Purification and molecular analysis of a monoamine oxidase isolated from Narcissus tazetta.

Semicarbazide-sensitive amine oxidase activity was detected in Narcissus tazetta. The enzyme was purified to homogeneity by the criterion of native polyacrylamide gel electrophoresis (PAGE) with DEAE-Sephacel, hydroxyapatite, and phenyl-Sepharose columns. The molecular mass of the enzyme, determined using a GS-520 HQ column, was estimated to be 135 kDa. SDS-PAGE yielded two bands of, 75 kDa and 65 kDa. The enzyme, which had catalytic activity for some aliphatic and aromatic monoamines, belongs to a class of monoamine oxidases (MAOs). The K(m) value for n-propylamine was 5.9 × 10⁻5 M. A substrate analog, 2-bromoethylamine, inhibited enzyme activity. Redox-cycling staining detected a quinone in the MAO protein. By inductively coupled plasma mass analysis, it was determined that there were 2.44 moles of copper atoms per mole of the enzyme. Protein sequence analysis revealed that there was no identity between two N-terminal residues of the 75 kDa and 65 kDa proteins of narcissus MAO.

Isolation of an Acremonium sp. capable of liquefying cross-linked poly(gamma-glutamic acid) hydrogels and the fungal enzyme involved in the disruption of gamma-ray irradiation-mediated cross-linking.

A microorganism capable of liquefying cross-linked poly(gamma-glutamic acid) (C-L PGA) was isolated and identified to be an Acremonium sp. The fungus produced an enzyme that can disrupt the cross-linkages of C-L PGA generated by gamma-ray irradiation. The enzyme can also liquefy C-L PGA prepared by chemical cross-linkage, suggesting the involvement of ester bonds in the gamma-ray irradiation-mediated cross-linking of PGA.

Biosynthesis of poly-gamma-glutamic acid in plants: transient expression of poly-gamma-glutamate synthetase complex in tobacco leaves.

Transient expression of genes coding for the poly-gamma-glutamate (gammaPGA) synthetase system (pgs) was investigated in tobacco plants. Three genes of the pgs, pgsA, pgsB and pgsC, were separately placed under the control of the CaMV 35S promoter and introduced into tobacco leaves via Agrobacterium infection. Synthesized gammaPGA in plant tissues was detected immunologically with mouse anti-gammaPGA antiserum which specifically reacts with gammaPGA on a nitrocellulose membrane. Confirmation of gammaPGA biosynthesis in the transient expression analysis in tobacco tissue indicates that subunits of pgs complex were expressed and reassembled in a functional form.

Flocculating activity of cross-linked poly-gamma-glutamic acid against bentonite and Escherichia coli suspension pretreated with FeCl3 and its interaction with Fe3+.

Cross-linked poly-gamma-glutamic acid (C-L gamma-PGA) at 5 microg/ml flocculated bentonite suspension pretreated with polyaluminum chloride (PAC) at 2 microg/ml Al3+-PAC to a transparency of approximately 30% after 30 min and more than 90% after 4 h, while Al3+ concentration in the upper phase of the suspension decreased with incubation time. When pretreated with FeCl3 at 16 microg/ml Fe3+-FeCl3, similar results were obtained. In the case of Escherichia coli suspension, the combination of C-L gamma-PGA and FeCl3 demonstrated a more marked flocculating activity with a satisfactory transparency occurring after 30 min of treatment, accompanied by a decrease in residual Fe3+ concentration. In the above two suspensions pretreated with FeCl3, small visible floats appeared in the early stage of incubation. These floats were found to be due to the direct interaction between FeCl3 and C-L gamma-PGA, indicating the formation of a water-insoluble complex. After allowing the suspension to stand for a long time, elemental analysis and inductively coupled plasma spectroscopy of the precipitates produced suggested that not only the complex was formed due to the interaction between Fe3+ in FeCl3 and COO- in the C-L gamma-PGA molecule, but also Fe2O3 and Fe(OH)3 might be entrapped in this complex. This could be applied to scavenge metal ions including Fe3+ from polluted water.

Physicochemical properties of cross-linked poly-gamma-glutamic acid and its flocculating activity against kaolin suspension.

