Precursor forms of substance P (SP) in nervous tissue: detection with antisera to SP, SP-Gly, and SP-Gly-Lys

Antisera generated to substance P-Gly (SP-G) and substance P-Gly-Lys (SP-G-K), the likely unamidated COOH-terminally extended forms of substance P, were used to quantify and localize substance P precursor forms in hamster brain stem and spinal cord. The precursor determinant SP-G-K was liberated from larger heterogeneous forms by mild trypsinization of tissue extracts and was converted into the second precursor determinant, SP-G, by subsequent treatment with carboxypeptidase B. The basal levels of SP-G-K in brain stem and spinal cord were approximately equal to 0.5 pg/mg of tissue and rose 43- to 64-fold after trypsinization. Basal levels of SP-G were comparable to those of SP-G-K and rose 10- to 29-fold after combined enzyme treatments. Immunohistochemical labeling of axons and somata with anti-SP-G-K increased dramatically after trypsinization. This labeling was eliminated by preadsorption with authentic SP-G-K but not substance P or SP-G. Gel-permeation chromatography revealed SP-G-K-like immunoreactivity in fractions corresponding to considerably higher molecular weight than mature substance P. Collectively, these results support the hypothesis that substance P is synthesized from larger precursors and demonstrate that extended precursor forms are normally present in the axons and somata of neural systems that synthesize substance P.