Brain α erythroid spectrin

identification, compartmentalization, and β spectrin associations

M. Blair Clark, Yupo Ma, Michael L. Bloom, Jane E. Barker, Ian S. Zagon, Warren E. Zimmer, Steven Goodman

Research output: Contribution to journalArticle

34 Citations (Scopus)

Abstract

Using isoform and subunit specific antibodies we have determined the presence, localization, and β spectrin associations of α erythroid spectrin, αSpIΣ*, as well as ga non-erythroid spectrin, αSpIIΣ1, in mouse brain. Peptide spcific antibodies against unique sequences within the βSpIIΣ1, non-erythroid β spectrin isoform, and within βSpIΣ1, erythrocyte β spectrin isoform were used to compare the immunolocalization of β spectrin subunit isoforms with that of α spectrin subunit isoforms and to immunoprecipitate spectrin tetramers in order to identify the subunit components by immunoblot analysis. The specificity and sensitivity of antibodies for isoform specific α and β subunits was determined by immunodot and immunoblot methods. Immunohistochemical analyses indicated that βSpIΣ2 is located in neuronal somata and dendrites in mouse cerebellum. βSpIIΣ1 is located in the medullary layer, chiefly composed of axonal tracts. Parallel immunohistochemical analysis with antibodies for the α and β spectrin isoforms revealed that antibodies specific for the α subunit of erythrocyte spectrin (αSpIΣ1) localized antigen to the somata and dendrites of cerebellar granule cell neurons, a pattern similar to that for the localization of the erythroid β subunit (βSpIΣ2). In contrast antibodies specific for the non-erythroid α subunit (αSpIIΣ1) localized antigen to axons in the cerebellum corresponding to the pattern for the non-erythroid β subunit (βSpIIΣ1). The distinct localization of antigens by antisera which recognize either the α subunit of red blood cell spectrin or the α subunit of non-erythroid brain spectrin, together with the correspondence of their localization with appropriate β subunits, clearly indicate that brain contains at least two species of spectrin each with distinct α and β subunits. Immunoprecipitation experiments of cerebellar extracts using β spectrin peptide specific antibodies follwoed by immunoblotting analysis confirmed the association of an erythroid α subunit isoform with a β erythroid subunit isoform, as well as the association of non-erythroid α and β subunits. In addition the immunoblot analysis of the immunoprecipitated material suggested there are minor populations of various hybrid tetramers in brain consisting of mixed erythroid and non-erythroid subunits. In summary these data collectively demonstrate that in mouse brain there are at least two α spectrin subunits, one erythroid αSpIΣ* and one non-erythroid αSpIIΣ1; these associate with an erythroid βSpIΣ1, and a non-erythroid βSpIIΣ1 in the cerebellum of mouse. Although mixed tetramers occur, spectrin tetramers occur predominantly as (αSpIΣ*/βSpIΣ1)2, a purely erythroid tetramer, and as (αSpIIΣ1/βSpIΣ1)2, a purely non-erythroid tetramer.

Original languageEnglish (US)
Pages (from-to)223-236
Number of pages14
JournalBrain Research
Volume663
Issue number2
DOIs
StatePublished - Nov 14 1994
Externally publishedYes

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Spectrin
Brain
Protein Isoforms
Antibodies
Cerebellum
Erythrocytes
Carisoprodol
Dendrites
Antigens
Peptides
Antibody Specificity

All Science Journal Classification (ASJC) codes

  • Neuroscience(all)
  • Molecular Biology
  • Clinical Neurology
  • Developmental Biology

Cite this

Brain α erythroid spectrin : identification, compartmentalization, and β spectrin associations. / Blair Clark, M.; Ma, Yupo; Bloom, Michael L.; Barker, Jane E.; Zagon, Ian S.; Zimmer, Warren E.; Goodman, Steven.

In: Brain Research, Vol. 663, No. 2, 14.11.1994, p. 223-236.

