Ovine placental lactogen and ovine prolactin: partial proteolysis and conformational stability

FERNÁNDEZ ML, CYMES GD, CURTO LM, WOLFENSTEIN-TODEL C

INTERNATIONAL JOURNAL OF BIOCHEMISTRY AND CELLULAR BIOLOGY

Elsevier

Año: 2000 vol. 32 p. 597 - 608

1357-2725

The high-resolution structure of ovine placental lactogen (oPL) and ovine prolactin (oPRL), not yet established indetail, was probed by limited proteolysis with the Glu-speci®c protease from Staphylococcus aureus V8. While inhGH there were no cleavage sites inside of any of the four a±helices, the analysis of the fragments obtained afterpartial proteolysis of oPL showed a site of cleavage at the putative third helix, suggesting that this helix is partiallyunwound at this point. The partial proteolysis of the rest of the molecule was compatible with a similar foldingpattern for oPL, hGH and pGH, on the basis of the crystal structure of these last hormones. In the case of oPRL,proteolytic cleavage occurred at Glu residues which would be located at the end of the ®rst helix and the beginningof the second in the hGH folding model, suggesting that these helices are shorter in oPRL than in hGH. In order togain further insight on the folding of these molecules, circular dichroism and intrinsic ¯uorescence measurementswere used to examine the e€ect of denaturing conditions on oPL and oPRL. After exposure to 6 M guanidine theunfolding of both proteins was completely reversed upon elimination of the denaturing agent. In contrast, exposureto pH 3.0 caused an irreversible decrease in the a-helical content in both hormones, most striking for oPL, indicating that this hormone is less stable than oPRL or hGH