Guanine deaminase in rat liver and mouse liver and brain

1. The guanine deaminase in rat liver supernatant preparations was resolved into two fractions, A and B, on DEAE-cellulose columns. The two differed in electrophoretic mobility and in various properties. The most noteworthy distinction between A and B components was that the enzyme A activity showed a sigmoid dependence on substrate concentration whereas the enzyme B showed classical Michaelis-Menten kinetics. The K(m) value of enzyme A for guanine was 5.3mum and that of enzyme B 20mum. 2. The entire guanine deaminase activity of mouse liver was contained in the 15000g supernatant of iso-osmotic homogenates. 3. A reinvestigation of the behaviour of rat brain 15000g supernatant guanine deaminase isoenzymes revealed that one enzyme had sigmoidal kinetics and the other enzyme showed a hyperbolic response. 4. Of the guanine deaminase in mouse brain iso-osmotic sucrose homogenate 80% was recovered in the 15000g supernatant and the rest from the particles. The supernatant guanine deaminase was resolvable into two fractions on DEAE-cellulose columns. One enzyme showed sigmoidal kinetics whereas the other showed a hyperbolic response to increasing substrate concentration; the K(m) values for the reaction with guanine were respectively 5 and 66mum. 5. The particulate fractions of mouse liver and brain were devoid of any overt inhibitory activity.     

Guanine-deaminase activity in rat brain and liver

1. Guanine deaminase in rat brain and liver was distributed among all the subcellular fractions: nuclei, `heavy' mitochondria, `light' mitochondria, microsomes and the supernatant fluid. The greater part of the activity passed into the soluble fraction. Among the particulate components, the `light' mitochondria constituted the richest fraction. 2. The sum of the enzymic activities of the component fractions obtained on differential centrifugation was considerably greater than the activity of guanine deaminase in the whole homogenate. 3. The `heavy'-mitochondrial fraction had a powerful inhibitory effect on the guanine-deaminase activity of the supernatant fraction. 4. All the sedimented fractions, except the microsomes, gave rise to higher guanine-deaminase activity on treatment with Triton X-100. 5. The inhibitory capacity of the `heavy' mitochondria increased on treatment with Triton X-100; the detergent-treated nuclear fraction also brought about inhibition of the 5000g supernatant. 6. Guanine-deaminase inhibitor from the `heavy' mitochondria was solubilized by high-speed grinding of the particles, followed by treatment with Triton X-100. The inhibitor appeared to be protein in nature, since it was precipitated by trichloroacetic acid and by half-saturation with ammonium sulphate, and was non-diffusible. It was inactivated by heating at 50° for 5min. 7. It is possible that the guanine deaminase associated with particles differs from the soluble enzyme in its response to inhibitor.     

Halothane binding proteome in human brain cortex

Inhaled anesthetics bind specifically to a wide variety of proteins in the brain. This set of proteins must include those that contribute to the physiological and behavioral phenotypes of anesthesia and the related side effects. To identify the anesthetic-binding targets and functional pathways associated with these targets in human brain, halothane photolabeling and two-dimensional (2D) gel electrophoresis were used. Both membrane and soluble proteins from human temporal cortex were prepared. More than 300 membrane and 400 soluble protein spots were detected on the stained blots, of which 23 membrane and 34 soluble proteins were labeled by halothane and identified by mass spectroscopy. Their functional classification reveals five groups, including carbohydrate metabolism, protein folding, oxidative phosphorylation, nucleoside triphosphatase, and dimer/kinase activity with different correlative stringency. When network analysis of the interaction between these protein molecules is used, the weighted interaction accentuates the cellular protein components important in cell growth and proliferation, cell cycle and cell death, and cell-cell signaling and interactions, although no pathway was specific. This study provides evidence for multiple anesthetic binding targets and suggests potential pathways involved in their actions.     

