Showing papers on "Enzyme assay published in 1988"

Purification and characterization of a rotenone-insensitive NADH:Q6 oxidoreductase from mitochondria of Saccharomyces cerevisiae.

Abstract: A mitochondrial NADH: Q6 oxidoreductase has been isolated from cells of Saccharomyces cerevisiae by a simple method involving extraction of the enzyme from the mitochondrial membrane with Triton X-100, followed by chromatography on DEAE-cellulose and blue Sepharose CL-6B. By this procedure a 2000-fold purification is achieved with respect to whole cells or a 150-fold purification with respect to the mitochondrion. The purified NADH dehydrogenase consists of a single subunit with molecular mass of 53 kDa as indicated by SDS/poly-acrylamide gel electrophoresis. The enzyme contains FAD, non-covalently linked, as the sole prosthetic group with Em,7.67.6 = -370 mV and no iron-sulphur clusters. The enzyme is specific for NADH with apparent Km= 31 μM and was found to be inhibited by flavone (I50= 95 mUM), but not by rotenone or piericidin. The purified enzyme can use ubiquinone-2, -6 or -10, menaquinone, dichloroindophenol or ferricyanide as electron acceptors, but at different rates. The greatest turnover of NADH was obtained with ubiquinone-2 as acceptor (2500 s−1). With the natural ubiquinone-6 this value was 500s-1. The NADH:Q2 oxidoreductase activity shows a maximum at pH 6.2, the NADH:Q6 oxidoreductase activity is constant between pH 4.5-9.0. The amount of enzyme in the cell is subject to glucose repression: it increases slightly when cells, grown on glucose or lactate, enter the stationary phase. The experiments performed so far suggest that the enzyme purified in this study is the external NADH:Q6 oxidoreductase, bound to the mitochondrial inner membrane and that it is involved in the oxidation of cytosolic NADH. The relation of this enzyme with respect to various other NADH dehydrogenases from yeast and plant mitochondria is discussed.

Evidence for a cytoplasmic pathway of oxalate biosynthesis in Aspergillus niger.

Abstract: Oxalate accumulation of up to 8 g/liter was induced in Aspergillus niger by shifting the pH from 6 to 8. This required the presence of Pi and a nitrogen source and was inhibited by the protein synthesis inhibitor cycloheximide. Exogenously added 14CO2 was not incorporated into oxalate, but was incorporated into acetate and malate, thus indicating the biosynthesis of oxalate by hydrolytic cleavage of oxaloacetate. Inhibition of mitochondrial citrate metabolism by fluorocitrate did not significantly decrease the oxalate yield. The putative enzyme that was responsible for this was oxaloacetate hydrolase (EC 3.7.1.1), which was induced de novo during the pH shift. Subcellular fractionation of oxalic acid-forming mycelia of A. niger showed that this enzyme is located in the cytoplasm of A. niger. The results are consistent with a cytoplasmic pathway of oxalate formation which does not involve the tricarboxylic acid cycle.

Isolation of a chymotrypsinlike enzyme from Treponema denticola.

Abstract: A chymotrypsinlike protease with an Mr of 95,000 was extracted from Treponema denticola ATCC 35405 and was partially purified by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The proteolytic activity was detected in an electrophoretogram containing polyacrylamide that was conjugated to bovine serum albumin. A single band of activity was detected when the T. denticola extract was solubilized and electrophoresed in the presence of sodium dodecyl sulfate. No activity was found in extracts of Treponema vincentii. The enzyme hydrolyzed transferrin, fibrinogen, alpha 1-antitrypsin, immunoglobulin A, immunoglobulin G, gelatin, bovine serum albumin, and a synthetic peptide containing phenylalanine. It did not degrade collagen or synthetic substrates containing arginine or proline. For the hydrolysis of azocoll, the pH optimum of the enzyme was 7.5. Heating at temperatures above 50 degrees C destroyed the activity. Reducing agents and the chelators EDTA and ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid increased the enzyme activity, while phenylmethylsulfonyl fluoride, L-1-tosylamide-2-phenylethyl chloromethyl ketone, sulfhydryl reagents, and human serum reduced activity. The ability of the enzyme to hydrolyze a number of humoral proteins suggests that it may be involved in spirochete invasiveness and tissue destruction.

