Binding of spectrin α22 tetramers to human erythrocyte membranes

Steven Goodman, S. A. Weidner

Research output: Contribution to journalArticle

27 Citations (Scopus)

Abstract

32P-labled spectrin α2-β2 tetramers were extracted from human erythrocyte ghosts at low ionic strength and 4°C. The [32P]spectrin tetramers were purified by column chromatography on Sepharose 4B, and appeared homogenous by the criteria of sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Column-purified spectrin tetramers were found to be 90% in the 12 S α2-β2 oligomeric form by rate zonal sedimentation. Incubation of the column-purified tetramers at 37°C (physiological ionic strength buffer, pH 7.5) for 1 h yielded 89% α-β heterodimeric spectrin. The spectrin tetramers and converted heterodimers bound to their high affinity membrane receptors (syndeins) with essentially the same affinity; the respective K(D) values were 17 ± 2 nM and 16 ± 2 nM. The maximal binding capacity for the spectrin tetramers was 270 μg of spectrin/mg of inverted vesicle membrane protein which is close to the physiological concentration of spectrin on the membrane. The maximal binding capacity determined for spectrin heterodimers was 132 μg of spectrin/mg of inverted vesicle protein or approximately one-half the physiological concentraton. Extraction of bound [32P]spectrin from the inverted vesicles at 4°C demonstrated that 90% of the bound column-purified tetramers remain in the α2-β2 tetrameric form; while 92% of the bound converted heterodimers are in the α-β heterodimeric form. Therefore, heterodimer-tetramer interconversion does not occur at the syndein binding site under the conditions of our binding studies (physiological ionic strength, pH 7.5, 4°C). We conclude that spectrin tetramers and heterodimers bind to the syndeins with indistinguishable affinity, and the stoichiometry of binding on the membrane is 1 α2-β2 spectrin tetramer/syndein binding site.

Original languageEnglish (US)
Pages (from-to)8082-8086
Number of pages5
JournalJournal of Biological Chemistry
Volume255
Issue number17
StatePublished - Dec 1 1980
Externally publishedYes

Fingerprint

Spectrin
Erythrocyte Membrane
Membranes
Ionic strength
Osmolar Concentration
Binding Sites
Column chromatography
Electrophoresis
Sedimentation
Sodium Dodecyl Sulfate
Stoichiometry
Sepharose
Chromatography
Polyacrylamide Gel Electrophoresis
Buffers

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Binding of spectrin α22 tetramers to human erythrocyte membranes. / Goodman, Steven; Weidner, S. A.

In: Journal of Biological Chemistry, Vol. 255, No. 17, 01.12.1980, p. 8082-8086.

