Identification of cytoskeletal elements enclosing the ATP pools that fuel human red blood cell membrane cation pumps

Abstract

The type of metabolic compartmentalization that occurs in red blood cells differs from the types that exist in most eukaryotic cells, such as intracellular organelles. In red blood cells (ghosts), ATP is sequestered within the cytoskeletal–membrane complex. These pools of ATP are known to directly fuel both the Na + /K + and Ca 2+ pumps. ATP can be entrapped within these pools either by incubation with bulk ATP or by operation of the phosphoglycerate kinase and pyruvate kinase reactions to enzymatically generate ATP. When the pool is filled with nascent ATP, metabolic labeling of the Na + /K + or Ca 2+ pump phosphoproteins (E Na -P and E Ca -P, respectively) from bulk [γ- 32 P]-ATP is prevented until the pool is emptied by various means. Importantly, the pool also can be filled with the fluorescent ATP analog trinitrophenol ATP, as well as with a photoactivatable ATP analog, 8-azido-ATP (N 3 -ATP). Using the fluorescent ATP, we show that ATP accumulates and then disappears from the membrane as the ATP pools are filled and subsequently emptied, respectively. By loading N 3 -ATP into the membrane pool, we demonstrate that membrane proteins that contribute to the pool’s architecture can be photolabeled. With the aid of an antibody to N 3 -ATP, we identify these labeled proteins by immunoblotting and characterize their derived peptides by mass spectrometry. These analyses show that the specific peptides that corral the entrapped ATP derive from sequences within β-spectrin, ankyrin, band 3, and GAPDH.

Identification of the Components of a Glycolytic Enzyme Metabolon on the Human Red Blood Cell Membrane

“…Evolutionarily, it would also make sense for the erythrocyte to localize an ATP-producing metabolon near the site of ATP consumption on the membrane (6, 42, 46, 58 -60). Consistent with this expectation, we have recently found that the ATP pool that fuels the Na/K Ϫ and Ca Ϫ pumps resides on the membrane within or near the band 3-ankyrin complex (9).…”

“…In the case of the human erythrocyte, complexes have been suggested to exist for gas transport (3)(4)(5)(6), cation efflux pumping (7)(8)(9), and glycolysis (10). Determination of whether additional complexes also exist to respond to oxidative stress (11), mechanical deformation (12), or hemostatic stimuli (13) may require further characterization.…”

Equilibrium physics breakdown reveals the active nature of red blood cell flickering

“…Interestingly, ATP-depletion of RBCs is not fully complete after 16h of incubation in glucosefree medium 17,37 . The effective energy coincides with the thermal energy at high frequency for all conditions, but it increases up to 23kBT at low frequencies for fresh RBCs (ref 27 ).…”

Hydrodynamic deformation reveals two coupled modes/time scales of red blood cell relaxation

“…ATP is known to bind to the cytoskeleton of the cell 50 and modify the elastic moduli of the cell. To study the effect of ATP, we metabolically depleted the ATP concentration.…”