Ribonucleotide reductase of Brevibacterium ammoniagenes is a manganese enzyme.

Abstract: Ribonucleotide reduction and not DNA replication is the site for the specific manganese requirement of DNA synthesis and cell growth in the coryneform bacterium Brevibacterium ammoniagenes. To characterize the metal effect we have isolated and purified ribonucleoside-diphosphate reductase from overproducing bacteria that were first deprived of and then reactivated by manganese ions. Purification on columns of Sephacryl S400, DEAE-cellulose and hydroxyapatite provided an apparently homogeneous enzyme consisting of two protein subunits. These were characterized by affinity chromatography on 2',5'-ADP-Sepharose as nucleotide-binding protein B1 (Mr = 80,000) and catalytic protein B2 (Mr = 100,000, composed of two Mr = 50,000 polypeptides), which were both necessary for activity. In vitro the purified enzyme does not require added metal ions except for an unspecific, twofold activity increase observed in the presence of Mg2+ and other divalent cations. Enzyme activity is inhibited by hydroxyurea (I50 = 2.5 mM). The electronic spectrum with maxima around 455 nm and 485 nm closely resembles that of manganese(III)-containing pseudocatalase and of oxo-bridged binuclear Mn(III) model complexes. Denaturation of the enzyme in trichloroacetic acid liberated an equimolar amount of Mn(II) which was detected by EPR spectroscopy. It was not possible to remove and reintroduce metal ions without loss of enzyme activity. Manganese-deficient cell cultures were also grown in the presence of 54MnCl2. Ribonucleotide reductase activity and radioactivity cochromatographed in several systems. Non-denaturing polyacrylamide gel electrophoresis showed that protein subunit B2 was specifically 54Mn-labeled. All these properties suggest that the ribonucleotide reductase of B. ammoniagenes is a manganese-containing analog of the non-heme-iron-containing reductases of Escherichia coli and eukaryotes.

Kinetics of inhibition of tissue-type and urokinase-type plasminogen activator by plasminogen-activator inhibitor type 1 and type 2

Abstract: Highly purified plasminogen-activator inhibitors of type 1 (PAI-1) and type 2 (PAI-2), low-Mr form, were compared with respect to their kinetics of inhibition of tissue-type (t-PA) and urokinase-type plasminogen activator (u-PA). The time course of inhibition of plasminogen activator was studied under second-order or pseudo-first-order conditions. Residual enzyme activity was measured by the initial rate of hydrolysis of a chromogenic t-PA or u-PA substrate or by an immunosorbent assay for t-PA activity. PAI-1 rapidly reacted with single-chain t-PA as well as with two-chain forms of t-PA and u-PA. The second-order rate constant k for inhibition of single-chain t-PA (5.5 x 10(6) M-1 s-1) was about three times lower than k for inhibition of the two-chain activators. PAI-2 reacted slowly with single-chain t-PA, k = 4.6 x 10(3) M-1 s-1. The association rate was 26 times higher with two-chain t-PA and 435 times higher with two-chain u-PA. The k values for inhibition of single-chain t-PA, two-chain t-PA and two-chain u-PA were respectively, 1200, 150 and 8.5 times higher with PAI-1 than with PAI-2. The removal of the epidermal growth factor domain and the kringle domain from two-chain u-PA did not affect the kinetics of inhibition of the enzyme, suggesting that the C-terminal proteinase part of u-PA (B chain) is responsible for both the primary and the secondary interactions with PAI-1 and PAI-2. The k values for inhibition of single-chain t-PA and endogenous t-PA in plasma by PAI-1 or PAI-2 were identical indicating that t-PA in blood consists mainly in its single-chain form.

Developmental control of N-CAM sialylation state by Golgi sialyltransferase isoforms.

