Avid O2-responsive HbS binding to RBC membrane defeats potential control of GAPDH activity. RBC membranes depleted of endogenous GAPDH were attached to silica beads. (A) To study membrane-based inhibition of free GAPDH, progressive numbers of membrane coated beads were incubated with a GAPDH activity buffer (containing a fixed amount of GAPDH). Beads were pelleted and residual enzyme activity in the supernatant was assayed (n = 3-9; mean ± SEM). *P < .05 for normal versus SS. (B) To quantitate GAPDH binding to membrane, progressive amounts of GAPDH (in activity buffer) were incubated with membrane-coated beads (bead number fixed at 3 × 108), beads were pelleted, and membrane-bound GAPDH was quantitated by immunoblot and densitometry. SSRBC membranes quenched and bound significantly less GAPDH than control RBC membranes (n = 4; mean ± SEM) *P < .05 for normal versus SS. In separate experiments, beads coated with membrane from oxygenated or deoxygenated normal RBCs were incubated with oxygenated (HbO2% > 85%) or deoxygenated (HbO2% < 15%) HbAo or HbS and then incubated with GAPDH. Enzyme activity was determined in the supernatant. (C) No difference was observed in GAPDH quenching after incubation of bead-attached membrane from oxygenated RBCs with either oxy-HbAo or HbS (n = 4; mean ± SEM). (D) However, beads coated with washed membrane from deoxygenated RBCs and incubated with deoxy-HbS quenched significantly less GAPDH than those incubated with deoxy-HbAo (n = 6; mean ± SEM). *P < .05 for deoxy-HbAo versus deoxy-HbS.
