Identification and characterization of a prolactin-like polypeptide synthesized by mitogen-stimulated murine...

Previously, we have reported that concanavalin A (Con A)-stimulated murine splenocytes synthesize and secrete into the medium a substance with prolactin (PRL)-like properties. Western blot analysis of the culture medium of Con A-stimulated murine splenocytes identified a PRL-like polypeptide (Ly-PLP) with an apparent molecular weight (Mr) of 46 kd. Rabbit anti-rat PRL antibody (S-9, NIDDK) was used for immunostaining. Specificity was proved by the absence of a band in properly preabsorbed primary and secondary antibodies. Electroeluted Ly-PLP enhanced the mitogenic response of lymphocytes to Con A. In situ hybridization analysis of dispersed lymphocyte smears demonstrated the presence of an mRNA that hybridized with a rat PRL cDNA probe. The size of the mRNA species was 1.4 kb on Northern blot analysis. Two-dimensional peptide map analysis of pituitary PRL and Ly-PLP showed three peptides with identical migration characteristics. Western blot analysis of lymphocyte culture medium following Con-A-affinity column treatment provided evidence that the Ly-PLP was a non-glycoprotein. Therefore, we conclude that Ly-PLP represents a structural variant of pituitary-PRL (Pit-PRL), and provide evidence to strongly suggest that it is a novel lymphokine.