Z-DNA-binding proteins from bull testis

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Abstract
Three Z-DNA-binding Droteins of Mr 31, 33 and 58 kO 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 chromatograohy on CM-Trisacryl M and two consecutive anion exchange FPLC runs on Mono Q. The proteins appeared virtually homogeneous by anion exchange FPLC, SDS polvacrylamide gel electrophoresis and reverse phase HPLC (58 kD protein only). Yields from 50 g of testis tissue were: 31 kD protein, 40 μg; 33 kD protein, 100 μg; and 58 kD protein, 150 μg. 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 × 10−7 M; 33 kD protein, 8 × 10−7 M; 58 kD protein, 6 × 10−8 M (primary binding site) and 6 × 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 × 10−8 M).