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

This article presents a fluorimetric study of the main properties of t he enzymes dinucleoside tetraphosphate (asymmetrical) hydrolase or din ucleoside tetraphosphatase (Ap(4)Aase, EC 3.6.1.17) and dinucleoside t riphosphate hydrolase or dinucleoside triphosphatase (Ap(3)Aase, EC 3. 6.1.29), both present in adrenal medulla cytosolic extracts. Diethenoa denosine polyphosphates, epsilon-(Ap(n)A), are used as artificial fluo rogenic substrates. Ap,Aase exhibits a molecular mass around 20 kDa an d neutral optimum pH (7.0-7.5). It requires Mg2+ and preferentially hy drolyzes 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 respec tively. 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). C a2+ 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. A p(3)Aase exhibits a molecular mass around 30 kDa and neutral optimum p H (7.0-7.5). It requires Mg2+ or Ca2+, but retains a low measurable ac tivity around 10% in the absence of these divalent cations. It only hy drolyzes 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, respe ctively. 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.