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Purification and partial characterization of brain adenosine deaminase: Inhibition by purine compounds and by drugs
Author(s) -
Centelles J. J.,
Franco R.,
Bozal J.
Publication year - 1988
Publication title -
journal of neuroscience research
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.72
H-Index - 160
eISSN - 1097-4547
pISSN - 0360-4012
DOI - 10.1002/jnr.490190212
Subject(s) - adenosine deaminase , adenosine , purine , pharmacology , chemistry , biochemistry , enzyme , medicine
Rat brain adenosine deaminase (E. C. 3.5.4.4.) was purified 667‐fold from the supernatant fraction by the following techniques: heat treatment (60°C), fraction‐ation with ammonium sulfate, column chromatography on DEAE‐Sepharose, and preparative gel electrophoresis. The purified enzyme was homogeneous by the criterion of polyacrylamide disc gel electrophoresis and isoelectric focusing. Amino acid composition is given. The isoelectric point of the enzyme (5.2) was determined by isoelectric focusing on agarose. The apparent molecular weight was estimated to be 39,000 (Stokes Radius [Rs] = 27.3 Å) using a calibrated Sephacryl S‐300 column. The study of the influence of the temperature on the initial reaction rates allowed calculation of Ea (8.9 Kcal/mole) and ΔH (5.0 Kcal/mole) values. The variation of V and Km with p H suggests the existence of a sulfhydryl group and an imidazole group in the enzyme‐substrate complex. The enzyme had a Km (adenosine) of 4.5 × 10 −5 M and was inhibited by inosine, guanosine, adenine, and hypoxanthine but not by other intermediates of purine metabolism. None of the inhibitors were active as substrates. The enzyme was also inhibited by dimethyl sulfoxide and ethanol. Inhibition by ethanol can account partially for the CNS depressant effects of levels 3 and 4 of alcohol intoxication. A number of drugs having therapeutic uses such as sedative, anxiolytic, analgesic, and relaxant are modulators of the enzyme. Among these, lidoflazine, phenylbutazone, and chlordiazepoxide are the most potent as inhibitors (K i 30, 54, and 83 μM, respectively), whereas medazepam is the most potent as activator (K a 0.32 mM). Thus, it is concluded that some drugs that inhibit adenosine uptake also modulate adenosine deaminase activity. Besides, since the enzyme is located extracellularly [Franco et al, 1986], these drugs can modulate the physiological effects exerted by extracellular adenosine.