Research output: Contribution to journalArticle

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title = "Binding of spectrin α2-β2 tetramers to human erythrocyte membranes",
abstract = "32P-labled spectrin α2-β2 tetramers were extracted from human erythrocyte ghosts at low ionic strength and 4°C. The [32P]spectrin tetramers were purified by column chromatography on Sepharose 4B, and appeared homogenous by the criteria of sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Column-purified spectrin tetramers were found to be 90{\%} in the 12 S α2-β2 oligomeric form by rate zonal sedimentation. Incubation of the column-purified tetramers at 37°C (physiological ionic strength buffer, pH 7.5) for 1 h yielded 89{\%} α-β heterodimeric spectrin. The spectrin tetramers and converted heterodimers bound to their high affinity membrane receptors (syndeins) with essentially the same affinity; the respective K(D) values were 17 ± 2 nM and 16 ± 2 nM. The maximal binding capacity for the spectrin tetramers was 270 μg of spectrin/mg of inverted vesicle membrane protein which is close to the physiological concentration of spectrin on the membrane. The maximal binding capacity determined for spectrin heterodimers was 132 μg of spectrin/mg of inverted vesicle protein or approximately one-half the physiological concentraton. Extraction of bound [32P]spectrin from the inverted vesicles at 4°C demonstrated that 90{\%} of the bound column-purified tetramers remain in the α2-β2 tetrameric form; while 92{\%} of the bound converted heterodimers are in the α-β heterodimeric form. Therefore, heterodimer-tetramer interconversion does not occur at the syndein binding site under the conditions of our binding studies (physiological ionic strength, pH 7.5, 4°C). We conclude that spectrin tetramers and heterodimers bind to the syndeins with indistinguishable affinity, and the stoichiometry of binding on the membrane is 1 α2-β2 spectrin tetramer/syndein binding site.",
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N2 - 32P-labled spectrin α2-β2 tetramers were extracted from human erythrocyte ghosts at low ionic strength and 4°C. The [32P]spectrin tetramers were purified by column chromatography on Sepharose 4B, and appeared homogenous by the criteria of sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Column-purified spectrin tetramers were found to be 90% in the 12 S α2-β2 oligomeric form by rate zonal sedimentation. Incubation of the column-purified tetramers at 37°C (physiological ionic strength buffer, pH 7.5) for 1 h yielded 89% α-β heterodimeric spectrin. The spectrin tetramers and converted heterodimers bound to their high affinity membrane receptors (syndeins) with essentially the same affinity; the respective K(D) values were 17 ± 2 nM and 16 ± 2 nM. The maximal binding capacity for the spectrin tetramers was 270 μg of spectrin/mg of inverted vesicle membrane protein which is close to the physiological concentration of spectrin on the membrane. The maximal binding capacity determined for spectrin heterodimers was 132 μg of spectrin/mg of inverted vesicle protein or approximately one-half the physiological concentraton. Extraction of bound [32P]spectrin from the inverted vesicles at 4°C demonstrated that 90% of the bound column-purified tetramers remain in the α2-β2 tetrameric form; while 92% of the bound converted heterodimers are in the α-β heterodimeric form. Therefore, heterodimer-tetramer interconversion does not occur at the syndein binding site under the conditions of our binding studies (physiological ionic strength, pH 7.5, 4°C). We conclude that spectrin tetramers and heterodimers bind to the syndeins with indistinguishable affinity, and the stoichiometry of binding on the membrane is 1 α2-β2 spectrin tetramer/syndein binding site.

AB - 32P-labled spectrin α2-β2 tetramers were extracted from human erythrocyte ghosts at low ionic strength and 4°C. The [32P]spectrin tetramers were purified by column chromatography on Sepharose 4B, and appeared homogenous by the criteria of sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Column-purified spectrin tetramers were found to be 90% in the 12 S α2-β2 oligomeric form by rate zonal sedimentation. Incubation of the column-purified tetramers at 37°C (physiological ionic strength buffer, pH 7.5) for 1 h yielded 89% α-β heterodimeric spectrin. The spectrin tetramers and converted heterodimers bound to their high affinity membrane receptors (syndeins) with essentially the same affinity; the respective K(D) values were 17 ± 2 nM and 16 ± 2 nM. The maximal binding capacity for the spectrin tetramers was 270 μg of spectrin/mg of inverted vesicle membrane protein which is close to the physiological concentration of spectrin on the membrane. The maximal binding capacity determined for spectrin heterodimers was 132 μg of spectrin/mg of inverted vesicle protein or approximately one-half the physiological concentraton. Extraction of bound [32P]spectrin from the inverted vesicles at 4°C demonstrated that 90% of the bound column-purified tetramers remain in the α2-β2 tetrameric form; while 92% of the bound converted heterodimers are in the α-β heterodimeric form. Therefore, heterodimer-tetramer interconversion does not occur at the syndein binding site under the conditions of our binding studies (physiological ionic strength, pH 7.5, 4°C). We conclude that spectrin tetramers and heterodimers bind to the syndeins with indistinguishable affinity, and the stoichiometry of binding on the membrane is 1 α2-β2 spectrin tetramer/syndein binding site.

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