Gyves P W, Gesundheit N, Taylor T, Butler J B, Weintraub B D
Endocrinology. 1987 Jul;121(1):133-40. doi: 10.1210/endo-121-1-133.
We have studied the carbohydrate structure of TSH as well as its response to TRH during postnatal ontogenesis in the rat using Concanavalin-A (Con A)-Sepharose chromatography of labeled glycopeptides. Pituitaries from neonatal (5-day-old) rats with low levels of endogenous TRH and mature (56-day-old) rats were incubated for 24 h in medium containing [3H] glucosamine in the presence or absence of 10(-7) M TRH. Both intracellular and secreted TSH were immunoprecipitated, treated with Pronase to generate glycopeptides, and analyzed by chromatography on Con A-Sepharose. The total amount of [3H]glucosamine-labeled TSH was greater per pituitary in mature rats compared to that in neonatal rats (P less than 0.05), while there was no significant difference between the groups in the concentration of total labeled TSH per microgram pituitary DNA. RIA determination of total TSH was greater in the older animals than in the younger animals when normalized both per pituitary and per microgram pituitary DNA (P less than 0.01 and P less than 0.02, respectively). However, for both labeled and unlabeled TSH the percentage of TSH secreted was greater in mature rats than in neonatal rats (P less than 0.02 and P less than 0.01, respectively), indicating a less active hormonal secretory process in the younger animals. In control animals, the proportion of labeled TSH glycopeptides that did not bind to Con A was greater in 56- than in 5-day-old animals for both intrapituitary and secreted forms (P less than 0.01), reflecting a shift toward more multiantennary and/or bisected biantennary complex carbohydrate structures in the older animals. In response to TRH in vitro, the total amount of labeled secreted TSH was increased more than 2-fold in both 5-day-old (P less than 0.05) and 56-day-old (P = NS) animals. However, there was a marked difference in the glycopeptide distribution between these two ages. Five-day-old animals showed a small but not significant decrease in the percentage of secreted TSH glycopeptides that bound to Con A-Sepharose, while 56-day-old animals had a specific increase in the glycopeptide fractions that bound and corresponded to biantennary complex and/or unusual hybrid forms (P less than 0.01). These studies in the rat suggest differences in TSH carbohydrate structure and secretion as well as a differential response to TRH during postnatal ontogenesis.
我们利用伴刀豆球蛋白A（Con A）-琼脂糖凝胶层析法对标记的糖肽进行分析，研究了大鼠出生后个体发育过程中促甲状腺激素（TSH）的碳水化合物结构及其对促甲状腺激素释放激素（TRH）的反应。将内源性TRH水平较低的新生（5日龄）大鼠和成熟（56日龄）大鼠的垂体，在含有[3H]葡糖胺的培养基中孵育24小时，孵育过程中分别添加或不添加10^(-7) M的TRH。对细胞内和分泌的TSH进行免疫沉淀，用链霉蛋白酶处理以生成糖肽，然后通过Con A-琼脂糖凝胶层析进行分析。与新生大鼠相比，成熟大鼠每个垂体中[3H]葡糖胺标记的TSH总量更多（P<0.05），而每微克垂体DNA中总标记TSH浓度在两组之间没有显著差异。当以每个垂体和每微克垂体DNA进行标准化时，放射免疫分析（RIA）测定的老年动物总TSH含量高于幼年动物（分别为P<0.01和P<0.02）。然而，对于标记和未标记的TSH，成熟大鼠中分泌的TSH百分比均高于新生大鼠（分别为P<0.02和P<0.01），这表明幼年动物的激素分泌过程活性较低。在对照动物中，无论是垂体内部还是分泌形式，56日龄动物中不与Con A结合的标记TSH糖肽比例均高于5日龄动物（P<0.01），这反映出老年动物中向更多的多天线和/或平分双天线复杂碳水化合物结构转变。在体外对TRH的反应中，5日龄（P<0.05）和56日龄（P=无显著性差异）动物中标记的分泌TSH总量均增加了2倍以上。然而，这两个年龄段的糖肽分布存在显著差异。5日龄动物中与Con A-琼脂糖凝胶结合的分泌TSH糖肽百分比略有下降，但无显著性差异，而56日龄动物中与双天线复杂和/或异常杂合形式相对应的结合糖肽部分有特异性增加（P<0.01）。这些在大鼠中的研究表明，出生后个体发育过程中TSH的碳水化合物结构和分泌存在差异，以及对TRH的反应也有所不同。
