Possible existence of high molecular precursor proteins for substance P biosynthesis in rabbit spinal ganglia

The presence of precursor protein for substance P (SP) was examined. Sephadex G-75 chromatography of extracts from rabbit spinal ganglia incubated with [³⁵S]methionine gave two radioactive peaks. In the lower molecular weight peak SP was identified by radioimmunoassay, Sephadex G-15 and TLC. When higher molecular weight proteins were incubated with spinal ganglia microsomal preparation and applied to Sephadex G-75, G-15, TLC and HPLC, ³⁵S-labeled SP was identified and characterized as authentic by immunoprecipitation followed by Sephadex G-15. The amount of ³⁵S-labeled SP was reduced by prior heating of ganglia homogenates, addition of N-ethylmaleimide or p-chloromercuribenzoic acid but not by cycloheximide. Characterization of higher molecular weight proteins by Sephadex G-200, gel-permeation chromatography and chromatofocusing revealed that the proteins were of approx. 100,000 and 7000 dalton with isoelectric points of 9.0, 8.4 and 7.8. These results suggest that the processing from a precursor protein to SP may involve several steps and our high molecular weight protein of 7000 dalton may be one of these intermediate precursor peptides for SP.