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 (Ap(n)A) in both intact cell or plasma m embrane preparations is described, Procedures are based on the use of the fluorogenic Ap(n)A analogs, e-(Ap(n)A), as artificial substrates, It is shown that these fluorogenic analogs behave as excellent substra tes of the ectoenzyme present in cultured chromaffin cells, The ectoen zyme hydrolyzed all e-(Ap(n)A) tested (n = 2-6), always producing e-AM P and e-Ado 5'(n - 1) phosphate moieties, These released nucleotide mo ieties were then further catabolized up to e-Ado by other ectonucleoti dases, e-(Ap(4)A) hydrolysis by cultured cells displayed K-m and V-max values of 4.1 +/- 1.5 mM and 13.2 +/- 1.3 pmol/min x 10(6) cells, res pectively, as measured by continuous fluorometric assays and 3.5 +/- 1 .6 mM and 10.0 +/- 1.9 pmol/min x 10(6) cells by chromatographic-fluor ometric assays, Using plasma membranes, values of 2.5 +/- 0.8 mM and 6 69 +/- 59 pmol/min x mg protein for K-m and V-max, respectively, were obtained through continuous fluorometric assays, Ap(n)A and Gp(n)G beh aved as competitors and K-i values for these dinucleotides ranged betw een 0.7 and 3.5 mhz. The ectoenzyme was activated by Mg2+ and Ca2+ and achieved maximal activity in the pH range 8.5-9.0. (C) 1995 Academic Press, Inc.