Kwast-Welfeld J, Jungmann R A
Department of Molecular Biology, Northwestern University Medical School, Chicago, Illinois 60611.
J Biol Chem. 1988 Oct 5;263(28):14343-50.
Biochemical as well as immunochemical studies were carried out to quantitatively and qualitatively evaluate the hormonal regulation of nuclear cAMP-dependent protein kinase subunits in ovaries from estrogen-treated hypophysectomized rats. Photoaffinity labeling of nuclear extracts with 8-azido-[32P]cAMP and electrophoretic analysis showed the existence of three variants of the regulatory subunit RI and of a 52,000-dalton RII variant (RII-52) in ovarian nuclei of estrogen-primed hypophysectomized rats. After follicle-stimulating hormone (FSH) stimulation, an additional variant of RII (RII-51, Mr = 51,000) was detected in nuclei. The cytosolic RII-54 variant (Mr = 54,000) could not be identified in nuclei by photoaffinity labeling. The FSH-mediated appearance of the nuclear RII-51 variant was accompanied by an approximate 2-fold increase of nuclear catalytic subunit activity. Using quantitation by enzyme-linked immunosorbent assay, we identified a marked FSH-mediated increase of nuclear RII variant(s) and confirmed the increase of nuclear catalytic subunit levels. Furthermore, morphometric analysis of nuclear and cytoplasmic antigen density by immunogold electron microscopy demonstrated a cell-specific modulation by FSH of RII and C subunit density. In granulosa cells, both nuclear as well as cytoplasmic RII density was increased by FSH, whereas catalytic subunit density was increased in the nuclear area only. In thecal cells, FSH increased only the nuclear catalytic subunit density. These results provide biochemical as well as immunochemical evidence for a cell-specific FSH regulation of nuclear RII and catalytic subunit levels which may be involved in the molecular events responsible for the FSH-mediated differentiation of the rat ovary.
进行了生化和免疫化学研究，以定量和定性评估雌激素处理的垂体切除大鼠卵巢中核环磷酸腺苷（cAMP）依赖性蛋白激酶亚基的激素调节。用8-叠氮基-[32P]cAMP对核提取物进行光亲和标记并进行电泳分析，结果显示在雌激素预处理的垂体切除大鼠的卵巢细胞核中存在调节亚基RI的三种变体以及一种52,000道尔顿的RII变体（RII-52）。在促卵泡激素（FSH）刺激后，在细胞核中检测到RII的另一种变体（RII-51，分子量=51,000）。通过光亲和标记在细胞核中未鉴定出胞质RII-54变体（分子量=54,000）。FSH介导的核RII-51变体的出现伴随着核催化亚基活性增加约2倍。使用酶联免疫吸附测定进行定量分析，我们确定了FSH介导的核RII变体显著增加，并证实了核催化亚基水平的增加。此外，通过免疫金电子显微镜对核和胞质抗原密度进行形态计量分析，结果表明FSH对RII和C亚基密度具有细胞特异性调节作用。在颗粒细胞中，FSH使核和胞质RII密度均增加，而催化亚基密度仅在核区域增加。在卵泡膜细胞中，FSH仅增加核催化亚基密度。这些结果提供了生化和免疫化学证据，表明FSH对核RII和催化亚基水平具有细胞特异性调节作用，这可能参与了大鼠卵巢FSH介导的分化过程中的分子事件。