Abstract: Summary A rat brain Golgi sialyltransferase activity capable of the differentiation-dependent control of N-CAM sialylation state is described. The specific activity of Golgi sialyltransferase was found to be developmentally regulated with respect to both endogenous and exogenous protein acceptors, with a particular elevation on postnatal days 10-12 when the heavily sialylated or 'embryonic' form of N-CAM is re-expressed. The subsequent developmental decrease in activity was associated with a significant decrease in apparent K m for the CMP-NeuNAc substrate, but not for the asialofetuin exogenous acceptor, which could not be attributed to the temporal expression of an endogenous competitive inhibitor. The apparent Vmax remained constant for CMP-NeuNAc but was significantly reduced for asialofetuin. Sialyltransferase activity, which was optimal at pH70-7-5, was also modulated by various cations. Zinc abolished enzyme function, in contrast to ferric ions which stimulated activity fourfold-sevenfold. The marked activation of the adult form of the enzyme by potassium and magnesium ions, together with the alterations in kinetic constants, suggested this activity to be distinct from that derived from postnatal day-12 tissue. The kinetics of [ l4 C]sialic acid incorporation into immunoprecipitated N-CAM demonstrated the individual polypeptides to be sialylated, possibly by addition of polysialosyl units, in a developmental sequence. The presence of four distinct sialyltransferase activities was demonstrated by non-denaturing gel electrophoresis followed by solid-phase enzyme assay. These isoforms were temporally expressed during development, two being correlated with the postnatal reexpression of the 'embryonic' form of N-CAM.

Mammalian tyrosinase: biosynthesis, processing, and modulation by melanocyte-stimulating hormone

Abstract: We have examined the rate of synthesis and degradation of tyrosinase (monophenol, 3,4-dihydroxyphenylalanine:oxygen oxidoreductase, EC 1.14.18.1), the critical enzyme involved in mammalian pigmentation, using pulse-chase metabolic labeling of murine melanoma cells and immunoprecipitation of protein extracts with antibodies directed specifically against the enzyme. We have found that tyrosinase is synthesized and glycosylated within melanocytes rapidly, since significant quantities of pulse-labeled enzyme could be detected within 30 min. The maximum amount of enzyme was processed within 4 hr, and the t1/2 of tyrosinase in vivo was 10 hr (compared to 120 hr with purified enzyme), suggesting that tyrosinase activity in melanocytes is at least in part regulated by rapid synthesis and active degradation. We also have examined the melanogenic stimulation caused by melanocyte-stimulating hormone, using metabolic labeling, radiometric assays, and immunofluorescence techniques; responding cells increased their melanogenic potential more than 7-fold within 4 days without increasing their levels of tyrosinase synthesis. The results demonstrate that a pool of inactive tyrosinase exists in melanocytes and that rapid increases in enzyme activity elicited by melanocyte-stimulating hormone reflect an alteration in the activity of a preexisting pool of intracellular tyrosinase.

Production, Distribution, and Kinetic Properties of Inulinase in Continuous Cultures of Kluyveromyces marxianus CBS 6556.

Abstract: From a screening of several Kluyveromyces strains, the yeast Kluyveromyces marxianus CBS 6556 was selected for a study of the parameters relevant to the commercial production of inulinase (EC 3.2.1.7). This yeast exhibited superior properties with respect to growth at elevated temperatures (40 to 45 degrees C), substrate specificity, and inulinase production. In sucrose-limited chemostat cultures growing on mineral medium, the amount of enzyme decreased from 52 U mg of cell dry weight at D = 0.1 h to 2 U mg of cell dry weight at D = 0.8 h. Experiments with nitrogen-limited cultures further confirmed that synthesis of the enzyme is negatively controlled by the residual sugar concentration in the culture. High enzyme activities were observed during growth on nonsugar substrates, indicating that synthesis of the enzyme is a result of a derepression/repression mechanism. A substantial part of the inulinase produced by K. marxianus was associated with the cell wall. The enzyme could be released from the cell wall via a simple chemical treatment of cells. Results are presented on the effect of cultivation conditions on the distribution of the enzyme. Inulinase was active with sucrose, raffinose, stachyose, and inulin as substrates and exhibited an S/I ratio (relative activities with sucrose and inulin) of 15 under standard assay conditions. The enzyme activity decreased with increasing chain length of the substrate.

