Specific dinucleoside polyphosphate cleaving enzymes from chromaffin cells: a fluorimetric study

Abstract

This article presents a fluorimetric study of the main properties of the enzymes dinucleoside tetraphosphate (asymmetrical) hydrolase or dinucleoside tetraphosphatase (Ap 4Aase, EC 3.6.1.17) and dinucleoside triphosphate hydrolase or dinucleoside triphosphatase (Ap 3Aase, EC 3.6.1.29), both present in adrenal medulla cytosolic extracts. Diethenoadenosine polyphosphates, ϵ-(Ap nA), are used as artificial fluorogenic substrates. Ap 4Aase exhibits a molecular mass around 20 kDa and neutral optimum pH (7.0–7.5). It requires Mg 2+ and preferentially hydrolyzes substrates with four phosphate groups. K m for ϵ-(Ap 4A) is 1.3 μM and K i for Ap 4A and Gp 4G are 1 and 0.2 μM respectively. K m for Ap 4A determined by HPLC is 1.6 μM. ϵ-(Ap 5A) and ϵ-(Ap 6A) are hydrolyzed at reduced rates. This enzyme is inhibited by Zn 2+ F − and very strongly by Ap 4 and ϵ-Ap 4. Ca 2+ cannot replace Mg 2+, but behaves as inhibitor in its presence. The substrate analogs dinucleoside triphosphates Ap 3A, Gp 3G, m 7Gp 3G and m 7Gp 3A and the periodate-oxidized nucleotides o-(Ap 4A), oge-(Ap 4A), o-Ap 4 and oϵ-Ap 4 behave as inhibitors. Ap 3Aase exhibits a molecular mass around 30 kDa and neutral optimum pH (7.0–7.5). It requires Mg 2+ or Ca 2+, 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 ϵ-(Ap 3A) is 11 μM and K i for Ap 3A and Gp 3G are 20 and 22 μM, respectively. K m for Ap 3A determined by HPLC is 16 μM. m 7Gp 3G and m 7Gp 3A are also good substrates for triphosphatase.