Isolation and characterization of a novel antifreeze protein from carrot (Daucus carota).
(49/6642)
A modified assay for inhibition of ice recrystallization which allows unequivocal identification of activity in plant extracts is described. Using this assay a novel, cold-induced, 36 kDa antifreeze protein has been isolated from the tap root of cold-acclimated carrot (Daucus carota) plants. This protein inhibits the recrystallization of ice and exhibits thermal-hysteresis activity. The polypeptide behaves as monomer in solution and is N-glycosylated. The corresponding gene is unique in the carrot genome and induced by cold. The antifreeze protein appears to be localized within the apoplast. (+info)
3-nitropropionic acid oxidase from horseshoe vetch (Hippocrepis comosa): a novel plant enzyme.
(50/6642)
A novel enzyme that catalyses the oxygen-dependent oxidation of 3-nitropropionic acid (3NPA) to malonate semialdehyde, nitrate, nitrite and H2O2 has been purified from leaf extracts of the horseshoe vetch, Hippocrepis comosa, and named 3NPA oxidase. The enzyme is a flavoprotein with a subunit molecular mass of 36 kDa containing 1 molecule of FMN and exhibits little specificity for all nitroalkanes tested other than 3NPA (apparent Km 620 microM). The maximum enzyme activity in vitro was expressed at pH4.8 and was inhibited strongly by the products nitrate and nitrite. 3NPA oxidase activity was detected in green shoots, which also contain high concentrations of 3NPA, from plants grown with nitrate, ammonium or N2 as sources of nitrogen. Enzyme activity was absent from roots and cell cultures, neither of which accumulate high levels of 3NPA. (+info)
Glyceraldehyde-3-phosphate dehydrogenase from Ehrlich ascites carcinoma cells its possible role in the high glycolysis of malignant cells.
(51/6642)
Glyceraldehyde-3-phosphate dehydrogenase has been purified to apparent homogeneity from Ehrlich ascites carcinoma (EAC) cells. The enzyme is quite active over a pH range of 7.5-9.0 with an optimum pH of 8.4-8.7. The specific activity of the enzyme is much higher than that from other normal sources. In contrast to enzyme obtained from rabbit muscle, the EAC cell enzyme is not significantly inhibited by physiological concentrations of ATP at physiological pH. Kinetic studies using different substrates and inhibitors indicate that the properties of the EAC cell enzyme are significantly different from those of glyceraldehyde-3-phosphate dehydrogenase obtained from other normal sources. The striking dissimilarity of the malignant cell glyceraldehyde-3-phosphate dehydrogenase compared with this enzyme from other normal sources, particularly in respect to the interaction with ATP, may in part explain the high glycolysis of malignant cells. (+info)
Phlorisovalerophenone synthase, a novel polyketide synthase from hop (Humulus lupulus L.) cones.
(52/6642)
Phlorisovalerophenone synthase (VPS), a novel aromatic polyketide synthase, was purified to homogeneity from 4.2 mg protein extract obtained from hop (Humulus lupulus L.) cone glandular hairs. The enzyme uses isovaleryl-CoA or isobutyryl-CoA and three molecules of malonyl-CoA to form phlorisovalerophenone or phlorisobutyrophenone, intermediates in the biosynthesis of the hop bitter acids (alpha- and beta-acids). VPS is an homodimeric enzyme, with subunits of 45 kDa. The pI of the enzyme was 6.1. Km values of 4 microm for isovaleryl-CoA, 10 microm for isobutyryl-CoA and 33 microm for malonyl-CoA, were found. The amino-acid sequences of two peptides, obtained by digestion of VPS, showed that the enzyme is highly homologous to plant chalcone synthases. The functional and structural relationship between VPS and other aromatic polyketide synthases is discussed. (+info)
Purification and biochemical characterization of two soluble alpha-mannosidases from Candida albicans.
