CHARACTERIZATION OF GROWTH HORMONE-BINDING PROTEIN IN CATTLE PLASMA -PROLACTIN-BINDING ACTIVITY AND 24-HOUR PROFILE

The purpose of this study was to characterize circulating growth hormo ne-binding proteins (GHBP) and prolactin-binding proteins (PRLBP) in c attle blood plasma. In particular, the 24-hr profile of these molecule s was investigated. The preincubation of bull plasma with iodinated bo vine growth hormone (bGH) or bovine prolactin (bPRL), followed by gel filtration chromatography (Superdex 200; 1.6 x 60 cm column), resulted in the formation of essentially two complexes. The majority of [I-125 ]bPRL eluted with the first one (M(r) similar to 600 kDa), whereas [I- 125]bGH mainly appeared in the second one (M(r) similar to 70 kDa). Th e fractions corresponding to these two peaks were analyzed by western ligand blotting (WLB), under reducing conditions. WLB revealed, respec tively, 190-, 56-, 52-, and 28-kDa bands for the first peak and only 5 2- and 28-kDa bands for the second one. The nature of the 600-kDa peak is at present undetermined, but the 70-kDa one was previously identif ied as high-affinity GHBP. Displacement studies demonstrated that bGH and bPRL were both able to bind to this GHBP, because the bGH- and bPR L-binding activities of this protein could be saturated by an excess o f either of these two hormones. This was indirectly confirmed by the c lose correlation (r = 0.615; P = 0.0001; n = 155) observed between pla sma bGH- and bPRL-binding activities, because this correlation could s uggest that both ligands are bound to the same proteins. The temporal concentrations of plasma GHBP were measured in samples collected at 20 -min intervals for 24 hr from 8 young bulls. The evaluation of GHBP wa s realized by WLB, followed by densitometric analysis. Some fluctuatio ns were observed, but these were not correlated with bGH release, even with a +/-2-hr lag period. In summary, we found that bovine high-affi nity GHBP binds not only bGH, but also bPRL. A second type of protein, of higher molecular weight, also binds these two hormones, but furthe r investigations are needed to determine its nature. Finally, GHBP con centrations in cattle blood plasma apparently show fluctuations over a 24-hr period, but no correlation was found between these fluctuations and plasma growth hormone concentrations.