814 ALTERED PATTERN OF SPECTRIN PHOSPHORYLATION IN SENESCENT HUMAN RED CELLS

Abstract

The membrane protein spectrin is important in the maintenance of red blood cell (RBC) membrane shape and stability. We have previously shown that it is phosphorylated at 4 sites clustered near the C-terminus of its smaller subunit. These phosphates may control spectrin's interaction with other membrane proteins. To test whether spectrin phosphorylation changed with RBC age, intact RBCs were labeled with 33PO4, fractionated by density, and assayed for age (using marker enzymes) and for the position and number of 33PO4's in spectrin dimer. 33p-spectrin prepared from old (average=90 d of age) and control RBCs had identical numbers of 33p phosphates; however the distribution of 33p was altered in old RBCs. 33P-label in the most distal phosphorylation site was reduced from 0.95±0.05 moles/mole spectrin dimer to 0.25±0.05(n=4) and was proportionally increased in a more proximal site(s). Similar changes occurred in RBCs incubated in the absence of substrate for 20 hrs (but not for 12 hrs). Phosphorylation of purified 33P-spectrin from old RBCs with spectrin kinase and γ-32P-ATP produced the same number and distribution of phosphates as control. We conclude that the location of spectrin phosphates in senescent or metabolically-depleted RBCs is altered. This may be caused by enzymatic redistribution of isotopic label or by a structural alteration of the distal phosphorylation site. This change may contribute to the membrane instability and eventual demise of the aged RBC.