大鼠睾丸中多核苷酸激酶的纯化及动力学特性

Purification and kinetic properties of polynucleotide kinase from rat testes.

作者信息

Bosdal T, Lillehaug J R

出版信息

Biochim Biophys Acta. 1985 Jun 18;840(2):280-6. doi: 10.1016/0304-4165(85)90129-1.

Abstract

Polynucleotide kinase (EC 2.7.1.78) has been purified from rat testes, and an approximately 2000-fold purification was obtained. The purified enzyme had an Mr of 38000 +/- 3800. The enzyme phosphorylated micrococcal nuclease-treated calf thymus DNA and (dT)10 while 5'-HO-tRNA was a very poor substrate. A certain degree of specificity towards purine-containing 5'-HO-nucleotides was observed. The polynucleotide kinase had an absolute requirement for a divalent cation. Both Mg2+ and Mn2+ could be used, but 10 mM MgCl2 gave optimal activity. The monovalent cations Na+, K+ and NH4+ all stimulated enzyme activity, and the optimal concentration was 0.1 M. The enzyme was inhibited by inorganic phosphate, pyrophosphate and sulphate. A 50% inhibition was obtained with 20, 0.3 and 2 mM, respectively. At 2 mM MgCl2, 1 mM spermine enhanced the enzyme activity 3-times. The apparent KATP was estimated to be 36 microM and KHO-DNA was found to be 2 microM.

摘要

多核苷酸激酶（EC 2.7.1.78）已从大鼠睾丸中纯化出来，获得了约2000倍的纯化效果。纯化后的酶的相对分子质量为38000±3800。该酶可使经微球菌核酸酶处理的小牛胸腺DNA和（dT）10磷酸化，而5'-羟基-tRNA是一种很差的底物。观察到该酶对含嘌呤的5'-羟基核苷酸具有一定程度的特异性。多核苷酸激酶绝对需要二价阳离子。Mg2+和Mn2+均可使用，但10 mM MgCl2可产生最佳活性。单价阳离子Na+、K+和NH4+均能刺激酶的活性，最佳浓度为0.1 M。该酶受到无机磷酸盐、焦磷酸盐和硫酸盐的抑制。分别在20 mM、0.3 mM和2 mM时可产生50%的抑制作用。在2 mM MgCl2条件下，1 mM精胺可使酶活性提高3倍。表观KATP估计为36 μM，KHO-DNA为2 μM。