Hinsch B, Kula M R
Nucleic Acids Res. 1980 Feb 11;8(3):623-33. doi: 10.1093/nar/8.3.623.
The site specific endonuclease Bam HI which is composed of subunits of a molecular weight of 22 000 [1] can aggregate to complexes of a molecular weight of 360 000. It is an acidic protein with an isoelectric point at pH 5.3. Optimal activity is reached at 13 mM MgCl2. A very simple method is presented to determine kinetic constants of restriction enzymes directly from agarose gel photographs without any further equipment applying the integrated Michaelis Menten equation. With pJC 80 DNA as a substrate KM was found to be 3.6 10(-10) M. The method can be used to redefine the unit activity of site specific endonucleases unambigously.
由分子量为22000的亚基组成的位点特异性内切酶Bam HI可聚合成分子量为360000的复合物。它是一种酸性蛋白质，等电点为pH 5.3。在13 mM MgCl₂时达到最佳活性。本文提出了一种非常简单的方法，无需任何其他设备，直接根据琼脂糖凝胶照片，应用积分米氏方程来测定限制酶的动力学常数。以pJC 80 DNA为底物时，发现米氏常数为3.6×10⁻¹⁰ M。该方法可用于明确重新定义位点特异性内切酶的单位活性。