Cross-linked poly-gamma-glutamic acid (C-L gamma-PGA) was prepared with gamma-PGA irradiated with gamma-ray at various kGy values. The physicochemical properties including viscosity and water absorption capacity were compared between C-L gamma-PGA and several typical flocculating agents. The viscosity of C-L gamma-PGA increased with the dose of gamma-irradiation, although the water absorption capacity of C-L gamma-PGA did not, which showed a maximum of 1005.6 ml/g at 20 kGy. Flocculating activity against kaolin suspension was not observed for any of the test compounds when used singly. However, the activity of C-L gamma-PGA markedly increased following the addition of polyaluminum chloride. The activity increased with temperature up to 80 degrees C and remained at 80 degrees C of heat pretreatment for 1 h, but did not at more than 50 degrees C of heat pretreatment for 24 h. The activity was also observed within a pH range of 4.5-10.0. Both the water absorption capacity and flocculating activity of C-L gamma-PGA decreased in parallel with increasing NaCl concentration, suggesting that the flocculating activity of C-L gamma-PGA was associated with its water absorption capacity, rather than viscosity. An investigation of the effects of various cations on the flocculating activity of C-L gamma-PGA showed that only trivalent cations had a synergistic effect. The mechanism of C-L gamma-PGA flocculating activity is discussed based on the results of preliminary experiments.

Proposals for wastewater treatment by applying flocculating activity of cross-linked poly-gamma-glutamic acid.

Cross-linked poly-gamma-glutamic acid (C-L gamma-PGA) markedly purified polluted water collected from rivers and ponds by flocculation and precipitation. This effect of C-L gamma-PGA occasionally required pretreatment with polyaluminum chloride (PAC). Components of polluted water in rivers or ponds are generally thought to be clay minerals, microorganisms and chemical compounds. In this study, the flocculating activities of C-L gamma-PGA against suspensions of bentonite, diatomaceous earth, Escherichia coli and Mycrocystis aeruginosa, and against solutions of crystal violet and bisphenol A were investigated. The mode of action of C-L gamma-PGA is thought to be based on electrostatic interaction between flocculants, C-L gamma-PGA and PAC, and the surface of polluted water components, which may lead to neutralization of the zeta-potential of those components.

Purification and properties of aminoaldehyde dehydrogenase from Avena sativa.

NAD-dependent aminoaldehyde dehydrogenase (AMADH, EC 1.2.1.-) from Avena shoots was purified by DEAE Sephacel, hydroxyapatite, 5'-AMP Sepharose 4B, Mono Q, and TSK-GEL column chromatographies to homogeneity by the criterion of native PAGE. SDS-PAGE yielded a single band at a molecular mass of 55 kDa. IEF studies showed a band with a p I value of 5.3. In contrast to AMADHs from other species, the TSK-GEL chromatography showed that AvenaAMADH exists as a monomer in the native state. The purified enzyme catalyzed the oxidations of 3-aminopropionaldehyde (APAL), 4-aminobutyraldehyde (ABAL) N-(3-aminopropyl)-4-aminobutyraldehyde (APBAL), and 4-guanidinobutyraldehyde (GBAL), but not of betaine aldehyde or indoleacetaldehyde. The K(m) values for APAL, ABAL, and GBAL were 1.5x10(-6), 2.2x10(-6), and 1.3x10(-5) M, respectively. Although N-terminal amino acid sequence of Avena AMADH could not be determined due to a modification of the amino residue, the sequence of the fragment of AMADH cleaved by V8 protease showed greater similarity to the barley BADH than to the pea AMADH.

Oxidative stress-dependent inhibition of yeast cell growth by farnesylamine and its possible relation to amine oxidase in the mitochondrial fraction.

