DIADENOSINE POLYPHOSPHATE HYDROLASE FROM PRESYNAPTIC PLASMA-MEMBRANESOF 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 tr iple prime-P-1,P-n-polyphosphate. The enzyme hydrolyses diadenosine po lyphosphates (Ap(n)A, where n = 3-5), producing AMP and the correspond ing adenosine (n-1) 5'-phosphate, Ap((n-1)). The K-m values of the enz yme were 0.543 +/- 0.015, 0.478 +/- 0.043 and 0.520 +/- 0.026 mu M, an d 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 pr ime-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 act ivators, 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 v alue of 1.38 +/- 0.19 mM, The ATP analogues adenosine 5'-tetraphosphat e and adenosine 5'-[gamma-thio]triphosphate are potent competitive inh ibitors and adenosine 5'-[alpha,beta-methylene]diphosphate is a less p otent 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 antagonis t 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 a lso exhibits a significant inhibitory effect on Torpeno apyrase activi ty. The effect of pH on the K-m and V-max values, together with inhibi tion by diethyl pyrocarbonate, strongly suggests the presence of funct ional histidine residues in Torpedo diadenosine polyphosphate hydrolas e. The enzyme from Torpedo shows similarities with that of neural orig in from neurochromaffin cells, and significant differences compared wi th that from endothelial vascular cells.