Purification and properties of benzoate-coenzyme A ligase, a Rhodopseudomonas palustris enzyme involved in the anaerobic degradation of benzoate.

Abstract: A soluble benzoate-coenzyme A (CoA) ligase was purified from the phototrophic bacterium Rhodopseudomonas palustris. Synthesis of the enzyme was induced when cells were grown anaerobically in light with benzoate as the sole carbon source. Purification by chromatography successively on hydroxylapatite, phenyl-Sepharose, and hydroxylapatite yielded an electrophoretically homogeneous enzyme preparation with a specific activity of 25 mumol/min per mg of protein and a molecular weight of 60,000. The purified enzyme was insensitive to oxygen and catalyzed the Mg2+ ATP-dependent formation of acyl-CoA from carboxylate and free reduced CoA, with high specificity for benzoate and 2-fluorobenzoate. Apparent Km values of 0.6 to 2 microM for benzoate, 2 to 3 microM for ATP, and 90 to 120 microM for reduced CoA were determined. The reaction product, benzoyl-CoA, was an effective inhibitor of the ligase reaction. The kinetic properties of the enzyme match the kinetics of substrate uptake by whole cells and confirm a role for benzoate-CoA ligase in maintaining entry of benzoate into cells as well as in catalyzing the first step in the anaerobic degradation of benzoate by R. palustris. Images

Thermostable alkaline protease produced by Bacillus thermoruber — a new species of Bacillus

Abstract: The proteolytic activity produced by a new species of Bacillus isolated in our laboratory was investigated. This enzyme was purified to homogeneity from cell-free culture liquids of B. thermoruber. The purification procedure included ion-exchange chromatography on DEAE-Sephadex A-50 and α-casein agarose affinity chromatography. The protease consists of one polypeptide chain with a molecular weight of 39000±800. the isoelectric point was 5.3; the optimum pH and temperature for proteolytic activity (on casein) was found to be pH 9 and 45°C respectively. Enzyme activity was inhibited by PMSF and EDTA. The stability was considerably increased by addition of Ca2+, and the protease exhibited a relatively high thermal stability. The alkaline protease shows a preference for leucine in the carboxylic side of the peptide bond of the substrate. The Kmvalue for benzyloxycarbonyl-Ala-Ala-Leu-p-nitroanilide was 2.5 mM.

Developmental Regulation of Enzymes of Indole Alkaloid Biosynthesis in Catharanthus roseus.

Abstract: Developing seedlings of Catharanthus roseus were analyzed for appearance of tryptophan decarboxylase (TDC), strictosidine synthase (SS), N-methyltransferase (NMT) and O-acetyltransferase (DAT) enzyme activities. SS enzyme activity appeared early after germination and was present throughout most of the developmental study. TDC activity was highly regulated and peaked over a 48 hour period achieving a maximum by day of 5 of seedling development. Both TDC and SS were present in all tissues of the seedling. NMT and DAT enzyme activities were induced after TDC and SS had peaked and these activities could only be found in hypocotyls and cotyledons. TDC, SS, and NMT did not require light for induction whereas DAT enzyme activity was increased approximately 10-fold after light treatment of dark grown seedlings.

New type of pullulanase from Bacillus stearothermophilus and molecular cloning and expression of the gene in Bacillus subtilis

Abstract: A new type of pullulanase which mainly produced panose from pullulan was found in Bacillus stearothermophilus and purified. The enzyme can hydrolyze pullulan efficiently and only hydrolyzes a small amount of starch. When pullulan was used as a substrate, the main product was panose and small amounts of glucose and maltose were simultaneously produced. By using pTB522 as a vector plasmid, the enzyme gene was cloned and expressed in Bacillus subtilis. Since the enzyme from the recombinant plasmid carrier could convert pullulan into not only panose but also glucose and maltose, we concluded that these reactions were due to the single enzyme. The new pullulanase, with a molecular weight of 62,000, was fairly thermostable. The optimum temperature was 60 to 65 degrees C, and about 90% of the enzyme activity was retained even after treatment at 60 degrees C for 60 min. The optimum pH for the enzyme was 6.0.

