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Molecular characterization of synaptophysin, a major calcium‐binding protein of the synaptic vesicle membrane.
Author(s) -
Rehm H.,
Wiedenmann B.,
Betz H.
Publication year - 1986
Publication title -
the embo journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 7.484
H-Index - 392
eISSN - 1460-2075
pISSN - 0261-4189
DOI - 10.1002/j.1460-2075.1986.tb04243.x
Subject(s) - synaptophysin , synaptic vesicle , vesicle , biology , synaptic membrane , membrane , neuraminidase , calcium , glutaraldehyde , biochemistry , biophysics , chromatography , chemistry , enzyme , immunohistochemistry , organic chemistry , immunology
Synaptophysin, a mol. wt 38 000 glycopolypeptide of the synaptic vesicle membrane, was solubilized using Triton X‐100 and purified by immunoaffinity or ion‐exchange chromatography. From gel permeation and sucrose‐density centrifugation in H2O/D2O, a Stokes radius of 7.3 nm, a partial specific volume of 0.830 and a total mol. wt of 119 000 were calculated for the native protein. Cross‐linking of synaptic vesicles with glutaraldehyde, dimethylsuberimidate, or Cu2+ ‐o‐phenantroline, resulted in the formation of a mol. wt 76 kd dimer of synaptophysin. Crosslinking of the purified protein in addition produced tri‐ and tetrameric adducts of the polypeptide. Native synaptophysin thus is a homooligomeric protein. Synaptophysin is N‐glycosylated, since cultivation of the rat phaeochromocytoma cell line PC12 in the presence of tunicamycin reduced its mol. wt by about 6 kd. Upon transfer to nitrocellulose and incubation with 45Ca2+, synaptophysin behaved as one of the major calcium‐binding proteins of the synaptic vesicle membrane. Pronase treatment of intact synaptic vesicles abolished this 45Ca2+ binding indicating that the Ca2+ binding site of synaptophysin must reside on a cytoplasmic domain of the transmembrane polypeptide. Based on these data, we propose that synaptophysin may play an important role in Ca2+‐dependent neurotransmitter release.

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