Glucocorticoid receptors in developing rat brain and liver

Dexamethasone receptors were measured by conventional equilibrium steroid binding studies in rat liver and brain cytosol, during late prenatal and postnatal development, Receptor binding could be detected in both cytosol preparations as early as the 17th day of gestation. Receptor levels in the cytosol from intact animals reached adult values by the 1st day after birth in both tissues. Using adrenalectomized animals an increase which reached adult values was observed during the first postnatal week for liver and the second postnatal week for brain. At physiological concentrations of endogenous glucocorticoids depletion of receptor from the cytosol of intact animals was minimal at postnatal day 1 and reached adult levels by day 7. chromatographie analysis in DEAE-Sephadex A50 minicolumns of unactivated and activated receptor constituents revealed the same pattern as that of adult animals. Glucocorticoid receptor complex from developing liver and brain was shown to be capable of binding to isolated adult liver nuclei after in vitro activation. However full capacity, for nuclear binding was observed in vivo, after injection of inducing doses of [³H]dexamethasone: By the end of the first week after birth adult nuclear binding capacity was observed in experiments in vivo while values peaked during the second week, in both tissues studied.     

Glycine transport in rat brain and liver mitochondria

Transport of glycine by rat brain and liver mitochondria has been investigated by both [¹⁴C]glycine uptake and swelling experiments. Glycine enters mitochondria passively down its concentration gradient by a respiratory-independent carrier-mediated process. This view is supported by the following observations: (a) glycine inside the mitochondria reaches the incubation medium concentration; (b) mitochondria swell in the presence of isoosmotic solutions of glycine in a concentration-dependent fashion; (c) the uptake of glycine is not influenced by respiratory inhibitors such as KCN or by uncouplers such as carbonylcyanide p-trifluoromethoxyphenylhydrazone; (d) initial rates of uptake approach saturation kinetics, the apparent K_m of the rat brain mitochondria for glycine being 1.7 mM and that of the liver mitochondria being 5.7 mM; (e) the rate of swelling is inhibited by methylmalonate, propionate and, at pH 6.5, by mersalyl, and (f) uptake is inhibited by phosphoserine, methylmalonate and propionate, but not by alanine or proline.     

Causes and prevention of tamoxifen-induced accumulation of triacylglycerol in rat liver

Tamoxifen can induce hepatic steatosis in women. In this study, we wanted to elucidate the mechanism behind the tamoxifen-induced accumulation of triacylglycerol in liver in female rats, and we hoped to prevent this development by combination treatment with the modified fatty acid tetradecylthioacetic acid (TTA). The increased hepatic triacylglycerol level after tamoxifen treatment was accompanied by decreased acetyl-coenzyme A carboxylase (ACC) and FAS activities, increased glycerol-3-phosphate acyltransferase (GPAT) activity, and a tendency to increased diacylglycerol acyltransferase (DGAT) activity. The activities and mRNA levels of enzymes involved in beta-oxidation, ketogenesis, and uptake of lipids from liver were unaffected by tamoxifen, whereas the uptake of lipoproteins was unchanged and the uptake of fatty acids was decreased. Combination treatment with tamoxifen and TTA (Tam+TTA) normalized the hepatic triacylglycerol level and increased the activities of ACC, FAS, GPAT, and DGAT compared with tamoxifen-treated rats. The activities and mRNA levels of enzymes involved in beta-oxidation, ketogenesis, and uptake of lipids were increased after Tam+TTA treatment. In conclusion, tamoxifen increased the hepatic triacylglycerol level, probably as a result of increased triacylglycerol biosynthesis combined with unchanged beta-oxidation. The tamoxifen-induced accumulation of triacylglycerol was prevented by cotreatment with TTA, through mechanisms of increased mitochondrial and peroxisomal beta-oxidation.     

