) of either substrate together with the addition of trub. The specific enzyme activity was determined by High Pressure Liquid Chromatography (HPLC) and expressed as using International Units based on total cyclodextrin formation. Optimum conditions for this determination were studied, finding that the best results are obtained at pH 5.0, 7.0 and 9.0, temperature 55 degrees C and 3 hours of incubation in 1% (w/v) of rice flakes as starch source.     

Sulphogalactolipid sulphohydrolase activity of arylsulphatase purified from a marine gastropod Charonia lampas

Sulphatide, cerebroside 3-sulphate was hydrolyzed at a considerable rate by arylsulphatase (aryl-sulphate sulphohydrolase, EC 3.1.6.1) purified from a marine gastropod, Charonia lampas. However, it was scarcely hydrolyzed by glycosulphatase (sugar-sulphate sulphohydrolase, EC 3.1.6.3) from the same origin. The same was observed with seminolipid, a sulphoglycerogalactolipid. The enzymatic characteristics of both sulphogalactolipid and sulphohydrolase activities of the arylsulphatase were determined as follows. The enzyme activities are stimulated by the addition of sodium taurodeoxycholate and MnCl2. The pH optimum of sulphatide sulphohydrolase activity was pH 5.0, while seminolipid sulphohydrolase activity had maximum activity at pH 5.5. Both of these pH versus activity curves were broad. The Km value was 6.22-10-5 M for both substrates. However, the V values were sulphatide were lower by a factor of one-third than those with seminolipid. These enzyme activities were inhibited by substrates of the arysulphatase, i.e., p-nitrophenyl sulphate, p-nitrocatechol sulphate, ascorbate 2-sulphate and each other sulphogalactolipid, but not by glucose 6-sulphate. Sulphate and phosphate anions inhibited both of the enzyme activities.     

Possible molecular mechanism of loss of homologous and heterologous gap junctional intercellular communication in rat liver epithelial cell lines

We have identified beta-galactosidase activity in purified bovine rod outer segments (ROS), using rho-nitrophenyl-beta-D-galactopyranoside (PNPG) and chlorophenol red-beta-D-galactopyranoside (CPRG) as substrates. This glycosylhydrolase activity did not appear to represent contamination from other retinal subcellular fractions, based upon the relative specific activities of beta-galactosidase vs. other hydrolases (N-acetyl-beta-glucosaminidase, alpha- and beta-mannosidase, alpha-fucosidase, and acid phosphatase) in bovine retina and ROS homogenates. Using PNPG as a substrate, two pH optima were observed (at 3.5 and 5.5), while the hydrolysis of CPRG exhibited a single, broad pH optimum centered at 5.5. In contrast, hydrolysis of PNPG and CPRG by retinal homogenates exhibited single pH optima, at 3.5 and 5.5., respectively. ROS beta-galactosidase activity increased linearly with time, temperature, and protein concentration, and obeyed Michaelis-Menten kinetics with both substrates. For PNPG, Vmax approximately 88 nmol/h/mg protein and the apparent Km approximately 147 microM. For CPRG, Vmax approximately 33 nmol/h/mg protein and the apparent Km approximately 50 microM. ROS beta-galactosidase activity was affected by carbohydrates and their derivatives: glucose, fucose, sucrose, maltose and N-acetyl-galactosamine were found to stimulate the activity, while D-galactono-gamma-lactone and, to a lesser extent, D-galactose were inhibitory. The enzyme activity also was slightly stimulated by [Cl-] and markedly by dithiothreitol (DTT), while rho-chloro-mercuribenzoic acid (PCMB) and rho-hydroxymercuribenzoic acid (PHMB) inactivated the enzyme. In addition, the enzymatic activity was also found to be differentially sensitive to various anionic and nonionic detergents. However, n-octyl-beta-D-glucoside was slightly stimulatory.(ABSTRACT TRUNCATED AT 250 WORDS)     

Immobilization of the tetrameric and monomeric forms of pigeon liver malic enzyme on Sepharose beads

