| The major family of plant acid phosphatases (APases), found in plant roots and most other tissues, are essential for growth and development. These APases play key roles in nutrient acquisition, metabolism and metabolic regulation. Although many APases have been identified, most studies have utilized impure enzyme preparations and tested activity with only a few substrates. No known gene sequences exist for APases.; We have purified the major isoform of potato tuber APase (PTAP) to homogeneity. With a P-Tyr hydrolyzing specific activity of 1917 units/mg, PTAP was determined to be one of the most active APases ever observed. PTAP also showed significant in vitro protein tyrosine phosphatase activity on potato tuber phosphotyrosyl polypeptides. Other properties of PTAP characterize it as a substrate 'non-specific' APase. These features include a pH optimum of 5.8, broad substrate range, thermostability, nonessential divalent cation requirement and potent inhibition by molybdate, vanadate and ZnCl{dollar}sb2.{dollar}; PTAP protein microsequence data was obtained from cyanogen bromide- and trypsin-digested fragments. These data facilitated the isolation of a number of putative APase genomic and cDNA clones by two different approaches. A degenerate PTAP probe was used to isolate two potato genomic clones and data base homology to a randomly sequenced rice cDNA clone led to the isolation of a rice cDNA clone and four potato genomic clones. Based on alignments with deduced amino acid sequences, two PTAP genomic clones (PTAP-1 and PTAP-2), three related APase genomic clones (PAP3, PAP7 and PAP11), and a rice cDNA clone (pRAP) were identified. Sequence identity among the deduced APase proteins ranged from 58% to 74%. From a data base search, Fe(III)/Zn(II) kidney bean purple acid phosphatase (KBPAP) exhibited up to 70% identity while Aspergillus ficuum aphA had limited homology to the APase coding sequences.; The full length 1.9 kb PTAP cDNA clone (pPTAP) was isolated using a PTAP-2 probe. It encodes a 451 amino acid protein including a 29 residue hydrophobic target peptide. The processed protein is 51.7 kD with no significant motifs but shows conservation of all the residues implicated in the active site of KBPAP (Asp161, Asp188, Tyr191, Asn226, His311, His348 and His350) and one glycosylation site (Asn422). All potato (dormant and sprouting tuber pith, tuber epidermis, stolon, root, stem and leaf) and rice (root and leaf) tissues exhibited APase activity and displayed immunologically-related PTAP polypeptides. However, northern blotting analysis showed PTAP-specific transcripts only occur in dormant potato tuber and stolon. These results indicate PTAP plays its role during tuber development and dormancy and is not likely involved in starch breakdown or phosphate acquisition from the soil. In addition, the northern blot and sequence data implies that numerous related APases are expressed in potato. |
