Activity In Rat Liver Cytosol Research Articles

Purification and Characterization of Mg2+-Dependent Glycogen Synthase Phosphatase (Phosphoprotein Phosphatase IA) from Rat Liver

Phosphoprotein phosphatase IA, which represents the major glycogen synthase phosphatase activity in rat liver cytosol, has been purified to apparent homogeneity by chromatography on DEAE-cellulose, histone - Sepharose-4B and Sephadex G-100. The molecular weight of the purified enzyme was 40 000 by gel filtration and 48 000 by sodium dodecyl sulfate gel electrophoresis, Phosphatase IA is therefore a monomeric protein. When treated with 80% ethanol at room temperature, phosphatase IA underwent an inactivation which was totally prevented by 2 mM MgCl2. Catalytically, phosphatase IA has a preference for glycogen synthase D compared with phosphatases IB and II and obligatorily requires Mg2+ or Mn2+ for activity. Maximum activity was attained at 5 mM MgCl2. Since Mg2+ does not activate other phosphoprotein phosphatases in rat liver cytosol, we propose the term 'Mg2+-dependent glycogen synthase phosphatase' for phosphatase IA.

Inhibition of rat hepatic thyroxine 5'-monodeiodinase by propylthiouracil: relation to site of interaction of thyroxine and glutathione.

When rat liver cytosol, dialyzed free of glutathione, was chromatographed on Sephadex G-100 after incubation with 35S-propylthiouracil, 2 peaks of bound radioactivity were observed, 1 of which contained nearly all the thyroxine 5'-monodeiodinase activity in rat liver cytosol. Binding of propylthiouracil to this peak was inhibited by glutathione but not by thyroxine. Approximately 25% of 35S-propylthiouracil initially bound to the thyroxine 5'-monodeiodinating activity peak remained bound after dialysis, precipitation with trichloroacetic acid, and multipe extractions with ethanol, methanol, and chloroform, suggesting that binding was at least in part covalent. Dialysis studies showed that the presumed covalent binding of 35S-propylthiouracil to the thyroxine 5'-monodeiodinase peak could be inhibited by glutathione, dithioerythritol, and unlabelled propylthiouracil but not by oxidized glutathione or thyroxine. Conversely, thyroxine binding was unaffected by thiol compounds. We studied the kinetics of thyroxine 5'-monodeiodination by radioimmunoassay techniques using rat liver homogenates as source of enzyme and observed the dependence of enzymic reaction upon glutathione (Km = 2.4 mM). Propylthiouracil inhibited the reaction and this inhibition could be overcome with increasing glutathione concentrations. We conclude that the thiol-dependent thyroxine 5'-monodeiodinase is inhibited by propylthiouracil through its covalent binding, probably as mixed disulfide, to s site on the enzyme at which glutathione interacts either as a cosubstrate or reducing agent. This binding site is separate from the site at which thyroxine binds.

Studies on NADPH-dependent chloral hydrate reducing enzymes in rat liver cytosol

Chloral hydrate, a sedative hypnotic and also a major metabolite of trichloroethylene in higher animals, is reduced to trichloroethanol by liver extracts. The reducing activity in rat liver cytosol could be separated into four fractions [one NADH- (F 1) and three NADPH-dependent (F 2, F 3 and F 4)] by DEAE-cellulose column chromatography. By several procedures, F 2 was purified over 1000-fold and F 4 was purified over 600-fold from liver cytosol. As judged from polyacrylamide gel electrophoresis performed with and without the addition of sodium dodecylsulfate, the final preparations were essentially homogeneous. They differed in molecular weight, mobility on polyacrylamide gel electrophoresis, pH optimum, substrate specificity, and sensitivity to inhibitors. The molecular weights were estimated to be 36,000 and 32,500 for F 2 and F 4, respectively, by polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate. The estimation of molecular weights by thin-layer gel chromatography indicated that the enzymes were monomers. An examination of over thirty substrates revealed that both enzymes catalyzed the reduction of long-chain aliphatic, alicyclic and aromatic aldehydes as well as halogenated acetaldehyde. The F 2 enzyme acted on d-glucuronate, indicating that it was identical to the aldehyde reductase recently reported by other workers ( l-gulonate: NADP + 1-oxidoreductase EC 1.1.1.19). The F 4 enzyme, on the other hand, preferentially acted on C 24 3-ketosteroids.

Effect of thioacetamide, growth hormone or partial hepatectomy on spermidine acetylase activity of rat liver cytosol.

Rat liver cytosol extracts catalyzed the formation of monoacetylspermidine when incubated with acetyl-CoA and spermidine. This activity was enhanced 15-fold by administration of thioacetamide (150 mg/kg). The peak of activity occurred 18-24 h after treatment with the drug and then declined reaching control levels by 76 h. Previous studies have shown that ornithine decarboxylase activity was also greatly increased over this time period. Putrescine content in the liver was increased 80-90-fold at 18-24 h and then declined. Spermidine levels were decreased significantly over the period 12-24 h after thioacetamide treatment and then increased substantially at later times. These results are consistent with the hypothesis that, at early times after administration of thioacetamide, the increase in putrescine content is brought about both by decarboxylation of ornithine and by degradation of monoacetylspermidine. Spermidine acetylase activity was also measured in liver extracts prepared after two other physiological stimuli known to enhance ornithine decarboxylase activity were used. Both growth hormone treatment and partial hepatectomy produced an early 2-3-fold increase in the cytosolic spermidine acetylase activity.

