钙和镁离子对牛胰脱氧核糖核酸酶A活性的一些影响。

Some effects of calcium and magnesium ions on the activity of bovine pancreatic deoxyribonuclease A.

作者信息

Douvas A, Price P A

出版信息

Biochim Biophys Acta. 1975 Jun 16;395(2):201-12. doi: 10.1016/0005-2787(75)90159-8.

Abstract

Bovine pancreatic deoxyribonuclease requires divalent metal cations for hydrolysis of DNA. The effects of calcium and magnesium, alone and combined, on the rate and kinetics of the reaction were examined. Divalent metal salts of DNA were used as substrates. The ratio of either Ca-2+ or Mg-2+ to DNA-P in these salts was 1:2. The Mg-2+ salt of DNA was found to have sufficient Mg-2+ for optimal DNAase activity. Addition of MgCl-2 to a large excess of Mg-2+ over DNA-P had no effect on the rate. Km for the hydrolysis of Mg-2+-DNA was 1.76 mM. Km for the hydrolysis of Ca-2+-DNA was too low to measure by our methods of assay, indicating a high affinity of enzyme for substrate. The rate of hydrolysis of Ca-2+-DNA, however, is slow compared to that of Mg-2+-DNA. By mixing the Ca-2+ and Mg-2+ salts of DNA, a synergistic effect on the activity of DNAase was observed. On the basis of kinetic studies the synergistic effect is attributed to an increased affinity of DNAase for DNA (Km equals 0.34 mM for the mixed Ca-2+, Mg-2+ salt of DNA). A 2-fold increase in DNAase activity was observed when DNAase was incubated in CaCl-2 before assay. This 2-fold stimulation was not related to the synergistic effect. DNAase I is inactive against the sodium salt of DNA. In experiments with mixed Na+ and Mg-2+ salts of DNA, Na+-DNA was found to be a competitive inhibitor of the action of DNAase against Mg-2+-DNA.

摘要

牛胰脱氧核糖核酸酶水解DNA需要二价金属阳离子。研究了钙和镁单独以及联合存在时对该反应速率和动力学的影响。DNA的二价金属盐用作底物。这些盐中Ca²⁺或Mg²⁺与DNA-P的比例为1:2。发现DNA的Mg²⁺盐含有足够的Mg²⁺以实现最佳的脱氧核糖核酸酶活性。向大大过量于DNA-P的Mg²⁺中添加MgCl₂对反应速率没有影响。Mg²⁺-DNA水解的Km为1.76 mM。Ca²⁺-DNA水解的Km太低，无法用我们的测定方法测量，表明酶对底物具有高亲和力。然而，Ca²⁺-DNA的水解速率与Mg²⁺-DNA相比很慢。通过混合DNA的Ca²⁺和Mg²⁺盐，观察到对脱氧核糖核酸酶活性有协同作用。基于动力学研究，这种协同作用归因于脱氧核糖核酸酶对DNA的亲和力增加（对于混合的Ca²⁺、Mg²⁺盐的DNA，Km等于0.34 mM）。在测定前将脱氧核糖核酸酶在CaCl₂中孵育时，观察到脱氧核糖核酸酶活性增加了2倍。这种2倍的刺激与协同作用无关。脱氧核糖核酸酶I对DNA的钠盐无活性。在使用DNA的Na⁺和Mg²⁺混合盐的实验中，发现Na⁺-DNA是脱氧核糖核酸酶作用于Mg²⁺-DNA的竞争性抑制剂。