Montague J W, Hughes F M, Cidlowski J A
Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill, North Carolina 27599, USA.
J Biol Chem. 1997 Mar 7;272(10):6677-84. doi: 10.1074/jbc.272.10.6677.
Previous work in our laboratory (Montague, J., Gaido, M., Frye, C., and Cidlowski, J. (1994) J. Biol. Chem. 269, 18877-18880) has shown that human recombinant cyclophilins A, B, and C have sequence homology with the apoptotic nuclease NUC18 and that denatured cyclophilins can degrade DNA. We have now evaluated the nucleolytic activity of recombinant cyclophilins under native conditions. We show that nuclease activity inherent to cyclophilins is distinct from cis-trans-peptidylprolyl isomerase activity and is similar to that described for apoptotic nucleases. Cyclophilin nucleolytic activity is stimulated by Ca2+ and/or Mg2+, with a combination of the two being optimal for cyclophilins A and B. Mg2+ alone is sufficient for cyclophilin C nuclease activity. pH optimums are in the range of pH 7.5-9.5. Cyclophilins can degrade both single-stranded and double-stranded DNA. Additionally, cyclophilins produce 3'-OH termini in linear double-stranded substrates, suggesting the cuts produced are similar to those of apoptotic cells. Cyclophilins also display endonucleolytic activity, demonstrated by their ability to degrade supercoiled DNA. In the absence of ions, cyclophilins bind linearized DNA. When added to nuclei from nonapoptotic cells, cyclophilin C induces 50-kilobase pair DNA fragmentation but not internucleosomal fragmentation. Together, these data suggest that cyclophilins are involved in degradation of the genome during apoptosis.
我们实验室之前的工作（蒙塔古，J.，盖多，M.，弗莱，C.，和齐德洛夫斯基，J.（1994年）《生物化学杂志》269卷，18877 - 18880页）表明，人重组亲环蛋白A、B和C与凋亡核酸酶NUC18具有序列同源性，并且变性的亲环蛋白能够降解DNA。我们现在评估了天然条件下重组亲环蛋白的核酸水解活性。我们发现亲环蛋白固有的核酸酶活性不同于顺反肽基脯氨酰异构酶活性，并且与凋亡核酸酶所描述的活性相似。亲环蛋白的核酸水解活性受到Ca2 +和/或Mg2 +的刺激，两者结合对亲环蛋白A和B最为适宜。单独的Mg2 +就足以支持亲环蛋白C的核酸酶活性。最适pH值在7.5 - 9.5范围内。亲环蛋白能够降解单链和双链DNA。此外，亲环蛋白在线性双链底物中产生3'-OH末端，这表明所产生的切割与凋亡细胞的切割相似。亲环蛋白还表现出内切核酸酶活性，这通过它们降解超螺旋DNA的能力得以证明。在没有离子的情况下，亲环蛋白结合线性化的DNA。当添加到非凋亡细胞的细胞核中时，亲环蛋白C诱导50千碱基对的DNA片段化，但不诱导核小体间片段化。总之，这些数据表明亲环蛋白参与凋亡过程中基因组的降解。
