Affinity chromatography was used to purify adenosine deaminase from calf thymus to apparent homogeneity. For preparation of the selective enzyme adsorbent 9 (p aminobenzyl) adenine was used. This compound is an ideal ligand because it is a strong competitive inhibitor of adenosine deaminase of thymus (K(i) 3 μM). It was synthesized by direct alkylation of adenine with p nitrobenzyl bromide followed by catalytic reduction of 9 (p nitrobenzyl) adenine. The enzyme preparation has a specific activity of 400 units/mg (37°C) and is homogeneous as shown by analytical gel electrophoresis and sucrose density gradient centrifugation. The molecular weight, determined by comparative elution from Sephadex gels, ranges from 39,000 to 44,000. The pH optimum is equal to 7 and the K(m) values for adenosine, 2' deoxyadenosine and 6 chloropurine riboside are 22, 4 and 310 μM respectively. Purine riboside behaves as a competitive inhibitor (K(i) 5 μM).
PURIFICATION AND SOME PROPERTIES OF CALF THYMUS ADENOSINE DEAMINASE
LUCACCHINI, ANTONIO
1974-01-01
Abstract
Affinity chromatography was used to purify adenosine deaminase from calf thymus to apparent homogeneity. For preparation of the selective enzyme adsorbent 9 (p aminobenzyl) adenine was used. This compound is an ideal ligand because it is a strong competitive inhibitor of adenosine deaminase of thymus (K(i) 3 μM). It was synthesized by direct alkylation of adenine with p nitrobenzyl bromide followed by catalytic reduction of 9 (p nitrobenzyl) adenine. The enzyme preparation has a specific activity of 400 units/mg (37°C) and is homogeneous as shown by analytical gel electrophoresis and sucrose density gradient centrifugation. The molecular weight, determined by comparative elution from Sephadex gels, ranges from 39,000 to 44,000. The pH optimum is equal to 7 and the K(m) values for adenosine, 2' deoxyadenosine and 6 chloropurine riboside are 22, 4 and 310 μM respectively. Purine riboside behaves as a competitive inhibitor (K(i) 5 μM).I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
