• BMB Reports
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• v.38 no.6
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• pp.703-708
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• 2005

We cloned and expressed human pyridoxal-5'-phosphate (PLP) phosphatase, the coenzymatically active form of vitamin $B_6$, in Escherichia coli using pET15b vector. Monoclonal antibodies (mAb) were generated against purified human brain PLP phosphatase in mice, and four antibodies recognizing different epitopes were obtained, one of which inhibited PLP phosphatase. The binding affinities of these four mAbs to PLP phosphatase, as determined using biosensor technology, showed that they had similar binding affinities. Using the anti-PLP phosphatase antibodies as probes, we investigated their cross-reactivities in various mammalian and human tissues and cell lines. The immunoreactive bands obtained on Western blots had molecular masses of ca. 33 kDa. Similarly fractionated extracts of several mammalian cell lines all produced a single band of molecular mass 33 kDa. We believe that these PLP phosphatase mAbs could be used as valuable immunodiagnostic reagents for the detection, identification, and characterization of various neurological diseases related to vitamin $B_6$ abnormalities.

• Journal of Life Science
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• v.18 no.4
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• pp.585-589
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• 2008

Chronophin is a phosphatase responsible for the dephosphorylation of cofilin, which regulates the rearrangement of actin cytoskeleton. It is also known as a phosphatase for pyrodoxal 5'-phosphate (PLP), an active form of vitamin $B_6$, and maintains the level of PLP in the cytoplasm. Since this phosphatase belongs to a HAD subfamily containing a cap domain, it is expected to undergo a conformational change for the binding of a substrate. However, the crystal structure of chronophin has a disulfide bridge between the cap and core domains preventing a movement of the cap domain against the core domain. It is possible that the disulfide bond between C91 and C221 was formed by an oxidation during the crystallization. Here, we obtained chronophin crystals under a reduced condition and determined the crystal structure. This reduced chronophin does not contain a disulfide bridge and shows a closed conformation like the oxidized form. It implies that an active chronophin binds its substrate under the closed conformation without the disulfide bond and shows a high substrate specificity in the cell.

• BMB Reports
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• v.41 no.5
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• pp.408-413
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• 2008

Pyridoxal-5'-phosphate phosphatase (PLPP) catalyzes the dephosphorylation of pyridoxal-5'-phosphate (PLP). A human brain PLPP gene was fused with a PEP-1 peptide and produced a genetic in-frame PEP-1-PLPP fusion protein. The purified PEP-1-PLPP fusion protein was efficiently transduced into PC12 cells in a time- and dose-dependent manner when added exogenously to culture media. Once inside the cells, the transduced PEP-1-PLPP fusion protein was stable for 36 h. The concentration of PLP was markedly decreased by the addition of exogenous PEP-1-PLPP to media pretreated with the vitamin $B_6$ precursors; pyridoxine, pyridoxal kinase and pyridoxine-5'-phosphate oxidase into cells. The results suggest that the transduction of the PEP-1-PLPP fusion protein can be one mode of PLP level regulation, and to replenish this enzyme in the various neurological disorders related to vitamin $B_6$.