The subcellular distribution and isotypes of a 49-kDa apyrase from pea (Pisum sativum L. var. Alaska).
Authors: Koichi Shibata1, Shunnosuke Abe1, Minoru Yamada1 *, and Eric Davies2
1Laboratory of Molecular Cell Biology, Department of Biological Resources, Faculty of Agriculture, Ehime University, Matsuyama, 790-8566 Japan
2Botany Department, Box 7612, North Carolina State University, Raleigh NC 27695-7612 U.S.A.
*Author to whom correspondence should be addressed (fax +81-89-946-9853; e-mail: email@example.com)
We isolated a 49 kDa protein from the cytoskeleton fraction from pea (Pisum sativum L. var. Alaska) stems using heparin affinity and cation exchange column chromatography, and identified the protein as apyrase (EC 188.8.131.52). The purified enzyme hydrolyzes both nucleoside triphosphates and nucleoside diphosphates into their monophosphates. Using an antibody raised against apyrase, we studied its sub-cellular distribution in isolated fractions and found significant amounts in the cell wall (50 %), the nuclei (3 %), the cytoskeleton (14 %), and the supernatant fractions (33 %). Immuno electron microscopy using gold-labeled antibody confirmed that apyrase was present in cell walls, nuclei, and in filamentous structures associated with ribosomes. Even though there is only one gene (with 2 alleles), 2D gels indicated there were at least five isotypes, three being major ones, and the relative abundance of these isotypes differed in different fractions. Enzyme from all fractions a) hydrolyzed nucleoside triphosphates and diphosphates, but not monophosphates, b) had similar activity against different substrates, c) were not sensitive to ATPase inhibitors (azide, fluoride, molybdate, ouabain, quercetin), and d) were all inhibited by vanadium pentoxide, especially the enzyme from the nuclei. Mass spectroscopy has confirmed the presence of five polypeptides, all of which are, indeed, isotypes of apyrase, and we are currently trying to determine what post-translational modifications occur in which isotypes. These results show the 49 kDa apyrase is located in various compartments within the cell and that it may be modified in various ways to furnish different forms with different functions and locations.
This work was supported by a grant from Ministry of Education, Culture, Sports, Science and Technology, Japan (Grant (#12660296).