Among various analogs of the isoprenoid farnesol (FOH), farnesylamine (FNH2) inhibited the growth of the budding yeast Saccharomyces cerevisiae by accelerating cellular reactive oxygen species (ROS) generation. Unlike the case with FOH, however, FNH2 did not cause mitochondrial transmembrane potential (mtDeltaPsi) hyperpolarization so that FNH2-treated cells were not protected against ROS production by inhibiting the proton pumping function of mitochondrial F(O)F1-ATPase. FNH2 promoted ROS generation even in cells of a respiration-deficient mutant, indicating a yeast metabolic pathway other than mitochondrial electron transport as the origin of ROS. FNH2 oxidase activity was detected in the yeast mitochondrial fraction, which produces hydrogen peroxide (H2O2) in the reaction with either FNH2 or geranylgeranylamine (GGNH2), in addition to polyamine oxidase activity specific for spermine. GGNH2 also exhibited the growth inhibitory effect with the accompanying induction of ROS generation, while such an activity was not detected with any of the polyamines tested or geranylamine. FNH2 oxidase, which was sensitive to a typical copper-chelating agent, diethyldithiocarbamic acid (DDC), could be solubilized with Triton X-100, and detected as a single band upon activity staining with FNH2 but not with spermine in polyacrylamide gel electrophoresis. FNH2-treated cells were partly protected against ROS production by the additional supplementation of DDC in the medium. Our results suggest the involvement of H2O2 production due to direct oxidation of FNH2 by copper amine oxidase in oxidative stress-dependent inhibition of yeast cell growth.

The vacuole-targeting fungicidal activity of amphotericin B against the pathogenic fungus Candida albicans and its enhancement by allicin.

In this study, the vacuole disruptive activity was evaluated as a cause of amphotericin B (AmB) lethality against the pathogenic fungus Candida albicans in terms of its enhancement by allicin, an allyl-sulfur compound from garlic. Vacuole disruption was observed in parallel to AmB-induced cell death when the antibiotic was used at a lethal concentration and at a non-lethal concentration in combination with allicin. Allicin did not enhance AmB-induced cell death and the accompanying vacuole disruption when the cells were incubated with exogenous ergosterol for its enrichment in the vacuole. The vacuoles isolated from intact cells could be directly disrupted by the action of AmB to the same extent in the absence and presence of allicin, whereas the organelles isolated from ergosterol-enriched cells were resistant to its direct disruptive action. AmB was similarly incorporated into the fungal cytoplasm in cells with or without ergosterol enrichment, supporting the fact that AmB-induced vacuole disruption depends on its direct disruptive action on the organelle. In agreement with these findings, allicin was found to inhibit ergosterol transport from the plasma membrane to the cytoplasm, which is considered to be a cellular protective response to AmB-induced vacuole disruption in S. cerevisiae. Our study suggests that AmB lethality against C. albicans depends at least in part on its vacuole disruptive activity under the physiological condition permissive for invasive growth of the fungus.

Signal transduction controlling the blue- and red-light mediated gene expression of S-adenosylmethionine decarboxylase in Pharbitis nil.

The signal transduction processes involved in the regulation of SAMDC gene expression by blue and red light were examined using pharmacological inhibitors of signalling pathways. Calcium and calmodulin positively regulated SAMDC gene expression in red light, whereas in blue light they regulated negatively. These results indicate that calcium homeostasis is involved in both red and blue light induction of SAMDC expression. Both signal transduction pathways also require new protein synthesis.

Purification and properties of betaine aldehyde dehydrogenase from Avena sativa.

Betaine aldehyde dehydrogenase (BADH; EC 1.2.1.8) is the enzyme that catalyzes the second step in the synthesis of the osmoprotectant, glycine betaine. NAD-dependent BADH was purified from Avena sativa shoots by DEAE Sephacel, hydroxyapatite, 5'-AMP Sepharose 4B, Mono Q and TSK-GEL column chromatographies to homogeneity by the criterion of native PAGE, and the properties of BADH were compared with those of aminoaldehyde dehydrogenase purified to homogeneity from A. sativa. The molecular mass estimated by both gel filtration using TSK-GEL column and Sephacryl S-200 was 120 and 115, kDa, respectively. The enzyme is a homodimer with a subunit molecular mass of 61 kDa as shown by SDS-PAGE. The pI value of the enzyme was found to be 6.3. The purified enzyme catalyzed not only the oxidation of betaine aldehyde (BAL), but also that of aminoaldehydes, 3-aminopropionaldehyde (APAL), 4-aminobutyraldehyde (ABAL), and 4-guanidinobutyraldehyde (GBAL). The K(m) values for BAL, APAL, ABAL and GBAL were 5x10(-6), 5.4x10(-7), 2.4x10(-5) and 5x10(-5) M, respectively. APAL showed substrate inhibition at a concentration of 0.1 mM. A fragment of BADH cleaved by V8 protease shared homology with other plant BADHs.