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

The high-resolution structure of ovine placental lactogen (oPL) and ovine prolactin (oPRL), not yet established in detail, was probed by limited proteolysis with the Glu-specific protease from Staphylococcus aureus V8. While in hGH there were no cleavage sites inside of any of the four alpha-helices, the analysis of the fragments obtained after partial proteolysis of oPL showed a site of cleavage at the putative third helix, suggesting that this helix is partially unwound at this point. The partial proteolysis of the rest of the molecule was compatible with a similar folding pattern 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 first helix and the beginning of the second in the hGH folding model, suggesting that these helices are shorter in oPRL than in hGH. In order to gain further insight on the folding of these molecules, circular dichroism and intrinsic fluorescence measurements were used to examine the effect of denaturing conditions on oPL and oPRL. After exposure to 6 M guanidine the unfolding of both proteins was completely reversed upon elimination of the denaturing agent. In contrast, exposure to pH 3.0 caused an irreversible decrease in the alpha-helical content in both hormones, most striking for oPL, indicating that this hormone is less stable than oPRL or hGH.

A sialic acid-binding lectin from ovine placenta: purification, specificity and interaction with actin.

A sialic-acid-specific lectin from ovine placental cotyledons was purified by affinity chromatography on bovine submaxillary mucin-agarose followed by gel filtration, and it showed a molecular weight of 65000 by sodium dodecylsulfate-polyacrylamide gel electrophoresis. This lectin has the capacity to interact with actin, since it binds to actin-F in a cosedimentation assay and it acts as a mediator in the binding of actin to the affinity column. The lectin agglutinated rabbit and rat erythrocytes, but not human A, B or O erythrocytes. Haemagglutination inhibition assays of different saccharides, glycoproteins and glycolipids indicate that this lectin has affinity for sialic acid, which is enhanced by its O-acetylation. The N-terminal sequence of the protein shows 92% identity with rabbit and porcine uterine calreticulin.

Detection and characterization of an ovine placental lactogen stable intermediate in the urea-induced unfolding process.

The urea-induced equilibrium unfolding of ovine placental lactogen, purified from ovine placenta, was followed by size-exclusion chromatography, far-UV CD, and intrinsic tryptophan fluorescence. The data obtained by each of these methods showed a poor fit to a two-state model involving only a native and an unfolded form. A satisfactory fit required, instead, a model that involved a stable, partially folded form in addition to the native and unfolded ones. The results obtained from the best-fitting theoretical curves for the three-state model indicated that this intermediate state, which is the predominant species in solution at 3.6 M of urea activity, is compact, largely alpha-helical, and changes considerably the native-like tertiary packing around its tryptophan residues. These findings suggest that this stable intermediate exhibits properties similar to those that characterize the molten globule state.

Identification of a tyrosine residue in ovine placental lactogen as essential for its binding to receptors.

Nitration of ovine placental lactogen (oPL) with a 10-fold molar excess of tetranitromethane over protein content resulted in the modification of 0.8 tyrosine residue. No conformational changes were observed by either fourth-derivative spectral analysis or circular dichroism. Nitration significantly decreased the binding capacity of the hormone to lactogenic and somatogenic rat liver receptors. This binding capacity was not restored by reduction of the nitro groups, thus indicating that the decrease was not due to the difference in pK between tyrosine and nitrotyrosine. The nitrotyrosine-containing peptide was isolated from a tryptic digest by HPLC and its modification extent was of 67%, which is consistent with the decrease in binding capacities (65% and 70%). Its amino acid sequence was determined and the modified tyrosine residue was identified as Tyr-46. These results provide the first evidence of the involvement of a tyrosine residue in the binding of oPL to both lactogenic and somatogenic receptors. This tyrosine appears to be a shared binding epitope between oPL and the prolactins.

Modification of arginine residues in ovine prolactin by 1,2-cyclohexanedione. Effect on binding capacity to lactogenic receptors.

The reactivity of arginine residues in ovine prolactin was studied by reaction with 1,2-cyclohexanedione. Kinetic analysis of the data showed a good fit with two simultaneous pseudo-first-order equations with apparent velocity constants of 0.28 and 1.2 x 10(-2) min-1, corresponding to 1.8 'fast' and 8.7 'slow' residues, respectively. Modification led to a decrease in binding capacity to lactogenic rat liver receptors, and apparently the modification of the two 'fast' reacting arginine residues is responsible for the rapid loss of this capacity. The presence of a non-reacting arginine has been described in human and bovine growth hormones, and it is located near the carboxy-terminus. This lack of reactivity is probably due to the formation of a salt bridge, since the arginine residue becomes susceptible to modification once the peptide is separated from the rest of the molecule. This salt bridge is absent in ovine prolactin, since the homologous arginine residue is reactive with cyclohexanedione. This result suggests that there could be a difference between the three-dimensional structure of ovine prolactin and of the growth hormones, at least near the carboxy-terminal region of the molecule.

Selective modification of tryptophan-150 in ovine placental lactogen.

1. Ovine placental lactogen was modified by reaction with o-nitrophenylsulfenyl chloride. Fluorescence measurements indicated that one of the two tryptophan residues of the molecule had reacted. Besides, there was some reagent not covalently bound. 2. The reagent was covalently bound to Trp-150. No evidence of modification of Trp-90 was found. 3. Binding capacity to lactogenic as well as somatogenic receptors was diminished but not abolished upon modification, indicating that absolute molecular integrity of Trp-150 is not required for binding. 4. This behavior is similar to that of the tryptophan residues of ovine prolactin.

Chemical modification of ovine prolactin with N-acetylimidazole.

Reaction of ovine prolactin (oPRL) with a 150-fold molar excess of N-acetylimidazole over protein content resulted in the modification of 2.5 tyrosine residues and 1.2 lysine residues. Acetylation greatly decreased the in vitro binding capacity to lactogenic sites. This binding capacity was partially restored by ammonium bicarbonate treatment, which removes O-acetyl groups from tyrosine residues but not N-acetyl groups from lysine residues. The modification extent of the tyrosine residues was determined. The results suggest that acetylation of tyrosine 44 or of tyrosine 96 is likely to be responsible for the decrease in binding activity of acetylated oPRL, and that one of these residues may play a role in the interaction of oPRL with lactogenic receptors.