Poster: Proteomics

Abs # 964: Changes in isotypes and enzyme activity of apyrase during germination of dark-grown pea (Pisum sativumL. var. Alaska) seedlings

Presenter:	Abe, Shunnosuke , abe@mcb.agr.ehime-u.ac.jp
Authors	Abe, Shunnosuke  (A)   Moustafa, Mahmoud F.M. (A)   Motohito,  Yoneda  (A)   Eric,  Davies  (B)
Affiliations:	(A): Department of Bioresources, faculty of Agriculture, Ehime University (B): Botany Department, North Carolina University

In the present study we used 2D-PAGE and Western blotting to investigate the expression of different isotypes of apyrase (EC 3.6.1.5) during imbibition, germination and initial growth of pea (Pisum sativum L. var. Alaska) seedlings in the dark. The 49 kDa apyrase was absent in the 10-hour imbibed embryos, but began to appear after 16 hours germination and increased with germination time. By 62 hours, there were five isotypes present at pI 5.8, 6.0, 6.3, 6.6 and 6.8, with those at pI 6.0, 6.3, and 6.6 being most abundant and the one at pI 6.3 predominating, while the most acidic and basic isotypes were only present in significant amounts in seedlings after 62 hours germination. Stems contained all five isotypes and had more pI 6.0, 6.3 and 6.6 isotype than the plumules, while in the roots there were very small amounts of all isotypes. Partial amino acid sequencing showed that all isotypes were identical with apy1, not the more recently-described apy2. Apyrase activity was absent in imbibed embryos, but increased sharply during germination and reached a maximum after 62 hours. Based upon the enzyme�fs capability to hydrolyze ATP, CTP, GTP, TTP, UTP, and ADP (but not AMP), its susceptibility to various ATPase inhibitors, and coincidence of expression of the protein and enzyme activity, we estimate that 50-70% of the ATPase activity results from the 49 kDa apyrase. The present results suggest that isotypes of pI 6.0, 6.3, and 6.6 are physiologically important and strongly indicate a crucial role for apyrase in the differentiation and development of pea seedlings.