Inhibition of urea-cycle activity by high concentrations of alanine.

1. Conditions are described in which high intracellular alanine concentrations inhibit urea-cycle flux in isolated hepatocytes. 2. Inhibition of urea-cycle flux by added alanine is DL-cycloserine-insensitive and is accompanied by an increase in intracellular citrulline and a decrease in ornithine. 3. Argininosuccinate synthetase (EC 6.3.4.5) activity in rat liver cytosol is inhibited by alanine in a competitive manner with respect to citrulline. It is concluded that this effects is the primary cause of inhibition of urea-cycle flux by alanine.

The influence of phenobarbital, 3-methylcholanthrene and 2,3,7,8-tetrachlorodibenzo- p-dioxin on glutathione s-transferase activity of rat liver cytosol

Specific activities and apparent Michaelis-Menten kinetic parameters were determined for glutathione (GSH) S-transferase activity (E.C. 2.5.1.18) in rat liver cytosol, towards styrene oxide (STOX), 1,2-butylene oxide (BOX) and 1-chloro-2,4-dinitrobenzene (CDNB) as electrophilic substrates, before and after pretreatment with the drug-metabolizing enzyme inducers phenobarbital (PB), 3-methylcholanthrene (MC) and 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD). The measured GSH S-transferase activities appear to obey Michaelis-Menten kinetics. In non-induced animals the apparent K m values of the transferase activities were equal for STOX vs GSH, but they differed by a factor of 2 for CDNB vs GSH and by a factor of 14 for BOX vs GSH. The apparent V max values in each combination of GSH and electrophilic substrate were equal, but differed by one order of magnitude for the mutual substrate combinations. Pretreatment of the rats with MC resulted in enhancement of all measured activities expressed in terms of cytosol protein, while TCDD only enhanced the activities expressed as per gram body wt. PB enhanced both activities when STOX was employed as substrate, but when CDNB was used as the substrate, only the activity per gram body wt increased. All pretreatments increased the V max values using CDNB as the substrate, while PB and MC had an enhancing effect using STOX; the V max using BOX was enhanced after TCDD administration only. The K m values using BOX as the substrate was lowered after MC pretreatment; TCDD pretreatment decreased the K m using STOX, while it increased the K m using CDNB. It is concluded that the GSH S-transferase system is inducible, but in contrast to the induction of the mixed function oxidase system, qualitative differences between the inducing effects of PB and MC were not observed. Use of TCDD as inducing agent, however. resulted in a different induction pattern, which may indicate that during induction with this agent different types of GSH S-transferases are involved.

Glutathione peroxidase: Inhibition by cyanide and release of selenium

Treatment of highly purified ovine erythrocyte glutathione peroxidase with KCN resulted in loss of enzyme activity and release of selenium from the enzyme. The inactivation by cyanide was time-dependent and the rate was strongly influenced by temperature, concentration of cyanide, oxidation state of the enzyme, and pH. The pH effect could be explained on the basis of the increasing proportion of cyanide ion with increasing pH. Inhibition could be prevented by prior reduction of the purified enzyme with glutathione, dithiothreitol, or dithionite. Oxidation with cumene hydroperoxide was necessary to demonstrate cyanide inhibition of glutathione peroxidase activity in rat liver cytosol. These observations explain why cyanide inhibition of glutathione peroxidase has not been noted previously and provide new approaches for studying the chemical nature of the enzyme-selenium.

Stabilization and characterization of the dexamethasone-binding proteins in rat liver cytosol

Conditions were worked out for maximal stabilization of dexamethasone binding activity of rat liver cytosol in the absence of the protective steroid ligand. Important stabilization factors are ionic strength, thiol-protecting agents, glycerol and pH. Maximal stability of the cytosol is observed in a buffer consisting of 20 mM Tris-HCl, pH 7.5, 50 mM KCl, 25 mM β-mercaptoethanol and 20% glycerol. Chromatography of cytosol on DEAE-cellulose revealed the existence of three dexamethasone receptors, binder DE-1, present in the flow-through fraction and binders DE-2 and DE-3, eluting from the column with salt concentrations of 100 and 190 mM, respectively. Binders DE-2 and DE-3 are not adsorbed on phosphocellulose at pH 7.5, whereas binder DE-1 is. All three receptors are retained to varying degrees on DNA-cellulose columns: binder DE-1 is eluted with salt concentrations of 270 mM, whereas binders DE-2 and DE-3 are eluted between 180 and 200 mM NaCl. The dexamethasone receptors also bind natural glucocorticoids, but to varying degrees, the highest binding being observed to binder DE-2. The receptors obtained after chromatography on DEAE-cellulose, but not on phosphocellulose, cannot be to an appreciable extent charged with dexamethasone.

