Inhibition of adenylyl cyclase activity in rat corpora luteal tissue by glycopeptides of human chorionic gonadotropin and the alpha-subunit of human chorionic gonadotropin.
Inhibition of adenylyl cyclase activity in rat corpora luteal tissue by glycopeptides of human chorionic gonadotropin and the alpha-subunit of human chorionic gonadotropin.
复制标题
人绒毛膜促性腺激素糖肽和人绒毛膜促性腺激素α亚基对大鼠黄体组织中腺苷酸环化酶活性的抑制。
DOI：
10.1021/bi00329a023
复制
发表时间：
1985
期刊：
影响因子：
2.9
通讯作者：
Ryan,RJ
中科院分区：
文献类型：
--
作者：
Calvo,FO;Ryan,RJ
摘要：间接证据表明，糖蛋白激素的碳水化合物部分参与了生殖组织受体-腺苷酸环化酶系统的激活。在本研究中，我们从人绒毛膜促性腺激素（hCG）、hCG的a亚基、胎儿素和牛7-球蛋白（b7G）中分离出糖肽（GP）。这些和一些合成的低聚糖一起测试了它们抑制腺苷酸环化酶（AC）的能力。hCG、hCGa、胎儿素和b7G的GP在双抗体hCG放射免疫试验或大鼠黄体放射受体试验中交叉反应性小于0.001%。当与100 ng /mL hCG （ED50）共孵育时，胎儿素、b7G和合成寡糖的GP均未抑制2000#黄体膜的AC活性。然而，当hCG和hCGa的GP与完整的hCG一起包含时，存在剂量相关的抑制作用。当hCG GP脱氮时，环化酶活性的抑制作用增强。这在hCG激活的延迟时间（计算为1-1.5分钟）没有变化。改变ATP和Mg2+的浓度不影响hCGa GP对hcg刺激的AC活性的抑制作用。hCG GP的抑制遵循非竞争动力学。GP对hCG的抑制作用似乎仅限于LH/hCG刺激的AC系统，因为当与胰高血糖素共孵育时，抑制大鼠黄体AC系统的相同剂量的hCG GP对大鼠肝细胞AC系统或黄体膜中naf刺激的活性没有任何影响。这些结果表明，膜凝集素可能参与调节黄体细胞hcg刺激的腺苷酸环化酶。我们进一步推测受体和凝集素的交联可能参与了微聚集、环化酶刺激和内化。糖蛋白激素，包括人绒毛膜促性腺激素（hCG）的作用机制已被广泛研究（Moyle, 1980; Ryan, 1982; Bahl & Kalyan, 1983）。例如，已知hCG与LH/hCG受体结合并随后在体内和/或体外充分激活乙酰基环化酶（AC）系统需要一种完整的激素或一种仅去除碳水化合物链唾液酸部分的激素。通过酶或化学手段进行的渐进式去糖基化会导致AC活性降低，同时细胞反应也会相应降低，但不会失去受体结合（Chen et al., 1982; Manjunath & Sairam & Manjunath, 1982; Sairam & Manjunath, 1983; Kalyan & Bahl, 1983； Moyle等，1975）。此外，去糖基化的制剂有效地与完整的hcg分子竞争受体位点并抑制生物活性（Keutmann et al., 1983; Sairam & Manjunath, 1983）。我们利用hCG、hCG的a-亚基（hCGa）、胎蛋白和牛7-免疫球蛋白（b7G）制备的糖肽（GP）进一步研究了这一现象。胎激素和b7G的GP在组成上与hCG大致相似，但也存在一些差异（Kornfeld & Kornfeld, 1976; Baezinger & Fiete, 1979）。此外，我们还研究了多种化学合成的低聚糖（Arnarp & Lonngren, 1981; Arnarp et al., 1982; Lonn & Lonngren, 1983）抑制黄体组织和肝脏制剂中AC活性的能力。t由美国国立卫生研究院（HD9140）和梅奥基金会资助。这些数据的初步报告在内分泌学会第65届年会上发表(1983…
Department of Cell Biology, Mayo Medical School, Rochester, Minnesota 55905 Received August 28, 1984 abstract: Indirect evidence has indicated that the carbohydrate moieties of the glycoprotein hormones are involved in the activation of the receptor-adenylyl cyclase system of reproductive tissues. In the present study, we have isolated the glycopeptides (GP) from human chorionic gonadotropin (hCG), the a-subunit of hCG, fetuin, and bovine 7-globulin (b7G). These along with a number of synthetic oligosaccharides were tested for their ability to inhibit adenylyl cyclase (AC). There was less than 0.001% cross-reactivity of the GP from hCG, hCGa, fetuin, and b7G when tested in a double-antibody hCG radioimmunoassay or rat corpora lutea radioreceptor assay. The GP of fetuin, b7G, and the synthetic oligosaccharides did not inhibit AC activity of 2000# corpora lutea membranes when coincubated with 100 ng of hCG/mL (ED50). However, when the GP of hCG and hCGa were included with intact hCG, there was a dose-related inhibition. Inhibition of cyclase activity was enhanced when the hCG GP were desialylated. This occurred without a change in thelag time of hCG activation which was calculated to be 1-1.5 min. Changing the concentration of ATP and Mg2+ did not affect the inhibitory effects of the hCGa GP on hCG-stimulated AC activity. Inhibition by hCG GP followed uncompetitive kinetics. The inhibition by the GP of hCG seems to be restricted to the LH/hCG-stimulatable AC system because the same dosage of hCG GP which inhibited the rat luteal AC system did nothave any effect on the rat hepatocyte AC system when coincubated with glucagon or on NaF-stimulated activity in luteal membranes. These results suggest that a membrane lectin may be involved in the regulation of luteal cell hCG-stimulated adenylyl cyclase. We further speculate that receptor and lectin cross-linking may be involved in microaggregation, cyclase stimulation, and internalization. e mechanism of action of the glycoprotein hormones, including human chorionic gonadotropin (hCG), 1 has been studied extensively (Moyle, 1980; Ryan, 1982; Bahl & Kalyan, 1983). It is known, for example, that binding of hCG to the LH/hCG receptor and subsequent full activation of the ade-nylyl cyclase (AC) system in vivo and/or in vitro require an intact hormone or one in which only the sialic acid moieties of the carbohydrate chains have been removed. Progressive deglycosylation by enzymatic or chemical means leads to decreased AC activation with a parallel decrease in cellular response but without loss of receptor binding (Chen et al., 1982; Manjunath & Sairam, 1982; Sairam & Manjunath, 1983; Kalyan & Bahl, 1983; Moyle et al., 1975). In addition, the deglycosylated preparations effectively compete with the intact hCGmolecule for receptor sites and inhibit biological activity (Keutmann et al., 1983; Sairam & Manjunath, 1983). We have studied this phenomenon furtherutilizing the glycopeptides (GP) prepared from hCG, the a-subunit of hCG (hCGa), fetuin, and bovine 7-immunoglobulin (b7G). The GP of fetuin and b7G are generally similar in composition to those of hCG, but there are some differences (Kornfeld & Kornfeld, 1976; Baezinger & Fiete, 1979). In addition, we have investigated the ability of a variety of chemically synthesized oligosaccharides (Arnarp & Lonngren, 1981; Arnarp et al., 1982; Lonn & Lonngren, 1983) to inhibit AC activity in luteal tissue and liver preparations. t Supported by funds from the National Institutes of Health (HD9140) and the Mayo Foundation. A preliminary report of these data was presented at the 65thAnnual Meeting of the Endocrine Society (1983 …