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Cloning of putative subunits of the soybean plasma membrane NADPH oxidase involved in the oxidative burst by antibody expression screening

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Summary

Plant cells respond to a variety of external signals with the production of reactive-oxygen species. The enzyme system generating these reactive-oxygen species is believed to be an NADPH oxidase located in the plasma membrane and sharing similarities with the NADPH oxidase from mammalian macrophages. Antibodies directed against individual subunits (p22phox, p47phox, p67phox) of the human NADPH oxidase cross-react with soybean proteins of a similar size and subcellular location. An extensive expression screening of a soybean cDNA-library with the anti-human NADPH oxidase antibodies gave a single class of cDNA-clones for each antibody. However, the sequence analysis of these clones clearly demonstrates that the different antibodies recognise proteins which are unrelated to the expected oxidase subunits. The anti-p22phox antibody recognised a microsomal protein with no significant homology to any known protein in the database. One anti-p47phox antibody cross-reacted with the UDP-glucose dehydrogenase and another antibody bound to the chaperon peptidyl prolyl-cis-trans isomerase, both soluble cytosolic proteins. The anti-p67phox antibody detected the soluble enzyme acetohydroxy acid reductoisomerase. Chromatography of soybean protein extracts on an ion-exchange column (MonoQ, FPLC) gave a perfect comigration of the enzyme activity with the antibody signal, thus confirming these unexpected results by independent biochemical experiments.

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Abbreviations

AARI:

acetohydroxy acid reductoisomerase

DPI:

diphenylene iodonium

GST:

glutathione-S-transferase

phox NADPH:

oxidase of phagocytes

ROS:

reactive-oxygen species

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  1. Fachbereich Biologie, Universität Kaiserslautern, Gebäude 22, D-67653, Kaiserslautern, Germany

    Raimund Tenhaken & Christine Rübel

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  1. Raimund Tenhaken
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  2. Christine Rübel
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Tenhaken, R., Rübel, C. Cloning of putative subunits of the soybean plasma membrane NADPH oxidase involved in the oxidative burst by antibody expression screening. Protoplasma 205, 21–28 (1998). https://doi.org/10.1007/BF01279289

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  • DOI: https://doi.org/10.1007/BF01279289

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