Biofactors - Volume 28, issue 2

Article Type: Other

Citation: BioFactors, vol. 28, no. 2, pp. 71-72, 2006

Authors: Schopfer, P. | Liszkay, A.

Article Type: Research Article

Abstract: Reactive oxygen species (ROS) produced as intermediates in the reduction of O_{2} to H_{2} O (superoxide radical, hydrogen peroxide, hydroxyl radical), are generally regarded as harmful products of oxygenic metabolism causing cell damage in plants, animals and microorganisms. However, oxygen radical chemistry can also play useful roles if it takes place outside of the protoplast. In plants, the production of these ROS initiated by the plasma membrane NAD(P)H oxidase can be used for …controlled polymer breakdown leading to wall loosening during extension growth. Backbone cleavage of cell wall polysaccharides can be accomplished by hydroxyl radicals produced from hydrogen peroxide and superoxide in a reaction catalyzed by cell wall peroxidase. Growing plant organs such as coleoptiles or roots of maize seedlings produce these ROS specifically in the apoplast of actively growing tissues, e.g. in the epidermis of the coleoptile and the growing zone of the root. Auxin promotes the release of hydroxyl radicals when inducing elongation growth. Experimental generation of hydroxyl radicals in the wall causes an increase in wall extensibility in vitro and replaces auxin in inducing growth. Auxin-induced growth can be inhibited by scavengers of ROS or inhibitors interfering with the formation of these molecules in the cell wall. These results provide the experimental background for a novel hypothesis on the mechanism of plant cell growth in which the generation of hydroxyl radicals, initiated by the plasma membrane NAD(P)H oxidase, plays a central role. Show more

Keywords: Cell growth, cell wall loosening, Haber-Weiss reaction, hydrogen peroxide, hydroxyl radical, NAD(P)H oxidase, peroxidase, reactive oxygen intermediates, superoxide radical

Citation: BioFactors, vol. 28, no. 2, pp. 73-81, 2006

Authors: Auderset, Guy | Morré, D. James

Article Type: Research Article

Abstract: Growth hormone-responsive and nucleoside triphosphate-dependent enlargement of inside-out vesicles of plasma membranes from soybeans prepared by aqueous two-phase partition and everted by freezing and thawing has been achieved in a cell-free system. In the presence of 100 μM ATP in 40 mM HEPES buffer, pH 7, enlargement of isolated plasma membrane vesicles was accelerated by the synthetic plant growth factor, 2,4-dichlorophenoxyacetic acid (2,4-D), compared to ATP alone, 2,4-D alone or no additions. …After 20 min with 1 μM 2,4-D, vesicles increased in diameter, 20% on average. Although vesicle diameters in the presence or absence of 2,4-D overlapped, the means were clearly separated. The 20% increases in diameter corresponded to a doubling of vesicle volume. Both 100 μM ATP and 1 μM 2,4-D were necessary to stimulate the cell-free vesicle enlargement. In the presence of 1 μM 2,4-D, enlargement observed with 100 μM ATP was greater than with either 10 μM ATP or 500 μM ATP alone. In the presence of 100 μM ATP, vesicle enlargement was proportional to the logarithm of 2,4-D concentration. With the growth-inactive 2,4-D analog, 2,3-D, no vesicle enlargement was observed either alone or in the presence of 100 μM ATP. Right side-out vesicles did not enlarge in response to either ATP, 2,4-D or the two in combination suggesting that the responsible ATP site was on the inside of the cell. Show more

Keywords: Cell enlargement, cell-free, ATP, 2,4-dichlorophenoxyacetic acid, 2,4-D, auxin, soybean, plant

Citation: BioFactors, vol. 28, no. 2, pp. 83-90, 2006

Authors: Hicks-Berger, Carrie | Morré, D. James

Article Type: Research Article

Abstract: The concept that the location of an AAA-ATPase associated with the plant plasma membrane may be indicative of a functional relationship to growth or cell enlargement by analogy with roles in physical membrane displacements as proposed for AAA-ATPases associated with internal membranes was tested. A plant growth hormone-responsive and nucleoside triphosphate-dependent enlargement of inside-out vesicles of plasma membranes from soybeans was utilized in a completely cell-free system. The rate of enlargement was accelerated by the …synthetic plant growth factor 2,4-dichlorophenoxyacetic acid (2,4-D) in a log dose-dependent manner and was increased approximately 2-fold with the addition of 1 μM 2,4-D plus 100 μM ATP compared to 100 μM ATP alone, 1 μM 2,4-D alone or no additions. The cell-free enlargement was inhibited by AAA-ATPase-specific antisera and by CoCl_{2} , an inhibitor specific for AAA-ATPases. The responsible ATP site appears to be on the inside of the cell, since right side-out vesicles did not enlarge in response to either ATP, 2,4-D or the two in combination. Show more

Keywords: Cell enlargement, cell-free, ATP, 2,4-dichlorophenoxyacetic acid (2,4-D), auxin, AAA-ATPase, soybean, plant

