Molecular Pharmacology, Vol 14, 199-209, Copyright © 1978 by the American Society for Pharmacology and Experimental Therapeutics

Studies on the Effects of Infusion of Enzyme Inhibitors on Mouse Adenosine Deaminase

¹ Memorial-Sloan-Kettering Cancer Center, New York, New York 10021

Two inhibitors of adenosine deaminase, 9-erythro-(2-hydroxy-3-nonyl)adenine (EHNA) and 9--D-arabinofuranosyl-6-hydroxylaminopurine (ara-HA), which represent relatively tight and weak binding substrate analogues, respectively, were infused into mice over a period of 5 days. Nine different tissues were excised, and adenosine deaminase activity levels, as well as selected kinetic parameters and electrophoretic variants, were characterized. Isoelectric focusing demonstrated two variants (adenosine deaminases I and II) that focused in the pH range 4.65-4.75 and exhibited an approximate apparent molecular weight of 36,000 by gel filtration. The ratio of these two forms, which differed from tissue to tissue, was unaffected by the inhibitor infusions. Striking increases in adenosine deaminase activity were observed after the infusions and were found to vary with the tissue as well as the nature and dose of inhibitor. Activities in the lung, stomach, liver, and jejunum increased by about 8-, 3-, 3-, and 2-fold, respectively, after infusion of no more than 0.6 mg/ml of EHNA. Other tissues displayed significant but smaller increases. The effects of ara-HA were generally smaller than those of EHNA, except in the jejunum, ileum, and thymus. Inhibition of adenosine deaminase in vitro by EHNA, and to a lesser extent by ara-HA, also varied from tissue to tissue, but did not correlate with the ratio of adenosine deaminases I and II. Adenosine deaminase partially purified from mice treated with a higher dose of EHNA (5.0-7.5 mg/ml) displayed a significantly lower K_i value for EHNA; smaller decreases were noted in the K_m, for adenosine. The most striking change was observed in the K_m of adenosine deaminase II for EHNA, which changed from 14.9 nM (control) to 3.1 nM (after infusion). By classical Ackermann-Potter plots EHNA was shown not to be a titrating or stoichiometric inhibitor either before or after the infusions. EHNA in all cases showed classical competition with substrate. The data may be of value in the development of combined chemotherapy regimens involving adenosine deaminase inhibitors as well as in understanding the molecular basis of the role of adenosine deaminase in regulating cell division.

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