Synaptic vesicles containing substance P purified by chromatography on controlled pore glass

Subsynaptosomal particles containing the peptide, substance P, detected by radioimmunoassay were prepared by osmotic lysis of rat brainstem synaptosomes and chromatographed on a calibrated column of controlled pore glass beads of nominal pore size 300 nm. Immunoreactive substance P migrated on this column with particles of apparent mean diameter 117 nm. This particulate, immunoreactive substance P was indistinguishable from synthetic substance P by gel filtration and reverse-phase high pressure liquid chromatography. The specific activity of substance P with respect to protein in peak fractions was 140 pmoles/mg protein, 75-fold higher than in the crude homogenate. Particulate substance P was resistant to attack by endogenous proteases in a crude preparation. Depolarization of synaptosomes with 75 muM veratridine prior to vesicle purification decreased the recovery of particulate substance P 37%. This depletion was dependent on external calcium ions and was blocked by 1 muM tetrodotoxin. The foregoing properties of particulate substance P are consistent with its identification with the large, intraterminal substance P-positive vesicles seen by immunocytochemistry and suggest that these vesicles are involved in the release of substance P.