Anti-ENTPD3 monoclonal antibody

The goal of this study was to determine the validity of using N-6-etheno-bridged adenine nucleotides to evaluate ecto-nucleotidase activity. We observed that the metabolism of N-6-etheno-ATP versus ATP was quantitatively similar when incubated with recombinant CD39, ENTPD2, ENTPD3, or ENPP-1, and the quantitative metabolism of N-6-etheno-AMP versus AMP was similar when incubated with recombinant CD73. This suggests that ecto-nucleotidases process N-6-etheno-bridged adenine nucleotides similarly to endogenous adenine nucleotides. Four cell types rapidly (t(1/2), 0.21 to 0.66 h) metabolized N-6-etheno-ATP. Applied N-6-etheno-ATP was recovered in the medium as N-6-etheno-ADP, N-6-etheno-AMP, N-6-etheno-adenosine, and surprisingly N-6-etheno-adenine; intracellular N-6-etheno compounds were undetectable. This suggests minimal cellular uptake, intracellular metabolism, or deamination of these compounds. N-6-etheno-ATP, N-6-etheno-ADP, N-6-etheno-AMP, N-6-etheno-adenosine, and N-6-etheno-adenine had little affinity for recombinant A(1), A(2A), or A(2B) receptors, for a subset of P2X receptors (H-3-alpha,beta-methylene-ATP binding to rat bladder membranes), or for a subset of P2Y receptors (S-35-ATP-alpha S binding to rat brain membranes), suggesting minimal pharmacological activity. N-6-etheno-adenosine was partially converted to N-6-etheno-adenine in four different cell types; this was blocked by purine nucleoside phosphorylase (PNPase) inhibition. Intravenous N-6-etheno-ATP was quickly metabolized, with N-6-etheno-adenine being the main product in naive rats, but not in rats pretreated with a PNPase inhibitor. PNPase inhibition reduced the urinary excretion of endogenous adenine and attenuated the conversion of exogenous adenosine to adenine in the renal cortex. The N-6-etheno-bridge method is a valid technique to assess extracellular metabolism of adenine nucleotides by ecto-nucleotidases. Also, rats express an enzyme with PNPase-like activity that metabolizes N-6-etheno-adenosine to N-6-etheno-adenine.

The neuroprotective role of adenosine and the deregulation of adenosine receptors in Alzheimer's disease (AD) have been extensively studied in recent years. However, little is known about the involvement of purine metabolism in AD. We started by analyzing gene expression in the entorhinal cortex of human controls and AD cases with whole-transcript expression arrays. Once we identified deregulation of the cluster purine metabolism, messenger RNA expression levels of 23 purine metabolism genes were analyzed with qRT-PCR in the entorhinal cortex, frontal cortex area 8, and precuneus at stages I-II, III-IV, and V-VI of Braak and Braak and controls. APRT, DGUOK, POLR3B, ENTPD3, AK5, NME1, NME3, NME5, NME7, and ENTPD2 messenger RNAs were deregulated, with regional variations, in AD cases when compared with controls. In addition, liquid chromatography mass spectrometry based metabolomics in the entorhinal cortex identified altered levels of dGMP, glycine, xanthosine, inosine diphosphate, guanine, and deoxyguanosine, all implicated in this pathway. Our results indicate stage- and region-dependent deregulation of purine metabolism in AD. (C) 2015 Elsevier Inc. All rights reserved.