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 postulated from the coding sequence of cloned cDNA and the results of cell-free translation studies, has been directly demonstrated in rat ovaries 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 connecting region. Immunoreactive bands of 18 and 16.5 kDa were shown in ovaries from day 20 pregnant rats. Partial amino acid sequence analysis of both peptides revealed that they had identical N-terminal sequences, corresponding to rat prorelaxin. Both 18- and 16.5-kDa bands were present only from midpregnancy until near term, when they declined sharply. These changes in the concentration of 18-kDa prorelaxin match changes in preprorelaxin mRNA levels, suggesting that relaxin synthesis is 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 might also be more than a precursor per se.
Soloff
M S
MS
Department of Biochemistry and Molecular Biology, Medical College of Ohio, Toledo 43699.
Shaw
A R
AR
Gentry
L E
LE
Marquardt
H
H
Vasilenko
P
P
D000595:Amino Acid Sequence; D000818:Animals; D001419:Bacteria / Q000378:metabolism*; D005260:Female; D007106:Immune Sera / Q000276:immunology*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010053:Ovary / Q000737:chemistry*; D011247:Pregnancy; D011270:Pregnancy, Animal / Q000378:metabolism*; D011498:Protein Precursors / Q000032:analysis* / Q000276:immunology / Q000302:isolation & purification; D012333:RNA, Messenger / Q000032:analysis; D011817:Rabbits; D051381:Rats; D012065:Relaxin / Q000032:analysis* / Q000276:immunology / Q000302:isolation & purification
D007106:Immune Sera; D011498:Protein Precursors; D012333:RNA, Messenger; C038656:prorelaxin; D012065:Relaxin