Osawa S, Betz G, Hall P F
J Cell Biol. 1984 Oct;99(4 Pt 1):1335-42. doi: 10.1083/jcb.99.4.1335.
Erythrocyte ghosts were loaded with pancreatic DNase I and fused with Y-1 adrenal tumor cells to test the possibility that this enzyme might inhibit the steroidogenic responses of the cells to ACTH and cyclic AMP. Fusion of erythrocyte ghosts loaded with DNase I, but not those containing albumin, ovalbumin, boiled DNase I, or DNase I with excess G-actin, inhibited the increase in production of 20 alpha-dihydroprogesterone produced by ACTH and dibutyryl cyclic AMP; inhibition was concentration-dependent with 50% inhibition by 3 X 10(7) molecules of DNase I per cell. It was found that inhibition by DNase I was exerted at the step in the steroidogenic pathway at which cholesterol is transported to mitochondria where steroidogenesis begins. This was shown by measuring transport of cholesterol into the inner mitochondrial membrane, by measuring the production of pregnenolone by isolated mitochondria and by demonstrating that DNase I was without effect on the conversion of pregnenolone to 20 alpha-dihydroprogesterone (an end-product of steroid synthesis). The actin content of Y-1 cells was measured by two methods based upon inhibition of DNase I and by SDS gels following centrifugation. The cells were found to contain 2-3 X 10(7) molecules of actin per cell of which two-thirds is present as G-actin. Since DNase I is known to bind to G-actin to give a one to one complex, these and other findings suggest that at least some of the G-actin in the cells may be necessary for the steroidogenic responses to ACTH and cyclic AMP.
将胰腺DNA酶I载入红细胞血影，并使其与Y-1肾上腺肿瘤细胞融合，以测试该酶是否可能抑制细胞对促肾上腺皮质激素（ACTH）和环磷酸腺苷（cAMP）的类固醇生成反应。载入DNA酶I的红细胞血影（而非含有白蛋白、卵清蛋白、煮沸的DNA酶I或与过量G-肌动蛋白结合的DNA酶I的红细胞血影）的融合，抑制了ACTH和二丁酰环磷酸腺苷所产生的20α-二氢孕酮产量的增加；抑制作用呈浓度依赖性，每个细胞3×10⁷个DNA酶I分子可产生50%的抑制效果。研究发现，DNA酶I的抑制作用发生在类固醇生成途径中胆固醇转运至线粒体（类固醇生成起始部位）的步骤。这通过测量胆固醇向内线粒体膜的转运、测量分离线粒体中孕烯醇酮的产生以及证明DNA酶I对孕烯醇酮转化为20α-二氢孕酮（类固醇合成的终产物）无影响得以证实。通过基于DNA酶I抑制作用的两种方法以及离心后的SDS凝胶法测量Y-1细胞的肌动蛋白含量。发现每个细胞含有2 - 3×10⁷个肌动蛋白分子，其中三分之二以G-肌动蛋白形式存在。由于已知DNA酶I与G-肌动蛋白结合形成1:1复合物，这些及其他发现表明，细胞中至少某些G-肌动蛋白可能是对ACTH和环磷酸腺苷产生类固醇生成反应所必需的。
