Synapsin I-mediated interaction of brain spectrin with synaptic vesicles

Aleksander F. Sikorski, Grzegorz Terlecki, Ian S. Zagon, Steven Goodman

Research output: Contribution to journalArticle

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Abstract

We have established a new binding assay in which 125I-labeled synaptic vesicles are incubated with brain spectrin covalently immobilized on cellulosic membranes in a microfiltration apparatus. We obtained saturable, high affinity, salt- (optimum at 50-70 mM NaCl) and pH- (optimum at pH 7.5-7.8) dependent binding. Nonlinear regression analysis of the binding isotherm indicated one site binding with a Kd = 59 μg/ml and a maximal binding capacity = 1.9 μg vesicle protein per μg spectrin. The fact that the binding of spectrin was via synapsin was demonstrated in three ways. (a) Binding of synaptic vesicles to immobilized spectrin was eliminated by prior extraction with 1 M KCl. When the peripheral membrane proteins in the 1 M KCl extract were separated by SDS-PAGE, transferred to nitrocellulose paper and incubated with 125I-brain spectrin, 96% of the total radioactivity was associated with five polypeptides of 80, 75, 69, 64, and 40 kD. All five polypeptides reacted with an anti-synapsin I polyclonal antibody, and the 80- and 75-kD polypeptides comigrated with authentic synapsin Ia and synapsin Ib. The 69- and 64-kD polypeptides are either proteolytic fragments of synapsin I or represent synapsin IIa and synapsin IIb. (b) Pure synapsin I was capable of competitively inhibiting the binding of radioiodinated synaptic vesicles to immobilized brain spectrin with a KI = 46 nM. (c) Fab fragments of anti-synapsin I were capable of inhibiting the binding of radioiodinated synaptic vesicles to immobilized brain spectrin. These three observations clearly establish that synapsin I is a primary receptor for brain spectrin on the cytoplasmic surface of the synaptic vesicle membrane.

Original languageEnglish (US)
Pages (from-to)313-318
Number of pages6
JournalJournal of Cell Biology
Volume114
Issue number2
DOIs
StatePublished - Jan 1 1991
Externally publishedYes

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Synapsins
Spectrin
Synaptic Vesicles
Brain
Peptides
Synaptic Membranes
Immunoglobulin Fab Fragments
Collodion
Radioactivity
Polyacrylamide Gel Electrophoresis
Membrane Proteins
Salts
Binding Sites
Regression Analysis

All Science Journal Classification (ASJC) codes

  • Cell Biology

Cite this

Synapsin I-mediated interaction of brain spectrin with synaptic vesicles. / Sikorski, Aleksander F.; Terlecki, Grzegorz; Zagon, Ian S.; Goodman, Steven.

In: Journal of Cell Biology, Vol. 114, No. 2, 01.01.1991, p. 313-318.

Research output: Contribution to journalArticle

Sikorski, Aleksander F. ; Terlecki, Grzegorz ; Zagon, Ian S. ; Goodman, Steven. / Synapsin I-mediated interaction of brain spectrin with synaptic vesicles. In: Journal of Cell Biology. 1991 ; Vol. 114, No. 2. pp. 313-318.
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