Guanine Deaminase from Rat Brain. Purification, Characteristics, And
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J. Bioche,n. 91, 167-176 (1982) Guanine Deaminase from Rat Brain. Purification, Characteristics, and Contribution to Ammoniagenesis in the Brain Syuzo MIYAMOTO, Hirofumi OGAWA,1 Hiroshi SHIRAKI, and Hachiro NAKAGAWA2 Division of Protein Metabolism, Institute for Protein Research, Osaka University, Suita, Osaka 565 Received for publication, June 18, 1981 1. Guanine deaminase [EC 3.5.4.3] was purified to a homogeneous state from rat brain by a procedure involving ammonium sulfate fractionation, DE-52 column chromatography, hydroxylapatite column chromatography, gel filtration on ACA-34 and isoelectric focusing. Homogeneity was shown by polyacrylamide gel electro phoresis in the presence and absence of SDS. 2. The molecular weight of the enzyme was determined by gel filtration (105,000), and that of its subunit by SDS-polyacrylamide gel electrophoresis (52,000). From these findings, we concluded that the native enzyme consisted of two identical sub- units. 3. The Km values for guanine and 8-azaguanine were calculated to be 0.17 mM and 0.67 mM, respectively. This enzyme was markedly inhibited by 5-amino 4- imidazolecarboxamide (AICA), a precursor of purine nucleotide synthesis, with a Ki value of 82 ƒÊM. 4. Guanine deaminase was purified from rat liver by the procedure used for purifi cation of the brain enzyme and anti-liver enzyme serum was raised in rabbits. This antiserum cross-reacted with the brain enzyme without spur formation in the Ouch terlony double diffusion test. 5. The ƒÁ-globulin fraction of the anti-guanine deaminase serum and AICA inhibited more than 50% of the ammoniagenesis in the brain system in which the purine nucleotide cycle operates. It was also shown that guanine nucleotides were degraded via guanosine and guanine liberating ammonia in the same brain system when sub strates for the purine nucleotide cycle were omitted. On the basis of these findings it is suggested that guanine deaminase contributes to ammoniagenesis in the brain. 1 Present address: Department of Biochemistry, Medical School, Toyama Medical and Pharmaceutical University, Sugitani, Toyama 930-01. 2 To whom reprint requests should be sent. Abbreviations: SDS, sodium dodecyl sulfate; AICA, 5-amino 4-imidazolecarboxamide; AICAR, 5-amino 4- imidazolecarboxamide ribonucleotide. Vol. 91, No. 1, 1982 167 168 S. MIYAMOTO, H. OGAWA, H. SHIRAKI, and H. NAKAGAWA The activity of guanine deaminase [guanine amino adenylate kinase (rabbit muscle) [EC 2.7.4.3], and hydrolase, EC 3.5.4.3], catalyzing the conversion cytochrome c (horse heart) were from Boehringer, of guanine to xanthine and ammonia, is especially W. Germany. DEAE-cellulose (DE-52) was from high in the brain and liver, although it is also Whatman Ltd., England. Hydroxylapatite was detected in various other organs of animals (1). from Clarkson Chemical Co., U.S.A. Ultrogel However, the physiological role of this enzyme is ACA-34 and Ampholines (pH range, 4-6 and 3- unknown. 10) were from LKB, Sweden. Freund's complete It was found that the ammonia concentration and incomplete adjuvants were products of Difco, in the brain increases on deprivation of paradoxical U.S.A. TSK-GEL G3000SW silica gel prepacked sleep (2), and decreases during sleep. These find columns for high pressure liquid chromatography ings suggested that the ammonia concentration were obtained from Toyo Soda. Other chemicals reflects the state of functional activity of the brain. were of analytical reagent grade. Many investigators have examined the sources of Enzyme Assays-Guanine deaminase was as ammonia in the brain to elucidate its correlation sayed by the method of Roush and Norris (4), with brain function. Recently, Schultz and spectrophotometric determination of decrease in Lowenstein suggested that the purine nucleotide guanine concentration. The reaction mixture con cycle accounts for at least 50% of the ammo tained enzyme solution, 0.16 mM guanine and 83 niagenesis occurring endogenously in brain slices mM Tris-HCI buffer, pH 8.0, in a final volume of (3). This raises the questions of what are the 1.2 ml. After preincubation for 10 min at 37•Ž, other sources of ammonia and of whether some the reaction was started by adding guanine, and ammonia formed in the cycle is derived from after incubation for 20 min it was stopped by guanine nucleotide, because GTP, a substrate of adding 0.3 ml of 15% HClO4. The absorbance of the cycle, is degraded to GDP by adenylosuccinate the supernatant obtained by centrifugation was synthetase [EC 6.3.4.4] during the operation of the measured at 248 nm. When nucleotides and cycle and a series of enzymes converting the latter related compounds that interfere with the spectro to guanine is thought to be present in the brain. photometric assay were present in the reaction We thought that some specific inhibitor(s) of the mixture, the activity was measured by determina enzyme, such as anti-guanine deaminase serum or tion of ammonia by the enzymatic method of Kun other chemical compounds, would be useful for (5). The sample (deproteinized and neutralized) solving these problems. Therefore, we attempted was added to the reaction medium consisting of to purify the enzyme from rat brain. 