Growth phase-dependent active transport of pyridoxine in a fission yeast,Schizosaccharomyces pombe

Abstract

Schizosaccharomyces pombe showed maximum pyridoxine uptake activity around 10 h after starting cultivation. High concentrations of thiamine and pyridoxine in the medium did not affect the activity or the time but changed intracellular levels of vitamin B6 compounds. Pyridoxine was taken up by a saturable mechanism with two kinds of affinity (K(m) 22.4 microM and 118 microM). The uptake depended on the energy produced anaerobically with an optimum pH of 4.5. The uptake was completely inhibited by amiloride, sodium azide or 2,4-dinitrophenol. The uptake system of the fission yeast was different in various respects from that of a budding yeast.

Amiloride Uptake and Toxicity in Fission Yeast Are Caused by the Pyridoxine Transporter Encoded by bsu1 + ( car1 + )

“…PN uptake was optimal at pH 4.5 and followed saturation kinetics. The apparent K m value for PN uptake was determined to be 24 M, which is in agreement with the lower of the two K m values previously reported for S. pombe cells (48).…”

“…Tpn1p and proteins with similarities to the related purine/cytosine permease Fcy2p are present in many yeast species, but searches of the completed genomic sequence failed to identify orthologues in S. pombe. This was unexpected because S. pombe was known to actively transport PN (48).…”

“…What remained unclear was why mutants lacking bsu1 ϩ were originally identified by their resistance to the drug amiloride. In S. pombe (48) and in human cell lines (33), PN uptake is drastically reduced in the presence of amiloride. Moreover, amiloride and PN are structurally related molecules (Fig.…”

“…Consistent with the presence of TPN1 orthologues in most yeast species, many yeast species can accumulate PN (47). In S. pombe, the apparent K m values for PN uptake were 22.4 and 118 M and PN uptake was sensitive to amiloride (48). Because amiloride affects many Na ϩ -transporting proteins (20), this observation has led to speculation that PN uptake in S. pombe cells is driven by Na ϩ symport (48).…”

Purification and Characterization of Pyridoxal 4-Dehydrogenase fromAureobacterium luteolum

“…Reversed-phase isocratic HPLC for measurement of vitamin B6 compounds, including 4-pyridoxolactone and 4-pyridoxic acid, was done as described previously. 8) Molecular weight of the native enzyme was measured by a gelˆltration (Hiprep 16 W 60 Sephacryl S-300 column) with FPLC. A calibration curve was made from the elution pattern of bovine liver catalase ( Mr ＝240,000), pig heart mitochondrial aspartate aminotransferase (90,000), malate dehydrogenase (67,000), and horse heart cytochrome c (12,000).…”