Pigeon liver malic enzyme was chemically attached to Sepharose 4B-CL beads. The enzyme lost approximately 50% of its original activity when immobilization was carried out with 5 mg CNBr/ml gel. Immobilization performed at pH 8.0 or pH 4.5 resulted in the formation of matrix-bound tetramer and monomer, respectively. Matrix-bound reconstituted tetramer was derived from matrix-bound monomer by mixing the latter with soluble enzyme at pH 4.5, then raised the pH of the solution to 8.0. The matrix-bound monomer was demonstrated to be enzymically fully active in terms of specific activity. The pH profile for the enzymic reaction was similar for both soluble and immobilized enzymes. However, the latter had a broader range for the optimum pH (pH 6.8-7.8). The Arrhenius plots for all immobilized enzyme forms were biphasic with inflection at approximately 27 degrees C. The apparent Michaelis constants for the substrates increased about 2-3-fold after immobilization. All immobilized enzyme forms, including the matrix-bound monomer, showed substrate inhibition at high concentrations of L-malate. Both high-affinity and low-affinity binding sites for Mn2+ existed for all immobilized enzyme forms. These results are consistent with an existing asymmetric model, but are not compatible with a sequential model for the enzyme tetramer. The immobilized enzyme was stable for at least four months at 4 degrees C. As compared to soluble enzyme, the immobilized enzyme was less inhibited by (NH4)2SO4 or NaCl. It was also resistant to inactivation with periodate-oxidized aminopyridine adenine dinucleotide phosphate, an affinity label for malic enzyme. Incubation of the immobilized enzyme (1.25 microM) with the reagent (5.6 mM) resulted in pseudo-first-order inactivation with a rate constant of 0.0108 min-1 that was at least an order of magnitude smaller than that for the soluble enzyme.     

Purification of glutamine transaminase K/cysteine conjugate beta-lyase from rat renal cytosol based on hydrophobic interaction HPLC and gel permeation FPLC

Cysteine conjugate beta-lyase (beta-lyase, EC 4.4.1.13) was purified to homogeneity from rat renal cytosol using a new and highly efficient method, based on C3-hydrophobic interaction (HI) high-performance liquid chromatography (HPLC) in combination with gel permeation fast protein liquid chromatography. The purity of the enzyme was judged from SDS-PAGE and C18-reversed-phase HPLC. The beta-lyase was estimated to be a homodimer consisting of a 47,400-Da subunit with absorption maxima at 280 and 420-430 nm. The specific activity of the purified beta-lyase toward S-(1,2-dichlorovinyl)-L-cysteine (1,2-DCVC) in the presence of alpha-keto-gamma-methiolbutyric acid (KMB) was 6.4 mumol/min/mg protein, which is by far the highest value so far reported. Kinetic analysis of 1,2-DCVC metabolism by the enzyme in the presence of KMB gave Km and Vmax values of 0.33 mM and 8.4 mumol/min/mg protein, respectively. No significant activity of the purified enzyme was detectable with S-2-benzothiazolyl-L-cysteine up to 2 mM. The purified enzyme also had glutamine transaminase K activity (EC 2.6.1.64) as assayed with phenylalanine and KMB as substrates. This specific activity was 16.0 mumol/min/mg. Amino acid analysis of the purified beta-lyase was carried out and was found to be closely similar to the amino acid composition of five other pyridoxal phosphate (PLP)-containing amino acid amino-transferases. This suggests that glutamine transaminase K/cysteine conjugate beta-lyase is a typical member of the PLP-containing aminotransferase group.     

A novel fungal endo-beta-N-acetylglucosaminidase that specifically acts on plant glycoproteins

An endo-beta-N-acetylglucosaminidase specific for plant glycoprotein oligosaccharides was purified from the culture fluid of a fungus. The Mr of the purified enzyme was 89,000. This enzyme was stable at pH 5.5-7.0, up to 30 degrees C, and showed the highest activity at pH 6.0. Among sugar chains tested, xylose-containing sugar chains (M3X, M3FX, and M2FX) were the most favored substrates. Oligomannose type (M3, M5, and M9) and hybrid type (GNM3) sugar chains were hydrolyzed much more slowly than xylose-containing sugar chains, and a complex type sugar chain (GN2M3) was not hydrolyzed at all by the enzyme. Moreover, the enzyme released sugar chains from native horseradish peroxidase and stem bromelain, which were not hydrolyzed by other endo-beta-N-acetylglucosaminidases (Endo H, D, and F). The enzyme could transfer the xylose-containing sugar chain from bromelain to DNS-Asn-GlcNAc-Fuc.     