Oxidative and reductive acetyl-CoA/carbon monoxide dehydrogenase pathway in Desulphovibrio autotrophicum. 2. Demonstration of the enzymes of the pathway and comparison of CO dehydrogenase.

Abstract: It has been proposed that in some anaerobic facultatively autotrophic bacteria the acetyl CoA/CO dehydrogenase pathway is operating both in the reductive and in the oxidative direction, depending on the growth conditions. One of these anaerobes, the Gram-negative sulfate-reducing cubacterium Desulfobacterium autotrophicum, was examined for enzymes of the proposed pathway. All the required enzyme activities were present in sufficient amounts both in autotrophically and in heterotrophically grown cells, provided that the cellular tetrahydropterin rather than tetrahydrofolate was used as cosubstrate in some of the enzyme assays. The question arises whether two sets of enzymes are operating in the reductive and oxidative direction, respectively. The key enzyme of this pathway, CO dehydrogenase, which was reasonably oxygen stable, was analysed by native polyacrylamide gel electrophoresis and anaerobic activity staining. Extracts from heterotrophically grown cells exhibited five enzyme activity bands. Extracts from autotrophically grown cells showed the same pattern but an additional activity band appeared.

Aspergillus ficuum phytase: partial primary structure, substrate selectivity, and kinetic characterization.

Abstract: Purified Aspergillus ficuum phytase's partial primary structure and amino acid and sugar composition were elucidated. Determination of kinetic parameters of the enzyme at different pH values and temperatures indicated no significant alteration of the Km for phytate while the Kcmt was affected. The enzyme was able to release more than 512 of the total available P1 from phytate in a 3.0 hr assay at 58°C, but the Kcmt dropped to 15Z of the initial rate. Substrate selectivity studies revealed phytate to be the preferred substrate. The pH optima of phytase was 5.0, 4.0, and 3.0 for phytate, ATP, and polyphosphate, respectively. The enzyme had varied sensitivity towards cations. While Ca±± and Fe±±produced no effect on the catalytic rate of the enzyme, Cu±, Cu±±, Zn±±, and Fe±±± were found to be inhibitory. Mn±± was observed to enhance enzyme activity by 33Z at 50 μM. Known inhibitors of acid phosphatases e. g. L (±)-tartrate, phosphomycin, and sodium fluoride had no effect on enzyme activity.

Deoxycytidine kinase from human leukemic spleen: preparation and characterization of the homogeneous enzyme

Abstract: Deoxycytidine kinase from human leukemic spleen has been purified 6000-fold to apparent homogeneity with an overall yield of 10%. The purification was achieved by using DEAE chromatography, hydroxylapatite chromatography, and affinity chromatography on dTTP-Sepharose. Only one form of deoxycytidine kinase activity was found during all the chromatographic procedures. The subunit molecular mass, as judged by sodium dodecyl sulfate--polyacrylamide gel electrophoresis, was 30 kilodaltons. The pure enzyme phosphorylates deoxycytidine, deoxyadenosine, and deoxyguanosine, demonstrating for the first time that the same enzyme molecule has the capacity to use these three nucleosides as substrates. The apparent molecular weight of the active enzyme, determined by gel filtration and glycerol gradient centrifugation, was 60,000. Thus, the active form of human deoxycytidine kinase is a dimer. The kinetic behavior of pure human deoxycytidine kinase was studied in detail with regard to four different phosphate acceptors and two different phosphate donors. The apparent Km values were 1, 20, 150, and 120 microM for deoxycytidine, arabinosylcytosine, deoxyguanosine, and deoxyadenosine, respectively. The Vmax values were 5-fold higher for the purine nucleosides as compared to the pyrimidine substrates. We observe competitive inhibition of the phosphorylation of one substrate by the presence of either of the three other substrates, but the apparent Ki values differed greatly from the corresponding Km values, suggesting the existence of allosteric effects. The double-reciprocal plots for ATP-MgCl2 as phosphate donor were convex, indicating negative cooperative effects. In contrast, plots with varying dTTP-MgCl2 concentration as phosphate donor were linear with an apparent Km of 2 microM. The enzyme activity was strongly inhibited by dCTP, in a noncompetitive way with deoxycytidine and in a competitive way with ATP-MgCl2.

