Synapsin I-mediated interaction of brain spectrin with synaptic vesicles.
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 micrograms/ml and a maximal binding capacity = 1.9 micrograms vesicle protein per microgram 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 Kl = 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.
Sikorski
A F
AF
Department of Structural and Cellular Biology, College of Medicine, University of South Alabama, Mobile 36688.
Terlecki
G
G
Zagon
I S
IS
Goodman
S R
SR
D000818:Animals; D015153:Blotting, Western; D001921:Brain / Q000378:metabolism*; D001923:Brain Chemistry; D002417:Cattle; D004591:Electrophoresis, Polyacrylamide Gel; D007425:Intracellular Membranes / Q000378:metabolism / Q000502:physiology / Q000648:ultrastructure; D009419:Nerve Tissue Proteins / Q000502:physiology*; D011485:Protein Binding / Q000502:physiology; D012044:Regression Analysis; D013049:Spectrin / Q000032:analysis / Q000378:metabolism* / Q000502:physiology; D016704:Synapsins; D013572:Synaptic Vesicles / Q000378:metabolism* / Q000502:physiology / Q000648:ultrastructure
D009419:Nerve Tissue Proteins; D016704:Synapsins; D013049:Spectrin