Purification and Characterization of Guanine Aminohydrolase from Rat Cerebrum.

Cha, Hee Joong; Jung, Hee Won; Koh, Young Cho; Sim, Bo Sung; Park, Joo Bae; Kim, Seung Woo

Original Article

Journal of Korean Neurosurgical Society 1984;13(4): 611-625.

Hee Joong Cha, Hee Won Jung, Young Cho Koh, Bo Sung Sim, Joo Bae Park, Seung Woo Kim

¹Department of Neurosurgery, College of Medicine, Seoul National University, Seoul, Korea.
²Department of Biochemistry, College of Medicine, Seoul National University, Seoul, Korea.

ABSTRACT

Guanine aminohydrolase(GAH;EC 3. 5. 4. 3.) was partially purified 122-fold from rat cerebrum to a specific activity of 7.22 in its per mg protein with a recovery of 7.47% by fractionation with ammonium sulfate, chromatography on DEAE-cellulose and hydroxyapatite, gel filtration on Sephadex G-200, and isoelectric focusing(pH4-6). The molecular weight of partially purified rat cerebral guanine aminohydrolase was estimated to be 110,000. But, in the cerebral cytosol, a rather higher molecular weight form of the enzyme was identified. The activity of the higher molecular weight form of guanine aminohydrolase was increased by dialyzing the cytosol, and it was converted into the lower molecular weight form(M.W.110,000) by addition of 2-mercaptoethanol. The reaction velocity of partially purified guanine aminohydrolase of rat cerebrum disclosed a hyperbolic curve, with its KM being 6.0uM at pH 8.0. The preparation showed high substrate specificity:among the purine nucleotides, nucleosides and bases with amino group, only guanosine and guanine were deaminated by the enzyme, and the reaction rate of the enzyme displayed by guanosine was less than 10% of that by guanine. When observed under the equimolar concentration of the substrate, hypoxanthine as well as inosine inhibited the activity of the rat cerebral guanine aminohydrolase by 9.4 and 7.8%, respectively, while 5-aminoimidazole-4-carboxamide inhibited the activity of it by 38%. The activity was inhibited by p-hydroxymercuric benzoate as well. Complete loss of its activity was observed after 30 minutes incubation at 60 degrees C, suggesting the preparation was heat labile.

Key Words: Purine; Guanine aminhydrolase; Neucleotide; Xanthine oxidase; Filtration; Purification