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Terminal deoxyribonucleotidyl transferase (EC 2.7.7.31; nucleoside triphosphate:DNA nucleotidyl.

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authors request that the following corrections be noted. A more detailed analysis of our genomic clones (suggested by J. P. Tillinghast, L. Fields, and -10 0 D. Loh) has revealed that exon 1 is located approximately in the middle of clone C1. As a consequence, the first intron spans about 15 kilobases (kb) (instead of 27 kb), and the entire gene spans about 37 kb (instead of 49 kb). The corrected structure of Fig. 2 with additional confirmed restriction sites

Hereditary deficiency of the enzyme adenosine deaminase (adenosine aminohydrolase, EC 3.5.4.4) results in an immunodeficiency syndrome characterized by a marked reduction in circulating lymphocytes. We have administered 2'-deoxycoformycin, a potent inhibitor of adenosine deaminase, to a patient with a lymphoproliferative malignancy. The Inherited deficiency of adenosine deaminase (ADA; adenosine aminohydrolase, EC 3.5.4.4) results in a severe immunodeficiency disease characterized by marked lymphopenia and loss of both T and B lymphocyte functions (1, 2). ADA catalyzes the deamination of adenosine and deoxyadenosine in the purine salvage pathway. Numerous studies have implicated the accumulation of metabolites derived from one or both of these substrates in the pathogenesis of the immune disorder (3-7).In the absence of ADA activity, deoxyadenosine is phosphorylated to dATP. Accumulation of this metabolite has been demonstrated in the erythrocytes from adenosine deaminasedeficient children (4, 5) and correlates with both inhibition of DNA synthesis and cytotoxicity in cultured lymphoid cells (8)(9)(10)(11). Deoxyadenosine is also known to inactivate S-adenosyl-L-homocysteine hydrolase (EC 3.3.1.1) (12) and, by this mechanism, may result in an increase in S-adenosylhomocysteine, a compound also associated with lymphocytotoxicity in vitro (13).We have administered 2'-deoxycoformycin (dCF), a potent ADA inhibitor, to a patient with a lymphoproliferative malignancy. Exogenous inhibition of ADA activity resulted in an increase in plasma deoxyadenosine, an increase in erythrocyte dATP, and an inhibition of S-adenosylhomocysteine hydrolase activity. In addition, we observed unexpected and profound depletion of ATP as dATP accumulated in the erythrocytes of patients treated with this drug. MATERIALS AND METHODS[14C]Adenosine (50.4 Enzyme Determinations. Heparinized whole blood was centrifuged at 500 X g and the plasma was removed. The cells were resuspended in an equal volume of Seligman's balanced salt solution, and dextran was added to a final concentration of 0.4%. The cells were allowed to stand in a 20-ml syringe placed upright in a 370C incubator for 1 hr. The plasma containing leukocytes was removed from the top of the syringe and centrifuged at 500 X g for 10 min. The pelleted leukocytes were collected and washed once in 10 mM Tris/140 mM NH4C1, pH 7.4, to lyse contaminating erythrocytes. Erythrocytes sedimented by dextran were collected by centrifugation. Leukocytes or erythrocytes were then washed three times in 10 mM Tris/150 mM NaCl, pH 7.4 (buffer A), and lysed by three freeze-thaw cycles. The lysates were centrifuged at 20,000 X g for 30 min, and the supernatants were dialyzed overnight at 4°C against buffer A. All assays for ADA activity were carried out by the method of Van (12).Plasma Adenosine and Deoxyadenosine Levels. Plasma obtained from heparinized whole blood was deproteinized by the method of Somogyi (16). Adenosine and deoxyadenosine were quantitated by high-pressure liquid chrom...