Investigation of molecular structures and subcellular localization of 49 kDa apyrasefrom plants

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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 3.6.1.5). 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. Immuno fluorescence microscopy and expression of GFP fusion protein also revealed their multi-localization. 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. These isotypes were successfully separated by an anion column chromatography with a pH and KOAc gradients. Mass spectroscopy and C-terminal amino acid sequencing the pea apyrase was cleaved at both the N- and C- termini, and clearly distinguished 4 apyrase isotypes of 46,491, 47,055, 47,260, and 47,450 kDa and that it may be modified in various ways to furnish different forms with different functions and locations.