0.3 M Tris-HCl buffer, pH 8.0, 30 mM ƒ¿-ketoglu This paper reports the purification and tarate, 0.36 mm NADH, and 12 units of glutamate characteristics of guanine deaminase of rat brain. dehydrogenase in a final volume of 1.5 ml. The The inhibitory effects of AICA, a specific inhibitor reaction was started by adding glutamate dehydro of guanine deaminase, and anti-guanine deaminase genase and allowed to proceed at 25•Ž for 90 min. serum on ammoniagenesis in a rat brain system The decrease in absorbance at 340 nm due to are also reported. oxidation of NADH by ammonia was measured. One unit of enzyme activity was defined as the amount catalyzing the formation of 1 ƒÊmol of MATERIALS AND METHODS ammonia per min under the standard assay con Materials-Male Wistar strain rats, weighing ditions; this is equivalent to a 1.17 decrease in 200 to 300 g, were killed by decapitation. The absorbance at 248 nm per min. brain and liver were removed and stored at -70•Ž Protein concentration was determined by the until use. method of Lowry et al. (6) with bovine serum NADH, AICA, AICAR, guanine, and dithio albumin as a standard. When the enzyme prepa threitol were purchased from Sigma Chemical Co., ration contained ampholine, which is nondialyzable U.S.A. Pyruvate kinase (rabbit muscle) [EC and interferes with protein determination, protein 2.7.1.40], lactate dehydrogenase (hog muscle) [EC was precipitated by adding HClO4 to a final con 1.1.1.27], glutamate dehydrogenase (beef liver) centration of 5%, and the precipitate was washed [EC 1.4.1.3], hexokinase (yeast) [EC 2.7.1.1], once with 5 % HClO4, dissolved in 0.1 N NaOH J. Biochem. GUANINE DEAMINASE IN RAT BRAIN 169 and subjected to quantitative determination. Step 1. Preparation of crude extract: Rat Gel Electrophoreses-Disc and SDS-poly brain (50 g) was homogenized with 200 ml of 0.2 acrylamide gel electrophoreses were performed by M KPB, pH 7.0, containing 0.1 mM dithiothreitol. the methods described by Williams and Reisfeld All solutions used in the following experiments (7) and Weber and Osborn (8), respectively. Pro contained 0.1 mM dithiothreitol. The homogenate tein was stained with 0.25% Coomassie brilliant was centrifuged at 20,000 x g for 30 min and the blue R-250. supernatant was used for further purification. Determination of Molecular Weight-Molec Step 2. AmMonium sulfate fractionation: ular weight was determined by gel filtration on a The supernatant was brought to 40% saturation TSK-GEL G3000SW column with a Hitachi 633 with amMonium sulfate slowly by adding the Liquid Chromatograph. The column was eluted powdered salt. The mixture was stood for 30 with 10 mM potassium phosphate buffer (KPB), min at 0°C and then centrifuged at 10,000 x g for pH 7.0, containing 0.3 M KCl at a flow rate of 20 min. The supernatant was brought to 70 0.5 ml per min. The molecular weight of sub- saturation with amMonium sulfate by adding the units of the enzyme was calculated from the powdered salt. The mixture was stood for 30 mobility on SDS-polyacrylamide gel in comparison min and then centrifuged at 10,000 x g for 20 min. with those of standard proteins. The resulting precipitate was dissolved in 100 ml Preparation of Anti-Rat Liver Guanine De of 50 mM KPB, pH 6.0, and dialyzed against 3 aminase Serum-Purified liver enzyme (1 mg of changes of 2 liters of the same buffer. protein in 2 ml) was emulsified with 2 ml of Step 3. DE-52 column chromatography: The Freund's adjuvant and injected subcutaneously dialysate was applied to a DE-52 column (1.5 x into various regions of two male rabbits, weighing 15 cm) equilibrated with 50 mM KPB, pH 6.0. The about 3 kg. Two weeks later, the same dose was column was washed with 100 ml of the same injected subcutaneously into other regions of the buffer, and then the enzyme was eluted with 200 same rabbits, and two weeks after this, 0.5 mg of ml of a linear gradient of 0-0.3 M KCl in the same enzyme protein emulsified with Freund's incom buffer. Fractions showing high guanine deaminase plete adjuvant was injected. One week after the activity were pooled and dialyzed against 3 changes third injection, blood was collected from the ear of 2 liters of 10 mM KPB, pH 7.0. vein and serum was separated by centrifugation.