Molecular characterization of synaptophysin, a major calcium-binding protein of the synaptic vesicle membrane.
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.
D000818:Animals; D001921:Brain / Q000378:metabolism; D002135:Calcium-Binding Proteins; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003902:Detergents; D007425:Intracellular Membranes / Q000378:metabolism*; D008565:Membrane Proteins / Q000302:isolation & purification* / Q000378:metabolism; D017830:Octoxynol; D011092:Polyethylene Glycols; D011487:Protein Conformation; D051381:Rats; D011919:Rats, Inbred Strains; D012995:Solubility; D013572:Synaptic Vesicles / Q000378:metabolism*; D016708:Synaptophysin
D002135:Calcium-Binding Proteins; D003902:Detergents; D008565:Membrane Proteins; D016708:Synaptophysin; D011092:Polyethylene Glycols; D017830:Octoxynol