Evidence for the existence of a prohormone for relaxin

Abstract

The existence of prohormone(s) for relaxin has been investigated both by purification from extracts of pregnant sow ovaries and by in vitro biosynthetic studies. An acid extract of fresh pregnant sow corpora lutea gave two major peaks (measured by UV absorbance at 280 nm) when gel filtered on a Sephadex G-50 (superfine) column. The first peak corresponded to the void volume, whereas the second peak corresponded to the 6,300 dalton, biologically active relaxin. However, radioimmunoassay of the column fractions detected six forms of relaxin-like materials with molecular weight ranging from above 30,000 to 6,300 daltons. Radioimmunoassay of column fraction from large scale extraction of frozen pregnant sow ovaries detected two major species of high molecular weight relaxin-like materials, besides the 6,300 dalton relaxin. On further purification these two species of relaxin-like materials were resolved into three forms with apparent molecular weights of 19,000, 13,000 and 10,000 daltons and correspond to the three predominant forms detected in fresh ovaries. Each of them can be converted to the 6,300 dalton relaxin by TPCK-trypsin. The biosynthesis of relaxin was studied by incubation of corpora luteal slices from 90 day pregnant sow ovaries in the presence of [3H]lysine for up to six hours. The tissue extracts and incubation media were then analyzed by gel filtration, followed by immunoprecipitation or radioimmunoassay of the column fractions. Two high molecular weight 3H-labeled fractions (39,500 and 28,000 dalton) were immunoprecipitated corresponding to the two largest of the six forms of immunoreactive materials detected by radioimmunoassay. No conversion of these high molecular weight forms to the lower molecular weight forms was observed even after six hours of incubation. The two 3H-labeled fractions obtained by immunoprecipitation were specific to corpus luteum, because they were not detected in stromal tissue treated similarly. Limited treatment with TPCK-trypsin converts these materials to a peak corresponding to relaxin. Although a precursor-product relationship has not been established, the 39,500 dalton component may represent a precursor for relaxin, from which subsequent proteolytic cleavage at different sites of the molecule may generate a family of intermediates and finally the 6,300 dalton relaxin.