Species-specific effects of chronic nerve stimulation upon tibialis anterior muscle in mouse, rat, guinea pig, and rabbit.

Abstract: Tibialis anterior (TA) muscle of mouse, rat, guinea pig, and rabbit was indirectly stimulated for 10 h/day at 10 Hz up to 28 days. Changes in the activity levels of hexokinase (HK), phosphofructokinase (PFK) glyceraldehydephosphate dehydrogenase (GAPDH), lactate dehydrogenase (LDH), creatine kinase (CK), citrate synthase (CS), malate dehydrogenase (MDH), 3-hydroxyacyl-CoA dehydrogenase (HADH), and β-hydroxybutyrate dehydrogenase (HBDH) were compared. Although the direction of changes in the enzyme activity pattern was in accordance with previous findings on rabbit TA, the magnitude of the responses varied markedly between themammals under study. Mouse TA was almost unaffected. A major effect of chronic stimulation in rat, guinea pig and rabbit was an increase in enzyme activities of aerobic-oxidative metabolism. According to intrinsic differences of the muscles under study, the increases varied among the species and appeared to be inversely related to the basal levels of these enzymes in the unstimulated muscles. Conversely, glycolytic enzyme activities (PFK, GAPDH, LDH) markedly decreased in rat, guinea pig, and rabbit, and were only slightly reduced in mouse. Changes in HK and HBDH activities displayed the largest variations in the induced change between species. These results indicate species-specific patterns of metabolic adaptation to increased contractile activity.

Effect of environmental pH on enzyme activity and growth of Bacteroides gingivalis W50.

Abstract: Since the pH of the gingival crevice increases from below neutrality in health to above pH 8 in disease, we decided to investigate the effect of environmental pH on the growth and enzyme activity of Bacteroides gingivalis W50. Cells were grown in a chemostat under hemin-excess conditions over a range of pH values; stable growth was observed only between pH 6.7 and 8.3, with the maximum yields obtained between pH 7.0 and 8.0. The enzyme profile of cells varied markedly with pH. Enzymes with a specificity for gingival connective tissue (collagenase, hyaluronidase) were produced optimally at or below neutral pH, whereas trypsinlike activity increased with the growth pH and was maximal at pH 8.0. Chymotrypsinlike activity was generally low, although its activity was highest at the extremes of growth pH, i.e., at pH 6.7 and 8.3. Inhibitor studies provided evidence that the breakdown of collagen involved the concerted action of both a collagenase and the trypsinlike enzyme. The ratio of trypsin to collagenolytic activity rose from 1:1 during growth at neutral pH and below to almost 7:1 during growth at pH 8.3. Thus B. gingivalis appears to be uniquely adapted as a periodontopathic organism in that under environmental conditions likely to prevail during the initial stages of pocket development it produces maximally those enzymes with a tissue-damaging potential. Then, as the pH of the pocket rises during the host inflammatory response, the activity of the trypsinlike enzyme increases markedly, which may enable cells to inactivate key components of the host defenses such as immunoglobulins and complement.

Dichloromethane dehalogenase with improved catalytic activity isolated from a fast-growing dichloromethane-utilizing bacterium.

Abstract: A methylotrophic bacterium, denoted strain DM11, was isolated from groundwater and shown to utilize dichloromethane or dibromomethane as the sole carbon and energy source. The new isolate grew at the high rate of 0.22 h-1 compared with 11 previously characterized dichloromethane-utilizing bacteria (micromax, 0.08 h-1). The dichloromethane dehalogenase from strain DM11 (group B enzyme) was purified by anion-exchange chromatography. It was shown to be substantially different from the set of dichloromethane dehalogenases from the 11 slow-growing strains (group A enzymes) that had previously been demonstrated to be identical. The Vmax for the group B enzyme was 97 mkat/kg of protein, some 5.6-fold higher than that of the group A enzymes. The group A dehalogenases showed hyperbolic saturation with the cosubstrate glutathione, whereas the group B enzyme showed positive cooperativity in glutathione binding. Only 1 of 15 amino acids occupied common positions at the N termini, and amino acid contents were substantially different in group A and group B dehalogenases. Immunological assays demonstrated weak cross-reactivity between the two enzymes. Despite the observed structural and kinetic differences, there is potentially evolutionary relatedness between group A and group B enzymes, as indicated by (i) hybridization of DM11 DNA with a gene probe of the group A enzyme, (ii) a common requirement for glutathione in catalysis, and (iii) similar subunit molecular weights of about 34,000. Images

Purification and properties of the inducible coenzyme A-linked butyraldehyde dehydrogenase from Clostridium acetobutylicum.