Isolation and some molecular properties of a trypsin-like enzyme from larvae of European corn borer Ostrinia nubilalis Hübner (Lepidoptera: Pyralidae)

A one-step high-yielding procedure is presented for the purification of a trypsin-like proteinase from Ostrinia nubilalis larvae, consisting of benzamidine-sepharose affinity chromatography. The purified enzyme was homogeneous as judged by SDS-PAGE. The enzyme presents a molecular mass of 24 650 Da, a maximum pH activity profile of 9.5, a remarkable thermal stability and an optimum temperature of about 53 degrees C Km values determined using N alpha-benzoyl-DL-arginine-ethylester and N alpha-benzoyl-DL-arginine-p-nitro-anilide were 3.2 x 10(-5) M and 4.1 x 10(-4) M respectively. The proteinase was inhibited by some typical serine proteinase inhibitors such as N alpha-tosyl-L-lysine chloromethyl ketone, soybean trypsin inhibitors, benzamidine and phenylmethylsulfonyl fluoride. In particular, it was competitively inhibited by benzamidine with a Ki of 1.2 x 10(-5) M, whereas it was not affected by cysteine proteinases inhibitors. Comparative analysis of the amino acid composition and N-terminal sequence of O. nubilalis proteinase confirmed that this enzyme is very similar to other serine proteinases from lepidopteran larvae.     

Purification and characterization of beta-glucosidase from Bacteroides JY-6, a human intestinal bacterium

A beta-glucosidase (EC 3.2.1.21.) was purified 2500-fold from Bacteroides JY-6, an intestinal anaerobic bacterium of human. The specific activity of the homogeneously purified enzyme was 210 mumol/min/mg protein. The enzyme (M(r) 75kDa) was an monomer whose pI and optimal pH values were 4.6 and 5.5-6, respectively. The best substrates were p-nitrophenyl beta-D-glucopyranoside and natural beta-bound glucosides, such as prunin and poncirenin. Puerarin, which is a C-glycoside, was weakly effective. However, cellobiose, alpha-bound glycosides and rhamnoglucosides were not effective. The apparent Kms for prunin and p-nitrophenyl-beta-D-glucopyranoside were determined to be 0.08 and 0.19 mM, respectively. The enzyme was strongly inhibited by p-chloromercuriphenylsulfonic acid and reaction products such as p-nitrophenol and glucose.     

The bifunctional enzyme leukotriene-A4 hydrolase is an arginine aminopeptidase of high efficiency and specificity

Leukotriene-A4 hydrolase (EC 3.3.2.6) cleaved the NH2-terminal amino acid from several tripeptides, typified by arginyl-glycyl-aspartic acid, arginyl-glycyl-glycine, and arginyl-histidyl-phenylalanine, with catalytic efficiencies (kcat/Km) > or = 1 x 10(6) M-1 s-1. This exceeds by 10-fold the kcat/Km for its lipid substrate leukotriene A4. Catalytic efficiency declined for dipeptides which had kcat/Km ratios 10-100-fold lower than tripeptides. Tetrapeptides and pentapeptides were even poorer substrates with catalytic efficiencies below 10(3) M-1 s-1. The enzyme preferentially hydrolyzed tripeptide substrates and single amino acid p-nitroanilides with L-arginine at the NH2 terminus. Peptides with proline at the second position were not hydrolyzed, suggesting a requirement for an N-hydrogen at the peptide bond cleaved. Peptides with a blocked NH2 terminus were not hydrolyzed. The specificity constant (kcat/Km) was optimal at pH 7.2 with pK values at 6.8 and 7.9; binding was maximal at pH 8.0. Serum albumins activated the peptidase, increasing tripeptide affinities (Km) by 3-10-fold and specificities (kcat/Km) by 4-13-fold. Two known inhibitors of arginine peptidases, arphamenine A and B, inhibited hydrolysis of L-arginine p-nitroanilide with dissociation constants = 2.0 and 2.5 microM, respectively. Although the primary role of LTA4 hydrolase is widely regarded as the conversion of the lipid substrate leukotriene A4 into the inflammatory lipid mediator leukotriene B4, our data are the first showing that tripeptides are "better" substrates. This is compatible with a biological role for the peptidase activity of the enzyme and may be relevant to the distribution of the enzyme in organs like the ileum, liver, lung, and brain. We present a model which accommodates the available data on the interaction of substrates and inhibitors with the enzyme. This model can account for overlap in the active site for hydrolysis of leukotriene A4 and peptide or p-nitroanilide substrates.     

