Adenosine ecto-deaminase (ecto-ADA) from porcine cerebral cortex synaptic membrane.
We have purified and investigated the role of adenosine ecto-deaminase (ecto-ADA) in porcine brain synaptic membranes and found a low activity of ecto-ADA in synaptic preparations from the cerebral cortex, hippocampus, striatum and medulla oblongata in the presence of purine transport inhibitors (NBTI, dipyridamole and papaverine). The purification procedure with affinity chromatography on epoxy-Toyopearl gel/purine riboside column as a crucial step of purification allowed a 214-fold purification of synaptic ecto-ADA with a yield of 30%. Gel filtration chromatography revealed a molecular mass estimated at 42.4+/-3.9 kDa. The enzyme had a broad optimum pH and was not affected by mono- and divalent cations. Ecto-ADA revealed a low affinity to adenosine (Ado) and 2'-deoxyadenosine (2'-dAdo) (K(M)=286.30+/-40.38 microM and 287.14+/-46.50 microM, respectively). We compared the affinity of ecto-ADA to the substrates with the physiological and pathological concentrations of the extracellular Ado in brains that do not exceed a low micromolar range even during ischemia and hypoxia, and with the affinity of adenosine receptors to Ado not exceeding a low nanomolar (A(1) and A(2A) receptors) or low micromolar (A(2B) and A(3)) range. Taken together, our data suggest that the role of synaptic ecto-ADA in the regulation of the ecto-Ado level in the brain and in the termination of adenosine receptor signaling is questionable. The porcine brain synapses must have other mechanisms for the ecto-Ado removal from the synaptic cleft and synaptic ecto-ADA may also play an extra-enzymatic role in cell adhesion and non-enzymatic regulation of adenosine receptor activity.
Romanowska
Małgorzata
M
Department of Biochemistry, Faculty of Biology and Earth Sciences, Nicolaus Copernicus University, 9 Gagarina St., 87-100 Toruń, Poland. malgromanowska@interia.pl
Ostrowska
Marta
M
Komoszyński
Michał A
MA
D015720:5'-Nucleotidase / Q000378:metabolism; D000243:Adenosine Deaminase / Q000302:isolation & purification / Q000378:metabolism*; D000818:Animals; D002540:Cerebral Cortex / Q000201:enzymology*; D003902:Detergents / Q000494:pharmacology; D007700:Kinetics; D013552:Swine; D013570:Synaptic Membranes / Q000201:enzymology*
D003902:Detergents; D015720:5'-Nucleotidase; D000243:Adenosine Deaminase