SUBCELLULAR LOCALIZATION OF TSH PROCESSING
SUBCELLULAR LOCALIZATION OF TSH PROCESSING
批准号:
3446184
负责人:
JAMES MAGNER
金额:
$5.13万
依托单位国家:
美国
项目类别:
财政年份:
1985
资助国家:
美国
项目状态:
已结题
起止时间:
1985-07-01 至 1988-06-30
关键词:
Golgi apparatus autoradiography carbohydrate structure electron microscopy endoplasmic reticulum fucose galactosyltransferases gel electrophoresis genetic translation hydrazones hypothyroidism mannose methionine microsomes oligosaccharides paper chromatography peptide hormone biosynthesis radiotracer secretion sulfates thyroid neoplasm thyroidectomy thyrotropin tissue /cell culture
中文摘要
拟议的研究的目标是确定亚细胞
促甲状腺激素翻译后加工的定位
激素(TSH)亚单位前体。将利用小鼠促甲状腺肿瘤
作为活跃分泌促甲状腺激素的来源;脑下垂体组织
正常甲状腺和切除甲状腺的小鼠将成为“休息”和
分别是“刺激”的促甲状腺激素细胞。肿瘤和垂体腺组织切片
将与[S-35]蛋氨酸、[H-3]岩藻糖、[S-35]体外孵育
硫酸盐、[H-3]N-乙酰甘露糖胺或[H-3]甘露糖,然后均质
用我们用过的蔗糖梯度法进行分馏
以前的研究。富含粗面内质网的细胞组分,
近端高尔基体、远端高尔基体和分泌颗粒的特征
通过电子显微镜,并分析“细胞器特有的”酶,如
半乳糖基转移酶。标记前驱体的顺序运动
将分析在脉冲追逐研究期间这些隔室之间的距离。
将使用羧基氰化物、间氯苯肼(CCCP)和莫能菌素
作为亚细胞分馏的辅助成分,以推断
处理事件的亚蜂窝位置。标记的TSH亚基将是
免疫沉淀来自细胞匀浆、细胞部分和培养液,以及
用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳法进行鉴定。[H-3]
甘露糖标记的低聚糖将从TSH亚基中释放出来
内切糖苷酶H及其纸法结构测定
层析法。我们将确定这三种组织类型是否在
新合成的TSH亚基在亚细胞间的分布
间隔、处理事件的位置或动力学,或
亚基在隔室之间的移位。我们将为每个人确定
每种组织类型的间隔α-β的范围和动力学
亚基组合、亚基的摩尔比、亚基含量
某些残基(岩藻糖、唾液酸和硫酸盐),以及
亚基的高甘露糖低聚糖。不同于对
对于糖蛋白的加工,我们建议研究一种
激素反应系统,其中主要的低聚糖
分泌性物种TSH被认为对生物学意义重大。
活动。我们的发现将在临床上与罕见的
下丘脑甲状腺功能减退症和垂体促甲状腺激素生成性肿瘤。首先,
我们希望为更好地了解基本的细胞生物学做出贡献
促甲状腺激素的生物合成。
英文摘要
The objective of the proposed research is to determine the subcellular
locations of the post-translational processing of thyroid-stimulating
hormone (TSH) subunit precursors. Mouse thyrotropic tumor will be utilized
as a source of actively secreting thyrotrophs; pituitary tissue of
euthyroid and thyroidectomized mice will be sources of "resting" and
"stimulated" thyrotrophs, respectively. Tumor and pituitary tissue minces
will be incubated in vitro with [S-35] methionine, [H-3] fucose, [S-35]
sulfate, [H-3]N- acetylmannosamine, or [H-3] mannose, and then homogenized
and fractionated by the sucrose step-gradient method that we have used for
previous studies. Cell fractions enriched in rough endoplasmic reticulum,
proximal Golgi, distal Golgi, and secretory granules will be characterized
by electron microscopy, and assayed for "organelle-specific" enzymes, such
as galactosyltransferase. The sequential movement of labeled precursors
between these compartments during pulse-chase studies will be analyzed.