Rapid purification and properties of betaine aldehyde dehydrogenase from Pseudomonas aeruginosa

Betaine aldehyde dehydrogenase (BADH) (EC 1.2.1.8) catalyzes the last, irreversible step in the synthesis of the osmoprotectant glycine betaine from choline. In Pseudomonas aeruginosa this reaction is also an obligatory step in the assimilation of carbon and nitrogen when bacteria are growing in choline or choline precursors. We present here a method for the rapid purification to homogeneity of this enzyme by the use of ion-exchange and affinity chromatographies on 2',5'-ADP-Sepharose, which results in a high yield of pure enzyme with a specific activity at 30 degreesC and pH 7.4 of 74.5 U/mg of protein. Analytical ultracentrifugation, gel filtration, chemical cross-linking, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis suggest that BADH from P. aeruginosa is a homodimer with 61-kDa subunits. The amino acid composition and the N-terminal sequence of 21 amino acid residues showed significant similarity with those of the enzymes from Xanthomonas translucens and Escherichia coli. Neither BADH activity nor BADH protein was found in cell extracts from bacteria grown in the absence of choline. In contrast to other BADHs studied to date, the Pseudomonas enzyme cannot use positively charged aldehydes other than betaine aldehyde as substrates. The oxidation reaction has an activation energy of 39.8 kJ mol-1. The pH dependence of the velocity indicated an optimum at pH 8.0 to 8.5 and the existence of two ionizable groups with macroscopic pK values of 7.0 +/- 0.1 and 9. 7 +/- 0.1 involved in catalysis and/or binding of substrates. The enzyme is inactivated at 40 degreesC, but activity is regained when the heated enzyme is cooled to 30 degreesC or lower. At the optimum pH of 8.0, the enzyme is inactivated by dilution, but it is stable at pH 6.5 even at very low concentrations. Also, P. aeruginosa BADH activity is rapidly lost on removal of K+. In all cases studied, inactivation involves a biphasic process, which was dependent on the enzyme concentration only in the case of inactivation by dilution. NADP+ considerably protected the enzyme against these inactivating conditions.     

Amino acid residues that define both the isoprenoid and CAAX preferences of the Saccharomyces cerevisiae protein farnesyltransferase. Creating the perfect farnesyltransferase

Studies of the yeast protein farnesyltransferase (FTase) have shown that the enzyme preferentially farnesylates proteins ending in CAAX (C = cysteine, A = aliphatic residue, X = cysteine, serine, methionine, alanine) and to a lesser degree CAAL. Furthermore, like the type I protein geranylgeranyltransferase (GGTase-I), FTase can also geranylgeranylate methionine- and leucine-ending substrates both in vitro and in vivo. Substrate overlap of FTase and GGTase I has not been determined to be biologically significant. In this study, specific residues that influence the substrate preferences of FTase have been identified using site-directed mutagenesis. Three of the mutations altered the substrate preferences of the wild type enzyme significantly. The ram1p-74D FTase farnesylated only Ras-CIIS and not Ras-CII(M,L), and it geranylgeranylated all three substrates as well or better than wild type. The ram1p-206DDLF FTase farnesylated Ras-CII(S,M,L) at wild type levels but could no longer geranylgeranylate the Ras-CII(M,L) substrates. The ram1p-351FSKN FTase farnesylated Ras-CIIS and Ras-CIIM but not Ras-CIIL. The ram1p-351FSKN FTase was not capable of geranylgeranylating the Ras-CII(M,L) substrates, giving this mutant the attributes of the dogmatic FTase that only farnesylates non-leucine-ending CAAX substrates and does not geranylgeranylate any substrate. These results suggest that the isoprenoid and protein substrate specificities of FTase are interrelated. The availability of a mutant FTase that lacked substrate overlap with the protein GGTase-I made possible an analysis of the role of substrate overlap in normal cellular processes of yeast, such as mating and growth at elevated temperatures. Our findings suggest that neither farnesylation of leucine-ending CAAX substrates nor geranylgeranylation by the FTase is necessary for these cellular processes.     

