Showing papers on "Enzyme assay published in 1991"

Purification and characterization of particulate endothelium-derived relaxing factor synthase from cultured and native bovine aortic endothelial cells.

Abstract: The particulate enzyme responsible for the synthesis of endothelium-derived relaxing factor has been purified from cultured and native (noncultured) bovine aortic endothelial cells. Purification of the solubilized particulate enzyme preparation by affinity chromatography on adenosine 2',5'-bisphosphate coupled to Sepharose followed by Superose 6 gel filtration chromatography resulted in a single protein band after denaturing polyacrylamide gel electrophoresis that corresponded to approximately 135 kDa. The enzyme activity in the various fractions was assayed by its stimulatory effect on soluble guanylyl cyclase of rat fetal lung fibroblasts (RFL-6 cells), by the formation of L-citrulline from L-arginine, by measuring nitrite/nitrate formation, and by bioassay on endothelium-denuded vascular strips. Endothelium-derived relaxing factor synthase was purified 3419-fold from the crude particulate fraction of cultured bovine aortic endothelial cells with a 12% recovery (RFL-6 assay). Purified endothelium-derived relaxing factor synthase required L-arginine, NADPH, Ca2+, calmodulin, and 5,6,7,8-tetrahydrobiopterin for full activity.

Type I iodothyronine deiodinase is a selenocysteine-containing enzyme

Abstract: Although thyroxine (3,5,3',5'-tetraiodothyronine, T4) is the principal secretory product of the vertebrate thyroid, its essential metabolic and developmental effects are all mediated by 3,5,3'-triiodothyronine (T3), which is produced from the prohormone by 5'-deiodination. The type-I iodothyronine deiodinase, a thiol-requiring propylthiouracil-sensitive oxidoreductase, is found mainly in liver and kidney and provides most of the circulating T3(1) but so far this enzyme has not been purified. Using expression cloning in the Xenopus oocyte, we have isolated a 2.1-kilobase complementary DNA for this deiodinase from a rat liver cDNA library. The kinetic properties of the protein expressed in transient assay systems, the tissue distribution of the messenger RNA, and its changes with thyroid status, all confirm its identity. We find that the mRNA for this enzyme contains a UGA codon for selenocysteine which is necessary for maximal enzyme activity. This explains why conversion of T4 to T3 is impaired in experimental selenium deficiency and identifies an essential role for this trace element in thyroid hormone action.

Purification of a soluble isoform of guanylyl cyclase-activating-factor synthase.

Abstract: The soluble form of guanylyl cyclase-activating-factor (GAF) synthase from rat cerebellum was purified to homogeneity by sequential affinity chromatographic steps on adenosine 2',5'-bisphosphate (2',5'-ADP)-Sepharose and calmodulin-agarose. Enzyme activity during purification was bioassayed by the L-arginine-, NADPH-, and Ca2+/calmodulin-dependent formation of a plasma membrane-permeable nitric oxide-like factor that stimulated soluble guanylyl cyclase in RFL-6 cells. With calmodulin and NADPH as cofactors, purified soluble GAF synthase induced an increase of 1.05 mumol of cGMP per 10(6) RFL-6 cells per 3 min per mg of protein. The coproduct of this signal-transduction pathway appeared to be L-citrulline. GAF synthase catalyzed the conversion of 107 nmol of L-arginine into L-citrulline per min per mg of protein. Based on these assays, this represents a purification of GAF synthase of approximately 10,076- and 8925-fold with recoveries of 16% and 19%, respectively. Rechromatography of the purified enzyme on Mono P (isoelectric point = 6.1 +/- 0.3), Mono Q, and Superose 12 or 6 resulted in no further purification or increase in specific activity. A Stokes radius of 7.9 +/- 0.3 nm and a sedimentation coefficient s20,w of 7.8 +/- 0.2 S were used to calculate a molecular mass of about 279 +/- 25 kDa for the native enzyme. SDS/PAGE revealed a single protein band with a molecular mass of about 155 +/- 3 kDa. These data suggest that soluble GAF synthase purified from rat cerebellum is a homodimer of 155-kDa subunits and that enzyme activity is dependent upon the presence of calmodulin.

