Haruyoshi Konno

Metal-tolerant moss Scopelophila cataractae accumulates copper in the cell wall pectin of the protonema.

The growth kinetics in the presence of copper (Cu) of the protonema of the moss Scopelophila cataractae and the matrix polysaccharides of its cell walls have been analyzed in this study. Protonemal cells cultured in a medium containing 0.2mM CuSO(4) showed a rapid accumulation of Cu, reaching a maximum between 30 and 60d at approximately 65 micromolg(-1) DW. Uronic acids were found in similar amounts in cell walls of both control and Cu-treated cells, whereas arabinose and galactose decreased to 61-67% in the presence of Cu. Cell wall polysaccharides were determined after successive extraction with 50mM CDTA, 50mM Na(2)CO(3), 1M KOH, and 4M KOH. The pectic fractions (CDTA- and Na(2)CO(3)-soluble) decreased to 47% and the hemicellulosic fractions (1M KOH- and 4M KOH-soluble) to 86% under Cu application. Approximately 43% of the Cu taken into cell walls was released following endo-pectate lyase treatment, suggesting that two-fifths of the total Cu accumulation was tightly bound to the homogalacturonan of the cell wall pectin.

An ice-nucleating active fungus isolated from the gut of the rice stem borer, Chilo suppressalis Walker (Lepidoptera: Pyralidae)

Abstract The existence of a fungus with the ability to nucleate ice formation in supercooled water was revealed. The fungus was isolated from the gut of larvae of the rice stem borer, Chilo suppressalis Walker, and from rice seedlings which were host plants of this insect. The fungus was identified as a Fusarium sp. on the basis of its morphology. Ice-nucleating activity, at around −5°C, was detected in the mycelial suspension of the fungus and also in the culture filtrate. The presence of the exogenous ice-nucleating active fungus in the gut and on the body surface caused an elevation in crystallization temperature of the larvae.

Tissue distribution of the ice-nucleating agents in larvae of the rice stem borer, Chilo suppressalis Walker (Lepidoptera: Pyralidae)

Abstract To investigate the tissue distribution of ice-nucleating agents in the rice stem borer, Chilo suppressalis Walker, the crystallization temperatures of the whole bodies and individual tissues of nondiapausing and diapausing larvae were measured. In nondiapausing mature larvae the crystallization temperature of the gut with its contents was the highest, being about − 8 °C, showing that a freezing site is present in the gut. As food particles in the alimentary canal of hibernating larvae were excreted in autumn, the larval supercooling capacity increased with lowering crystallization temperature of the gut. In diapausing larvae the crystallization temperatures of the muscle and epidermis were the highest, being above − 15 °C, which is similar to that of the whole larvae, and the hemolymph crystallization temperature was the lowest, being below − 25 °C. Furthermore, the crystallization temperatures of the nervous system, trachea, silk gland, salivary gland, and ovary were below − 20 °C, which was equivalent to those of 0.9% NaCl solution. Consequently, in diapausing larvae a primary site of freezing is present in the muscle and epidermis, indicating that the potent ice-nucleating agents exist in these tissues. However, since the epidermis could not be completely divided from the muscle, it was not concluded whether the potent ice-nucleating agents existed in the epidermis or not.

Enzymatic degradation of pectic substances and cell walls purified from carrot cell cultures

Abstract The purified pectic substances from carrot ( Daucus carota ) cell cultures were depolymerized by purified exo -polygalacturonase and endo -pectate lyase. The degraded pectic fractions were fractionated by gel filtration chromatography, and the degree of polymerization and glycosyl composition determined for each fraction. The results indicate that subfractionated pectic substances contain a homogalacturonan region with a degree of polymerization of ca 70 and neutral glycosyl residues such as side-chains arranged in blocks (‘hairy’ regions) of different M r s. In addition, the endo -pectate lyase released four pectic fragments from isolated cell walls. Based on the analysis of glycosyl composition of each fragment, the pectic substances of carrot cell walls are characterized.

Purification and properties of α-glucosidase from millet seeds

Abstract Two forms of α-glucosidase (EC 3.2.1.20), designated as I and II, have been isolated from millet ( Panicum miliaceum L.) seeds by a procedure including fractionation with ammonium sulphate, CM-cellulofine column chromatography, Sephadex G-100 column chromatography, preparative isoelectric focusing and preparative disc gel electrophoresis. The two enzymes showed identical M r , calculated to be 85 000 on SDS-PAGE and 93 000 on gel filtration. The two enzymes readily hydrolysed maltose and malto-oligosaccharides, and native starch weakly. The two enzymes hydrolysed amylose liberating α-glucose.

A β-glucosidase associated with cell walls from cell suspension cultures of carrot

Abstract The activity of β-glucosidase (EC 3.2.1.21) in the protein fraction solubilized with 3 M LiCl from cell walls of carrot cell cultures was found to be much higher than those of the other glycan-hydrolases. The cell wall-associated β-glucosidase was purified to electrophoretic homogeneity. The M r of the purified enzyme was estimated to be 46 000 by Sephacryl S-200HR gel-permeation, and 48 000–52 000 by SDS-PAGE under denaturing conditions. The enzyme contained carbohydrate and protein in a ratio of 1 : 15 (w/w) and was rich in Ser, Gly, Glx and Ala. The isoelectric point was pH 8.2, the pH optimum 4.6–5.2 and the temperature optimum 50°. The activity was inhibited by Cu 2+ , Ag + , Hg 2+ , p -chloromercuribenzoate, and d -glucono-1,5-lactone. The K m and V max values for p -nitrophenyl (PNP)-β-glucopyranoside were 0.12 mM and 0.13 mmol (mg protein) −1 hr −1 , respectively. The enzyme also acted on PNP-β-cellobioside, lichenan and laminarin, but was not capable of hydrolysing the glucose-containing polymers isolated from cell walls of carrot cell cultures.