Purification and partial characterization of a neutral protease from a virulent strain of Bacillus cereus

The factors involved in the pathogenesis of Bacillus cereus (B. cereus) in non-gastrointestinal diseases are poorly investigated. Some researchers suggest that B. cereus proteases may be involved in these illnesses. The aim of this work was to purify and characterize a protease isolated from a virulent strain of B. cereus to explain its assumptive damaging effect. The enzyme was purified in a four-step procedure involving ammonium sulfate fractionation, acetone precipitation, Bio-Gel filtration and column chromatography on DEAE-cellulose (DE-52 cellulose). The enzyme appeared homogenous using disc electrophoresis. The specific activity of the protease was 72 U/mg of protein. The enzyme was shown to have a relative molecular mass of 29 kDa. The protease was most active at pH 7.0 and 40 degrees C with haemoglobin as the substrate. The enzyme was made completely inactive by ethylenediaminetetraacetic acid (EDTA), beta-mercaptoethanol, dithiothreitol (DTT) and benzamidine (at a concentration of 1 mM) and by diisopropylfluorophosphate (DIPF), L-cysteine, L-histidine, 1,10-phenanthroline (at a concentration of 10 mM). Divalent cations, especially Ca2+ increased enzyme activity. The enzyme hydrolysed haemoglobin, albumin and casein as the substrates. With haemoglobin and albumin as the substrates Michaelis-Menten kinetics was observed. The obtained Km values were 86 +/- 40 microM (SD, n = 3) and 340 +/- 100 microM (SD, n = 3) for haemoglobin and albumin, respectively. The corresponding Vmax values were 1.26 +/- 0.1 (SD, n = 3) and 0.38 +/- 0.07 (SD, n = 3) mumol of tyrosine liberated per min, per ml, and per mg, while those for casein were not determined. It is concluded that this enzyme is a metal-chelator-sensitive, neutral protease damaging haemoglobin and albumin.     

Purification, characterization, and substrate specificity of a novel highly glucose-tolerant beta-glucosidase from Aspergillus oryzae

Aspergillus oryzae was found to secrete two distinct beta-glucosidases when it was grown in liquid culture on various substrates. The major form had a molecular mass of 130 kDa and was highly inhibited by glucose. The minor form, which was induced most effectively on quercetin (3,3',4',5,7-pentahydroxyflavone)-rich medium, represented no more than 18% of total beta-glucosidase activity but exhibited a high tolerance to glucose inhibition. This highly glucose-tolerant beta-glucosidase (designated HGT-BG) was purified to homogeneity by ammonium sulfate precipitation, gel filtration, and anion-exchange chromatography. HGT-BG is a monomeric protein with an apparent molecular mass of 43 kDa and a pI of 4.2 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and isoelectric focusing polyacrylamide gel electrophoresis, respectively. Using p-nitrophenyl-beta-D-glucoside as the substrate, we found that the enzyme was optimally active at 50 degreesC and pH 5.0 and had a specific activity of 1,066 micromol min-1 mg of protein-1 and a Km of 0.55 mM under these conditions. The enzyme is particularly resistant to inhibition by glucose (Ki, 1. 36 M) or glucono-delta-lactone (Ki, 12.5 mM), another powerful beta-glucosidase inhibitor present in wine. A comparison of the enzyme activities on various glycosidic substrates indicated that HGT-BG is a broad-specificity type of fungal beta-glucosidase. It exhibits exoglucanase activity and hydrolyzes (1-->3)- and (1-->6)-beta-glucosidic linkages most effectively. This enzyme was able to release flavor compounds, such as geraniol, nerol, and linalol, from the corresponding monoterpenyl-beta-D-glucosides in a grape must (pH 2.9, 90 g of glucose liter-1). Other flavor precursors (benzyl- and 2-phenylethyl-beta-D-glucosides) and prunin (4',5,7-trihydroxyflavanone-7-glucoside), which contribute to the bitterness of citrus juices, are also substrates of the enzyme. Thus, this novel beta-glucosidase is of great potential interest in wine and fruit juice processing because it releases aromatic compounds from flavorless glucosidic precursors.     