K(+)-p-nitrophenylphosphatase activity in rat brain and liver

K(+)-p-nitrophenylphosphatase (K(+)pNPPase) is the enzyme, which is considered to be involved in K(+)-dependent hydrolysis of the phosphoenzyme in the reaction cycle of Na(+), K(+)ATPase. The aim of our present study was to characterize some features of K(+)pNPPase in homogenates of the rat brain and liver. We determined p-nitrophenylphosphatase (pNPPase) activity in the presence of various ion combinations (Mg(2+)+ K(+), Mg(2+), K(+)). We found a higher total pNPPase activity in the brain (0.8+/-0.079 nkat/mg protein) than in the liver (0.08+/-0.01 nkat/mg protein). Contrary to the liver, the main part of the total brain activity was K(+)-dependent. The activity of K(+)pNPPase was significantly higher in cerebral cortex homogenates (0.86+/-0.073 nkat/mg protein) in comparison to those of the whole brain (0.57+/-0.075 nkat/mg protein). The specific K(+)pNPPase activity was two times higher in the isolated pellet fraction (0.911+/-0.07 nkat/mg protein), rich in synaptosomes, compared to the whole brain homogenate (0.57+/-0.075 nkat/mg protein). Our results demonstrate the high activity of K(+)pNPPase in the brain tissue and its distribution mainly into the pellet fraction, what might indicate a possible role of K(+)pNPPase in specific structures of the brain, e.g. in synaptosomes.     

Effect of biliary ligation on manganese accumulation in rat brain

Neurologic and radiologic disorders have been reported to occur in miners inhaling manganese (Mn)-laden dust and in humans receiving long-term parenteral nutrition. These abnormalities have been attributed to Mn intoxication because of elevated serum Mn concentrations. Because the liver, by way of the bile, is the major route of Mn excretion, it is possible that anything that decreases biliary excretion could increase accumulation of Mn in the brain. The purpose of this study was to determine whether biliary ligation would increase Mn accumulation in the brain of rats that were exposed to deficient or adequate amounts of dietary manganese. The first experiment had a 2 x 3 factorial design, two levels of Mn (0 or 45 μg/g diet) and three surgical treatments (control, sham, or bile-ligation). Animals were sacrificed 10 d after being fed⁵⁴Mn. In experiment 2, animals that had a sham operation or bile-ligation were sacrificed at 8 time points after being injected intraportally with⁵⁴Mn complexed to albumin. The biliaryligated animals had a significantly (p < 0.001) smaller percentage of the⁵⁴Mn in their brains (when expressed as a percentage of whole animal⁵⁴Mn) than the sham-operated animals. Mn deficiency had a similar effect. However, we did observe an increased accumulation of the radioisotope in the brain over time. Therefore, in short-term studies, biliary-ligated rats do not appear to be a good model for Mn accumulation in the brains of people with cholestatic liver disease. The U.S. Department of Agriculture, Agriculture Research Service, Northern Plains Area, is an equal opportunity/affirmative action employer and all agency services are available without discrimination.     

Synthesis of taurine in rat liver and brain in vivo

The in vivo formation of taurine and the analysis of labeled taurine precursors was examined in rat brain and liver at different times after an intracisternal injection of [³⁵S]cysteine and an intraperitoneal injection of [³H]cysteine, simultaneously administered. The distribution pattern of radioactivity was similar in liver and brain. Most of the labeling in both organs (85% in brain and 80% in liver) was recovered in glutathione (oxidized and reduced), cysteic acid, cysteine sulfinic acid, hypotaurine, cystathionine, and a mixed disulfide of cysteine and glutathione. The relative rates of labeling of cysteine sulfinic acid and taurine in liver and brain suggest than in vivo, liver possesses a higher capacity for taurine synthesis than brain. A small amount of [³H]taurine was detected in brain after intraperitoneal injection of [³H]cysteine. The time of appearance of this [³H]taurine as well as the fact that it occurs when [³H]cysteine is not detectable in brain or plasma suggests that it was probably not synthesized in brain from labeled precursors but formed elsewhere and transported into the brain through an exchange process.