Molecular cloning, expression, and induction of berberine bridge enzyme, an enzyme essential to the formation of benzophenanthridine alkaloids in the response of plants to pathogenic attack.

Abstract: The berberine bridge enzyme [(S)-reticuline: oxygen oxidoreductase (methylene-bridge-forming), EC 1.5.3.9] is a vesicular plant enzyme that catalyzes the formation of the berberine bridgehead carbon of (S)-scoulerine from the N-methyl carbon of (S)-reticuline in a specific, unparalleled reaction along the biosynthetic pathway that leads to benzophenanthridine alkaloids. Cytotoxic benzophenanthridine alkaloids are accumulated in certain species of Papaveraceae and Fumariaceae in response to pathogenic attack and, therefore, function as phytoalexins. The berberine bridge enzyme has been purified to homogeneity from elicited cell-suspension cultures of Eschscholtzia californica, and partial amino acid sequences have been determined. A cDNA, isolated from a Agt11 cDNA bank of elicited E. californica cell-suspension cultures, coded for an open reading frame of 538 amino acids. The first 22 amino acids constitute the putative signal peptide. The mature protein has a Mr of 57,352, excluding carbohydrate. The berberine bridge enzyme was heterologously expressed in a catalytically active form in Saccharomyces cerevisiae. Southern hybridization with genomic DNA suggests that there is only one gene for the enzyme in the E. californica genome. Hybridized RNA blots from elicited E. californica cell-suspension cultures revealed a rapid and transient increase in poly(A)+ RNA levels that preceded both the increase in enzyme activity and the accumulation of benzophenanthridine alkaloids, emphasizing the integral role of the berberine bridge enzyme in the plant response to pathogens.

Expression of a maize sucrose phosphate synthase in tomato alters leaf carbohydrate partitioning.

Abstract: We isolated a complementary DNA sequence for the enzyme sucrose phosphate synthase (SPS) from maize utilizing a limited amino acid sequence. The 3509-bp cDNA encodes a 1068-amino acid polypeptide. The identity of the cDNA was confirmed by the ability of the cloned sequence to direct sucrose phosphate synthesis in Escherichia coli. Because no plant-specific factors were necessary for enzymatic activity, we can conclude that SPS enzyme activity is conferred by a single gene product. Sequence comparisons showed that SPS is distantly related to the enzyme sucrose synthase. When expressed from a ribulose bisphosphate carboxylase small subunit promoter in transgenic tomatoes, total SPS activity was boosted up to sixfold in leaves and appeared to be physiologically uncoupled from the tomato regulation mechanism. The elevated SPS activity caused a reduction of starch and increase of sucrose in the tomato leaves. This result clearly demonstrates that SPS is involved in the regulation of carbon partitioning in the leaves.

Rapid Modulation of Spinach Leaf Nitrate Reductase Activity by Photosynthesis I. Modulation in Vivo by CO2 Availability

Abstract: It has been shown recently that in spinach leaves (Spinacia oleracea) net photosynthesis and nitrate reduction are closely linked: when net photosynthesis was low because of stomatal closure, rates of nitrate reduction decreased (WM Kaiser, J Forster [1989] Plant Physiol 91: 970-974). Here we present evidence that photosynthesis regulates nitrate reduction by modulating nitrate reductase activity (NRA, EC 1.6.6.1). When spinach leaves were exposed to low CO2 in the light, extractable NRA declined rapidly with a half-time of 15 minutes. The inhibition was rapidly reversed when leaves were brought back to air. NRA was also inhibited when leaves were wilted in air; this inhibition was due to decreased CO2 supply as a consequence of stomatal closure. The modulation of NRA was stable in vitro. It was not reversed by gel filtration. In contrast, the in vitro inhibition of nitrate reductase (NR) by classical inhibitors such as cyanide, hydroxylamin, or NADH disappeared after removal of free inhibitors by gel filtration. The negative modulation of NRA in —CO2-treated leaves became manifest as a decrease in total enzyme activity only in the presence of free Mg2+ or Ca2+. Mg2+ concentrations required for observing half-maximal inhibition were about 1 millimolar. In the presence of EDTA, the enzyme activity was always high and rather independent of the activation status of the enzyme. NRA was also independent of the pH in the range from pH 7 to pH 8, at saturating substrate and Mg2+ concentrations. The apparent substrate affinities of NR were hardly affected by the in vivo modulation of NR. Only Vmax changed.

