Adenosine Deaminase from Takadiastase

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Abstract
A procedure was described for the purification of adenosine deaminase [EC 3.5.4.4, adenosine aminohydrolase, Aspergillus oryzae] from Takadiastase. The enzyme preparation was monodisperse as judged by ultracentrifugation and Tiselius electrophoresis. The sedimentation coefficient was 11.9S, and the molecular weight calculated from s20, w and D20, w was 214, 000, and from amino acid analysis 221, 000. The enzyme was dissociated into subunits by guanidine-HCl. In 2 m guanidine, the enzyme showed s20, w of 8.2 and in 3 m, 2.6 and 9.0. After dialysis against ammonium acetate buffer (pH5.5) for 48 hr, the 8.2S subunit was associated and its s20, w was 11.9. It seems that the deaminase might consist of eight subunits. Results of amino acid analysis and measurement of carbohydrate contents suggest that the deaminase is made from 75% of protein and 25% of carbohydrate. Experiments of N-bromosuccinimide inhibition indicate that the tryptophan residue may be involved in the active center of the enzyme. Although the enzyme activity was inhibited by PGMB, cysteine residue was not detected by amino acid analysis. The most preferred substrates of the deaminase were adenosine and 5'-AMP. Moreover, ApUp, 8-azaadenosine, and formycin were also de-aminated at a considerable rate. 6-Methylpurine riboside, adcnosine-1-oxide, and 2-chloroadenosine did not serve as substrate, but markedly inhibited the deamination of adenosine or 5'-AMP. The structures of the substrates and the inhibitors are also discussed.

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