Guanine deaminase inhibitor from rat liver. Isolation and characterization

1. An inhibitor of cytoplasmic guanine deaminase of rat liver was isolated from liver ;heavy mitochondrial' fraction after freezing and thawing and treatment with Triton X-100. 2. Submitochondrial fractionation revealed that the inhibitor was localized in the outer-membrane fraction. 3. The method of purification of inhibitor, involving precipitation with (NH(4))(2)SO(4) and chromatography on DEAE-cellulose, its precipitability by trichloroacetic acid and the pattern of absorption in the u.v. indicated that the inhibitor was a protein. In confirmation, tryptic digestion of the isolated material resulted in destruction of the inhibitor activity. The inhibitor was stable to acid, but labile to heat. 4. The isolated inhibitor required phosphatidylcholine (lecithin) for activity. Phosphatidylcholine also partially protected the inhibitor against heat inactivation. 5. When detergent treatment was omitted, the inhibitor activity of frozen mitochondria was precipitated by (NH(4))(2)SO(4) in a fully active form without supplementation with phosphatidylcholine, indicating that Triton X-100 ruptured the linkage between inhibitor and lipid. 6. A reconstituted sample of inhibitor-phosphatidylcholine complex was precipitated in a fully active form by dialysis against 2-mercaptoethanol, but treatment of the precipitate with NaCl yielded an extract which was inactive unless supplemented with fresh phosphatidylcholine. 7. We interpret the results as evidence that the inhibitor was present in vivo as a lipoprotein and that once the complex was dissociated by the action of detergent and the protein precipitated, there was an absolute need for exogenous phosphatidylcholine for its activity. The manner in which inhibitor associated with the outer membrane of rat liver mitochondria might regulate the activity of the enzyme in the supernatant has been suggested.     

Primary structure of rat liver L-threonine deaminase

The primary structure of rat liver L-threonine deaminase has been studied utilizing a highly purified preparation (S.A. = 940 U/mg protein) obtained from Wistar male rats. These data have been compared with the predicted sequences obtained by other Authors, showing a considerable concordance with the Noda's prediction and difference with the Ogawa's results. The FAB-MS analysis has demonstrated the presence of an acetyl group as blocking agent on the N-terminal alanine.     

Characterization of porphobilinogen deaminase from rat liver

Porphobilinogen deaminase (porphobilinogen ammonia-lyase, EC 4.3.1.8) was isolated from rat liver. The final preparation was homogeneous according to polyacrylamide gel electrophoresis and immunodiffusion criteria. Electrophoresis of the native enzyme revealed a single band of activity which was distributed into three bands after incubation with porphobilinogen. When electrophoresed under denaturing condition it displayed a single polypeptide band with a molecular weight of 42,000 confirmed by exclusion chromatography and by sucrose density gradient centrifugation. The enzyme showed a pH optimum of 7.5 both in 0.1 M sodium phosphate and 0.05 M Tris-HCl buffer, when assayed at 37 degrees C. An isoelectric point of 4.9 for the native purified protein was found. Hepatic porphobilinogen deaminase was remarkably heat-stable showing maximum activity at 55-60 degrees C with one break in the Arrhenius plot. The kinetic behaviour of the purified enzyme followed the typical Michaelis-Menten kinetics with values of Km = 17 microM and Vmax = 29.4 units power mg in 0.1 M phosphate buffer at 37 degrees C. The amino acid composition was determined, showing that the enzyme had a low content of sulphur-containing amino acids and a considerable number of acidic residues per mol of polypeptide chain. Reagents known to interact with sulphydryl groups have small effect on rat liver enzyme activity.     

Isoenzymicity in mouse liver guanine deaminase demonstrable under substrate stress

Two isoenzymes of guanine deaminase could be demonstrated in the liver of mice subjected to guanine stress while the salinetreated controls showed only one. The one appearing under stress was a regulatory protein showing a sigmoidal substrate saturation curve, but was not influenced by GTP, allantoin or Mg²⁺     

Guanine derivatives modulate L-glutamate uptake into rat brain synaptic vesicles

Glutamate uptake into synaptic vesicles is driven by a proton electrochemical gradient generated by a vacuolar H(+)-ATPase and stimulated by physiological concentrations of chloride. This uptake plays an important role in glutamatergic transmission. We show here that vesicular glutamate uptake is selectively inhibited by guanine derivatives, in a time- and concentration-dependent manner. Guanosine, GMP, GDP, guanosine-5'-O-2-thiodiphosphate, GTP, or 5'-guanylylimidodiphosphate (GppNHp) inhibited glutamate uptake in 1.5 and 3 min incubations, however, when incubating for 10 min, only GTP or GppNHp displayed such inhibition. By increasing ATP concentrations, the inhibitory effect of GTP was no longer observed, but GppNHp still inhibited glutamate uptake. In the absence of ATP, vesicular ATPase can hydrolyze GTP in order to drive glutamate uptake. However, 5mM GppNHp inhibited ATP hydrolysis by synaptic vesicle preparations. GTP or GppNHp decreased the proton electrochemical gradient, whereas the other guanine derivatives did not. Glutamate saturation curves were assayed in order to evaluate the specificity of inhibition of the vesicular glutamate carrier by the guanine derivatives. The maximum velocity of the initial rate of glutamate uptake was decreased by all guanine derivatives. These results indicate that, although GppNHp can inhibit ATPase activity, guanine derivatives are more likely to be acting through interaction with vesicular glutamate carrier.     

Induction of guanine deaminase and its inhibitor in rodent liver and brain

1. Guanine deaminase activities in homogenates and supernatant fractions of liver and brain of rat and mouse were elevated by administration of guanine to the animals. The maximum induction in mouse tissues occurred within 24h and in rat tissues within 48h. 2. Mitochondria of rat (but not mouse) liver and brain contain an inhibitor of supernatant guanine deaminase, and this was also increased by guanine treatment. 3. Administration of ethionine, cycloheximide or actinomycin D prevented the guanine-dependent increase in deaminase activity and also the increase in mitochondrial inhibitory activity; chloramphenicol suppressed only the latter.     

Effect of benzodiazepines on the activity of AMP deaminase and adenosine deaminase in rat brain tissue in vivo

Phenazepam (5 mg/200 g) and seduxen (3 mg/200 g) injected intraperitoneally to 184 rats altered AMP-deaminase and adenosine deaminase brain activity. Seduxen was observed to increase AMP-deaminase and adenosine deaminase activity by 89.1% and 32.4%, respectively an hour after the injection. Phenazepam increased the activity of the enzymes by 35.5% and 38.5%, respectively two hours after the injection. The effect is suggested to be due to de novo benzodiazepine-induced enzyme synthesis.