Signal peptide sequence processing site of purple acid phosphatase from kidney bean (Phaseolus vulgaris L. Ohfuku) seeds.

Abstract

SUMMARYWe purified purple acid phosphatase (PAPase) from kidney bean (Phaseolus vulgaris L. Ohfuku) seeds, a Japanese cultivar of the kidney bean. The molecular mass of the purified native enzyme was estimated approximately 110kDa by gel filtration. Following SDS-PAGE in the presence of 2-mercaptoethanol, glycosylated polypeptides of major 61kDa and minor 59kDa were observed. The N-terminal amino acid sequences for both bands were determined to be GKSSNFVRKTNKNRDMPLDS, suggesting they were two different subunits with the same N-terminal but probably different C-terminal or degree of glycosylation.The partial nucleotide sequences covering the N-terminal region were also determined using polymerase chain reaction (PCR). Comparison with the deduced amino acid sequence from the nucleotide sequence and that of the purified enzyme revealed that the N-terminal amino acid residue corresponded to Gly 23 from the initiation colon (Met). For the deduced amino acid sequence of the PAPase gene, a SignalP (ver. 2.0) analysis program predicted that a 22 amino acid sequence at the N-terminus of a precursor protein was signal peptide. This prediction was in agreement with the fact that PAPase with a signal peptide at the N-terminus was cleaved between Gly 22 and Gly 23 by putative signal peptidase.

Characterization of a Novel Acid Phosphatase from Embryonic Axes of Kidney Bean Exhibiting Vanadate-dependent Chloroperoxidase Activity

“…Through all column chromatography purification steps, a single chromatographic peak of KeACP activity was observed, and the final preparation was colorless. This suggests that neither purple color ACP, which has usually been found in dry seeds (20), nor multiple ACP forms are present in embryonic axes of kidney beans. The pH dependence of phosphatase activity was also investigated for KeACP, yielding a curve with a pH optimum at pH 6.0 (data not shown) that was almost identical to that of other ACPs.…”

“…In the absence of vanadate, the apoenzyme revealed phosphatase activity at the same level as the native enzyme but no chloroperoxidase activity. On the other hand, vanadate-substituted purple color ACP prepared from kidney bean seed (20) revealed no chloroperoxidase activity under the same conditions, such as dialysis against EDTA buffer followed by incubation with vanadate. This means that the vanadatesubstituted KeACP uniquely catalyzes the chloroperoxidation reaction.…”