Effects of local anaesthetics on phospholipases

1. 1. The effects of six local anaesthetics have been studied on the activities of soluble phospholipases A 2 (EC 3.1.1.4) and lysophospholipase (EC 3.1.1.5). 2. 2. Phospholipase A 2 activity in human seminal plasma towards sonicated radioactively-labelled phosphatidylethanolamine was slightly stimulated at low and inhibited at high concentrations of all anaesthetic compounds employed. The order of decreasing potency was chlorpromazine, dibucaine, tetracaine, lidocaine, cocaine and procaine. In line with previous findings, the mode of inhibition was seen to be competitive with respect to Ca 2+. 3. 3. Phospholipase A 2 activity in crude venom of Crotalus adamanteus was not affected or slightly stimulated by local anaesthetics up to 10 −2 M concentrations, when egg yolk was used as substrate. However, with sonicated radioactively-labelled phosphatidylcholine or phosphatidylethanolamine as substrate, stimulation of phospholipase activity was seen with all local anaesthetics up to 10 −2 M, the order of decreasing potency again being chlorpromazine, dibucaine, tetracaine, lidocaine, cocaine and procaine. The mode of stimulation was seen to be un-competitive with respect to substrate and probably independent of any involvement of Ca 2+. 4. 4. As in seminal plasma phospholipase A 2, the activity in crude Naja naja venom towards sonicated radioactively labelled phosphatidylcholine was stimulated at low and inhibited at high concentrations of dibucaine and chlorpromazine, for example. The mode of inhibition was seen to be competitive with respect to Ca 2+, whereas stimulation by the anaesthetic drugs was independent of Ca 2+. Binding between drug and enzyme was demonstrated by equilibration filtration of purified phospholipase A 2 of Naja naja venom through a Sephadex G 25-fine column, previously equilibrated with 0.5 mM radioactively labelled chlorpromazine. 5. 5. Lysophospholipase activity in rat liver cytosol towards radioactively labelled lysophosphatidylcholine was inhibited by all local anaesthetics used; the order of decreasing potency was chlorpromazine, dibucaine, tetracaine, cocaine, lidocaine and procaine. The inhibition was un-competitive with respect to substrate. 6. 6. The inhibitory and stimulatory potencies of the local anaesthetics employed closely parallel their lipid solubilities and anaesthetic potencies.

Further studies on the site and mechanism of action of [formula omitted] in rat liver

DCVC, but not DCVMP, inhibits lipoyl dehydrogenase activity of isolated rat liver mitochondria and glutathione reductase activity of rat liver cytosol. DCVC can be degraded to pyruvic acid and ammonia by the cytosolic fraction of rat liver. Metabolism of DCVC by the cytosolic fraction appears necessary for inhibition of glutathione reductase activity.

Identification and partial characterization of a rat liver cytosol protein with high affinity to RNA and DNA

The RNA-binding activity of rat liver cytosol has been separated into two fractions of high and low anionic migration at pH 8.3 in free-flow electrophoresis. 1. 1. The low-mobility peak contains five components which have been resolved using polyacrylamide disc electrophoresis in the presence of sodium dodecylsulfate. The polypeptide molecular weights are: 50 000 (Protein 1); 43 000 (Protein 2); 39 000 (Protein 3); 36 000 (Protein 4) and 23 000 (Protein 5). 2. 2. Only Protein 2 appears to be active in the standard RNA-binding assay. 3. 3. The same protein also binds to salmon sperm DNA at pH 8.3 in a fast reaction independent of temperature between 0 and 37°C. The resulting complex precipitates as visible fibrous structures. 4. 4. Amino acid compositions of Proteins 1, 3 and 5 isolated by density gradient centrifugation and Protein 2 recovered from the DNA complex have been determined.

Studies on the RNA-binding activity of rat liver cytosol

Labelled RNA's from rat liver nuclei or yeast were incubated with protein fractions derived from rat liver cytoplasm and the amount of protein-bound RNA was measured in a filter assay using cellulose nitrate membranes. 1. 1. An “RNA-binding factor” which is rather labile and tends to form insoluble aggregates was purified about 80-fold with the aid of free-flow electrophoresis and density gradient centrifugation at a pH of 8.5. 2. 2. The binding factor is confined to the high-speed supernatant of the cytoplasm and the purified preparation has a sedimentation coefficient of 9 S. It is free from nucleic acids and shows slightly basic properties. 3. 3. From its behaviour during the purification, it is concluded that the binding factor consists of not more than a few components which are possibly arranged in a complex structure. Disc electrophoresis at pH 4.5 of the most purified preparation revealed three discrete bands showing similar electrophoretic mobilities as Bands A, B, and C of the protein of nuclear informosomes. 4. 4. The binding reaction is presumably a nonenzymatic process mediated by electrostatic forces as it proceeds at a high velocity independent of temperature between 0 and 37°. 5. 5. In the presence of inorganic phosphate (0.01 M), SH-blocking chemicals as p-chloromercuribenzoate (10 −4 M), sodium deoxycholate or Triton WR 1339 (1%), the formation of the complex was inhibited.