Tight binding inhibitors—VI: Interactions of deoxycoformycin and adenosine deaminase in intact human erythrocytes and sarcoma 180 cells

Abstract

The inactivation and reactivation of adenosine deaminase (ADA) by deoxycoformycin was studied in intact human erythrocytes and murine Sarcoma 180 cells in vitro. The second-order rate constant ( k 1) for the association reaction between deoxycoformycin and intraerythrocytic ADA was calculated to be 5.1 × 10 3M −1 sec −1. This is about 300 to 500-fold lower than the k 1 values determined either with hemolyzed human erythrocytes ( k 1 = 1.4 × 10 6 M −1 sec −1) or with partially purified human erythrocytic ADA ( k 1 = 2.6 × 10 6 M −1 sec −1). In intact erythrocytes only slight reactivation (<10 per cent) of the inhibited ADA ( EI complex) was detectable over 24 hr, whereas with hemolysates about 50 per cent reactivation of the inhibited ADA was observed in about 25 hr ( k 2 = 7.7 × 10 −6sec −1). The k 1 values with intact and supernatant fractions from homogenized Sarcoma 180 cells were determined to be 1.1 × 10 4M −1 sec −1 and 4.2 × 10 6 M −1 sec −1 respectively. With intact Sarcoma 180 cells, negligible reactivation of ADA was seen during a 48-hr period. Preliminary studies indicate an important role for the erythrocytic nucleoside transport system on the apparent k 1 values and the rate of inactivation of ADA by deoxycoformycin in intact cells.