The core element of the EcoRII methylase as defined by protease digestion and deletion analysis.

Abstract: Binding of the EcoRII DNA methyltransferase to azacytosine-containing DNA protects the enzyme from digestion by proteases. The limit digest yields a product having a Mr on SDS-PAGE 20% less than the intact protein. The N terminus of the tryptic digestion product was sequenced and found to be missing the N terminal 82 amino acids. Under the conditions used unbound enzyme was digested to small peptides. Protection of the enzyme from protease digestion implies that the enzyme undergoes major conformational changes when bound to DNA. The trypsin sensitive region of the EcoRII methyltransferase occurs prior to the first constant region shared with other procaryotic DNA(cytosine-5)methyltransferases. To determine if this region played a role in substrate binding or specificity, N-terminal deletion mutants were studied. Deletion of 97 amino acids resulted in a decrease of enzyme activity. Further deletions caused a complete loss of activity. Enzyme deleted through amino acid 85 was purified and found to have the same specificity as wild type however there was an increase in Km for both S-adenosylmethionine (AdoMet) and DNA of 27 and 18 fold respectively. The N-terminus of the EcoRII methylase, although a variable region present in many procaryotic DNA(cytosine-5)methylases, plays no role in determining enzyme specificity, although it does contribute to the interaction with both AdoMet and DNA.

Induction of nitric oxide synthase in rat peritoneal neutrophils and its inhibition by dexamethasone.

Abstract: Rat peritoneal polymorphonuclear leukocytes (PMN) elicited with oyster glycogen contain a Ca(2+)-independent nitric oxide (NO) synthase which is induced in vivo in a time-dependent manner. When washed PMN containing low levels of enzyme activity were cultured ex vivo further expression of NO synthase was observed. This was inhibited by cycloheximide indicating that de novo synthesis of the enzyme occurred during the ex vivo incubation. Enzyme activity was enhanced by interferon (IFN)-gamma, but not by tumor necrosis factor (TNF)-alpha when added ex vivo. However, IFN-gamma and TNF-alpha synergized to increase further the expression of NO synthase. Treatment of rats with dexamethasone inhibited the induction of NO synthase in elicited PMN. This treatment reduced the accumulation of PMN by approximately 30%, without affecting cell viability. Dexamethasone also inhibited the induction of the NO synthase ex vivo in a concentration-dependent manner. Furthermore, the enhanced enzyme activity following treatment of PMN with cytokines was also inhibited by dexamethasone. Once induced, dexamethasone did not affect enzyme activity. These data indicate that PMN elicited in the rat peritoneum with oyster glycogen express an NO synthase in vivo and ex vivo. The induction of the enzyme can be further stimulated ex vivo with IFN-gamma and TNF-alpha and inhibited by dexamethasone. The inhibition of the induction of NO synthase in the PMN by dexamethasone may contribute to the anti-inflammatory activity of this and other glucocorticoids.

Primary structures of the precursor and mature forms of stearoyl-acyl carrier protein desaturase from safflower embryos and requirement of ferredoxin for enzyme activity.

