The Uptake of Pyridoxal Phosphate by Human Red Blood Cells

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In order to examine whether Pyridoxal phosphate could or could not be transferred into red blood cells in the form of a phosphate ester, the red blood cells were incubated with 14C-32P-pyridoxal phosphate and the following results were obtained.1. The radioactivity of the extracellular medium decreased with time, and the same amount of radioactivity was found in the cell fraction. The ratio of 14C:32P in the medium or in the cells did not change throughout the reaction. 2. Most of the radioactivities present in the cells were recovered in the form of pyridoxal phosphate. The ratio of 14C:32P of pyridoxal phosphate in the cells was the same as that in the medium. A slight amount of radioactivity of 14C of pyridoxal and 32P of the inorganic phosphate were observed in the cells. 3. NaF and CH2ICOOH inhibited the transfer of pyridoxal phosphate to the extent of 50%. At low temperature, the transfer of the radioactivity into the cell was remarkably depressed. 4. After hemolysis, the radioactivity in the red blood cells was liberated in the supernatant. By starch-gel electrophoresis and Sephadex-gel filtration, pyridoxal phosphate was found to be combined with hemoglobin. 5. The red blood cells containing pyridoxal phosphate was incubated with hemoglobin solution, but pyridoxal phosphate was not liberated from the cells. The pyridoxal phosphate was not hydrolyzed by alkaline phosphatase. These results suggested that pyridoxal phosphate was not merely adsorbed on the surface of cell membrane, but was transferred through the cell membrane. These observations lead to the conclusion that pyridoxal phosphate was transferred into the red blood cells without being hydrolyzed to pyridoxal and inorganic phosphate.
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The Uptake of Pyridoxal Phosphate by Human Red Blood Cells

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