Use of Fluorogenic Substrates for Detection and Investigation of Ectoenzymatic Hydrolysis of Diadenosine Polyphosphates: A Fluorometric Study on Chromaffin Cells

A set of procedures to assay and investigate ectoenzymatic hydrolysis of diadenosine polyphosphates (ApnA) in both intact cell or plasma membrane preparations is described. Procedures are based on the use of the fluorogenic ApnA analogs, ϵ-(ApnA), as artificial substrates. It is shown that these fluorogenic analogs behave as excellent substrates of the ectoenzyme present in cultured chromaffin cells. The ectoenzyme hydrolyzed all ϵ-(ApnA) tested (n = 2-6), always producing ϵ-AMP and ϵ-Ado 5′(n − 1) phosphate moieties. These released nucleotide moieties were then further catabolized up to ϵ-Ado by other ectonucleotidases, ϵ-(Ap4A) hydrolysis by cultured cells displayed Km and Vmax values of 4.1 ± 1.5 mM and 13.2 ± 1.3 pmol/min × 106 cells, respectively, as measured by continuous fluorometric assays and 3.5 ± 1.6 mM and 10.0 ± 1.9 pmol/min × 106 cells by chromatographic-fluorometric assays. Using plasma membranes, values of 2.5 ± 0.8 mM and 669 ± 59 pmol/min × mg protein for Km and Vmax, respectively, were obtained through continuous fluorometric assays. ApnA and GpnG behaved as competitors and Ki values for these dinucleotides ranged between 0.7 and 3.5 mM. The ectoenzyme was activated by Mg2+ and Ca2+ and achieved maximal activity in the pH range 8.5-9.0.