Abstract: Stearoyl-acyl carrier protein (ACP) desaturase (EC 1.14.99.6) catalyzes the principal conversion of saturated fatty acids to unsaturated fatty acids in the synthesis of vegetable oils. Stearoyl-ACP desaturase was purified from developing embryos of safflower seed, and extensive amino acid sequence was determined. The amino acid sequence was used in conjunction with polymerase chain reactions to clone a full-length cDNA. The primary structure of the protein, as deduced from the nucleotide sequence of the cDNA, includes a 33-amino-acid transit peptide not found in the purified enzyme. Expression in Escherichia coli of a gene encoding the mature form of stearoyl-ACP desaturase did not result in an altered fatty acid composition. However, active enzyme was detected when assayed in vitro with added spinach ferredoxin. The lack of significant activity in vitro without added ferredoxin and the lack of observed change in fatty acid composition indicate that ferredoxin is a required cofactor for the enzyme and that E. coli ferredoxin functions poorly, if at all, as an electron donor for the plant enzyme.

Purification and characterization of a molluscan egg-specific NADase, a second-messenger enzyme.

Abstract: An egg-specific NADase has been purified to homogeneity from the ovotestis of the opisthobranch mollusk Aplysia californica. Unlike other NADases, the Aplysia enzyme generates primarily cyclic-ADP-ribose (cADPR) rather than ADP-ribose from NAD. cADPR has been shown to stimulate the release of Ca2+ from microsomes prepared from sea urchin egg and, when injected into intact eggs, to activate the cortical reaction, multiple nuclear cycles, and DNA synthesis. The Aplysia enzyme was initially identified as an inhibitor of cholera and pertussis toxin-catalyzed ADP-ribosylation. By the use of an NADase assay, it was purified from the aqueous-soluble fraction of ovotestis by sequential column chromatography. The enzyme has an apparent molecular mass of 29 kDa, a Km for NAD of 0.7 mM, and a turnover rate of approximately 27,000 mol NAD.min-1.mol enzyme-1 at 30 degrees C. Monoclonal antibodies were generated to the NADase. Immunoblots of two-dimensional gels revealed multiple isoforms of the enzyme, with pls ranging from 8.1 to 9.8. The multiple isoforms were resolved with a cation exchange high-pressure liquid chromatography column and shown to generate cADPR. Immunohistochemical analysis of cryostat sections of Aplysia ovotestis shows that the enzyme is specific to the eggs and restricted to large 5- to 10-microns granules or vesicles. To date the cADPR-generating enzyme activity has been identified in various organisms, including mammals. The Aplysia enzyme is the first example in which the enzyme that generates cADPR has been purified. All of the available evidence indicates that this NADase is a second-messenger enzyme, implying that other NADases may serve a similar function.

Expression of Ascorbic Acid Oxidase in Zucchini Squash (Cucurbita pepo L.)

Abstract: The expression of ascorbic acid oxidase was studied in zucchini squash (Cucurbita pepo L.), one of the most abundant natural sources of the enzyme. In the developing fruit, specific activity of ascorbic acid oxidase was highest between 4 and 6 days after anthesis. Protein and mRNA levels followed the same trend as enzyme activity. Highest growth rate of the fruit occurred before 6 days after anthesis. Within a given fruit, ascorbic acid oxidase activity and mRNA level were highest in the epidermis, and lowest in the central placental region. In leaf tissue, ascorbic acid oxidase activity was higher in young leaves, and very low in old leaves. Within a given leaf, enzyme activity was highest in the fast-growing region (approximately the lower third of the blade), and lowest in the slow-growing region (near leaf apex). High expression of ascorbic acid oxidase at a stage when rapid growth is occurring (in both fruits and leaves), and localization of the enzyme in the fruit epidermis, where cells are under greatest tension during rapid growth in girth, suggest that ascorbic acid oxidase might be involved in reorganization of the cell wall to allow for expansion. Based on the known chemistry of dehydroascorbic acid, the end product of the ascorbic acid oxidase-catalyzed reaction, we have proposed several hypotheses to explain how dehydroascorbic acid might cause cell wall "loosening."

