Characterization of striped bass growth hormone receptors by disulfide-bond reduction and cross-linking studies.
Characterization of striped bass growth hormone receptors by disulfide-bond reduction and cross-linking studies.
复制标题
通过二硫键还原和交联研究表征条纹鲈鱼生长激素受体。
DOI：
10.1002/jez.1402680603
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发表时间：
1994
期刊：
影响因子：
--
通讯作者：
Tsai,RW
中科院分区：
文献类型：
--
作者：
Gray,ES;Tsai,RW
通过在放射性受体测定中加入二硫键还原剂，并通过将条纹鲈鱼和银鲑（Oncorhynchus kisutch）粗膜制备物与放射性标记激素交联，分析条纹鲈鱼（Morone saxatilis）中的生长激素（GH）受体。二硫苏糖醇（DTT）导致125 I-罗非鱼（Oreochromis mossambicus）GH与条纹鲈鱼膜制备物特异性结合的剂量依赖性增加。使用1 mM DTT和0.03胰蛋白酶抑制剂单位/ml抑肽酶获得3.4倍的最大增强。加入N-乙基马来酰亚胺（NEM）（其与游离巯基共价结合）可降低特异性结合。条纹低音膜制剂的Scatchard分析表明一个单一的类GH受体。添加DTT和抑肽酶使GH结合位点浓度从278 fmol/mg增加至507 fmol/mg，而解离常数0.56 nM保持不变。将125 I-罗非鱼GH与条纹鲈鱼肝膜制备物和125 I-鲑鱼GH与银鲑膜制备物交联，在十二烷基硫酸钠（SDS）-聚丙烯酰胺凝胶电泳上产生了2至3个特异性标记的蛋白质。内切糖苷酶H处理对来自两个物种的特异性标记蛋白没有影响。用N-糖苷酶F消化后，特异性标记的125 I-GH复合物的相对分子量降低，表明条纹鲈鱼和鲑鱼中的肝脏GH结合蛋白是N-连接糖蛋白。© 1994 Wiley利斯公司
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 of125I‐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‐linking125I‐tilapia GH to striped bass hepatic membrane preparations and125I‐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 labeled125I‐GH complexes were reduced, suggesting that hepatic GH‐binding proteins in striped bass and salmon are N‐linked glycoproteins. © 1994 Wiley‐Liss, Inc.