Isolation and properties of the natural and the recombinant sialidase from Clostridium septicum NC 0054714

The natural sialidase of Clostridium septicum was purified and characterized in parallel with the recombinant enzyme expressed by Escherichia coli. The two enzymes exhibit almost identical properties. The maximum hydrolytic activity was measured at 37 degrees C in 60 mM sodium acetate buffer, pH 5.3. Glycoproteins like fetuin and saponified bovine submandibular gland mucin, most of them having alpha(2-6) linked sialic acids, are preferred substrates, while sialic acids from gangliosides, sialyllactoses, or the alpha(2-8) linked sialic acid polymer (colominic acid) are hydrolysed at lower rates. alpha(2-3) Linkages are more rapidly hydrolysed than alpha(2-6) bonds of sialyllactoses. The cleavage rate is markedly reduced by O-acetylation of the sialic acid moiety. These properties are similar to those of other secreted clostridial sialidases. The enzyme exists in mono-, di- and trimeric forms, the monomer exhibiting a molecular mass of 125 kDa, which is close to the protein mass of 111 kDa deduced from the nucleotide sequence of the cloned gene.     

Detection of enzymes active on various beta-1,3-glucans after denaturing polyacrylamide gel electrophoresis

Enzymes were assayed for glucanase activity after denaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in gels containing beta-1,3-glucans embedded as substrate. Lentinan, curdlan, paramylon, baker's yeast alkali-insoluble glucan, baker's yeast alkali-soluble glucan and carboxymethyl (CM)-pachyman were compared to oligomeric laminarin, which is the usual substrate for assaying beta-1,3-glucanase activities. Detecting enzyme activities by aniline blue fluorescent staining was also compared with the staining of released reducing sugars by 2,3,5-triphenyltetrazolium chloride (TTC). For the nonreduced proteins, the Driselase extract exhibited one major band at 32.5 kDa and one less intense band at 23 kDa for most substrates with the two detection procedures. No Lyticase enzyme was detected in either detection procedures for all tested substrates. For barley enzymes, no activity was revealed after aniline blue staining while one undescribed 19 kDa glucanase activity was best shown after TTC staining with curdlan, paramylon and CM-pachyman as substrates. In the case of reduced proteins, the Lyticase extract yielded three bands (33, 36 and 46 kDa) on several substrates with both detection procedures. This was the same for the barley leaf extract (32, 36 and 39 kDa). The Driselase extract showed one 42 kDa band. Many enzymes active on beta-1,3-glucans are thus best revealed when proteins are denatured and reduced and when protein renaturation after SDS-PAGE involves a pH 8.0 treatment and the inclusion of 1 mM cysteine in buffers. However, some enzymes are only detected when proteins are denatured without reduction. Finally, the use of various polymeric beta-1,3-glucan substrates different from oligomeric laminarin is necessary to detect new types of enzymes such as the 19 kDa barley glucanase.     

Human thioredoxin reductase from HeLa cells: selective alkylation of selenocysteine in the protein inhibits enzyme activity and reduction with NADPH influences affinity to heparin

Human thioredoxin reductase (TR) contains selenocysteine (Secys) in a redox center [cysteine (Cys)-497,Secys-498] near the C-terminus. The essential role of Secys in TR isolated from HeLa cells was demonstrated by the alkylation studies. Reaction of native NADPH reduced enzyme with bromoacetate at pH 6.5 inhibited enzyme activity 99%. Of the incorporated carboxymethyl (CM) group, 1.1 per subunit, >90% was in CM-Secys-498. Alkylation at pH 8 increased the stoichiometry to 1.6 per subunit with additional modification of the Cys-59, Cys-64 disulfide center. A minor tryptic peptide containing both CM-Cys-497 and CM-Secys-498 was isolated from enzyme alkylated at pH 6.5 or at pH 8. Preparations of TR isolated from HeLa cells grown in a fermentor under high aeration contained selenium-deficient enzyme species that had 50% lower activity. Decreasing oxygen to an optimal level increased cell yield, and fully active TR containing one Se per subunit was present. Reduction of fully active enzyme with tris-(2-carboxyethyl) phosphine converted it from a low to a high heparin affinity form. The tris-(2-carboxyethyl) phosphine-reduced enzyme was oxygen-sensitive and lost selenium and catalytic activity unless maintained under strictly anaerobic conditions. This enzyme could be converted to an oxygen-insensitive species by addition of NADPH, indicating that bound pyridine nucleotide is important for enzyme stability. An induced enzyme conformation in which the essential Secys is shielded from oxidative damage could explain these effects.