Citation: BioFactors, vol. 28, no. 2, pp. 91-104, 2006

Authors: Morré, D. James | Kim, Chinpal | Hicks-Berger, Carrie

Article Type: Research Article

Abstract: A recombinant ECTO-NOX (tNOX) and a recombinant plasma membrane associated AAA-ATPase (ATPase Associated with Different Cellular Activities) were combined in stoichometric proportions into liposomes together with albumin as a source of protein thiols. Large lamellar vesicles were formed from phosphatidylcholine, cholesterol and dicetyl phosphate in a molar ratio of 50:45:5, where the phosphatidylcholine was a 2:1 mixture of synthetic dimyristoyl and dipalmitoyl phosphatidylcholines. The lipids were dried to a film and reconstituted …into vesicles by resuspension in buffer containing the recombinant proteins in equimolar ratios of 0.04 nmoles/mg lipid. In the presence of ATP, these vesicles enlarged in an ATP-dependent manner based on light-scattering measurements. Because the drug-inhibited ECTO-NOX protein, tNOX was utilized, the enlargement was inhibited by capsaicin, a quinone site tNOX inhibitor specific for tNOX. With the lipid vesicle systems, the recombinant ECTO-NOX, the recombinant AAA-ATPase, a source of protein thiols and ATP all were required. In control experiments, no ATP-dependent vesicle enlargement was observed with the AAA-ATPase or the ECTO-NOX protein alone. Also addition of ATP was without any effect when only the single proteins were incorporated into the lipid vesicles. A model has been developed whereby the plasma membrane AAA-ATPase is linked via disulfide bonds, formed and broken by the ECTO-NOX protein, to membrane structural proteins. Binding of ATP and subsequent hydrolysis and release of ADP would advance the ATPase hexamer ratchet thereby both thinning the membrane and increasing the vesicle surface. Show more

Citation: BioFactors, vol. 28, no. 2, pp. 105-117, 2006

Authors: Chen, Chun-Feng | Huang, Shing | Liu, Shan-Chi | Chueh, Pin Ju

Article Type: Research Article

Abstract: Previous reports have described a tumor-associated NADH oxidase (tNOX) and its continuous activation in transformed culture cells. Certain anticancer drugs have been shown to inhibit preferentially both the tNOX activity and the growth of transformed culture cells and the cytotoxicity is associated with the induction of apoptosis. To investigate the biological function of tNOX protein, we have raised polyclonal antisera against bacterial expressed tNOX protein and the antisera are able to recognize protein bands in …transformed cells but not the non-transformed cells tested. With tNOX antisera treatment, the survival in transformed cell lines is decreased but not the non-transformed cells. In addition, tNOX antisera-induced cytotoxicity is accompanied by the induction of apoptosis. However, slightly higher amount of PARP cleavage and activation of caspase-9 are observed in tNOX antisera treated HCT116 cells. Further experiments have demonstrated the activation of JNK and phosphorylation of p53 by treatment. In addition, tNOX antisera treatment leads to an impressive increase in reactive oxygen species in COS cells but not the control sera. Our data suggest that (a) tNOX antisera treatment may inhibit the growth of transformed cells by inducing apoptosis and (b) the apoptotic mechanism might be through modulating ROS production and JNK pathway. Show more

Keywords: Apoptosis, polyclonal antisera, reactive oxygen species (ROS), transformed cells, tumor-associated NADH oxidase (tNOX)

Citation: BioFactors, vol. 28, no. 2, pp. 119-133, 2006

Authors: Hicks-Berger, Carrie | Sokolchik, Irina | Kim, Chinpal | Morré, D. James

Article Type: Research Article

Abstract: An AAA-ATPase (ATPases Associated with a Variety of Cellular Activities) localized to the plasma membrane of soybean (Glycine max) was isolated, partially sequenced and cloned (SBPM AAA-ATPase). The protein with an apparent monomer molecular mass of about 97 kDa was isolated using a combination of anion exchange, preparative SDS-PAGE, reverse phase HPLC, and ATP affinity chromatography. The cDNA for the full-length SBPM AAA-ATPase was cloned by screening an expression library using an …antibody against the highly conserved Walker B AAA-ATP-binding motif. Northern blot analysis detected one transcript of ∼2700 bp. The full-length cDNA sequence was that previously obtained (GenBank Database; U20213) encoding a protein with two copies of the conserved AAA-ATP-binding motif and regions of sequence homology with other AAA-ATPases. Electron microscopic preparations of the recombinant SBPM AAA-ATPase revealed hexamers typically formed by these proteins. The cloned and expressed protein was identical to the protein isolated from the soybean plasma membrane as confirmed using antisera raised to a non-conserved region of the derived protein sequence and by N-terminal sequencing of peptides derived from the isolated protein. Show more

Keywords: Soybean, plasma membrane, AAA-ATPase, cell enlargement, physical membrane displacement, p97

Citation: BioFactors, vol. 28, no. 2, pp. 135-149, 2006