Tight binding inhibitors—VIII: Studies of the interactions of 2′-deoxycoformycin and transport inhibitors with the erythrocytic nucleoside transport system

Abstract

Studies were performed to extend earlier observations that the rate-limiting step in the inactivation of intraerythrocytic human adenosine deaminase (ADA) by 2'-deoxycoiormycin (dCF) is the nucleoside transport system (NTS). The NTS inhibitors 2-amino-6-[(2-hydroxy-5-nitrobenzyl)thio]-9-β-D-ribofuranosyl purine (HNBTGR), 6-[(4-nitrobenzyl)thio]-9-β- d-ribofuranosyl purine (NBMPR), 2-amino-6-[(4-nitrobenzyl)Seleno]-9-β- D-ribofuranosyl purine (NBSeGR), dipyridamole and the competitive permeant, uridine, all decreased the rate of ADA inactivation by dCF in a concentrationdependent manner. Lineweaver-Burk plots of 1 k γ (where k γ is the pseudo first-order rate constant for the inactivation of ADA) 1/dCF concentrations were linear, giving a K m tor dCF tor the NTS of 6 × 10 −7 M. The maximal k γ calculated by extrapolation to infinite dCF concentrations was 6 × 10 −3 per sec which corresponds to a T 1 2 of about 115 sec. Similar plots for experiments with the NTS inhibitors and uridine yielded classic patterns of competitive inhibition for NBMPR, HNBTGR, NBSeGR and uridine, whereas with dipyridamole a pattern of non-competitive inhibition was obtained. Dissociation or inhibition constants have been reported for several of these compounds (determined by other methods) and values similar to these were obtained. Inhibition by dipyridamole was non-competitive ( k l = 2.5 × 10 −7 M) and was of a bi-phasic nature with respect to time. Dipyridamole caused rapid and irreversible inhibition for the first 7–15 min with slow and progressive but reversible inhibition thereafter. These observations are consistent with the hypothesis that NBMPR, HNBTGR, NBSeGR and uridine interact with the same site on a macromolecular component of the NTS that forms ligands with dCF. The behavior of dipyridamole appears more complex and will require more extensive study.