Abstract: The coenzyme A (CoA)-linked butyraldehyde dehydrogenase (BAD) from Clostridium acetobutylicum was characterized and purified to homogeneity. The enzyme was induced over 200-fold, coincident with a shift from an acidogenic to a solventogenic fermentation, during batch culture growth. The increase in enzyme activity was found to require new protein synthesis since induction was blocked by the addition of rifampin and antibody against the purified enzyme showed the appearance of enzyme antigen beginning at the shift of the fermentation and increasing coordinately with the increase in enzyme specific activity. The CoA-linked acetaldehyde dehydrogenase was copurified with BAD during an 89-fold purification, indicating that one enzyme accounts for the synthesis of the two aldehyde intermediates for both butanol and ethanol production. Butanol dehydrogenase activity was clearly separate from the BAD enzyme activity on TEAE cellulose. A molecular weight of 115,000 was determined for the native enzyme, and the enzyme subunit had a molecular weight of 56,000 indicating that the active form is a homodimer. Kinetic constants were determined in both the forward and reverse directions. In the reverse direction both the Vmax and the apparent affinity for butyraldehyde and caproaldehyde were significantly greater than they were for acetaldehyde, while in the forward direction, the Vmax for butyryl-CoA was fivefold that for acetyl-CoA. These and other properties of BAD indicate that this enzyme is distinctly different from other reported CoA-dependent aldehyde dehydrogenases.

Purification and characterization of human salivary peroxidase.

Abstract: Human salivary peroxidase (SPO) has been purified to homogeneity by subjecting human parotid saliva to immunoaffinity, cation exchange, and affinity chromatography. These procedures resulted in a 992-fold purification of the enzyme. When purified SPO was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), three Coomassie stainable bands were apparent, all of which stained positive for enzyme activity. The apparent molecular weights of the three bands were 78,000, 80,000, and 280,000 as analyzed by SDS-PAGE. Reduction with 2-mercaptoethanol resulted in a decreased mobility of these bands, and enzyme activity could no longer be detected on the gels. The SPO preparation had the characteristic peroxidase heme spectrum in the range 405-420 nm. The ratio between the absorbance of the Soret band (412 nm) and the absorbance at 280 nm was 0.81. The enzyme activity was inhibited by the classical peroxidase inhibitors cyanide and azide. Salivary peroxidase is similar to bovine lactoperoxidase (LPO) in amino acid composition, in ultraviolet and visible spectrum, in reaction with cyanide, in susceptibility to 2-mercaptoethanol inactivation, and in thermal stability. The two enzymes differ in carbohydrate composition and content. SPO contains 4.6% and LPO 7% total neutral sugars. The ratio of glucosamine to galactosamine is 2:1 in SPO and 3:1 in LPO. SPO contains mannose, fucose, and galactose in a molar ratio of 1.5:1.5:1.0, while the ratio was 14.9:0.5:1.0 in LPO. Glucose was present in both preparations in minor amounts. The concentration of azide required for 50% inhibition of enzyme activity was 20-fold greater for LPO than for SPO.(ABSTRACT TRUNCATED AT 250 WORDS)

Glutathione synthetase in tobacco suspension cultures: catalytic properties and localization

