PURIFICATION AND CHARACTERIZATION OF A POTATO-TUBER ACID-PHOSPHATASE HAVING SIGNIFICANT PHOSPHOTYROSINE PHOSPHATASE-ACTIVITY

The major acid phosphatase (APase) from potato (Solanum fuberosom L. c v Chiefton) tubers has been purified 2289-foId 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 poly acrylamide gel electrophoresis of the final preparation resolved a sin gle protein-staining band that comigrated with APase activity. Followi ng sodium dodecyl sulfate polyacrylamide gel electrophoresis, glycosyl ated 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) immunog lobulin C. Immunoblotting studies revealed that the 55-kD subunit did not arise via proteolytic cleavage of the 57-kD subunit after tissue e xtraction. The native molecular mass was approximately 100 kD, suggest ing that the holoenzyme could exist as either a homodimer or a heterod imer. 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 phosphomonoester s including p-nitrophenylphosphate, O-phospho-L-serine, phosphoenolpyr uvate, 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 ph ysiological significance of the potato APase's substantial in vitro ac tivity with P-Tyr remains obscure, the possibility that this APase may function to dephosphorylate certain protein-located P-Tyr residues in vivo is suggested.