8-Azido-nucleotides as substrates of Torpedo electric organ apyrase. effect of photoactivation on apyrase activity.
Ecto-apyrase is a widespread enzymatic activity that hydrolyses tri- and diphosphonucleotides and consequently controls the amount of available extracellular ATP and ADP. In the nervous system, purines have important neuromodulatory actions, acting at pre- and postsynaptic sites, and consequently, ecto-apyrase may play an indirect role in the modulation of nucleotide- and nucleoside-mediated processes. The azido-nucleotides have been largely employed to characterize the nucleotide binding sites of several proteins. In the present work the azido-nucleotides are described as putative substrates for apyrase activity in a presynaptic plasma membrane preparation (PSPM) from the Torpedo electric organ. Both 8-N3-ATP and 8-N3-ADP were hydrolyzed in a calcium-dependent manner showing Vmax of 23.8 +/- 4.8 and 14.5 +/- 3 U/mg of protein, and Km values (in microM) of 116 +/- 39 and 119 +/- 4, respectively. Vmax for calcium-dependent hydrolysis of ATP and ADP were significantly higher: 59.2 +/- 3.9 and 32.9 +/- 3.5 U/mg of protein respectively, while Km values did not show any significant differences regarding azido-nucleotides: 83.8 +/- 12 microM for Ca2+-ATP and 121 +/- 34 microM for Ca2+-ADP. The photoactivation of the PSPM in the presence of the azido-derivatives results in an irreversible inactivation of apyrase activity, showing an IC50 of 10 microM and a maximal inhibitory effect of 38 and 60% on Ca2+-ATPase and Ca2+-ADPase activities. Apyrase was protected from inactivation by nucleotides that are natural substrates for this enzymatic activity and also by AMP while adenosine did not protect from apyrase inhibition.
Martí
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E
Dept. Biologia Cellular i Anatomia Patològica, Facultat de Medicina, Hospital de Bellvitge, Universitat de Barcelona, Spain.
Gómez de Aranda
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I
Solsona
C
C
D000244:Adenosine Diphosphate / Q000031:analogs & derivatives* / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000378:metabolism; D000345:Affinity Labels; D000818:Animals; D001081:Apyrase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D001386:Azides / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors / Q000378:metabolism; D002462:Cell Membrane / Q000201:enzymology; D004557:Electric Organ / Q000201:enzymology*; D004789:Enzyme Activation / Q000502:physiology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D066298:In Vitro Techniques; D009419:Nerve Tissue Proteins / Q000378:metabolism; D010775:Photic Stimulation; D010777:Photochemistry; D014101:Torpedo / Q000378:metabolism*
D000345:Affinity Labels; D001386:Azides; D004791:Enzyme Inhibitors; D009419:Nerve Tissue Proteins; C017040:8-azidoadenosine 5'-triphosphate; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D001081:Apyrase; D000252:Calcium-Transporting ATPases; C021622:8-azidoadenosine diphosphate