Characterization of striped bass growth hormone receptors by disulfide-bond reduction and cross-linking studies.
Growth hormone (GH) receptors were analyzed in striped bass (Morone saxatilis) by addition of disulfide-bond reducing agents to radioreceptor assays and by cross-linking both striped bass and coho salmon (Oncorhynchus kisutch) crude membrane preparations to radiolabeled hormone. Dithiothreitol (DTT) caused a dose-dependent increase in specific binding of 125I-tilapia (Oreochromis mossambicus) GH to striped bass membrane preparations. Maximal enhancement of 3.4-fold was obtained with 1 mM DTT and 0.03 trypsin inhibitor units/ml of aprotinin. Addition of N-ethylmaleimide (NEM), which binds covalently to free sulfhydryl groups, decreased specific binding. Scatchard analysis of striped bass membrane preparations indicated a single class of GH receptors. Addition of DTT with aprotinin increased GH-binding site concentration from 278 to 507 fmol/mg, while the dissociation constant of 0.56 nM remained unchanged. Cross-linking 125I-tilapia GH to striped bass hepatic membrane preparations and 125I-salmon GH to coho salmon membrane preparations yielded two to three specifically labeled proteins on sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Endoglycosidase H treatment was without effect on specifically labeled proteins from either species. Following digestion with N-glycosidase F, relative molecular weights of specifically labeled 125I-GH complexes were reduced, suggesting that hepatic GH-binding proteins in striped bass and salmon are N-linked glycoproteins.
Gray
E S
ES
Department of Integrative Biology, University of California, Berkeley 94720.
Tsai
R W
RW
D000818:Animals; D001492:Bass / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D003432:Cross-Linking Reagents; D004220:Disulfides / Q000378:metabolism; D004229:Dithiothreitol / Q000494:pharmacology; D006026:Glycoside Hydrolases / Q000378:metabolism; D017684:Oncorhynchus kisutch; D011986:Receptors, Somatotropin / Q000378:metabolism*
D003432:Cross-Linking Reagents; D004220:Disulfides; D011986:Receptors, Somatotropin; D006026:Glycoside Hydrolases; D004229:Dithiothreitol