Gut S H, Bischoff M, Hobi R, Kuenzle C C
Institut für Pharmakologie und Biochemie, Universität Zürich-Irchel, Switzerland.
Nucleic Acids Res. 1987 Dec 10;15(23):9691-705. doi: 10.1093/nar/15.23.9691.
Three Z-DNA-binding proteins of Mr 31, 33 and 58 kD were isolated from mature bull testis. They were obtained in a native state suitable for binding studies. These are the first examples of Z-DNA-binding proteins from a mammalian tissue. Purification involved tissue extraction with 0.35 M NaCl, cation exchange chromatography on CM-Trisacryl M and two consecutive anion exchange FPLC runs on Mono Q. The proteins appeared virtually homogeneous by anion exchange FPLC, SDS polyacrylamide gel electrophoresis and reverse phase HPLC (58 kD protein only). Yields from 50 g of testis tissue were: 31 kD protein, 40 micrograms; 33 kD protein, 100 micrograms; and 58 kD protein, 150 micrograms. Z-DNA binding was determined by Scatchard analysis of filter binding data using brominated poly(dG-dC).poly(dG-dC) as a conformation-specific ligand. Dissociation constants (Kz, in mol nucleotide/liter) were: 31 kD protein, 7 X 10(-7) M; 33 kD protein, 8 X 10(-7) M; 58 kD protein, 6 X 10(-8) M (primary binding site) and 6 X 10(-7) M (secondary binding site). B-DNA binding to poly(dG-dC).poly(dG-dG) was too low for reliable determination under the conditions of assay. This attested to a high degree of conformational specificity of the three proteins. The 58 kD protein bound Z-DNA at the primary site with an affinity almost equivalent to that of a polyclonal anti-Z-DNA antiserum raised in a rabbit (Kz, 4 X 10(-8) M).
从成熟公牛睾丸中分离出了分子量分别为31kD、33kD和58kD的三种Z-DNA结合蛋白。它们以适合结合研究的天然状态获得。这些是来自哺乳动物组织的Z-DNA结合蛋白的首个实例。纯化过程包括用0.35M NaCl进行组织提取、在CM-Trisacryl M上进行阳离子交换层析以及在Mono Q上连续进行两次阴离子交换快速蛋白质液相色谱（FPLC）。通过阴离子交换FPLC、SDS聚丙烯酰胺凝胶电泳和反相HPLC（仅针对58kD蛋白）分析，这些蛋白质几乎呈现均一状态。50g睾丸组织的产量分别为：31kD蛋白40微克；33kD蛋白100微克；58kD蛋白150微克。使用溴化聚（dG-dC）·聚（dG-dC）作为构象特异性配体，通过对滤膜结合数据进行Scatchard分析来测定Z-DNA结合。解离常数（Kz，单位为mol核苷酸/升）分别为：31kD蛋白，7×10⁻⁷M；33kD蛋白，8×10⁻⁷M；58kD蛋白，6×10⁻⁸M（主要结合位点）和6×10⁻⁷M（次要结合位点）。在测定条件下，B-DNA与聚（dG-dC）·聚（dG-dG）的结合太低，无法进行可靠测定。这证明了这三种蛋白质具有高度的构象特异性。58kD蛋白在主要位点结合Z-DNA的亲和力几乎等同于在兔体内产生的多克隆抗Z-DNA抗血清（Kz，4×10⁻⁸M）。
