Effect of protein 4.1 peptides on enzyme activity.
(A) Cleavage of human erythrocyte membrane ankyrin and protein 4.1. Five microliters of human erythrocyte IOVs (2 mg/mL) were added to 25-μL reactions in 5 mM sodium phosphate buffer, pH 7.5, 1 mM DTT containing no enzyme (lane 1), or 0.1 μg rFP-2 in the absence (lane 2) or presence (lanes 3-9) of peptides. Inhibition of ankyrin cleavage was calculated from the intensity of the truncated ankyrin (155-kd band) obtained by densitometry and is given below in parentheses. Lanes 3 to 7: 25 μM (0%), 50 μM (0%), 500 μM (98%), 1000 μM (100%), or 1500 μM (100%), respectively of peptide P1. Lane 8: 1500 μM (5%) peptide P2. Lane 9: 1500 μM (5%) peptide P3. Lanes 10 and 11: 1000 μM (0%) and 1500 μM (5%), respectively, of peptide P4. Lanes 12 and 13: 1000 μM (0%) and 1500 μM (0%), respectively, of peptide P5. Reactions were incubated for 30 minutes at 37°C. Vesicles were analyzed by reducing SDS-PAGE and were stained with Coomassie blue. The sizes of truncated ankyrin and protein 4.1 are shown on the left. (B) Cleavage of human hemoglobin. Three micrograms human hemoglobin was added to 25-μL reactions in 100 mM sodium acetate, pH 5.5, 1 mM DTT containing no enzyme (lane 1), or 100 nM recombinant FP-2 in the absence (lane 2) or presence of increasing amounts of the peptide P1. Inhibition of hemoglobin cleavage is given in parentheses: 100 μM (0%), 500 μM (50%), and 1000 μM (80%), respectively in lanes 3, 4, and 5. Reactions were incubated for 60 minutes at 37°C, analyzed by 15% SDS-PAGE under reducing conditions, and stained with Coomassie blue. (C) Mechanical stability of erythrocyte ghosts. Human erythrocyte ghosts (300 μL) were incubated with 0 μg FP-2 (i), 0.1 μg FP-2 (ii), 0.1 μg FP-2 + 80 μM P1 (iii), or 0.1 μg FP-2 + 80 μM P2 (iv) for 10 minutes at 0°C. Ghosts were subsequently resealed in isotonic solution and subjected to a constant shear stress of 750 dyne/cm2. The rate of change of deformability index was measured in an ektacytometer.
