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, N6-ethenoadenosine) 5',5'''-P1,Pn-polyphosphate. The enzyme hydrolyses diadenosine polyphosphates (ApnA, where n=3-5), producing AMP and the corresponding adenosine (n-1) 5'-phosphate, Ap(n-1). The Km values of the enzyme were 0.543+/-0.015, 0.478+/-0.043 and 0. 520+/-0.026 microM, and the Vmax values were 633+/-4, 592+/-18 and 576+/-45 pmol/min per mg of protein, for the etheno derivatives of Ap3A (adenosine 5',5'''-P1,P3-triphosphate), Ap4A (adenosine 5',5"'-P1,P4-tetraphosphate) and Ap5A (adenosine 5',5'''-P1,P5-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 Ki values being 0.29+/-0.03, 0.43+/-0.05 and 7.18+/-0.8 microM respectively. The P2-receptor antagonist pyridoxal phosphate 6-azophenyl-2',4'-disulphonic acid behaves as a non-competitive inhibitor with a Ki value of 29.7+/-3.1 microM, and also exhibits a significant inhibitory effect on Torpedo apyrase activity. The effect of pH on the Km and Vmax 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.
Mateo
J
J
Departamento de Bioqu approximately ímica, Facultad de Veterinaria, Universidad Complutense de Madrid, Av. Puerta de Hierro s/n, E-28040 Madrid, Spain.
Rotllan
P
P
Marti
E
E
Gomez De Aranda
I
I
Solsona
C
C
Miras-Portugal
M T
MT
D017766:Acid Anhydride Hydrolases / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D000249:Adenosine Monophosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D002413:Cations, Divalent / Q000378:metabolism; D004047:Diethyl Pyrocarbonate / Q000494:pharmacology; D015226:Dinucleoside Phosphates / Q000378:metabolism; D004557:Electric Organ / Q000201:enzymology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009419:Nerve Tissue Proteins / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D017729:Presynaptic Terminals / Q000737:chemistry*; D011732:Pyridoxal Phosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D014101:Torpedo / Q000378:metabolism*
D002413:Cations, Divalent; D015226:Dinucleoside Phosphates; D004791:Enzyme Inhibitors; D009419:Nerve Tissue Proteins; C077792:pyridoxal phosphate-6-azophenyl-2',4'-disulfonic acid; D000249:Adenosine Monophosphate; C012275:P(1),P(5)-di(adenosine-5'-)pentaphosphate; C020733:diadenosine tetraphosphate; C011440:diadenosine triphosphate; D011732:Pyridoxal Phosphate; D000255:Adenosine Triphosphate; D017766:Acid Anhydride Hydrolases; C075829:diadenosine polyphosphate hydrolase; D004047:Diethyl Pyrocarbonate