Calcium-dependent phosphorylation of symbiosome membrane proteins from nitrogen-fixing soybean nodules : evidence for phosphorylation of nodulin-26.

Abstract: By using a peptide (CK-15) based on the COOH-terminal sequence of nodulin-26, we have demonstrated the presence of a Ca(2+)-dependent protein kinase in soluble as well as particulate fractions of nitrogen-fixing soybean (Glycine max) root nodules. Substantial enzyme activity was found in symbiosome membranes. The soluble enzyme was purified 1570-fold. The enzyme was fractionated from endogenous calmodulin and yet was fully activated by Ca(2+) (K(0.5) = 0.4 micromolar) in the absence of exogenous calmodulin, phosphatidylserine and 1,2-dioleylglycerol, oleic acid, and platelet activating factor. CK-15 was used to generate a site-specific antibody to nodulin-26. The antibody reacted with a protein in the symbiosome membrane with an apparent molecular mass of 27,000 daltons, consistent with the molecular mass predicted for nodulin-26 from the deduced amino acid sequence. A symbiosome membrane protein with an identical electrophoretic mobility was phosphorylated in vitro in a Ca(2+)-dependent manner. Additionally, this symbiosome membrane protein was phosphorylated when nodules were incubated with (32)P-phosphate. Overall, the results show the existence of a Ca(2+)-dependent and calmodulin/lipid-independent enzyme in nitrogen-fixing soybean root nodules and suggest that nodulin-26 is a substrate for Ca(2+)-dependent phosphorylation.

Tunicamycin-inhibited carrot somatic embryogenesis can be restored by secreted cationic peroxidase isoenzymes

Abstract: Somatic embryogenesis of carrot (Daucus carota L.) is inhibited by the glycosylation inhibitor tunicamycin. This inhibition is reversible by the addition of correctly glycosylated glycoproteins which have been secreted into the culture medium. To identify the proteins responsible for complementation, glycoproteins present in the medium of embryo cultures were purified and tested for their activity in the tunicamycin inhibition/ complementation assay. A 38-kDa glycoprotein was purified that could restore embryogenesis to more than 50% of that in untreated controls. This 38-kDa glycoprotein was identified as a heme-containing peroxidase on the basis of its A405/A280 ratio (Reinheit Zahl or RZ) and enzyme activity. The 38-kDa peroxidase consisted of four different cationic isoenzymes of which only one or possibly two appeared active in the complementation assay. The cationic peroxidase isoenzymes from the carrot medium could be effectively replaced by cationic horseradish peroxidases which depended on their catalytic properties for their ability to restore tunicamycin-inhibited somatic embryogenesis.

Novel cyanide-hydrolyzing enzyme from Alcaligenes xylosoxidans subsp. denitrificans.

Abstract: A cyanide-metabolizing bacterium, strain DF3, isolated from soil was identified as Alcaligenes xylosoxidans subsp. denitrificans. Whole cells and cell extracts of strain DF3 catalyzed hydrolysis of cyanide to formate and ammonia (HCN + 2H2O----HCOOH + NH3) without forming formamide as a free intermediate. The cyanide-hydrolyzing activity was inducibly produced in cells during growth in cyanide-containing media. Cyanate (OCN-) and a wide range of aliphatic and aromatic nitriles were not hydrolyzed by intact cells of A. xylosoxidans subsp. denitrificans DF3. Strain DF3 hydrolyzed cyanide with great efficacy. Thus, by using resting induced cells at a concentration of 11.3 mg (dry weight) per ml, the cyanide concentration could be reduced from 0.97 M (approximately 25,220 ppm) to less than 77 nM (approximately 0.002 ppm) in 55 h. Enzyme purification established that cyanide hydrolysis by A. xylosoxidans subsp. denitrificans DF3 was due to a single intracellular enzyme. The soluble enzyme was purified approximately 160-fold, and the first 25 NH2-terminal amino acids were determined by automated Edman degradation. The molecular mass of the active enzyme (purity, greater than 97% as determined by amino acid sequencing) was estimated to be greater than 300,000 Da. The cyanide-hydrolyzing enzyme of A. xylosoxidans subsp. denitrificans DF3 was tentatively named cyanidase to distinguish it from known nitrilases (EC 3.5.5.1) which act on organic nitriles.

