Biochemical characterization of growth hormone receptor in rainbow trout (Oncorhynchus mykiss) before and after purification

The aim of this work was to verify if, compared to mammals, the lower molec ular weight of GH-R previously reported in salmonid is real or due to the e xperimental process. For this purpose, we compared the apparent molecular w eight of GH-R, obtained by SDS-PAGE after cross-linking with I-125-rtGH, ob tained from rainbow trout crude liver membrane preparation, incubated in di fferent buffers with those obtained after purification with affinity chroma tography. Using crude liver membrane preparation, two specific bands of I-125-rtGH-pr otein complex were observed: the major one corresponds to a MW of 70 kDa an d the minor one to 45 kDa. However, the pattern of electrophoresis varied a ccording to the different incubation buffers tested. Digestion of the cross -linked complex with beta-galactosidase and phospholipase did not significa ntly modify the position of bands, whilst N-glycosidase F induced a large s mear including 4 more pronounced bands (50, 65, 97 and > 130 kDa), the heav ier band corresponding to the most intensive signal. GH receptors were purified using solubilisation and affinity chromatography . The yield of the liver GH-R from crude liver membrane preparation by the solubilization technique was optimized (48%) using Triton 1% for 1 h (12 de grees C ). Specific binding sites in the solubilized membrane proteins were saturable when incubated with increasing I-125-rtGH concentrations, and re vealed a high affinity constant (Ka=0.7x10(9) M-1). After affinity chromato graphy, specific binding activity was increased 64,000 fold. However, the p urity of the preparation was partial and the purification yield was very lo w (about 0.3%). This enriched fraction, analysed by SDS-PAGE after cross-li nking, showed a very intense band (about 63 kDa) which disappeared with an excess of cold rtGH. These results suggest that the lower molecular weight observed in salmonid (41 kDa), compared to mamals, is not due to the experimental process. The s ignificance of GH-R size difference between salmonids and mammals is discus sed.