Figure 5.
Effects of the E91K substitution on mini-spectrin tetramerization and stability of the membrane skeleton. (A) Representative elution profiles of gel permeation chromatography (GPC) for tetramer-dimer formation of mini-Sp. The mini-SpE91 (E) and mini-SpK91 (K) stored at 4°C were incubated at 37°C for 1 hour at a concentration of 0.2 mg/mL separately (E/E and K/K) or after 1:1 combined (E/K), chilled on ice for 10 minutes, and then loaded onto a Superdex 200 10/300 GL column. Eluting positions of tetramer, dimer, and marker proteins (thyroglobulin, 86 Å; ferritin, 61 Å; aldolase, 48 Å; ovalbumin, 28 Å) are indicated. (B) The abundance of the tetramer relative to the total amount of tetramer and dimer in GPC analysis in panel A is found in the percentage for each of E/E, E/K, and K/K. Data are expressed as the means ± SD, n = 3. ∗P < .05 by 1-way ANOVA with the Tukey multiple comparison test. (C-D) Representative profiles of (C) SDS-PAGE and (D) immunoblotting to detect spectrin in vesicles generated from RBCs with or without DIDS treatment. RBCs from cattle with SPTA1 genotypes SpαB/SpαB (E/E) or SpαB/SpαBK91 (E/K) were incubated in the presence (+) or absence (−) of 50 μM of DIDS at 37°C for 30 minutes, washed, and suspended in PBS, followed by filtration as described in the legend for Figure 1. Proteins in vesicles obtained (Vesicle) and RBC ghosts (Ghost) were separated by SDS-PAGE on 8% SDS gels followed by staining with Coomassie brilliant blue in panel C or immunoblotting using the anti-spectrin antibody in panel D. Migrating positions of α- and β-spectrin (α- and β-Sp), band 3 (B3), and size marker polypeptides in kDa are indicated. (E-G) The contents of band 3 (E) and spectrin (F, total of α and β) in vesicles obtained from filtrates of 1 mL 10% RBC suspension were determined by densitometric scanning of Coomassie blue–stained gels. The relative abundance of spectrin was illustrated as spectrin/band 3 in (G). Unpaired Student t test was used to determine statistical significance between E/E and E/K and between with or without DIDS treatment. ∗∗∗P < .001; ∗∗∗∗P < .0001. (H) The vesicles from E/K RBCs (E/K) with (+) or without (−) DIDS treatment (described previously) were analyzed for protein 4.1R by immunoblotting using the anti-4.1R antibody (anti-4.1R) in parallel with spectrin (anti-Sp). Signals for 4.1R (4.1R) and spectrin (Sp) and migrating positions of size markers in kDa are indicated.

Effects of the E91K substitution on mini-spectrin tetramerization and stability of the membrane skeleton. (A) Representative elution profiles of gel permeation chromatography (GPC) for tetramer-dimer formation of mini-Sp. The mini-SpE91 (E) and mini-SpK91 (K) stored at 4°C were incubated at 37°C for 1 hour at a concentration of 0.2 mg/mL separately (E/E and K/K) or after 1:1 combined (E/K), chilled on ice for 10 minutes, and then loaded onto a Superdex 200 10/300 GL column. Eluting positions of tetramer, dimer, and marker proteins (thyroglobulin, 86 Å; ferritin, 61 Å; aldolase, 48 Å; ovalbumin, 28 Å) are indicated. (B) The abundance of the tetramer relative to the total amount of tetramer and dimer in GPC analysis in panel A is found in the percentage for each of E/E, E/K, and K/K. Data are expressed as the means ± SD, n = 3. ∗P < .05 by 1-way ANOVA with the Tukey multiple comparison test. (C-D) Representative profiles of (C) SDS-PAGE and (D) immunoblotting to detect spectrin in vesicles generated from RBCs with or without DIDS treatment. RBCs from cattle with SPTA1 genotypes SpαB/SpαB (E/E) or SpαB/SpαBK91 (E/K) were incubated in the presence (+) or absence (−) of 50 μM of DIDS at 37°C for 30 minutes, washed, and suspended in PBS, followed by filtration as described in the legend for Figure 1. Proteins in vesicles obtained (Vesicle) and RBC ghosts (Ghost) were separated by SDS-PAGE on 8% SDS gels followed by staining with Coomassie brilliant blue in panel C or immunoblotting using the anti-spectrin antibody in panel D. Migrating positions of α- and β-spectrin (α- and β-Sp), band 3 (B3), and size marker polypeptides in kDa are indicated. (E-G) The contents of band 3 (E) and spectrin (F, total of α and β) in vesicles obtained from filtrates of 1 mL 10% RBC suspension were determined by densitometric scanning of Coomassie blue–stained gels. The relative abundance of spectrin was illustrated as spectrin/band 3 in (G). Unpaired Student t test was used to determine statistical significance between E/E and E/K and between with or without DIDS treatment. ∗∗∗P < .001; ∗∗∗∗P < .0001. (H) The vesicles from E/K RBCs (E/K) with (+) or without (−) DIDS treatment (described previously) were analyzed for protein 4.1R by immunoblotting using the anti-4.1R antibody (anti-4.1R) in parallel with spectrin (anti-Sp). Signals for 4.1R (4.1R) and spectrin (Sp) and migrating positions of size markers in kDa are indicated.

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