PURIFICATION TO HOMOGENEITY OF RAT-LIVER DINUCLEOSIDE TETRAPHOSPHATASE BY AFFINITY ELUTION WITH ADENOSINE 5'-TETRAPHOSPHATE

Starting rom a partially purified dinucleoside tetraphosphatase (Np4 Nase; EC 3.6.1.17), we developed an affinity elution purification protocol involving the strong competitive inhibitor adenosine 5′-tetraphosphate. Np4 Nase bound to Cibacron Blue F3G-A-Sepharose 4B or to Reactive Blue 2-Sepharose CL-6B was specifically eluted with 10 μM adenosine 5′-tetraphosphate and 5 mM MgCl2, but not by either of them separately. The final Np4 Nase preparation was homogeneous by sodium dodecyl sulfate polyacrylamide gel electrophoresis followed by Coomassie blue or silver staining. The protein band showed an apparent 18 kDa molecular mass. The specific activity of the homogeneous Np4 Nase was about 150 units/mg, meaning a 45 000-fold increase and a 10% recovery with respect to the crude extract. After preparative polyacrylamide gel electrophoresis, protein visualization with KCl, fragmentation of the gel lane, and extraction, all the renatured Np4 Nase activity was found associated to the 18 kDa band. The renatured enzyme showed the same Km value for diadenosine 5′,5′″-P1,P4-tetraphosphate as the partially purified or the native homogeneous Np4 Nase. © 1990.