Proteolytic processing of secretory proteins in Paramecium: Immunological and biochemical characterization of the precursors of trichocyst matrix proteins (original) (raw)

We used polyclonal serum raised against mature trichocyst matrix proteins to detect their unprocessed precursors, a group of proteins (45-55 kDa) present in the whole-cell extract. These precursor proteins were partially purified from the soluble fraction of wild-type cells by ammonium sulfate precipitation and anion-exchange chromatography. Using monoclonal antibodies against each of four families of mature (processed) matrix proteins, we showed that each family was derived from a separate group of precursors. Our results also suggest that in three of four precursors, those in which the mature proteins consist of disulfide-linked heterodimers, intrachain disulfide bonds form before proteolytic processing. Purified precursors eluted from preparative SDS-gels were used to raise rabbit antiserum, which after preadsorption with mature processed proteins specifically recognized precursors, as judged by ELISA and immunoblots. In cross-sections of developing trichocysts, the anti-precurso...