J Korean Neurosurg Soc.  1984 Dec;13(4):611-625.

Purification and Characterization of Guanine Aminohydrolase from Rat Cerebrum

• Cha HJ
• Jung HW
• Koh YC
• Sim BS
• Park JB
• Kim SW

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.

Keyword

Purine; Guanine aminhydrolase; Neucleotide; Xanthine oxidase; Filtration; Purification

MeSH Terms

Ammonium Sulfate
Animals
Benzoates
Cerebrum*
Chromatography
Chromatography, Gel
Cytosol
DEAE-Cellulose
Durapatite
Filtration
Guanine Deaminase*
Guanine*
Guanosine
Hot Temperature
Hydrogen-Ion Concentration
Hypoxanthine
Inosine
Mercaptoethanol
Molecular Weight
Nucleosides
Purine Nucleotides
Rats*
Xanthine Oxidase
Ammonium Sulfate
Benzoates
DEAE-Cellulose
Durapatite
Guanine
Guanine Deaminase
Guanosine
Hypoxanthine
Inosine
Mercaptoethanol
Nucleosides
Purine Nucleotides
Xanthine Oxidase