Investigation of the preferred Mg(II)‐adenine‐nucleotide complex at the active site of ectonucleotidases in intact vascular cells using phosphorothioate analogues of ADP and ATP

Abstract

In the presence of Mg' + the ecto-(nucleoside diphosphatase) on intact vascular endothelial or smooth muscle cells in culture selectively catabolises the PS diastereoisomer of adenosine 5'-[~-thio]diphosphate, (PSI-ADP [aS], and the ecto-(nucleoside triphosphatase) selectively catabolises the PS isomer of adenosine 5'- [P-thio] [l]) have proved to be powerful tools with which to analyse the stereochemical course of phosphorylation of phosphohydrolase reactions. The presence of stereoselectivity towards one isomer makes it likely that the enzyme under investigation recognises an Mg-nucleotide complex at its active site, selectivity being imposed because Mg2+ preferentially binds to oxygen rather than to sulphur in the analogues. This can be demonstrated unequivocally, and the conformation of the Mg(I1)-nucleotide complex involved at the active site assigned, by comparing the observed stereoselectivity in the presence of different bivalent metal cations with known preferences for co-ordination to oxygen or sulphur ligands. Previous studies of this type have used purified enzymes, usually kinases (e. g. [2 -51). In our study of the characteristics of ectonucleotidases on vascular endothelial and smooth muscle cells we found that each cell type possesses distinct nucleoside triphosphatase, nucleoside diphosphatase and 5'-nucleotidase ectoenzymes whose activity can be assayed using intact cells in culture. We also noted that catabolism of ATP [pS] Enzymes. Nucleoside triphosphatase (EC 3.6.1.15); nucleoside diphosphatasc (EC 3.6.1.6); 5'-nucleotidase (EC 3.1.3.5). MATERIALS AND METHODS Cell cultureEndothelial and smooth muscle cells were separately isolated from the aortas of newborn pigs and cultured as previously described [9, 101. Cells were used for experiments after 2 -17 passages. They were grown to confluence on the surface of 16-mm-diameter wells of 24-well tissue-culture trays (Nunc) and used after briefly rinsing twice with bivalentcation-free phosphate-buffered saline. Replicate incubations at 37°C were then carried out in the same saline containing the appropriate phosphorothioate analogue (100 pM) and 0.8 mM M g Z f , CO" or Cd' '. Subsamples of the incubation solutions were removed at timed intervals and stored at -20 "C until required. Analogues and measurement of metabolitesThe PR and PS diastereoisomers of ADP [aS], ATP [aS] and ATP [pS] were synthesised and purified as previously described [l, 7, 111. Catabolism of each isomer was assessed directly by HPLC separation of non-extracted subsamples of the incubation solutions, using reversed-phase HPLC [7]. Rates of catabolism were measured by analysis of the peak heights or areas on the HPLC recordings. RESULTSEssentially similar results were found for both endothelial cells and smooth muscle cells; we have therefore presented data for endothelial cells alone. Since we were working with limited numbers of intact cells, rather than purified enzymes, we could not practicably determine K , and V,,, values for each isomer (both of which are likely to ...

The [Ca2+]i increase induced in murine thymocytes by extracellular ATP does not involve ATP hydrolysis and is not related to phosphoinositide metabolism

“…In both cases the ATP effect was dose dependent; in the absence of Mg2+ its effect occurred at a nucleotide concentration lower than in the presence of Mg'+: the half-maximal concentration of ATP shifted from 0.25 to 0.16 mM with 5 mM MgCl2 ( fig.2A). These data establish that MgATP2-is not the entity involved in [Ca2']i enhancement and consequently demonstrate that ATP hydrolysis is not required, this complex being the preferential substrate of ATPases and kinases [16]. [lo].…”

Two Distinct P2 Purinergic Receptors, P2Y and P2U, Are Coupled to Phospholipase C in Mouse Pineal Gland Tumor Cells

“…However, in PGT-~3cells the concentrationdependence curves for UTP4 indicate that the P 2r eceptors are also responsive to the tetrabasic form of UTP. Other studies have also shown that P2ñucleotide receptors can be activated by UTP 4 as well as ATP4 (Pearson and Cusack, 1985;Lustig et al, 1992).…”

Mechanisms of Flow-Mediated Signal Transduction in Endothelial Cells: Kinetics of ATP Surface Concentrations

“…This conclusion was based on the observation that most of the nonhydrolyzable ATP analogs are much less potent, on a molar basis, than ATP; thus, ectonucleotidase activity may be required for complete P2y-purinoceptor activa tion. Subsequent work by Pearson and Cusack [32], has demonstrated that the P2y-purinoceptorand ecto-ATPase have distinct characteristics regarding stereospecific re quirements for the ATP molecule, which led them to con clude that the two are separate molecules. For example the P2y-purinoceptor demonstrates the greatest stcreospecific requirement for the ribose moiety, while ectoATPase is selective for the phosphate chain and requires a nucleotide/Mg2+ complex for activation [27], These con siderations.…”