Research output: Contribution to journalArticle

Blair Clark, M. ; Ma, Yupo ; Bloom, Michael L. ; Barker, Jane E. ; Zagon, Ian S. ; Zimmer, Warren E. ; Goodman, Steven. / Brain α erythroid spectrin : identification, compartmentalization, and β spectrin associations. In: Brain Research. 1994 ; Vol. 663, No. 2. pp. 223-236.
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abstract = "Using isoform and subunit specific antibodies we have determined the presence, localization, and β spectrin associations of α erythroid spectrin, αSpIΣ*, as well as ga non-erythroid spectrin, αSpIIΣ1, in mouse brain. Peptide spcific antibodies against unique sequences within the βSpIIΣ1, non-erythroid β spectrin isoform, and within βSpIΣ1, erythrocyte β spectrin isoform were used to compare the immunolocalization of β spectrin subunit isoforms with that of α spectrin subunit isoforms and to immunoprecipitate spectrin tetramers in order to identify the subunit components by immunoblot analysis. The specificity and sensitivity of antibodies for isoform specific α and β subunits was determined by immunodot and immunoblot methods. Immunohistochemical analyses indicated that βSpIΣ2 is located in neuronal somata and dendrites in mouse cerebellum. βSpIIΣ1 is located in the medullary layer, chiefly composed of axonal tracts. Parallel immunohistochemical analysis with antibodies for the α and β spectrin isoforms revealed that antibodies specific for the α subunit of erythrocyte spectrin (αSpIΣ1) localized antigen to the somata and dendrites of cerebellar granule cell neurons, a pattern similar to that for the localization of the erythroid β subunit (βSpIΣ2). In contrast antibodies specific for the non-erythroid α subunit (αSpIIΣ1) localized antigen to axons in the cerebellum corresponding to the pattern for the non-erythroid β subunit (βSpIIΣ1). The distinct localization of antigens by antisera which recognize either the α subunit of red blood cell spectrin or the α subunit of non-erythroid brain spectrin, together with the correspondence of their localization with appropriate β subunits, clearly indicate that brain contains at least two species of spectrin each with distinct α and β subunits. Immunoprecipitation experiments of cerebellar extracts using β spectrin peptide specific antibodies follwoed by immunoblotting analysis confirmed the association of an erythroid α subunit isoform with a β erythroid subunit isoform, as well as the association of non-erythroid α and β subunits. In addition the immunoblot analysis of the immunoprecipitated material suggested there are minor populations of various hybrid tetramers in brain consisting of mixed erythroid and non-erythroid subunits. In summary these data collectively demonstrate that in mouse brain there are at least two α spectrin subunits, one erythroid αSpIΣ* and one non-erythroid αSpIIΣ1; these associate with an erythroid βSpIΣ1, and a non-erythroid βSpIIΣ1 in the cerebellum of mouse. Although mixed tetramers occur, spectrin tetramers occur predominantly as (αSpIΣ*/βSpIΣ1)2, a purely erythroid tetramer, and as (αSpIIΣ1/βSpIΣ1)2, a purely non-erythroid tetramer.",
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T1 - Brain α erythroid spectrin

T2 - identification, compartmentalization, and β spectrin associations

AU - Blair Clark, M.

AU - Ma, Yupo

AU - Bloom, Michael L.

AU - Barker, Jane E.

AU - Zagon, Ian S.

AU - Zimmer, Warren E.

AU - Goodman, Steven

PY - 1994/11/14

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N2 - Using isoform and subunit specific antibodies we have determined the presence, localization, and β spectrin associations of α erythroid spectrin, αSpIΣ*, as well as ga non-erythroid spectrin, αSpIIΣ1, in mouse brain. Peptide spcific antibodies against unique sequences within the βSpIIΣ1, non-erythroid β spectrin isoform, and within βSpIΣ1, erythrocyte β spectrin isoform were used to compare the immunolocalization of β spectrin subunit isoforms with that of α spectrin subunit isoforms and to immunoprecipitate spectrin tetramers in order to identify the subunit components by immunoblot analysis. The specificity and sensitivity of antibodies for isoform specific α and β subunits was determined by immunodot and immunoblot methods. Immunohistochemical analyses indicated that βSpIΣ2 is located in neuronal somata and dendrites in mouse cerebellum. βSpIIΣ1 is located in the medullary layer, chiefly composed of axonal tracts. Parallel immunohistochemical analysis with antibodies for the α and β spectrin isoforms revealed that antibodies specific for the α subunit of erythrocyte spectrin (αSpIΣ1) localized antigen to the somata and dendrites of cerebellar granule cell neurons, a pattern similar to that for the localization of the erythroid β subunit (βSpIΣ2). In contrast antibodies specific for the non-erythroid α subunit (αSpIIΣ1) localized antigen to axons in the cerebellum corresponding to the pattern for the non-erythroid β subunit (βSpIIΣ1). The distinct localization of antigens by antisera which recognize either the α subunit of red blood cell spectrin or the α subunit of non-erythroid brain spectrin, together with the correspondence of their localization with appropriate β subunits, clearly indicate that brain contains at least two species of spectrin each with distinct α and β subunits. Immunoprecipitation experiments of cerebellar extracts using β spectrin peptide specific antibodies follwoed by immunoblotting analysis confirmed the association of an erythroid α subunit isoform with a β erythroid subunit isoform, as well as the association of non-erythroid α and β subunits. In addition the immunoblot analysis of the immunoprecipitated material suggested there are minor populations of various hybrid tetramers in brain consisting of mixed erythroid and non-erythroid subunits. In summary these data collectively demonstrate that in mouse brain there are at least two α spectrin subunits, one erythroid αSpIΣ* and one non-erythroid αSpIIΣ1; these associate with an erythroid βSpIΣ1, and a non-erythroid βSpIIΣ1 in the cerebellum of mouse. Although mixed tetramers occur, spectrin tetramers occur predominantly as (αSpIΣ*/βSpIΣ1)2, a purely erythroid tetramer, and as (αSpIIΣ1/βSpIΣ1)2, a purely non-erythroid tetramer.

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