Subunit structure and amino acid analyses of β-galactosidase purified from carrot cell cultures

Abstract β-Galactosidase has been purified to electrophoretic homogeneity from cell suspension cultures of carrot. It has an apparent Mr of 104 000 and consists of two identical subunits with an apparent Mr of 50 000. The amino acid composition of the enzyme is characterized by a relatively large content of Asx and Leu, and a small content of His and Met. The amino-terminal amino acid of the enzyme is Glu.

Characteristics, hydrolysis of cell wall polymers, and response to calcium deficiency of a cell wall-associated β-galactosidase from carrot cells

Summary The activity of β-galactosidase (EC 3.2.1.23) in the protein fraction solubilized with 3 mol/L LiCl from cell walls of suspension-cultured carrot ( Daucus carota ) cells has been found to be higher than those of the other glycosyl-hydrolytic enzymes. The cell wall-associated β-galactosidase/exo-galactanase was purified to electrophoretic homogeneity. Analysis by denaturing PAGE revealed a single polypeptide chain with a molecular mass of 50 kDa, similar to 52 kDa estimated for the native protein by size-exclusion chromatography. The enzyme contained carbohydrate and protein in a ratio of 1 : 10 (w/w), and was rich in Gly and Phe, followed by Ser, Ala and Thr. The isoelectric point was pH 6.5, and pH and temperature optima 4.4 and 45-50 °C, respectively. The β-galactosidase activity was inhibited by Mg 2+ , Ag + , Hg 2+ , p -chloromercuribenzoate and D-galactono-1,4-lactone. The K m and V max values for p -nitrophenyl-β-D-galactopyranoside were 0.77 mmol/L and 0.32 mmol (mg protein) −1 h −1 , respectively. The enzyme hydrolyzed citrus galactan in an exo-fashion and was capable of hydrolysing an acidic pectic polymer containing galactosyl and arabinosyl residues from carrot cell walls and, therefore, is an exo-galactanase. However, even after an exhaustive reaction, the enzyme had no effect on a galactose-rich hemicellulosic polymer from carrot cell walls. Under calcium deficiency the activities of a cell wall-associated α-galactosidase (EC 3.2.1.22) and β-galactosidase increased until 15 days of culture and then decreased gradually, whereas β-glucosidase (EC 3.2.1.21) activity increased markedly with time despite poor cell growth.

Purification and characterization of β-galactosidase from cell suspension cultures of Marchantia polymorpha

Abstract A β-galactosidase (β-D-galactoside galactohydrolase, EC 3.2.1.23) has been isolated and purified to homogeneity from cell homogenates of cell suspension cultures of a thalloid liverwort, Marchantia polymorpha . The enzyme in 0.1 M phosphate buffer soluble protein fraction was dialyzed at pH 5.2 and further purified by a combination of chromatographic techniques including DEAE-Sephadex A-50, p -aminophenyl β- d -thiogalactopyranoside-linked Sepharose 4B and Sephadex G-200. The molecular weight of β-galactosidase by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and by Sephadex G-200 gel filtration is 62 000 and 67 000, respectively. The isoelectric point of the enzyme is found to be pH 4.2. The optimal activity occurs at pH 3.4 with McIlvaine buffer, and at pH 2.6 with glycineHCl buffer. The K m - and V max -values are 0.625 mM and 658 units/mg protein for p -nitrophenyl β- d -galactopyranoside. The enzyme catalyzes the transfer of the d -galactosyl residues from p -nitrophenyl β- d -galactopyranoside to d -glucose. Furthermore, the enzyme acts on the galactan extracted from citrus pectic polysaccharides in an exo-fashion.

acrB Mutation Located at Carboxyl-terminal Region of Gyrase B Subunit Reduces DNA Binding of DNA Gyrase

Mutations that exhibit susceptibility to acriflavine have been isolated and classified as acrmutations in Escherichia coli. We cloned theacrB gene, which has been identified as a mutation of thegyrB gene, and found a double point mutation altering two consecutive amino acids (S759R/R760C) in the COOH-terminal region of the gyrase B subunit. The mutant B subunit was found to associate with the A subunit to make the quaternary structure, and the reconstituted gyrase showed an 80-fold reduction of specific activity in DNA supercoiling assay; the sensitivity to acriflavine was not different in the same unit of wild-type and mutant gyrases. The mutant enzyme retained intrinsic ATPase activity, but DNA-dependent stimulation was observed infrequently. A gel shift assay showed that acriflavine inhibited the DNA binding of gyrase. The acrBmutation also reduced significantly the DNA binding of gyrase but did not change the sensitivity to acriflavine. These results revealed that the acrB mutation is related to the inhibitory mechanism of acriflavine; and the acriflavine sensitivity of the mutant, at leastin vitro, is caused mainly by reduction of the enzyme activity. Further, our findings suggest that the COOH-terminal region of the B subunit is essential for the initial binding of gyrase to the substrate DNA.