SPECIFIC DINUCLEOSIDE POLYPHOSPHATE CLEAVING ENZYMES FROM CHROMAFFIN CELLS - A FLUOROMETRIC STUDY

This article presents a fluorimetric study of the main properties of the enzymes dinucleoside tetraphosphate (asymmetrical) hydrolase or dinucleoside tetraphosphatase (Ap(4)Aase, EC 3.6.1.17) and dinucleoside triphosphate hydrolase or dinucleoside triphosphatase (Ap(3)Aase, EC 3.6.1.29), both present in adrenal medulla cytosolic extracts. Diethenoadenosine polyphosphates, epsilon-(Ap(n)A), are used as artificial fluorogenic substrates. Ap,Aase exhibits a molecular mass around 20 kDa and neutral optimum pH (7.0-7.5). It requires Mg2+ and preferentially hydrolyzes substrates with four phosphate groups. K-m for epsilon-(Ap(4)A) is 1.3 mu M and K-i for Ap(4)A and Gp(4)G are 1 and 0.2 mu M respectively. K-m for Ap(4)A determined by HPLC is 1.6 mu M. epsilon-(Ap(5)A) and epsilon-(Ap(6)A) are hydrolyzed at reduced rates. This enzyme is inhibited by Zn2+, F- and very strongly by Ap(4) and epsilon-Ap(4). Ca2+ cannot replace Mg2+, but behaves as inhibitor in its presence. The substrate analogs dinucleoside triphosphates Ap(3)A, Gp(3)G, m(7)Gp(3)G and m(7)Gp(3)A and the periodate-oxidized nucleotides o-(Ap(4)A), o epsilon-(Ap(4)A), o-Ap(4) and o epsilon-Ap(4) behave as inhibitors. Ap(3)Aase exhibits a molecular mass around 30 kDa and neutral optimum pH (7.0-7.5). It requires Mg2+ or Ca2+, but retains a low measurable activity around 10% in the absence of these divalent cations. It only hydrolyzes substrates with three phosphate groups. K-m for epsilon-(Ap(3)A) is 11 mu M and K-i for Ap(3)A and Gp(3)G are 20 and 22 mu M, respectively. K-m for Ap(3)A determined by HPLC is 16 mu M m(7)Gp(3)G and m(7)Gp(3)A are also good substrates for triphosphatase.