Radioimmunoreactivity and receptor-binding activity of the recombined molecule obtained by complementation of two fibrinolysin fragments of ovine prolactin.

Abstract

The recombined molecule obtained by complementation of two fibrinolysin fragments of ovine prolactin (oPRL) has been characterized by radioimmunoassay and radioreceptor assay. The recombinant alone exhibits very low radioimmunoreactivity and radioreceptor activity. However, in the presence of excess fragment oPRL-(1-53), which does not compete with oPRL in either assay, the recombinant has 101% of the radioimmunoreactivity and only 13% of the radioreceptor activity. The result shows that the low activity of the recombinant when assayed alone is due to dissociation ofthe molecule. When the molecule is completely in the recombined form in the presence of excess oPRL-(1-53), it retains full immunoreactivity but only part of the radioreceptor activity.It has been reported (1) that fibrinolysin (plasmin; EC 3.4.21.7) specifically cleaves the peptide bond of ovine prolactin (oPRL) between Met-53 and Ala-54, giving rise to two fragments: oPRL-(1-53) and oPRL-(54-199). These two fragments have been shown to recombine by noncovalent interaction to form a recombinant indistinguishable from the native molecule im, munologically as revealed by gel double diffusion and microcomplement fixation. Exclusion chromatography and circular dichroism spectra of the recombinant also exhibited patterns nearly identical to those of the native molecule. However, the recombined molecule possesses very low biological activity in the pigeon crop-sac assay (1). In an attempt to explain this apparent dichotomy, we further characterize the immunoreactivity by radioimmunoassay (RIA) and the receptor-binding activity by radioreceptor assay (RRA) of the recombined molecule. Results ofthese investigations are reported herein.