A spectrophotometric assay for hydroperoxide lyase

Abstract: Spectrophotometric assays for hydroperoxide lyase have traditionally measured the loss in absorption at 234 nm due to the disruption of conjugated diene in the fatty acid hydroperoxide However, that assay does not distinguish between hydroperoxide lyase and hydroperoxide dehydrase activities, both of which cause a loss of conjugation in the substrate hydroperoxide A new assay has been developed which is specific for hydroperoxide lyase It is based on the ability of hydroperoxide lyase products, aldehydes and ω-oxoacids, to serve as substrates for yeast alcohol dehydrogenase Thus, the new hydroperoxide lyase assay is a coupled enzyme assay, which is conducted spectrophotometrically at 340 nm and measures the oxidation of nicotinamide adenenine dinucleotide, reduced form (NADH) In addition to its specificity for hydroperoxide lyase, the coupled assay allows higher concentrations of both fatty acid hydroperoxide substrate and crude enzyme extracts than the assay conducted at 234 nm

Induced plant responses to pathogen attack. Analysis and heterologous expression of the key enzyme in the biosynthesis of phytoalexins in soybean (Glycine max L. Merr. cv. Harosoy 63).

Abstract: In soybean (Glycine max L.), pathogen attack induces the formation of glyceollin-type phytoalexins. The biosynthetic key enzyme is a reductase which synthesizes 4,2', 4'-trihydroxychalcone in co-action with chalcone synthase. Screening of a soybean cDNA library from elicitor-induced RNA in lambda gt11 yielded two classes of reductase-specific clones. The deduced proteins match to 100% and 95%, respectively, with 229 amino acids sequenced in the purified plant protein. Four clones of class A were expressed in Escherichia coli, and the proteins were tested for enzyme activity in extracts supplemented with chalcone synthase. All were active in 4,2',4'-trihydroxychalcone formation, and the quantification showed that shorter lengths of the cDNAs at the 5' end correlated with progressively decreasing enzyme activities. Genomic blots with DNA from plants capable of 4,2',4'-trihydroxychalcone synthesis revealed related sequences in bean (Phaseolus vulgaris L.) and peanut (Arachis hypogaea L.), but not in pea (Pisum sativum L.). No hybridization was observed with parsley (Petroselinum crispum) and carrot (Daucus carota) which synthesize other phytoalexins. The reductase protein contains a leucine-zipper motif and reveals a marked similarity with other oxidoreductases most of which are involved in carbohydrate metabolism.

Regional distribution of 11 beta-hydroxysteroid dehydrogenase in rat brain

Abstract: The activity and distribution of 11 beta-hydroxysteroid dehydrogenase in rat brain is described. Oxidation of corticosterone to 11-dehydrocorticosterone was significantly increased by NADP; the reverse reaction was increased by NADPH. Cortisol was a poor substrate. Both 11-dehydrogenase (11-DH) and 11-oxoreductase (11-OR) activities were found in brains of rats from 6 days to adult, and 11-DH and 11-OR activities were positively correlated with each other. Highest enzyme activities were found in pituitary, cerebellum, hippocampus, and cortex. Lower levels were found in the olfactory region, hypothalamus, brain stem, preoptic nucleus, and amygdala. Two antisera to 11-DH, designated 56-125 and 56-126, reacted with a 34K component corresponding in mass to rat liver 11-DH on Western blots. The dominant species of protein in all brain regions reacting with rat liver 11-DH antibody 56-125, was at 26K mol wt. Antiserum 56-126 did not cross-react with the 26K protein. The 26K component was not a 34K degradation product. In each region of the brain, Western blot analysis showed that the 26K band intensity was directly proportional to enzyme activity. However, the 26K protein was devoid of 11-DH activity. All 11-DH and 11-OR activities were associated with the 34K antigen. The data demonstrate the nonuniform distribution of 11-DH in brain tissue. They are consistent with the notion that 11-DH may confer upon brain the ability to control intracellular levels of active glucocorticoids and in this way mediate steroid function within the cell.

