Diadenosine polyphosphate hydrolase from presynaptic plasma membranes of Torpedo electric organ

The diadenosine polyphosphate hydrolase present in presynaptic plasma membranes from the Torpedo electric organ has been characterized using fluorogenic substrates of the form di-(1,N-6-ethenoadenosine) 5',5 triple prime-P-1,P-n-polyphosphate. The enzyme hydrolyses diadenosine polyphosphates (Ap(n)A, where n = 3-5), producing AMP and the corresponding adenosine (n-1) 5'-phosphate, Ap((n-1)). The K-m values of the enzyme were 0.543 +/- 0.015, 0.478 +/- 0.043 and 0.520 +/- 0.026 mu M, and the V-max values were 633 +/- 4, 592 +/- 18 and 576 +/- 45 pmol/min per mg of protein, for the etheno derivatives of Ap(3)A (adenosine 5',5 triple prime-P-1,P-3-triphosphate), Ap(4)A (adenosine 5',5 triple prime-P-1,P-4-tetraphosphate) and Ap(5)A (adenosine 5',5 triple prime-P-1,P-5-pentaphosphate) respectively. Ca2+, Mg2+ and Mn2+ are enzyme activators, with EC50 values of 0.86 +/- 0.11, 1.35 +/- 0.24 and 0.58 +/- 0.10 mM respectively. The fluoride ion is an inhibitor with an IC50 value of 1.38 +/- 0.19 mM, The ATP analogues adenosine 5'-tetraphosphate and adenosine 5'-[gamma-thio]triphosphate are potent competitive inhibitors and adenosine 5'-[alpha,beta-methylene]diphosphate is a less potent competitive inhibitor, the K-i values being 0.29 +/- 0.03, 0.43 +/- 0.05 and 7.18 +/- 0.8 mu M respectively. The P2-receptor antagonist pyridoxal phosphate 6-azophenyl-2',4'-disulphonic acid behaves as a non-competitive inhibitor with a K-i value of 29.7 +/- 3.1 mu M, and also exhibits a significant inhibitory effect on Torpeno apyrase activity. The effect of pH on the K-m and V-max values, together with inhibition by diethyl pyrocarbonate, strongly suggests the presence of functional histidine residues in Torpedo diadenosine polyphosphate hydrolase. The enzyme from Torpedo shows similarities with that of neural origin from neurochromaffin cells, and significant differences compared with that from endothelial vascular cells.