(53/6642)
Two soluble alpha-mannosidases, E-I and E-II, were purified from C. albicans yeast cells by a three-step procedure consisting of size exclusion and ion exchange chromatographies in Sepharose CL6B and Mono Q columns, respectively, and preparative nondenaturing electrophoresis. E-I and E-II migrated as monomeric polypeptides of 54.3 and 93.3 kDa in SDS-PAGE, respectively. Some biochemical properties of purified enzymes were investigated by using 4-methylumbelliferyl-alpha-D-mannopyranoside and p-nitrophenyl-alpha-D-mannopyranoside as substrates. Hydrolysis of both substrates by either enzyme was optimum at pH 6.0 with 50 mM Mes-Tris buffer and at 42 degrees C. Apparent Kmvalues for hydrolysis of 4-methylumbelliferyl-alpha-D-mannopyranoside and p-nitrophenyl-alpha-D-mannopyranoside by E-I were 0.83 microM and 2. 4 mM, respectively. Corresponding values for E-II were 0.25 microM and 1.86 mM. Swansonine and deoxymannojirimicin strongly inhibited the hydrolysis of 4-methylumbelliferyl-alpha-D-mannopyranoside by both enzymes. On the contrary, hydrolysis of p-nitrophenyl-alpha-D-mannopyranoside by E-I and E-II was slightly stimulated or not affected, respectively, by both inhibitors. E-I and E-II did not depend on metal ions although activity of the latter was slightly stimulated by Mn2+and Ca2+in the range of 0.5-2 mM. At the same concentrations, Mg2+was slightly inhibitory of both enzymes. Substrate specificity experiments revealed that both E-I and E-II preferentially cleaved alpha-1,6 and alpha-1,3 linkages, respectively. (+info)
The measurement of nitrite in adulterated urine samples by high-performance ion chromatography.
(54/6642)
With the increased availability of nitrite-containing commercial products that are used for the adulteration of urine samples in the workplace, it is necessary for laboratories to be able to detect and confirm the presence of nitrite in these samples. We have developed a method to confirm the presence of nitrite in urine samples. The method uses the IonPac AS 14 analytical column with the Dionex series 45001 Bio LC system equipped with an anion self-generating suppressor and conductivity detector. Using a single-point calibration, the method is linear and accurately quantitates nitrite to 12,000 microg/mL. The limit of detection is 30 microg/mL, and the day-to-day precision of the assay has a coefficient of variation (CV) of 4.3% at 1200 microg/mL and 3.8% at 2700 microg/mL of nitrite. (+info)
Purification and characterization of D-glucosaminitol dehydrogenase from Agrobacterium radiobacter.
(55/6642)
D-Glucosaminitol dehydrogenase, which catalyzes the conversion of D-glucosaminitol to 3-keto-D-glucosaminitol, was purified to apparent homogeneity from extracts of Agrobacterium radiobacter. This organism has constitutively depressed levels of the enzyme but expression of the enzyme is induced by addition of D-glucosamine to the medium. Purification included ammonium sulfate fractionation and chromatography on columns of DEAE-Sephacel, Octyl-Sepharose CL-4B, and Cellulofine. The purified enzyme migrated as a single band, coinciding with dehydrogenase activities specific for D-glucosaminitol and ethanol, when electrophoresed on a 7.5% polyacrylamide gel at pH 8.0. Electrophoresis on a 12.5% PAGE in the presence of 1% SDS also yielded a single band. The enzyme had an apparent molecular mass of 79 kDa, as measured by the pattern of elution from a column of Cellulofine. The results indicated that the enzyme was a dimer of identical (or nearly identical) subunits of 39.5 kDa. D-Glucosaminitol dehydrogenase required NAD+ as a cofactor and used ethanol as the preferred substrate, as well as aliphatic alcohols with 2 to 4 carbon atoms, D-glucosaminitol, D-glucosaminate, DL-allothreonine, glycerol, and erythritol as additional substrates. In 50 mM Tris-HCl buffer (pH 9.0) at 25 degrees C, the K(m) for D-glucosaminitol, ethanol, and NAD+ were 2.2, 2.0, and 0.08 mM, respectively. The enzyme had a pH optimum of 10 for D-glucosaminitol and 8.5 for ethanol. The enzyme lost substantial activity when treated with pyrazole, with certain reagents that react with sulfhydryl groups and with Zn2+ ion. The various results together suggest that the enzyme exploits different amino acid residues for the dehydrogenation of ethanol and of D-glucosaminitol. (+info)
Soluble antigens of virulent and attenuated biotypes of Brucella abortus.
(56/6642)
Several methods were used to characterize three Brucella abortus biotypes (1, 5, and 7), including the attenuated vaccine strain S-19. Chemical analysis did not reveal remarkable differences among these strains, and only minor differences were noted in elution patterns of soluble extracts subjected to column chromatography. Qualitative and quantitative differences in extract components were demonstrated, however, by polyacrylamide gel isoelectric focusing. A distinctive difference was the presence of components in extracts from one or more of the virulent biotypes that were absent in similar preparations from the attenuated strain. In addition, one component common to all virulent strains was absent in strain S-19. Results of immunodiffusion experiments employing adsorbed and unadsorbed antisera also suggested that the quantity, quality, and surface distribution of various cellular antigens differed among the biotypes studied. (+info)