title: Purification and Characterization of a Potato Tuber Acid Phosphatase Having Significant Phosphotyrosine Phosphatase Activity.
abstract: The major acid phosphatase (APase) from potato (Solanum tuberosom L. cv Chiefton) tubers has been purified 2289-fold to near homogeneity and a final O-phospho-L-tyrosine (P-Tyr) hydrolyzing specific activity of 1917 [mu]mol Pi produced min-1 mg-1 of protein. Nondenaturing polyacrylamide gel electrophoresis of the final preparation resolved a single protein-staining band that co-migrated with APase activity. Following sodium dodecyl sulfate polyacrylamide gel electrophoresis, glycosylated polypeptides of 57 and 55 kD were observed. The two polypeptides are immunologically closely related, since both proteins cross-reacted on immunoblots probed with rabbit anti-(Brassica nigra APase) immunoglobulin G. Immunoblotting studies revealed that the 55-kD subunit did not arise via proteolytic cleavage of the 57-kD subunit after tissue extraction. The native molecular mass was approximately 100 kD, suggesting that the holoenzyme could exist as either a homodimer or a heterodimer. The enzyme displayed a pH optimum of 5.8, was activated 40% by 4 mM Mg2+, and was potently inhibited by molybdate, vanadate, and ZnCl2. The final preparation displayed the highest activity and specificity constant with P-Tyr, but also dephosphorylated other phosphomonoesters including p-nitrophenylphosphate, O-phospho-L-serine, phosphoenolpyruvate, PPi, and ATP. Antibodies to P-Tyr were used to demonstrate that several endogenous phosphotyrosylated tuber polypeptides could serve as in vitro substrates for the purified APase. Although the precise physiological significance of the potato APase's substantial in vitro activity with P-Tyr remains obscure, the possibility that this APase may function to dephosphorylate certain protein-located P-Tyr residues in vivo is suggested.
authors.affiliation: Departments of Biology and Biochemistry, Queen's University, Kingston, Ontario, Canada K7L 3N6.
authors.forename: K. S.
authors.initials: KS
authors.lastname: Gellatly
authors.forename: GBG.
authors.initials: G
authors.lastname: Moorhead
authors.forename: SMG.
authors.initials: S
authors.lastname: Duff
authors.forename: D. D.
authors.initials: DD
authors.lastname: Lefebvre
authors.forename: W. C.
authors.initials: WC
authors.lastname: Plaxton