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 × 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.
Reduction and carbamidomethylation of two of the three disulfide bridges of ovine placental lactogen was accomplished by the use of 20‐fold molar excess of dithiothreitol over protein disulfide content. The derivative retained its binding capacity to somatogenic as well as lactogenic rat liver receptors, although the latter was somewhat diminished. The two disulfide bonds exposed to the reducing agent are those located near the carboxy‐ and amino‐terminus. while the larger loop remained intact after reduction. This behaviour is similar to that of bovine growth hormone, where the larger loop was also more resistant to reduction.
The α‐amino group of ovine prolactin (oPRL) and human growth hormone (hGH) was selectively modified by transamination with glyoxylic acid. No difference was found in the binding capacity of transaminated oPRL to rat liver lactogenic receptors with respect to its control, although both samples showed a decrease in its binding capacity with reference to the native hormone. This decrease was due to conformational changes caused by the reaction conditions and not by the transamination itself, as shown by the circular dichroism spectra. Transaminated hGH retained the full binding capacity of the hormone. These results suggest that the α‐amino group is not relevant for the binding to lactogenic liver receptors in both lactogenic hormones.
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