Carboxyl cyanide m-chlorophenylhydrazone (CCCP) and monensin will be used
as adjuncts to subcellular fractionation to make inferences about the
subcellular location of processing events. Labeled TSH subunits will be
immunoprecipitated from cell homogenates, cell fractions, and media, and
will be characterized by SDS-polyacrylamide gel electrophoresis. [H-3]
mannose-labeled oligosaccharides will be released from TSH subunits by
endoglycosidase H, and their structures determined by paper
chromatography. We will determine whether the three tissue types differ in
their distribution of newly synthesized TSH subunits among subcellular
compartments, the locations or kinetics of processing events, or the
translocation of subunits between compartments. We will determine for each
compartment of each tissue type the extent and kinetics of alpha-beta
subunit combination, the molar ratios of subunits, the subunit content of
certain residues (fucose, sialic acid, and sulfate), and the structures of
the high mannose oligosaccharides of subunits. Unlike studies of
glycoprotein processing by others, we propose to study a
hormonally-responsive system in which the oligosaccharides of a major
secreted species, TSH, are believed to be important for biological
activity. Our findings will be clinically relevant to rare patients with
hypothalamic hypothyroidism and pituitary TSH-producing tumors. Primarily,
we hope to contribute to a better understanding of the basic cell biology
of TSH biosynthesis.
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Rates of processing of the high mannose oligosaccharide units at the three glycosylation sites of mouse thyrotropin and the two sites of free alpha-subunits.
小鼠促甲状腺素的三个糖基化位点和游离 α 亚基的两个位点处的高甘露糖寡糖单元的加工速率。
DOI:
10.1210/endo-123-3-1296
发表时间:
1988
期刊:
Endocrinology
影响因子:
4.8
作者:
[Miura,Y, Perkel,VS, Magner,JA]
通讯作者:
Magner,JA
Blockade by brefeldin A of intracellular transport of secretory proteins in mouse pituitary cells: effects on the biosynthesis of thyrotropin and free alpha-subunits.
布雷菲德菌素 A 阻断小鼠垂体细胞中分泌蛋白的细胞内转运:对促甲状腺素和游离 α 亚基生物合成的影响。
DOI:
10.1210/endo-122-3-912
发表时间:
1988
期刊:
Endocrinology
影响因子:
4.8
作者:
[Magner,JA, Papagiannes,E]
通讯作者:
Papagiannes,E
Differential susceptibility to N-glycanase at the individual glycosylation sites of mouse thyrotropin and free alpha-subunits.
小鼠促甲状腺素和游离 α 亚基的各个糖基化位点对 N-聚糖酶的敏感性不同。
DOI:
10.1210/endo-123-5-2207
发表时间:
1988
期刊:
Endocrinology
影响因子:
4.8
作者:
[Miura,Y, Perkel,VS, Magner,JA]
通讯作者:
Magner,JA
Subcellular localization of fucose incorporation into mouse thyrotropin and free alpha-subunits: studies employing subcellular fractionation and inhibitors of the intracellular translocation of proteins.
岩藻糖掺入小鼠促甲状腺素和游离 α 亚基的亚细胞定位:采用亚细胞分级和蛋白质细胞内易位抑制剂的研究。
DOI:
10.1210/endo-119-3-1315
发表时间:
1986
期刊:
Endocrinology
影响因子:
4.8
作者:
[Magner,JA, Novak,W, Papagiannes,E]
通讯作者:
Papagiannes,E
Metabolic labeling of human thyroglobulin with [35S]sulfate: incorporation into chondroitin 6-sulfate and endoglycosidase-F-susceptible carbohydrate units.
[35S]硫酸盐对人甲状腺球蛋白的代谢标记:掺入软骨素 6-硫酸盐和糖苷内切酶-F-敏感碳水化合物单位。
DOI:
10.1210/endo-122-6-2428
发表时间:
1988
期刊:
Endocrinology
影响因子:
4.8
作者:
[Schneider,AB, McCurdy,A, Chang,T, Dudlak,D, Magner,J]
通讯作者:
Magner,J
共 9 条
SUBCELLULAR LOCALIZATION OF TSH PROCESSING
-
批准号:3462841
-
项目类别:
-
资助金额:$10.43万
-
财政年份:1987
-
负责人:JAMES MAGNER
-
依托单位:
SUBCELLULAR LOCALIZATION OF TSH PROCESSING
-
批准号:3462843
-
项目类别:
-
资助金额:$9.0万
-
财政年份:1987
-
负责人:JAMES MAGNER
-
依托单位:
SUBCELLULAR LOCALIZATION OF TSH PROCESSING
-
批准号:3462842
-
项目类别:
-
资助金额:$8.83万
-
财政年份:1987
-
负责人:JAMES MAGNER
-
依托单位:
SUBCELLULAR LOCALIZATION OF TSH PROCESSING
-
批准号:3447369
-
项目类别:
-
资助金额:$6.49万
-
财政年份:1985
-
负责人:JAMES MAGNER
-
依托单位:
海外基金