Long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency.

Abstract: We describe the clinical features and biochemical findings of two patients with long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency. Both children presented with an acute metabolic crisis. Both had hypoglycemia and excreted even-chain unsubstituted dicarboxylic and 3-hydroxy-dicarboxylic acids in the urine. Measurement of the enzymes of fatty acid oxidation in cultured skin fibroblasts showed low activity of long-chain 3-hydroxyacyl-CoA dehydrogenase, but normal activity of short-chain 3-hydroxyacyl-CoA dehydrogenase. The defect was further characterized by immunoprecipitating the short-chain enzyme using monospecific antibodies. It is probably inherited as an autosomal recessive trait, inasmuch as intermediate enzyme activity was found in the fibroblasts from the parents of one child.

Cloning and sequencing of the 7 alpha-hydroxysteroid dehydrogenase gene from Escherichia coli HB101 and characterization of the expressed enzyme

Abstract: The 7 alpha-hydroxysteroid dehydrogenase (EC 111159) gene from Escherichia coli HB101 was cloned and expressed in E coli DH1 The hybrid plasmid pSD1, with a 28-kbp insert of chromosomal DNA at the BamHI site of pBR322, was subcloned into pUC19 to construct plasmid pSD3 The entire nucleotide sequence of an inserted PstI-BamHI fragment of plasmid pSD3 was determined by the dideoxy chain-termination method Within this sequence, the mature enzyme protein-encoding sequence was found to start at a GTG initiation codon and to comprise 765 bp, as judged by comparison with the protein sequence The deduced amino acid sequence of the enzyme indicated that the molecular weight is 26,778 The transformant of E coli DH1 harboring pSD3 with a 18-kbp fragment showed about 200-fold-higher enzyme activity than the host The enzyme was purified by a single chromatography step on DEAE-Toyopearl and obtained as crystals, with an activity yield of 39% The purified enzyme was homogeneous, as judged by sodium dodecyl sulfate gel electrophoresis The enzyme was most active at pH 85 and stable between pH 8 and 9 The enzyme was NAD+ dependent and had a pI of 43 The molecular mass was estimated to be 120 kDa by the gel filtration method and 28 kDa by electrophoresis, indicating that the enzyme exists in a tetrameric form

Detoxification of organophosphate pesticides using an immobilized phosphotriesterase from Pseudomonas diminuta.

Abstract: A purified phosphotriesterase was successfully immobilized onto trityl agarose in a fixed bed reactor. A total of up to 9200 units of enzyme activity was immobilized onto 2.0 mL of trityl agarose (65 micromol trityl groups/mL agarose), where one unit is the amount of enzyme required to catalyze the hydrolysis of one micromole of paraoxon in one min. The immobilized enzyme was shown to behave chemically and kinetically similar to the free enzyme when paraoxon was utilized as a substrate. Several organophosphate pesticides, methyl parathion, ethyl parathion, diazinon, and coumaphos were also hydrolyzed by the immobilized phosphotriesterase. However, all substrates exhibited an affinity for the trityl agarose matrix. For increased solubility and reduction in the affinity of these pesticides for the trityl agarose matrix, methanol/water mixtures were utilized. The effect of methanol was not deleterious when concentrations of less than 20% were present. However, higher concentrations resulted in elution of enzyme from the reactor. With a 10-unit reactor, a 1.0 mM paraoxon solution was hydrolyzed completely at a flow rate of 45 mL/h. Kinetic parameters were measured with a 0.1-unit reactor with paraoxon as a substrate at a flow rate of 22 mL/h. The apparent K(m) for the immobilized enzyme was 3-4 times greater than the K(m) (0.1 mM) for the soluble enzyme. Immobilization limited the maximum rate of substrate hydrolysis to 40% of the value observed for the soluble enzyme. The pH-rate profiles of the soluble and immobilized enzymes were very similar. The immobilization of phosphotriesterase onto trityl agarose provides an effective method esterase onto trityl agarose provides an effective method for hydrolyzing and thus detoxifyuing organophosphate pesticides and mammalian acetylcholinesterase inhinbitors.