Abstract: Glutathione synthetase activity (EC 6.3.2.3) was analysed in ammonium sulfate precipitates of extracts l'rom photohetevotrophically grown cells of Nicotiana tabactm L. cv. Samsun by determination of glutathione as its monobromobimane derivative. Maximal enzyme activity was obtained at pH 8.0–9.0 in Tris-HCl and CHES as buffer systems. The enzyme showed an absolute requirement for Mg2+ and was slightly stimulated by K+. When Mg2+ was replaced by Mn2+ less synthetase activity was observed, and above 30 mM Mn2+ no activity was found. The enzyme was specific for glycine (KM = 0.308 mM). No product formation was observed with s-alanine and γy-aminobutyrate using substrate conccntrations of 10 mM. The apparent KM values for γ-glutamylcysteine and γ-glutamyl-l-α-aminobutyrate were, respectively, 0.022 and 0.033 mM. By chloroplast Isolation ca 24% of the total glutathione synthetase activity of the cells could be shown to be localized in the chloroplasts, the rest being attributed to the cytoplasm of the tobacco cells.

A plasma membrane-bound enzyme oxidizes L-tryptophan to indole-3-acetaldoxime.

Abstract: The in vitro conversion of [14C]-tryptophan to [14C]-indole-3-acetaldoxime (IAOX) by microsomal membranes of Chinese cabbage (Brassica campestris ssp. pekinensis cv. Granat) has been studied. The reaction product was identified by thin-layer chromatography (TLC) and high performance liquid chromatography (HPLC). Furthermore. IAOX was identified as an endogenous compound of Chinese cabbage by mass spectroscopy. The tryptophan-oxidizing enzyme (TrpOxE) was characterized. MnCl2 was required as cofactor, H2O2, and 2,4-dichlorophenol (DCP) stimulated the reaction. The enzyme showed a pH optimum at pH 8–9 and a Km for l-tryptophan of 20 μM. The membranes containing TrpOxE activity were identified as plasma membranes by means of aqueous polymer two-phase partitioning. The TrpOxE from Chinese cabbage was purified 3-fold from plasma membranes by solubilization followed by (NH4)2SO4-fractionation, affinity-chromatography with concanavalin A, and native gel electrophoresis. Enzyme activity was reduced by a tunicamycin pretreatment. Several other plant species, e.g. maize (Zea mays L. Inrakorn), sunflower (Helianthus annuus L. cv. Hohes Sonnengold), tobacco (Nicotiana tabacum L. cv. White Burley), and pea (Pisum sativum L. cv. Krombeck) showed a similar conversion of [14C]-tryptophan to [14C]-IAOX by phase-partitioned plasma membranes.

The stromacentre inAvena plastids: an aggregation ofβ-glucosidase responsible for the activation of oat-leaf saponins.

Abstract: The stromacentre, a particular structure in the plastids of mostAvena species, was isolated from etioplasts ofAvena sativa and then characterized to determine its biological function. When comparing differentAvena species with or without stromacentre, it was shown that the stromacentre, a 63-kDa protein, and saponins (characteristic compounds ofAvena sativa) either occur together or not at all. This linkage was confirmed by demonstrating a transformation of saponins by the isolated stromacentre protein: avenacosides were hydrolyzed to 26-desgluco-avenacosides. Therefore, the stromacentre protein had to be regarded as aβ-glucosidase. Enzyme assays usingp-nitrophenyl-β-d-glucopyranoside as substrate showed that thisβ-glucosidase has a pH optimum at pH 6.0. The calculatedK m value for this substrate was 2.2·10-3 M. Antibodies against the stromacentre protein inhibitedβ-glucosidase activity. The determination of the molecular weight of theβ-glucosidase by sodium dodecyl sulfate-gel electrophoresis showed that it consists of subunits of 63 kDa. After gel electrophoresis under non-denaturing conditions, enzymatically active molecules were shown to consist of at least two of these subunits. Molecules aggregated up to about 106 Da also had enzyme activity. Enzyme assays using avenacosides as substrate showed a pH optimum at pH 6.0. The calculatedK m value for this substrate was 1.2·10-5 M. The high affinity to the avenacosides and the high specificity for the C-26 bound glucose indicate that avenacosides are the natural substrates for thisβ-glucosidase. Assuming that the avenacosides in oat leaves play a role as preformed chemical inhibitory substances against phytopathogenic microorganisms, a model is presented showing the stromacentre with a central role in activating the fungitoxicity of avenacosides.