Cooke B A, Lindh M L, Janszen F H
Biochem J. 1977 Oct 15;168(1):43-8. doi: 10.1042/bj1680043.
The effect of lutropin on phosphorylation of endogenous proteins in testis Leydig cells was investigated, by incubating purified Leydig cells with lutropin and [(32)P]P(i) followed by sodium dodecyl sulphate/polyacrylamide-slab gel electrophoresis of the [(32)P]phosphoproteins. The radioactivity of the proteins was quantified by densitometry of the radio-autograms obtained. The following results were obtained. 1. Lutropin increased the amount of (32)P incorporated into three proteins (A, B and C) with apparent mol.wts. of 14300, 57000 and 77600 respectively. 2. The increase in incorporation of (32)P into these proteins was detectable within 5min, reaching a maximum in approx. 20min. 3. The (32)P incorporated into protein B (but not proteins A and C) was significantly increased with 0.1 and 1.0ng of lutropin/ml. Incorporation of (32)P into all three proteins was significantly increased with 10ng of lutropin/ml, reaching a maximum with 100ng/ml. 4. Testosterone production was significantly increased with 1ng of lutropin/ml, and between 10 and 1000ng/ml the degree of stimulation of testosterone production and incorporation of (32)P into proteins A, B and C was similar. 5. Cyclic AMP production was significantly increased with 10ng of lutropin/ml and had not reached a maximum with 1000ng/ml. 6. In Leydig cells isolated from hypophysectomized rats 3h after injection of choriogonadotropin in vivo, phosphoproteins with the same molecular weights as proteins A, B and C were found. No further increases in incorporation of (32)P into these proteins were obtained when lutropin was added to the Leydig cells in vitro. 7. Dibutyryl cyclic AMP (but not follitropin or testosterone) also stimulated the incorporation of (32)P into proteins A, B and C in Leydig cells.
通过将纯化的睾丸间质细胞与促黄体生成素和[³²P]磷酸无机焦磷酸共同孵育，随后对[³²P]磷蛋白进行十二烷基硫酸钠/聚丙烯酰胺平板凝胶电泳，研究了促黄体生成素对睾丸间质细胞内源性蛋白质磷酸化的影响。通过对所得放射自显影片进行光密度测定来定量蛋白质的放射性。获得了以下结果。1.促黄体生成素增加了掺入三种蛋白质（A、B和C）中的³²P的量，其表观分子量分别为14300、57000和77600。2.在5分钟内可检测到³²P掺入这些蛋白质的量增加，约20分钟时达到最大值。3.当促黄体生成素浓度为0.1和1.0纳克/毫升时，掺入蛋白质B（而非蛋白质A和C）中的³²P显著增加。当促黄体生成素浓度为10纳克/毫升时，³²P掺入所有三种蛋白质的量均显著增加，在100纳克/毫升时达到最大值。4.当促黄体生成素浓度为1纳克/毫升时，睾酮分泌显著增加，在10至1000纳克/毫升之间，睾酮分泌的刺激程度以及³²P掺入蛋白质A、B和C的程度相似。5.当促黄体生成素浓度为10纳克/毫升时，环磷酸腺苷的生成显著增加，在1000纳克/毫升时未达到最大值。6.在体内注射绒毛膜促性腺激素3小时后从垂体切除大鼠分离得到的睾丸间质细胞中，发现了分子量与蛋白质A、B和C相同的磷蛋白。当在体外向这些睾丸间质细胞中添加促黄体生成素时，³²P掺入这些蛋白质的量没有进一步增加。7.二丁酰环磷酸腺苷（而非促卵泡生成素或睾酮）也刺激了³²P掺入睾丸间质细胞中的蛋白质A、B和C。