Purification and characterization of a new k-carrageenase from a marine Cytophaga-like bacterium

Abstract: A bacterial strain able to degrade various sulfated galactans (carrageenans and agar) was isolated from the marine red alga Delesseria sanguinea. From the cell-free supernatant of cultures grown on crude lambda-carrageenan, a kappa-carrageenase was purified by ammonium sulfate fractionation, gel filtration on Sephacryl S 200 HR and ion-exchange chromatography on DEAE--Sepharose-CL6B. The purified kappa-carrageenase was detected as a single protein upon SDS/PAGE. Its molecular mass was estimated at 40 kDa. Activity was observed against kappa-carrageenan over the pH range 5.0-8.5 and was optimal at pH 7.2 in Tris buffer or 7.0 in Mops buffer. The enzyme activity remained stable at 30 degrees C, but only for up to 1 h at 40 degrees C. Analysis of the degradation products of the kappa-carrageenase by gel filtration and 13C-NMR spectroscopy indicated that the enzyme degrades kappa-carrageenan down to the level of the kappa-neocarratetraose sulfate. The properties of this new enzyme are compared with those of previously characterized carrageenases.

Purification and characterization of methionine gamma-lyase from Trichomonas vaginalis.

Abstract: Methionine gamma-lyase (EC 4.4.1.11) was purified to homogeneity from the anaerobic protozoan parasite Trichomonas vaginalis by a series of f.p.l.c. procedures. The enzyme catalyses alpha gamma- and alpha beta-elimination reactions of a number of derivatives of methionine and cysteine. It also catalyses gamma-replacement reactions of the thiomethyl group of methionine, homocysteine and ethionine to yield the corresponding S-substituted homocysteine derivative. The enzyme is pyridoxal 5'-phosphate-dependent, has a native molecular mass of approx. 160 kDa and consists of four apparently identical subunits of molecular mass 43-45 kDa. The absorption spectrum of the enzyme is typical of those obtained for other pyridoxal 5'-phosphate-dependent enzymes, and the holoenzyme can be resolved to the apoenzyme by incubation with hydroxylamine and reconstituted by addition of the cofactor. The enzyme activity is significantly affected by carbonyl and thiol reagents, is competitively inhibited by a number of substrate analogues and is completely inactivated by the suicide inhibitor DL-propargylglycine. The T. vaginalis enzyme is similar, in terms of activity and properties, to the enzymes found in a number of species of bacteria that metabolize methionine under anaerobic conditions. It is suggested that methionine catabolism may be of particular importance to the survival of T. vaginalis under microaerophilic conditions in its host.

Inhibition of ribonucleotide reductase by gallium in murine leukemic L1210 cells.

Abstract: Our previous studies of the mechanism of cell growth inhibition by gallium have suggested that the block in cellular iron uptake induced by transferrin-gallium results in an inhibition of the iron-dependent M2 subunit of ribonucleotide reductase. However, it is not known whether the inhibitory effect of gallium on ribonucleotide reductase is solely the result of limiting iron availability for enzyme activity or whether a direct effect of intracellular gallium on the enzyme is also involved. In the present study, utilizing a cell-free assay, we show that gallium nitrate directly inhibits CDP and ADP reductase activity. Inhibition of DNA synthesis by gallium nitrate thus appears to be due to a combination of a block in iron availability to ribonucleotide reductase and a direct inhibition of the enzyme by gallium.