Mehansho H, Henderson L M
J Biol Chem. 1980 Dec 25;255(24):11901-7.
The mechanisms of transport of pyridoxine (PN) and pyridoxal (PL) into erythrocytes have been investigated using the rapid-mixing technique with 3H-labeled substrates. Erythrocytes take up PN by passive diffusion as indicated by the fact that the initial influx process is nonsaturable and is not affected by pyridoxamine (PM), PL, or 4'-deoxypyridoxine. Transport of PL has the same characteristics. The transport of both PN and PL is temperature-dependent with a sharp increase in rate of uptake between 15 and 20 degrees C. The accumulation of 3H from [3H]PN against a concentration gradient was the result of phosphorylation. When the phosphorylation of [3H]PN was reduced by adding 160 microM unlabeled PN, PL, PM, or 4'-deoxypyridoxine, the cell:medium concentration ratio for 3H did not exceed unity. In contrast, with [3H]PL, 1.6 mM concentrations of these unlabeled substrates did not reduce the cell:medium concentration ratio of 3H. This kinase-in-dependent accumulation of PL against a concentration gradient, in the absence of evidence for mediated transport, suggested that binding by an intracellular protein was responsible. Evidence for such binding was obtained from Sephadex G-75 gel filtration, ultrafiltration, and equilibrium dialysis studies with [3H]PL. Reduction of the PL x protein complex with NaBH4 resulted in a stable pyridoxyl protein. The high binding capacity and the molecular weight by gel filtration suggest that hemoglobin is the binding protein.
