Identification of a synaptic vesicle-specific 38,000-dalton protein by monoclonal antibodies

Abstract

Synaptic vesicles were purified from the guinea pig cerebrum by sucrose density gradient centrifugation, and monoclonal antibodies (MAbs) were produced against this vesicle fraction. Seven MAbs (171B5, 171E8, 174D12, 174H11, 177A2, 177H11 and 178D4) recognized a novel acidic protein of about 38,000 daltons which was specific to synaptic vesicles. In immunofluorescence microscopy, the staining pattern of these MAbs corresponded to the distribution of the synapses in the guinea pig central nervous system. These MAbs appeared to stain all synaptic regions, irrespective of their synaptic function or type of neurotransmitters. MAb 171B5 and 174H11 stained the rat, rabbit and bovine synapses similarly to the guinea pig. Two other MAbs (171E8 abd 177H11) stained other mammals weakly but the remaining 3 MAbs reacted only with the guinea pig. In immunoelectron microscopy of both the cerebellar tissue and isolated vesicle fraction, these MAbs selectively labeled the synaptic vesicles but not other structures. Immunoblot analysis was performed on electrophoretically separated proteins in vesicle fraction and brain homogenate. All of 7 MAbs reacted with a band at a molecular weight of about 38,000 from the guinea pig. Isoelectric focussing disclosed that this protein was acidic (pI 4.5–5).