Glyceraldehyde-3-phosphate dehydrogenase in the isolated human erthrocyte membrane: selective displacement by bilirubin.

When unsealed erythrocyte ghosts in 6 mm phosphate buffer (pH 8.0, 4 °C) were incubated with bilirubin in excess of 0.1 mm and washed with buffer, a single polypeptide component (band 6 in sodium dodecyl sulfate-polyacrylamide-gel electrophoresis) diminished and was recovered in the supernatant fraction. Release of this component was virtually complete at 1 mm initial bile pigment. Since band 6 was believed to be the protomer of membrane-bound glyceraldehyde-3-phosphate dehydrogenase (G3PD), assays for this enzyme in bilirubin-treated ghosts were carried out. These revealed that enzymatic activity decreased concurrently with the disappearance of band 6. The molecular weight of the eluted polypeptide was found to be 36,000, in agreement with the known value for the G3PD protomer. When Mg²⁺-resealed ghosts were washed after exposure to 1 mm bilirubin, less than 20% of the G3PD was eluted, which is consistent with the fact that the enzyme is attached to the cytoplasmic face of the membrane. NAD⁺ in concentrations up to 2 mm displaced no more than 15% of the G3PD from unsealed ghosts. However, after preincubation with NAD⁺ (1 mm) followed by bilirubin (1 Mm) and washing, loss of G3PD was similar to that observed in the absence of cofactor. Since NAD⁺ did not attenuate release of the enzyme, it appears unlikely that such release is attributable to binding of bilirubin at the active site. Protoporphyrin acted similarly to bilirubin on unsealed ghosts, whereas rose bengal had a more pronounced effect, removing all enzymatic activity when the dye concentration reached 0.2 mm. Electrophoretic analysis of ghosts after rose bengal treatment, however, revealed a diminution not only of band 6 but bands 1, 2, and 5 as well.