DEMONSTRATION OF RELAXIN PRECURSORS IN PREGNANT RAT OVARIES WITH ANTISERA AGAINST BACTERIALLY EXPRESSED RAT PRORELAXIN

The existence of rat 18-kilodalton (kDa) prorelaxin, which has been po stulated from the coding sequence of cloned cDNA and the results of ce ll-free translation studies, has been directly demonstrated in rat ova ries with antibodies against bacterially expressed rat prorelaxin. The peptide expressed in E. coli from a rat prorelaxin cDNA construct was comprised of the B- and A-chains of relaxin and a 105-amino acid conn ecting region. Immunoreactive bands of 18 and 16.5 kDa were shown in o varies from day 20 pregnant rats. Partial amino acid sequence analysis of both peptides revealed that they had identical N-terminal sequence s, corresponding to rat prorelaxin. Both 18- and 16.5-kDa bands were p resent only from midpregnancy until near term, when they declined shar ply. These changes in the concentration of 18-kDa prorelaxin match cha nges in preprorelaxin mRNA levels, suggesting that relaxin synthesis i s regulated at the transcriptional level and not by protein processing . Prorelaxin was transiently secreted by COS-1 cells transfected with preprorelaxin cDNA. Treatment of culture medium with trypsin resulted in the appearance of material corresponding in size to mature relaxin. Thus, correctly folded prorelaxin appears to be a suitable precursor for relaxin. The combined concentrations of 18- and 16.5-kDa peptides in ovaries on day 20 of pregnancy were considerably more than 30 times greater than that of relaxin, however, suggesting that prorelaxin mig ht also be more than a precursor per se.