Characterization of apyrases in pea, arabidopsis, and cotton
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Apyrases are proteins that bind to and hydrolyze most nucleoside di- and triphosphates, but not nucleoside monophosphates. Some function inside the cell; others hydrolyze extracellular nucleotides in the extracellular matrix. In Arabidopsis, apyrases and eATP have been implicated in diverse responses, ranging from phosphate mobilization to herbicide and toxin resistance to overall plant growth. Isolated garden pea nuclei exhibit phytochrome-dependent, red/far red-reversible changes in apyrase activity and in protein phosphorylation. O-linked β-N-acetylglucosamine (O-GlcNAc) is a post-translational modification that functions in a manner analogous but often antagonistic to O-phosphorylation. Given the known red light effects in pea nuclei and the link between O-GlcNAc and O-phosphorylation, we tested whether O-GlcNAc modification in pea nuclei might change in response to red light (Rc). Western blots showed that anti-O-GlcNAc antibody could sometimes recognize modifications in pea nuclei proteins, but not consistently. Experiments testing red light irradiation, O-GlcNAc transferase inhibitors, and GlcNAcase inhibitors all yielded similarly inconsistent results. As the project progressed we learned that the O-GlcNAc modification is very labile, and that this lability was the probable basis of the inconsistent results. Methods to overcome this technical problem were not readily available and so this project was not pursued further. A second project focused on identifying the members of the protein complexes associated with apyrase in partially purified nuclear preparations. The apyrase complex was isolated from etiolated seedlings of both Arabidopsis and pea by co-immunoprecipitation using antibodies raised against purified protein from each system. Apyrase activity was also compared in etiolated and red-light irradiated nuclei in both systems. For both experiments and in both systems, the complex of proteins associated with apyrase in etiolated and Rc-irradiated nuclei were not consistently different. The discovery of a cotton EST with a sequence similar to other plant apyrases opened a new line of investigation in a model system with fibers that are suited for the study of growth kinetics of single cells. This putative apyrase is expressed at high levels in elongating fibers but not in fibers that are not elongating or in ovules, suggesting that apyrase may influence cotton fiber elongation. Studies of fiber growth kinetics in the presence of inhibitors that block apyrase activity, chemical inhibitors of animal P2X-type purinoceptors, and anti-apyrase antibodies also favor this hypothesis. I cloned the full length cDNAs of GhApy1 and GhApy2. Antibodies raised against a suspected antigenic and variable sequence of GhApy1 were produced and confirmed to recognize native cotton apyrase. Crude protein isolated from pollen exhibited apyrase activity.