Purification and partial characterization of brain adenosine deaminase: Inhibition by purine compounds and by drugs

Abstract

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 degrees C), fractionation 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 A) 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 delta H (5.0 Kcal/mole) values. The variation of V and Km with pH 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 X 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 (Ki 30, 54, and 83 microM, respectively), whereas medazepam is the most potent as activator (Ka 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.

Transition-State Variation in Human, Bovine, and Plasmodium falciparum Adenosine Deaminases

“…All solids were dissolved when the reaction was heated to 90 °C. The reaction mixture was then stirred under these conditions for another 12 h and volatile residues were removed under reduced pressure, followed by oil pump vacuum for 24 h. The solid residues were subject to recrystallization in hot water to give pure [6-13 C], [6][7][8][9][10][11][12][13][14][15] C]-labeled adenosines were prepared from radiolabeled riboses 7 and isotopically labeled adenines through two one-pot enzymatic reactions. Specifically labeled ATPs were synthesized as described previously with some modification.…”

“…Generation of Meisenheimer complex is followed by [ Table 2). The primary [6][7][8][9][10][11][12][13][14][15] (Figure 3). The hydroxyl attack and sp 3 rehybridization at C6 make the N6 electrostatic potential more negative in the transition states with overall charges of -0.912 to -0.925.…”

“…15,19,[24][25][26][27][28] Our interest in enzymatic transition states led us to determine the transition state structures of PfADA, HsADA and BtADA via the KIE approach. We also report a new synthetic method for making [6][7][8][9][10][11][12][13] C], [6][7][8][9][10][11][12][13][14][15] N], [6-13 C, 6-15 N]-labeled adenines. These adenines, together with [1-15 N] adenine, were combined with [1'-14 C] or [1'-3 H] riboses to give isotopically-labeled adenosines as substrates.…”

“…The set of KIEs for the three ADAs were determined under competitive conditions. The intrinsic [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15] N], [6-13 C], and [6- 30 Adenylate kinase (AK), pyruvate kinase (PK), hexokinase, and bovine spleen adenosine deaminase (BtADA) were purchased from Sigma. Phospho-D-ribosyl-1-pyrophosphate (PRPP) synthase and adenine phosphoribosyltransferase (APRTase) were purified according to the methods reported previously.…”

A Product Inhibition Study on Adenosine Deaminase by Spectroscopy and Calorimetry

“…Therefore, *To whom correspondence should be addressed. Tel: 0098-21-6498819; Fax: 0098-21-6404680 E-mail: saboury@chamran.ut.ac.ir the modulation of ADA with the use of highly specific inhibitors might modify the action of endogenous adenosine under various physiological and pathological conditions (Agarwal, 1982;Phillis et al, 1985;Centelles et al, 1988). In this study, we investigated the inhibitory effect of the inosine product on the enzymatic reaction of ADA by spectroscopy and calorimetric methods.…”