Human Placental ( Asymmetrical) Diadenosine 5′,5‴- P 1, P4-Tetraphosphate Hydrolase: Purification to Homogeneity and Some Properties

Abstract

The diadenosine 5′,5‴- P 1, P 4-tetraphosphate ( asymmetrical) hydrolase (EC 3.6.1.17) from human placenta has been purified to homogeneity by ammonium sulfate fractionation, ion-exchange chromatography on DEAE-Sephecel, gel filtration on Sephadex G-100, and affinity elution from red Sepharose. The enzyme is a single polypeptide of M r 19,200. It exhibits maximum (100%) activity at pH 7.3 in the presence of 3mM MgCl 2 and 60, 50, and 40% of the activity in 1 mM CoCl 2, 0.1 mM ZnCl 2, and 0.5 mM MnCl 2, respectively. The K m value calculated for diadenosine tetraphosphate in the presence of Mg 2+ is 10 μM and in the presence of Zn 2+ 40 μM. Adenosine 5′-tetraphosphate, guanosine 5′-tetra-phosphate, and fluoride proved to he inhibitors of the diadenosine tetraphosphate hydrolase; the I 50 values were 6, 10, and 20 μM, respectively. Diguanosine tetraphosphate, bis-2,6-diaminopurine β-D-ribofuranoside tetraphosphate, and diadenosine pentaphosphate were substrates for the hydrolase; relative velocities of hydrolysis estimated for 0.5 mM diadenosine tetraphosphate and these other substrates were 1:0.51:0.44:0.20, respectively. Diadenosine tetraphosphate analogues with P 2 P 3 bridges such as CF 2, CCI 2, and CH 2 were hydrolyzed to adenosine 5′-phosphate and the corresponding adenosine 5′-triphosphate analogue. Interaction of the human placental diadenosine tetraphosphate hydrolase with diadenosine triphosphate and its phosphonate analogues was also tested. These compounds appeared to be competitive inhibitors with K i values lower than the K m for diadenosine tetraphosphate.