Characterization of striped bass growth hormone receptors by disulfide-bond reduction and cross-linking studies
✍ Scribed by Gray, Elisabeth S. ;Tsai, Robert W.
- Book ID
- 102893098
- Publisher
- John Wiley and Sons
- Year
- 1994
- Tongue
- English
- Weight
- 825 KB
- Volume
- 268
- Category
- Article
- ISSN
- 0022-104X
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✦ Synopsis
Abstract
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 ^125^I‐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 ^125^I‐tilapia GH to striped bass hepatic membrane preparations and ^125^I‐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 ^125^I‐GH complexes were reduced, suggesting that hepatic GH‐binding proteins in striped bass and salmon are N‐linked glycoproteins. © 1994 Wiley‐Liss, Inc.