In vivo and in vitro turnover of spectrin phosphate in erythrocytes.
Erythrocyte deformability may be modulated by regulatory phosphorylation of spectrin. Most investigators have assumed that the phosphate attached to spectrin turns over in vivo. We tested that assumption using dog erythrocytes. Erythrocytes were labeled in vitro with solutions containing 32P. After 1 week at 4 degrees C, 60% of the 32P had been incorporated into the erythrocytes, the erythrocytic gamma-32P-ATP had a specific activity of 0.285 muCi/mumol (S.E. = 0.0138), and a membrane extract enriched in spectrin had 15,200 +/- 2390 cpm/mg of protein. When these erythrocytes were labeled with 51Cr, washed with saline, and infused autologously, they had a normal lifespan (t1/2 = 24.9 +/- 1.24 days). The gamma-32P-ATP disappeared exponentially, with a t1/2 of 7.84 +/- 0.506 hr. The radioactivity in low-ionic-strength membrane extracts and Triton X-100 pellets declined exponentially, with a t1/2 of 33.4 +/- 0.336 and 39.1 +/- 0.806 hr, respectively. When 32P-labeled erythrocytes were incubated in vitro in a 15 mM phosphate buffer, the gamma-32P-ATP (t1/2 = 3.97 hr) and the radioactivity of low-ionic-strength extracts (t1/2 = 4.18 hr) and Triton pellets (t1/2 = 4.54 hr) declined rapidly. These rates of spectrin phosphorylation-dephosphorylation suggest that this process may be physiologically important and that in vitro turnover rates are significantly higher than those observed in vivo.
D000818:Animals; D005591:Chemical Fractionation; D004285:Dogs; D004492:Edetic Acid; D004912:Erythrocytes / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism*; D010766:Phosphorylation; D011092:Polyethylene Glycols; D013049:Spectrin / Q000032:analysis / Q000378:metabolism*
D008565:Membrane Proteins; D013049:Spectrin; D011092:Polyethylene Glycols; D004492:Edetic Acid