Guanine deaminase from rat brain. Purification, characteristics, and contribution to ammoniagenesis in the brain.
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 electrophoresis 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 subunits. 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 K1 value of 82 micrometer. 4. Guanine deaminase was purified from rat liver by the procedure used for purification 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 Ouchterlony double diffusion test. 5. The gamma-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 substrates 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.
D000619:Aminohydrolases / Q000378:metabolism*; D000620:Aminoimidazole Carboxamide / Q000494:pharmacology; D000641:Ammonia / Q000378:metabolism*; D000818:Animals; D001375:Azaguanine / Q000378:metabolism; D001921:Brain / Q000201:enzymology*; D002845:Chromatography; D006147:Guanine / Q000378:metabolism; D006148:Guanine Deaminase / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000378:metabolism; D007158:Immunologic Techniques; D008099:Liver / Q000201:enzymology; D008297:Male; D008970:Molecular Weight; D051381:Rats; D011919:Rats, Inbred Strains
D000620:Aminoimidazole Carboxamide; D006147:Guanine; D000641:Ammonia; D006160:Guanosine Triphosphate; D000619:Aminohydrolases; D006148:Guanine Deaminase; D001375:Azaguanine